i- i- m- %-'^:-^-^^j&'^W:. v r-i m% >.>i >M rn';«w- .iu.r^ >■ V ^■H / ir:> - ',' * «jip' )(Sjjm £2 >->-<.- BIOCHEMICAL BULLETIN ISSUED QUARTERLY BY THE COLUMBIA UNIVERSITY BIOCHEMICAL ASSOCIATION PRESS OF THE NEW ERA PRINTINQ COMPANY LANCASTEK, PA. BiocHEMiCAL Bulletin Edited, for the Columbia University Biochemical Association, by the EDITORIAL COMMITTEE: iJuly, igiz-June, 1913) ALFRED P. LOTHROP, Chairman, PAUL E. HOWE, Secretary, WILLIAM J. GIES, Treasurer, WALTER H. EDDY, MAX KAHN, EMILY C. SEAMAN, Oct., 1912-July, 191 3 NELLIS B. FOSTER, ARTHUR KNUDSON, CLAYTON S. SMITH, July-September, 19 12 F. G. GOODRIDGE, EDGAR G. MILLER, Jr., ETHEL WICKWIRE, TULA L. HARKEY, H. O. MOSENTHAL, LOUIS E. WISE, Oct., 1912-July, 1913 JOSEPH S. HEPBURN, JACOB ROSENBLOOM, JuIy-September, 1912 ALL OK THE StAFF OF THE BlOCHEMICAL DEPARTMENT OF COLUMBIA UnIVERSITY VOLUME II : Nos. 5-8 1912-1913 WITH EIGHT PORTRAITS, EIGHT PLATES AND TWO ADDITIONAL ILLUSTRATIONS LIBRARY NnvV YORK NEW YORK Columbia University Biochemical Association 1913 Entered as second-class matter in the Post Ofiice at Lancaster, Pa. MEMBERS OF THE COLUMBIA UNIVERSITY BIOCHEMICAL ASSOCIATION Honorary Members PROF, R. H. CHITTENDEN, First Director of ihe Columbia University De- partment of Biological (Physiological) Chemistry; Director of the Shef- field Scientific School of Yale University PROF. HUGO KRONECKER, Director of the Physiological Institute, Uni- versity of Bern, Switserland PROF. SAMUEL W. LAMBERT, Dean of the Columbia University School of Mediane DR. JACQUES LOEB, Member of the Rockef eller Institute for Medical Re- search; Head of the Department of Experimental Biology PROF. ALEXANDER SMITH, Head of the Department of Chemistry, Co- lumbia University Corresponding Members PROF. LEON ASHER, University of Bern, Switserland PROF. FILIPPO BOTTAZZI, University of Naples, Italy PROF, ROBERT B. GIBSON, University of the Philippines, P. I. PROF. VLADIMIR S. GULEVIC, University of Moscow, Russia PROF. W. D. HALLIBURTON, King's College, London PROF. S. G. HEDIN, University of Upsala, Sweden PROF. FREDERICO LANDOLPH, University of La Plata, Argentina PROF. A. B. MACALLUM, University of Toronto, Canada PROF. D. McCAY, Medical College, Calcutta, India PROF. C. A. PEKELHARING, University of Utrecht, Holland PROF. S. P. L. SÖRENSEN, Carlsberg Laboratory, Copenhagen, Denmark Members Resident in New York City Brooklyn Botanic Garden. — C. Stuart Gager. College of the City of New York. — Wm. B. Boyd, Louis J. Curtman, Benj. G. Feinberg, A. J. Gold färb. Columbia University: Departments. — Anatomy: Alfred J. Brown, H. von W. Schulte; Bacteriology: James G. Dwyer; Biological Chemistry: Walter H. Eddy, Nellis B. Foster, William J. Gies, F. G. Goodridge, Tula L. Harkey, Joseph S. Hepburn, Benjamin Horowitz, Paul E. Howe, Max Kahn, Arthur Knudson, Alfred P. Lothrop, Edgar G. Miller, Jr., H. O. Mosenthal, Emily C. Seaman, Chris. Seifert, Ethel Wickwire, Louis E. Wise; Botany: E. R. Alten- burg, C. A. Darling, Fred D. Fromme; Cancer Research: W. H. Woglom; Chem- istry: A. M. Buswell, R. P. Calvert, Gustave Egloff, H. L. Fisher, P. W. Punnett, A. W. Thomas; Clinical Pathology: Edward Cussler, Peter Irving; Diseases of Children: Herbert B. Wilcox; Gynecology: Wilbur Ward; Mediane: T. Stuart Hart, I. Ogden Woodruff; Pathology: B. S. Oppenheimer, Alwin M. Pappen- heimer; Pharmacology: Charles C. Lieb; Physiology: Russell Burton-Opitz, Donald Gordon, Leander H. Shearer, Wm. K. Terriberry; Surgery: Hugh Auchincloss, William Darrach, Rolfe Kingsley, Adrian V. S. Lambert, F. T. Van Buren, Jr. ; Therapeutics: Maximilian Schulman; University Physician: iv Members resident in New York (con.) Wm. H. McCastline; Vanderbilt Clinic: F. Morris Class, Julius W. Weinstein; Zoology: H. B. Goodrich, John D. Haseman, H. J. Muller, Charles Packard. Colleges. — Barnard: Helene M. Boas, Ella H. Clark, Ruth S. Finch, Louise H. Gregory; College of Pharmacy: Charles W. Ballard; Teachers College: Mary G. McCormick, Mrs. A. P. McGowan, Sadie B. Vanderbilt. Students. — Graduate: Cora J. Beckwith, Sidney Born, O. C. Bowes, Helen B. Davis, Mary C. de Garmo, Frank R. Eider, Louis J. Hirschleifer, Mildred A. Hoge, Shojiro Kubushiro, Victor E. Levine, Darwin O. Lyon, W. A. Perlzweig, Edward Plaut, Geo. S. Rosenthal, Edward C. Stone, Fred L. Thompson, Jennie A. Walker, Charles Weisman, C. A. Wells, Isabel Wheeler. — Teachers College: Anna M. Connelly, Ula M. Dow, Ada M. Field, Helen McClure, Alice H. McKinney, Elizabeth Rothermel, Mary B. Stark, Helen B. Thompson. — Medical: Louis Berman, Ernst Boas, David C. Bull, Will H. Chapman, Robert T. Corry, Calvin B. Coulter, Joseph Felsen, Joseph Goldstone, Julius Gottesman, Leon M. Herbert, Martin Holzman, Walter F. Hume, Julius Hyman, M. V. Miller, Nathan Rosenthal, A. V. Salomon, Harry J. Seiflf, Jacob Shulansky, H. J. Spencer, Henry A. Sussman, Wm. W. Tracey, Grover Tracy. CoRNELL Univeesity Medical COLLEGE. — Stanley R. Benedict, Ernest D. Clark, Robert A. Cooke, Jessie A. Moore, Charles R. Stockard, Geo. W. Vandegrift. EcLECTic Medical College. — David Alperin. Harriman Research Laboratory. — Marston L. Hamlin. Hospitals. — Babies': Morris Stark; Bellevue: Edward C. Brenner, Edward M. Colie, Jr., Ralph W. Lobenstine; Beth Israel: Charles J. Brim and Alfred A. Schwartz; City: Henry H. Janeway, Louis Pine; Flower: Henry L. Weil; Flushing: Eimer W. Baker; General Memorial: Clinton B. Knapp; German: H. G. Baumgard, Alfred M. Hellman, Melvin G. Herzfeld, Frederick B. Humphries, Charles H. Sanford, Fred S. Weingarten; Jewish: Abraham Ravich; Lebanon: Samuel Gitlow, M. J. Gottlieb, William Weinberger; Lutheran: Daniel R. Lucas; Mt. Sinai: George Baehr, Samuel Bookman, Leo Buerger, Burrill B. Crohn, Simon S. Friedman, David J. Kaliski, John L. Kantor, Leo Kessel, Reuben Otten- berg, Harry Wessler; TV. Y.: James C. Greenway, Ralph G. Stillman; N. Y. Nursery and Child's: Oscar M. Schloss; Presbyterian: Herbert S. Carter, Russell L. Cecil, Arthur W. Swann; Roosevelt: J. Buren Sidbury; St. Luke's: Norman E. Ditman, Edward C. Kendali, W. S. Schley, Chas. H. Smith. Long Island Medical College. — Matthew Steel. MoNTEFioRE HoME. — Isidor GrecHwald. Museum of Natural History. — Louis Hussakof, Israel J. Kligler. N. Y. Aquarium. — Raymond C. Osburn. N. Y. Association for Improving the Condition of the Poor. — Donald B. Armstrong. N. Y. Botanical Garden. — Fred J. Seaver. N. Y. City Department of Education. — Boys" High School: Frank T. Hughes; Brooklyn Training School: C. A. Mathewson ; Commercial High School: ' W. J. Donvan, B. C. Gruenberg, Edgar F. Van Buskirk; DeWitt Clinton High School: Frank M. Wheat; Eastern District High School: Gertrude S. Burling- ham; Girls' High School: Marguerite T. Lee; High School of Commerce: Harvey B. Clough, Fred W. Hartwell; Jamaica High School: Ella A. Holmes, Charles H. Vosburgh; Manual Training High School: Anna Everson; Morris Members resident in New York (con.) High School: Charles A. Wirth; Newtown High School: Nellie P. Hewins; Wadleigh High School: Helen Gavin, Elsie A. Kupfer, Helen G. Russell, Helen S. Watt. N. Y. City Department of Health. — Charles F. Bolduan, Alfred F. Hess. N. Y. City Normal College. — Beatrix H. Gross. N. Y. Eye and Ear Infirmary. — Harold M. Hays. N. Y. Milk Committee. — Philip Van Ingen. N. Y. Polyclinic Medical School. — Jesse G. M. Bullowa, Mabel C. Little. Post Graduate Medical School. — Louis E. Bisch, Arthur F. Chace. Pratt Institute. — Grace MacLeod. Rockefeller Institute. — Alfred E. Cohn, George W. Draper, Frederic M. Hanes, Michael Heidelberger, Gustave M. Meyer. Russell Sage Institute of Pathology. — Eugene F. DuBois. TuRCK Institute. — Anton R. Rose. Vettin School. — Laura I. Mattoon. E. V. Delphey, 400 West 57th Street, Manhattan; Leopold L. Falke, 5316 Thirteenth Avenue, Brooklyn; Mabel P. Fitzgerald, 416 East 6sth Street, Man- hattan; Abraham Gross, c/o Arbuckle Sugar Co., Brooklyn; Alfred H. Kropff, 619 Kent Avenue, Brooklyn. Non-Resident Members Agnes Scott College (Decatur, Ga.). — Mary C. de Garmo. Allegheny General Hospital (Pittsburgh). — James P. McKelvy. Carnegie Institution (Cold Spring Harbor, L. I.). — Ross A. Gortner. Cornell University (Ithaca). — Jean Broadhurst. Drake University Medical School (Des Meines, la.). — E. R. Posner. Forest School (Biltmore, N. C). — Homer D. House. Iowa University Hospital (Iowa City). — Louis Baumann. Isolation Hospital (San Francisco, Cal.). — L. D. Mead. Jefferson Medical College (Phila.). — P. B. Hawk, Edward A. Spitzka. Johns Hopkins University (Baltimore). — John Howland, W. M. Kraus, Burton E. Livingston, Edwards A. Park. Lehigh University (Bethlehem, Pa.). — William H. Welker. Leland Stanford University (Palo Alto, Cal.). — Hans Zinsser. MacDonald College (Quebec). — Kathryn Fisher. Mass. Agricultural College (Amherst). — H. D. Goodale. New Mexico Agricultural College (State College). — R. F. Hare. N. J. Agricultural Experiment Station (New Brunswick). — Carl A. Schwarze, Guy West Wilson. N. Dakota Agricultural College (Agricultural College). — H. L. White. Ohio Agricultural Experiment Station (Wooster). — A. D. Selby. Princeton University (N. J.). — E. Newton Harvey. Psychopathic Hospital (Boston). — Herman M. Adler. Rensselaer Polytechnic Institute (Troy, N. Y.). — Fred W. Schwartz. Rochester A and M Institute. — Elizabeth G. Van Hörne. Secondary Schools. — Brockport State Normal School (N. Y.) : Ida C. Wads- worth; Hermon High School (N. Y.) : Sidney Liebovitz; Indiana State Normal School (Terre Haute): Roscoe R. Hyde; Ingleside School (New Milford, Conn.) : Mary L. Chase; Knox School (Tarrytown, N. Y.) : Clara G. Miller; New Bedford Industrial School (Mass.): Constance C. Hart; North Texas vi Non-resident members (con.) State Normal School (Benton) : Blanche E. Shaffer; Passate High School (N. J.) : Hazel Donham, Helene M. Pope; Rochester High School (N. Y.) : David F. Renshaw; State Normal School (Truro, N. S.) : Blanche E. Harris. Texas A and M College (College Station). — M. K. Thornton. Trinity College (Hartford, Conn.). — Max Morse, R. M. Yergason. TuLANE University (New Orleans, La.). — Allan C. Eustis. U. S. Department of Agriculture (Wash.). — Carl L. Aisberg, W. N. Berg, H. E. Buchbinder, William Salant, Clayton S. Smith. U. S. Food and Drug Inspection Laboratory (Phila.). — Harold E. Woodward. U. S. FooD- Research Laboratory (Phila.). — Joseph S. Hepburn. University of Alabama Medical School (Birmingham). — Richard A. Bliss. University of California (Berkeley). — William T. Home, University of Chicago. — Mathilde Koch. University of Georgia Medical School (Atlanta). — ^William D. Cutter. University of Illinois (Urbana). — George D, Beal, Isabel Bevier, A. D. Emmett. University of Indiana (Bloomington). — Clarence E. May. University of Kentucky (Louisville). — Mary E. Sweeny. University of Manitoba (Winnipeg, Can.). — A. T. Cameron. University of Michigan (Ann Arbor). — A. Franklin Shull. University of Montana (Missoula). — J. E. Kirkwood. University of Pennsylvania (Phila.). — A. N. Richards. University of Porto Rico (Las Pietras). — L. A. Robinson. University of Tennessee (Memphis). — Edwin D. Watkins. University of Texas (Austin). — Mary E. Gearing, Anna E. Richardson. University of Toronto (Canada). — Olive G. Patterson. i University of Utah (Salt Lake City). — H. A. Mattill. University of Wisconsin (Madison). — ^W. H. Peterson. Vassar College ( Poughkeepsie, N. Y.). — Winifred J. Robinson. Washington State College (Pullman). — Josephine T. Berry, Louise McDanell. Wesleyan University (Middletown, Conn.). — David D. Whitney. West Pennsylvania Hospital (Pittsburgh). — J. Bronfen Brenner, Jacob Rosenbloom. Williams College (Williamstown, Mass.). — John S. Adriance, Yale University (New Haven, Conn.). — Lorande Loss Woodruff. Albert H. Allen, Saranac Lake, N. Y. ; Emma A. Buehler, Newark, N. J. ; George A. Geiger, West Orange, N. J. ; Edward G. Griffin, Albany, N. Y. ; F. C. Hinkel, Utica, N. Y.; Cavalier H. Joüet, Roselle, N. J.; A. E. Olpp, West Hoboken, N. J. ; Adeline H. Rowland, Pittsburgh, Pa. ; William A. Taltavall, Redlands, Cal. ; David C. Twichell, Saranac Lake, N. Y. Vll EDITORS OF THE BIOCHEMICAL BULLETIN The editorial committee with the coUaboration of the members and the SPECIAL CONTRIBUTORS: DR. JOHN AUER, Rockef eller Institute for Medical Research PROF. WILDER D. BANCROFT, Cornell University, Ithaca DR. WALTER L. GROLL, Elizabeth Steel Magee Hospital, Pittshurgh, Pa. DR. CHARLES A. DOREMUS, 55 W. 53d St., New York City DR. ARTHUR W. DOX, Iowa State College Agric. Experiment Station, Arnes PROF. JOSEPH ERLANGER, Washington Univ. Medical School, St. Louis DR, LEWIS W. FETZER, U. S. Dep't of Agriculture, Washington, D. C. PROF. MARTIN H. FISGHER, University of Cincinnati DR. MARY LOUISE FOSTER, Smith College, Northampton, Mass. PROF. J. E. GREAVES, Utah Agricultural College, Logan DR. V. J. HARDING, McGill University, Montreal, Canada DR. R. H. M. HARDISTY, McGill University, Montreal, Canada DR. J. A. HARRIS, Carnegie Sta. for Exp. Evolution, Cold Spring Harbor, L. I. DR. K. A. HASSELBALCH, Einsen Institute, Copenhagen, Denmark PROF. G. O. HIGLEY, Ohio Wesleyan University, Delaware DR. VERNON K. KRIEBLE, McGill University, Montreal, Canada PROF. FRANCIS E. LLOYD, McGill University, Montreal, Canada PROF. JOHN A. MANDEL, A''. Y. Univ. and Bellevue Hospital Med. College PROF. ALBERT P. MATHEWS, University of Chicago PROF. SHINNOSUKE MATSUNAGA, University of Tokyo, Japan PROF. LAFAYETTE B. MENDEL, Yale University PROF. VICTOR C. MYERS, N. Y. Post-Graduate Med. School and Hospital DR. THOMAS B. OSBORNE, Conn. Agric. Experiment Station, New Haven DR. AMOS W. PETERS, The Training School, Vineland, N. J. PROF. R. F. RUTTAN, McGill University, Montreal, Canada DR. E. E. SMITH, 50 East 4ist St., New York City DR. A. E. SPAAR, City Hospital, Trincomalee, Ceylon PROF. UMETARÖ SUZUKI, University of Tokyo, Japan MISS ANNA W. WILLIAMS, University of Illinois, Urbana, III. PROF. E. WINTERSTEIN, Polytechnic Institute, Zürich, Switserland DR. JULES WOLFF, Pasteur Institute, Paris vm OFFICERS OF THE COLUMBIA UNIVERSITY BIOCHEMICAL ASSOCIATION 1912-1913 HONORARY OFFICERS Post President (1910-1912) : Prof. Alfred N. Richards, University of Pennsylvania, Phila. President: Prof. Philip B. Hawk, Jefferson Medical College, Phila. Vice Presidents: Dr. Herman M. Adler, Psychopathie Hospital, Boston, Mass. Prof. Allan C. Eustis, Tulane University, New Orleans, La. Miss Olive G. Patterson, Toronto University, Toronto, Can. Prof. Winifred J. Robinson, Vassar College, Poughkeepsie, N. Y. Prof. Lorande Loss Woodruff, Yale University, New Haven, Conn. ACTIVE OFFICERS President, Dr. Walter H. Eddy. Vice President, Prof. Stanley R. Benedict. Secretary, Dr. Alfred P. Lothrop. Treasurer, Prof. William J. Gies. Executive Commiftee — Prof. Stanley R. Benedict, Dr. Wal- ter H. Eddy, Dr. Nellis B. Foster, Prof. William J. Gies, Dr. Frederic G. Goodridge, Prof. Paul E. Howe and Dr. Alfred P. Lothrop. Editorial Committee: See the title page. ix SUMMARY OF CONTENTS: VOL. II, Nos. 5-8. No. 5. September, 19 12. PAGE Ernst ScHxn-ZE. Biography and Bibliography (with portrait). Ernst Winterstein. i A ReSUME OF THE LiTERATURE ON InOSITE-PhoSPHORIC AciD ("PhYTIN"), WITH Special Reference to the Relation of that Substance to Plants. Anton Richard Rose 21 A New Type of Artificial Cell Suitable for Permeability and Other BioCHEMicAL Studies. E. Ncwtou Hürvey 50 On a New Function of the Catalyzer Called Peroxidase and on the Biochemical Transformation of Orcin into Orcein. Jules Wolff . ... 53 Studies of Diffusion through Rubber Membranes : 1. Preliminary Observations on the Diffusibility of Lipins and Lipin- soluble Substances. William J. des SS 2. Dififusibility of Lipins from Ether through Rubber Membranes into Ether. Jacob Rosenbloom 64 3. Diffusibility of Protein through Rubber Membranes, with a Note on the Disintegration of Collodion Membranes by Common Ethyl Ether and Other Solvents. William H. Welker 70 4. The Comparative DiffusibiHty of Various Pigments in Different Sol- vents. George D. Beal and George A. Geiger 78 The Colloidal Nitrogen in the Urine from a Dog with a Tumor of the Breast. Max Kahn and Jacob Rosenbloom 87 General Aspects of Fasting. Paul E. Hozue 90 The Physico-chemical Basis for the Contraction of Striated Muscle: 2. Surface Tension. William N. Berg loi A Study of Some Protein Compounds. Walter H. Eddy iii Effects of Intraperitoneal Injections of Epinephrin on the Partition OF Nitrogen in Urine from a Dog. Jacob Rosenbloom and William Weinberger. 123 The Biochemical Society, England. W. D. Halliburton 128 Proceedings of the Section (II) ON DiETETic Hygiene and Hygienic Physiology OF the 15TH International Congress on Hygiene and Demography, with Abstracts of Some of the Papers. Lafayette B. Mendel, Secretary. 129 Program of the Proceedings of the Section on Biochemistry Including Pharm acology (viii, d), of the 8th International Congress of Applied Chemistry. John A. Mandel, Secretary 150 Proceedings of the Sixth Scientific Meeting of the Columbia Univer- siTY Biochemical Association. Alfred P. Lothrop, Secretary 156 Biochemical News, Notes and Comment: General 188 X PAGE Columbia University Biochemical Association 200 Columbia Biochemical Department 201 Editori ALS : Ernst Schulze 205 Important though Unknown Factors in Nutrition 205 The Coming of Age of the Babcock Test 207 Organotherapy 208 Biochemical Society, England 20g "Baustein" or " Construction Unit"? 209 " Splitting Products " or Cleavage Products ? 209 A Rare Compliment 210 X-rays 210 No. 6. January, 19 13. PAGE Carl L. Alsberg. Biography and Bibliography (with portrait). H.M.A.. 211 A Differential Chemical Study of Glucoses from a Case of Pancreatic Diabetes. Frederic Landolph 217 The Detection of Aceto-acetic Acid by Sodium Nitroprussid and Ammonia. V. J. Harding and R. F. Ruttan. 223 Ortho-tolidin as an Indicator for Occult Blood. R. F. Ruttan and R. H. M. Hardisty. 225 Synthetical Properties of Emulsin. Vernon K. Krieble 227 On the Occurrence of Nicotinic Acid in Rice Bran. U. Suzuki and S. Matsunaga. 228 A Study of the Influence of Cancer Extracts on the Growth of Lupin Seedlings. Jacob Rosenhloom 229 The Biochemistry of the Female Genitalia: 3. A Quantitative Study of Certain Enzymes of the Ovary, Uterus, and Bladder, of Pregnant and Non-pregnant Sheep. Thuisco A. Erpf-Lefkovics and Jacob Rosenbloom. 233 4. On the Absence of Certain Enzymes from the Human Chorion. Jacob Rosenbloom. 236 A Department of Biochemical Research at Vineland, New Jersey. Arnos W. Peters. 238 Biochemistry in New York Twenty Years Ago. E. E. Smith 243 Immunity in Some of its Biochemical Aspects. Charles Frederick Bolduan. 247 A Plan for the Organization of the American Biological Society. Albert P. Mathews. 261 Organization of the Federation of American Societies for Experimental BlOLOGY, COMPRISING THE AmERICAN PhYSIOLOGICAL SoCIETY, AmERICAN Society of Biological Chemists, and American Society for Pharma- COLOGY and Experimental Therapeutics. John Auer 269 Annual Meetings of the Organizations Comprising the Federation of American Societies for Experimental Biology : I. The American Physiological Society. Joseph Erlanger, Acting Secretary. 271 PAGB 2. The American Society of Biological Chemists. Alfred N. Richards, Secretary. 275 3. The American Society for Pharmacology and Experimental Thera- peutics. John Aner, Secretary 27g Meeting of the American Society of Animal Nutrition (American Society of Animal Production) . Lewis W. Fetzer 282 Proceedings of the Eighth Scientific Meeting of the Columbia Univer- siTY BiocHEMicAL ASSOCIATION. Alfred P. Lothrop, Secretary ;.. 284 Folio Microbiologica. C. A. Pekelharing 297 BiocHEMiCAL Bibliography AND Index. William J. Gies 298 BiocHEMicAL News, Notes and Comment: General 307 Columbia University Biochemical Association 321 Columbia Biochemical Department 324 Editorials : New Plan of Quarterly Issue of the Bulletin 329 Carl L. Aisberg 329 Stock Poisoning Due to Spoiled Silage. Help ! 330 Demand for Biological Chemists in the Hospitals 330 Federation of American Societies for Experimental Biology 331 Electrons 332 No. 7. April, 19 13. PAGE Heinrich Ritthausen (Portrait) 334 Appreciation. Thomas B. Osborne 335 Bibliography. Lewis W. Fetzer 339 Dinner to Professor Chittenden: Testimonial by his Pupils. '94S 349 Society for Experimental Biology and Medicine: Tenth Anniversary Meeting and Dinner. Nineteen O. Three 358 Methods for the Electrometric Determination of the Concentration of Hydrogen Ions in Biological Fluids. K. A. Hasselbalch 367 A Method for the Determination of Tryptophan Derived from Pro- teins. Jesse A. Sanders and Clarence E. May 373 Physical Chemistry of Muscle Plasma. Filippo Bottazzi 379 Fasting Studies : IL A Note on the Composition of Muscle from Fast- iNG DoGs. H. C. Biddle and Paul E. Howe 386 SoME Notes on the Form of the Curve of Carbon-dioxide Excretion Re- suLTiNG from Muscular Work Following Forced Breathing. G. O. Higley. 390 The Influence of Barometric Pressure on Carbon-dioxide Excretion in Man. G. O. Higley 393 The Relation of Acapniato Shock, andaConsideration of the Mechan- iCAL Effects of Artificial Hyper-respiration upon the Circulation. Henry H. Janeway and Ephraim M. Ewing. 403 Cleavage of Pyromucuric Acid by Mold Enzymes. Arthur W. Dax and Ray E. Neidig. 407 Analysis of the Ash of the Castor Bean. Marston Lovell Hamlin 410 Notes on the Chemical Nature of the " Tannin Masses " in the Fruit of the Persimmon. Ernest D. Clark 412 xü PAGE HisTON AND iTS Prep ARATION. Walter H. Eddy 419 DiD VON Wittich Antedate Ostwald in the Definition of Enzyme Action ? William N. Berg. 441 The Biochemical Society, England 446 Scientific Proceedings of the Columbia University Biochemical Asso- ciation. Alfred P. Lothrop, Secretary 452 Biochemical Bibliography and Index. William J. Gies 470 Biochemical News, Notes and Comment: General 476 Columbia University Biochemical Association 484 Editorials : Biochemical Society, England 487 The Bleached Flour Decision 487 Occupational Diseases in Chemical Trades 488 The Mathews Plan for an American Biological Society 490 Antigens 508 No. 8. July, 19 13. PAGE An Investigation to Determine the Accuracy of a Modified Meigs Method for the Quantitative Determination of Fat in Milk, with A Description of an Improved Form of Apparatus. Walter Lewis Croll. 509 The Occurrence of Arsenic in Soils. /. E. Greaves 519 Further Notes on the Relationship between the Weicht of the Sugar Beet and the Composition of its Juice. /. Arthur Harris and Ross Atken Gortner. 524 Note on the Relationship between Barometric Pressure and Carbon- dioxide ExcRETioN IN Man. /. Arthur Harris 530 The Bleached Flour Decision. Ross Aiken Gortner 532 Emil Chr. Hansen Fund. 5". P. L. Sörensen 535 Biological Chemistry in the Philippines. Robert Banks Gibson 536 Doctorates in Biological Chemistry. Conferred by American Univer- sities, 1912-13. P. H. D 538 Scientific Proceedings of the Columbia University Biochemical Asso- ciation. Alfred P. Lothrop, Secretary 541 Biochemical Bibliography and Index. William J. Gies 559 Biochemical News, Notes and Comment: General 567 Columbia University Biochemical Association 574 Columbia Biochemical Department 578 Editorials : Peroxides and Nitrites in Plants 582 Mathews Plan for the Organization of an American Biological Society. 582 Crystals 588 Index: Volume II. (Includes names of authors, and impersonal and per- sonal subj ects) 589 Title Page for Volume II, with Summary of Contents, List of Illus- TRATIONS, ETC i-Xvi . xiii Alphabetic list of authors named in the foregoing summary of Contents (See author index — page 589 — for additional names of authors of abstracts, quotations, comment, etc.) Adler, HM, 211 AuER, J, 269, 279 Beal, GD, 78 Berg, WN, ioi, 441 BiDDLE, HC, 386 BOLDUAN, CF, 247 BoTTAzzi, F, 379 Clark, ED, 412 Croll, WL, 509 Dox, AW, 407 Eddy, WH, III, 419 Erlanger, J, 271 Erpf-Lefkovics, TA, 233 EwiNG, EM, 403 Fetzer, LW, 282, 339 Geiger, GA, 78 GiBSON, RB, 536 Gies, WJ, 5s, 298, 349, 358, 470, 559 GoRTNER, RA, 524, 532 Greaves, JE, 519 Halliburton, WD, 128 Hamlin, ml, 410 Harding, VJ, 223 Hardisty, RHM, 225 Harris, JA, 524, 530 Harvey, EN, 50 Hasselbalch, KA, 367 HiGLEY, GO, 390, 393 Howe, PE, 90, 386 Janeway, HH, 403 Kahn, M, 87 Kribble, VK, 227 Landolph, F, 217 LoTHROP, AP, 156, 284, 452, 541 Mandel, JA, 150 Mathews, AP, 261 Matsunaga, S, 228 May, CE, 373 Mendel, LB, 129 Neidig, RE, 407 OsBORNE, TB, 335 Pekelharing, CA, 297 Peters, AW, 238 P. H. D., 538 Richards, AN, 275 Rose, AR, 21 ROSENBLOOM, J, 64, 87, 123, 229, 233, 236 Ruttan, RF, 223, 225 Sanders, JA, 373 Smith, EE, 243 Sörensen, SPL, 535 Suzuki, W, 228 Weinberger, W, 123 Welker, WH, 70 Winterstein, E, i WOLFF, J, 53 XIV LIST OF ILLUSTRATIONS Eight portraits, eight plates (inserts), and two additional illustrations No. 5- SEPTEMBER, 1912 PAGE Portrait. Ernst Schulze i Plate I. Structure of muscle fibrils (Berg) 107 Portrait. Paul E. Howe 201 No. 6. JANUARY, 1913 Portrait. Carl L. Aisberg 211 Plate 2. Receptors of three kinds (Bolduan) 254 No 7. APRIL, 1913 Portrait. Heinrich Ritthausen 335 Portrait. Russell H. Chittenden 349 Engrossed Greetings to Prof. Chittenden by his colleagues of the Governing Board of th^^effield Scientific School 351 Faces of the gol(^medal presented to Prof. Chittenden by the National Insti- tute of Social Sciences 353 Portrait (group). Testimonial dinner to Prof. Chittenden by his pupils, Mar. I, 1913 355 Portrait. Samuel J. Meltzer 359 Portrait (group). Dinner of the Society for Experimental Biology and Medicine, tenth anniversary, Feb. 19, 1913 363 Plate 3. Apparatus for the electrometic determination of the concentration of hydrogen ions (Hasselbalch) 371 Plate 4. Curve of carbon-dioxide excretion resulting from muscular work after forced breathing. (Higley) 390 Plate 5. Influence of barometric pressure on the excretion of carbon-dioxide (Higley) 396 Plate 6. Micro-Kjeldahl apparatus (Morse) 458 No. 8. JULY, 1913 Plate 7. Apparatus for use with the Meigs method for the determination of fat in milk (Croll) 517 Plate 8. Relationship between the weight of the sugar beet and the compo- sition of its juice (Harris and Gortner) 526 XV Vol. II September, 1912 No. 5 Biochemical Bulletin Edited, for the Columbia University Biochemical Association, by the EDITORIAL COMMITTEE: ALFRED P. LOTHROP, Chöirman, PAUL E. HOWE, Secretary, WILLIAM J. GIES, Treasurer, WALTER H. EDDY, EDGAR G. MILLER, JR., NELLIS B FOSTER, HERMAN O. MOSENTHAL, FREDERIC G. GOODRIDGE, JACOB ROSENBLOOM, TULA L. HARKEY, EMILY C. SEAMAN, JOSEPH S. HEPBURN, CLAYTON S. SMITH, ARTHUR KNUDSON, ETHEL WICKWIRE, all of the Staff of the Biochemical Department of Columbia University. CONTENTS PAGB Ernst Schulze. Biography and Bibliography (with portrait) Ernst Winterstein i A ReSUME of the LiTERATURE OX InOSITE-PhOSI'HORIC AcID (" PhYTIN "), WITH Special Reference to the Relation of that Substaxce to Plants. Anton Richard Rose 21 A New Type of Artificial Cell Suitable für Permeability and other Bio- chemical Studies. E. Newton Harvey . 50 On a New Function of the Catalyzer Called Peroxidase and on the Bio- chemical Transformation of Orcin into Orcein. ßiles Wolff. 53 Studies of Diffusion Through Rubber Membranes : i. Preliminary observations on the diffusibility of lipins and lipin-soluble sub- stances. Williajn J. Gies 55 2. Diffusibility of lipins from ether through rubber membranes into ether. Jacob Rosenbloom 64 3. Diffusibility of protein through rubber membranes, with a note onthe dis- integration of collodion membranes by common ethyl ether and other solvents. William H. Welker 7° 4. The comparative diffusibility of various pigments in different solvents. George D. Beal and George A. Geiger 78 The Colloidal Nitrogen in the Urine from a Dog with a Tumor of the Breast. Max Kahn and Jacob Roseitbloom 87 General Aspects of Fasting. Paul E. Hoive 90 The "Physico-Chemical Basis for the Contraction of Striated Muscle: 2. Surface tension (with plate i). William N. Berg lOi A Study of some Protein Compounds. Walter H. Eddy iii Effects of Intraperitoneal Injections of Epinephrin on the Partition of Nitrogen IN Urine from A Dog. Jacob Rosenbloom and William Weinberger 123 The Biochemical Society, England. W. D. Halliburton 128 PrOCEEDINGS of the SeCTION (II) ON DiETETIC HYGIENE AND HYGIENIC PhYSI- ology of the 15TH International Congress on Hygiene and Demog- raphy, with Abstracts of some of the Papers. Lajayette B. Mendel, Secretary 129 Program of the Proceedings of the Section on Biochemistry Including Pharmacology (viii, d), of the Sth International Congress of Applied Chemistry. John A. Mandel, Secretary 150 Proceedings of the Sixth Scientific Meeting of the Columbia University Biochemical Association. Alfred P. Lothrop, Secretary 156 Biochemical News, Notes and Comment 188 Editorials 205 NEW YORK Columbia University Biochemical Association. Entered as second-class matter in the Post Office at Lancaster, Pa. Honorary Members of the Columbia University Biochemical Association PROF. R. H. CHITTENDEN, First Director of the Columbia University De- partment of Biological (Physiological) Chemistry; Director of the Shef- field Scientific School of Yale University PROF. SAMUEL \V. LAMBERT, Dean of the Columbia University School of Medicine PROF. ALEXANDER SMITH, Head of the Department of Chemistry, Co- lumbia University Editors of the Biochemical Bulletin EDITORIAL COMMITTEE (See names on the outside of this cover) CONTRIBUTING EDITORS OF THE BIOCHEMICAL BULLETIN PROF. LEON ASHER, University of Bern, Switserland PROF. FILIPPO BOTAZZI, University of Naples, Italy PROF. W. D. HALLIBURTON, King's College, London PROF. S. G. HEDIN, University of Upsala, Sweden PROF. FREDERICO LANDOLPH, University of La Plata, Argentina PROF. C. A. PEKELHARING, University of Utrecht, Holland DR. S. P. L. SÖRENSEN, Carlsberg Laboratory, Copenhagen, Denmark SPECIAL CONTRIBUTORS TO THE CONTENTS OF VOLUMES I AND II PROF. WILDER D. BANCROFT, Cornell University, Ithaca DR. CHARLES A. DOREMUS, 55 W. 52d St., New York City PROF. MARTIN H. FISCHER, University of Cincinnati DR. MARY LOUISE FOSTER, Smith College, Northampton, Mass. PROF. FRANCIS E. LLOYD, McGill University, Montreal, Canada PROF. JOHN A. MANDEL, A^. Y. Univ. and Bellevue Hospital Med. College PROF. ALBERT P. MATHEWS, University of Chicago PROF. LAFAYETTE B. MENDEL, Yale University PROF. VICTOR C. MYERS, N. Y. Post-Graduate Med. School and Hospital DR. E. E. SMITH, 50 Last 4ist St., New York City DR. A. E. SPAAR, City Hospital, Trincomalee, Ceylon MISS ANNA W. WILLIAMS, University of Illinois, Urbana, III. PROF. E. WINTERSTEIN, Polytechnic Institute, Zürich, Switserland DR. JULES WOLFF, 26 Rue Dutot, Paris ASSOCIATE EDITORS DAVID ALPERIN, Eclectic Medical College EDGAR ALTENBURG, Department of Botany, Columbia University HUGH AUCHINCLOSS, Department of Surgery, Columbia University GEORGE BAEHR, Mount Sinai Hospital ELMER W. BAKER, Flushing Hospital CHARLES W. BALLARD, College of Pharmacy, Columbia University HANS G. BAUMGARD, German Hospital Dispensary CORA J. BECKWITH, Department of Zoology, Columbia University Associate editors (continued) STANLEY R. BENEDICT, Cornell University Medical College LOUIS E. BISCH, Manhattan State Hospital HELENE M. BOAS, Barnard College, Columbia University CHARLES F. BOLDUAN, Health Department of New York City SAMUEL BOOKMAN, Mount Sinai Hospital SIDNEY BORN, Department of Chemistry, Columbia University WILLIAM BALLANTINE BOYD, College of the City of New York EDWARD C. BRENNER, Bellevue Hospital JACOB J. BRONFENBRENNER, Rockefeiler Institute for Medical Research LEO BUERGER, Mt. Sinai Hospital JESSE G. M. BULLOWA, New York Polyclinic Medical School GERTRUDE S. BURLINGHAM, Rastern District High School, Brooklyn RUSSELL BURTON-OPITZ, Department of Physiology, Columbia University HERBERT S. CARTER, Presbyterian Hospital RUSSELL L. CECIL, Presbyterian Hospital ARTHUR F. CHACE, New York Post-Graditate Medical School ERNEST D. CLARK, Cornell University Medical College ALFRED E. COHN, Rocke feller Institute for Medical Research EDWARD M. COLIE, Jr., Bellevue Hospital BURRILL B. CROHN, Mt. Sinai Hospital LOUIS J. CURTMAN, College of the City of New York EDWARD CUSSLER, Department of Clinical Pathology, Columbia University CHESTER A. DARLING, Department of Botany, Columbia University WILLIAM DARRACH, Department of Surgery, Columbia University NORMAN E. DITMAN, St. Luke's Hospital GEORGE DRAPER, Hospital of the Rockef eller Institute BENJAMIN G. FEINBERG, College of the City of New York HARRY L. FISHER, Department of Chemistry, Columbia University SIMON S. FRIEDMAN, Mt. Sinai Hospital C. STUART GAGER, Brooklyn Botanic Garden HELEN GAVIN, Wadleigh High School SAMUEL GITLOW, Lebanon Hospital Dispensary A. J. GOLDFARB, College of the City of New York DONALD GORDON, Department of Physiology, Columbia University MARK I. GOTTLIEB, Fordham University ISIDOR GREENWALD, Montefiore Home Laboratory LOUISE HOYT GREGORY, Barnard College, Columbia University ABRAHAM GROSS, Arbuckle Sugar Co., Brooklyn BEATRIX H. GROSS, N. Y. City Normal College BENJAMIN C. GRUENBERG, Brooklyn Commercial High School MARSTON L. HAMLIN, Harriman Research Laboratory, Roosevelt Hospital FREDERIC M. HANES, Rockef eller Institute for Medical Research JOHN D. HASEMAN, Department of Zoology, Columbia University HAROLD M. HAYS, New York Eye and Ear Infirmary MICHAEL HEIDELBERGER, Rockefeller Institute for Medical Research ALFRED M. HELLMAN, German Hospital MELVIN G. HERZFELD, German Hospital ALFRED F. HESS, Health Department of New York City NELLIE P. HEWINS, Newtown High School, L. I. ELLA A. HOLMES, Jamaica High School, L. I. Associate editors (continued) FRANK T. HUGHES, Boys High School, Brooklyn FREDERICK B. HUMPHRIES, Gertnan Hospital LOUIS HUSSAKOF, American Museum of Natural History PETER IRVING, Department of Clinical Pathology, Columbia University HENRY H. JANEWAY, City Hospital, New York CAVALIER H. JOÜET, Roselle, N. J. DAVID J. KALISKI, Mt. Sinai Hospital JOHN L. KANTOR, Mt. Sinai Hospital EDWARD C. KENDALL, St. Lukc's Hospital LEO KESSEL, Mt. Sinai Hospital ROLFE KINGSLEY, Department of Surgery, Columbia University ISRAEL J. KLIGLER, American Museum of Natural History CLINTON B. KNAPP, General Memorial Hospital ALFRED H. KRÖPFE, Hoffman and Kropff Chemical Co., Brooklyn ELSIE A. KUPFER, Wadleigh High School ADRIAN VAN S. LAMBERT, Department of Surgery, Columbia University MARGUERITE T. LEE, Girls High School, Brooklyn CHARLES C. LIEB, Department of Pharmacology, Columbia University MABEL C. LITTLE, New York Polyclinic Hospital RALPH W. LOBENSTINE, Bellevue Hospital DANIEL R. LUCAS, St. Joseph's Hospital CHESTER A. MATHEWSON, Brooklyn Training School for Teachers LAURA I. MATTOON, Vcttin School, i6o W. 74th Street WILLIAM H. McCASTLINE, University Physician, Columbia University MARY G. McCORMICK, Teachers College, Columbia University MRS. ELLEN BEERS McGOWAN, Teachers College, Columbia University GUSTAVE M. MEYER, Rockefeiler Institute for Medical Research JESSIE A. MOORE, Loomis Laboratory, Cornell University Medical College HERMANN J. MULLER, Cornell University Medical Collegs B. S. OPPENHEIMER, Department of Pathology, Columbia University RAYMOND C. OSBURN, New York Aquarium REUBEN OTTENBERG. Mt. Sinai Hospital CHARLES PACKARD, Department of Zoology, Columbia University ALWIN M. PAPPENHEIMER, Department of Pathology, Columbia University F. W. PUNNETT, Department of Chemistry, Columbia University ABRAHAM RAVICH, Jewish Hospital, Brooklyn ANTON R. ROSE, Department of Chemistry, Columbia University CHARLES H. SANFORD, German Hospital WINFIELD S. SCHLEY, St. Luke's Hospital OSCAR M. SCHLOSS, New York Nursery and Chlld's Hospital MAX SCHULMAN, Department of Applied Therapeutics, Columbia University H. VON W. SCHULTE, Department of Anatomy, Columbia University FRED J. SEAVER, Netv York Botanical Garden LEANDER H. SHEARER, Department of Physiology, Columbia University JAMES B. SIDBURY, Roosevelt Hospital MORRIS STARK, Babies Hospital MATTHEW STEEL, Long Island Medical College RALPH G. STILLMAN, Nezv York Hospital CHARLES R. STOCKARD, CorncU Univcrsilv Medical College ARTHUR W. SWANN, Presbyterian Hospital Associate editors (continued) WM. K. TERRIBERRY, Department of Physiology, Columbia University F. T. VAN BEUREN, Jr., Department of Surgery, Columbia University GEORGE W. VANDEGRIFT, Cornell University Medical College SADIE B. VANDERBILT, Teachers College, Columbia University CHARLES H. VOSBURGH, Jamaica High School WILBUR WARD, Department of Gynecology, Columbia University HELEN S. WATT, Wadleigh High School WILLIAM WEINBERGER, Lebanon Hospital FRED S. WEINGARTEN, German Hospital JULIUS W. WEINSTEIN, Vanderbilt Clinic, Columbia University HARRY WESSLER, Mt. Sinai Hospital H. B. WILCOX, Department of Diseases of Children, Columbia University LOUIS E. WISE, Standard Varnish Works, Staten Island, N. Y. WILLIAM H. WOGLOM, Dep't. of Cancer Research, Columbia University I, OGDEN WOODRUFF, Department of Mediane, Columbia University (Locol members of the Columbia University Biochemical Association) ASSISTANT EDITORS HERMAN M. ADLER, Psychopathie Hospital, Bos.ton, Mass. JOHN S. ADRIANCE, Williams College, Williamstozvn, Mass. CARL L. ALSBERG, Bureau of Plant Industry, U. S. Dep't. of Agriculture D. B. ARMSTRONG, Massachusetts Institute of Technology, Boston LOUIS BAUMANN, University Hospital, Iowa City, Iowa GEORGE D. BEAL, University of Illinois, Urbana, III. WILLIAM N. BERG, Bureau of Animal Industry, U. S. Dep't of Agriculture JOSEPHINE T. BERRY, State College, Pullman, Washington ISABEL BEVIER, University of Illinois, Urbana, III. A. RICHARD BLISS, Birmingham Medical College, Birmingham, Ala. JEAN BROADHURST, Cornell University, Ithaca, N. Y. WILLIAM D. CUTTER, Medical College of Georgia, Augusta, Ga. A. D. EMMETT, University of Illinois, Urbana, III. ALLAN C. EUSTIS, Tulane University, Nezv Orleans, La. KATHARINE A. FISHER, MacDonald College, Quebec, Canada MARY E. GEARING, University of Texas, Austin, Texas GEORGE A. GEIGER. Marcus Hook, Fa. H. D. GOODALE, Carnegie Sta'n. for Exp. Evolut'n, Cold Spring Harhor, L. I. R. A. GORTNER, Carnegie Sta'n for Exp. Evolu'tn, Cold Spring Harbor, L. I. R. F. HARE, New Mex. Coli, of Agric. and Mech. Arts, Agric. College, N. M. E. NEWTON HARVEY, Princeton University, Princeton, N. J. BLANCHE R. HARRIS, State Normal School, Truro, Nova Scotia CONSTANCE C. HART, New Bedford Industrial School, New Bedford, Mass. P. B. HAWK, JeffersoH Medical College, Philadelphia WILLIAM T. HÖRNE, University of California, Berkeley, Cal. HOMER D. HOUSE, Forest School, Bilfmore, N. C. J. E. KIRKWOOD, University of Montana, Missoula, Mont. MATHILDE KOCH, University of Chicago, Chicago, III. W. M. KRAUS, Johns Hopkins Medical School, Baltimore, Md. SIDNEY LIEBOVITZ, Hermon High School, Hermon, N. Y. BURTON E. LIVINGSTON, Johns Hopkins University, Baltimore, Md. J. P. McKELVY, Allegheny General Hospital, Pittsburgh, Pa. H. A. MATTILL, University of Utah, Salt Lake City, Utah CLARENCE E. MAY, Indiana University, Bloomington, Ind. Assistant editors (continued) L. D. MEAD, Isolation Hospital, San Francisco, Cal. CLARA G. MILLER, Knox School, Tarrytczvn, N. Y. MAX W. MORSE, Trinity College, Hartford, Conn. EDWARDS A. PARK, Johns Hopkins Medical School OLIVE G. PATTERSON, Toronto University, Toronto, Canada W. H. PETERSON, University of Wisconsin, Madison, Wis. E. R. POSNER, Drake University Medical School, Des Maines, la. DAVID F. RENSHAW, West High School, Rochester, N. Y. ALFRED N. RICHARDS, University of Pennsylvania^ Philadelphia ANNA E. RICHARDSON, University of Texas, Austin WINIFRED J. ROBINSON, Vassar College, Poughkeepsie, N. Y. WILLIAM SALANT, Bureau of Chemistry, U. S. Department of Agriculture CARL A. SCHWARZE, N. J. Agricultural Experiment Station, New Briuiswick FREDERICK W. SCHWARTZ. Rensselaer Polytechnic Institute, Troy, N. Y. A. D. SELBY, Ohio Agricultural Experiment Station, Wooster, Ohio BLANCHE E. SHAFFER, North Texas State Normal School, Benton, Texas A. FRANKLIN SHULL, University of Michigan, Ann Arbor, Mich. EDWARD A. SPITZKA, Jcfferson Medical College, Philadelphia EDWARD C. STONE, Trinity College, Hartford, Conn. MARY E. SWEENY, University of Kentucky, Lexington, Ky. WILLIAM A. TALTAVALL, Redlands, Cal. IDA C. WADSWORTH, Brockport State Normal School, Brockport, N. Y. WILLIAM H. WELKER, Red Hill, Pa. DAVID D. WHITNEY, Wesleyan University, Middletown, Conn. LORANDE LOSS WOODRUFF, Yale University, New Haven, Conn. HAROLD E. WOODWARD, U. S. Food and Drug Inspection Lahoratory, Philadelphia HANS ZINSSER, Leland Stanford University, Palo Alto, Cal. {Non-resident members of the Columbia University Biochentical Association) ^r^^'i^ ^^>^^^Sz^ BiocHEMiCAL Bulletin Volume II SEPTEMBER, 191 2 No. 5 IN MEMORIAM ERNST SCHULZE Born July 31, 1840 Died June 15, 19 12 The death o£ Ernst Schulze is an irreparable loss to biology. Wherever the biochemistry of plants is appreciated, Schulze's death causes profound sorrow. Schulze was one of the founders of our present exact biochemical investigation. His researches in phyto- chemistry are classical and they have been charged with funda- mental ideas that continue to influence research in this great field. Dr. Ernst Schulze, professor of agricultural chemistry in the Eidgenössischen Technischen Hochschule at Zürich, was born July 31, 1840, in the hamlet of Bovenden, near Göttingen. In 1858 Schulze studied chemistry under Wöhler in Göttingen and also spent a Semester with Bunsen in Heidelberg. In 1861 he was assistant to Lehman, and subsequently to Geuther, at the Chemical Institute in Jena. His scientific activity began at the Agricultural Experi- ment Station in Weende, under the direction of Henneberg. In 1871 Schulze was appointed director of the newly founded Agri- cultural Experiment Station in Darmstadt. Even while he was at Weende, his ability had attracted the attention of the Eidgenös- sischen institution. In June, 1872, he was called to Zürich, where his activities continued fruit fully for forty years. Schulze's first important research was published with the collab- oration of his friend Märcker, in 1870, in the Journal für Land- zmrtschaft. In this paper it was shown that the principles of pro- 2 Ernst Schuhe [Sept. tein metabolism, as they were stated by Voit on the basis of experi- ments on carnivorous animals, applied to the ruminants as well. In Zürich, Schulze brought his researches in animal physiology to an end by a thorough investigation of wool-fat. He succeeded in preparing typical cholesterol in a pure State and in isolating an isomer, isocholesterol. Since 1872 Schulze had concerned himself exclusively with phy- tochemical research ; and forty years of activity in this field f ortified the conclusion that plants and animals contain the same classes of substances and that the chemical composition of animals is in many ways identical with that of plants. Schulze developed new methods for the quantitative determina- tion of nitrogenous substances and showed how to separate them in pure forms from the complex mixtures in plant Juices and extracts. With his collaborators Schulze made classical discoveries of the fol- lowing nitrogenous Compounds and, by masterly methods, estab- lished their Constitution : Glutamin, an amide of glutamic acid ; Arginin, guanido-ot-aminovalerianic acid ; Phenylalanin, yS-phenyl-ct-aminopropionic acid ; Vernin (identical with the guanosin subsequently obtained by Levene from nucleic acid) ; Stachydrin, the dimethylbetain of a-prolin ; Lupinin, an alkaloid from lupins. Schulze found the following nitrogenous substances in different plant materials and studied their role in plant metabolism: amino- valerianic acid, leucin, isoleucin, prolin, glutamin, asparagin, Phenyl- alanin, tyrosin, arginin, histidin, lysin, vicin, convicin, xanthin, hy- poxanthin, guanidin, vernin, allantoin, cholin, betain, trigonellin, and stachydrin. Schulze was working with the betains during his last illness but, unfortunately, he was unable to complete this re- search. Considerable interest was aroused by his discoveiy of the presence of allantoin in plants. Schulze was the first to make a successful investigation of phyto- lecithins ( Phosphatids) and their cleavage products. He found that the lecithin in many seeds can be extracted only with hoiling alcohol. For this reason he believed that lecithin exists in such 1912] ■ Ernst Winterstein 3 seeds in some sort of combination with proteins. In this connection he investigated the plant cholesterols, or phytosterols. Schulze then began his thorough studies of the carbohydrates and nitrogen-free reserve materials in plants. A paper entitled: "Untersuchungen über die stickstofffreien Reservestoffe der Samen von Lupinus Intens und über die Umwandlung derselben während des Keimungsprozesses," was given a prize by the Königlichen Gesellschaft der Wissenschaften in Göttingen. In this connection Schulze studied the constituents of cell mem- branes in plants. He showed that the walls of various plant-cells contain carbohydrates which resemble cellulose to a certain extent but differ from it by dissolving easily in warm dilute Solutions of acids and alkalis. These cell-wall constituents proved to be xylans, arabans, galactans, and mannans. They play the part of food reserves in seeds. Schulze called them " hemi-celluloses." He showed, further, that ordinary cellulose on hydrolysis yields other glucoses besides dextrose. Stachys tubers were found to contain stachyose, a tetrasaccharid. All these researches yielded data and experience that proved useful to Schulze in his discussions and de- velopment of analytical methods for phytochemical research. The role of asparagin and glutamin in the protein metabolism and synthesis in plants greatly interested Schulze to the end of his life. Although he was not able fully to explain the process of protein synthesis, he made fundamental contributions to the subject. He clarified our knowledge of protein metabolism in seedlings. What chemist or biologist has not heard of the investigations which were begun in 1876, and whose results were usually published in Prus- sian agricultural year books and also in the Zeitschrift für physio- logische Chemie? Even in his second paper on the subject, in 1878, Schulze showed the importance of the characteristic composition of etiolated seedlings and their high asparagin content. He concluded from his observations that the protein decomposition products do not persist, in seedlings, in the proportions in which they were originally produced from protein, hut, that after such protein cleav- age, these nitro genotis suhstances seem to he changed for the most part into asparagin. Schulze prepared a great many plant proteins and studied their 4 Ernst Schulze [Sept decomposition products. After his pupils, working outside our Institute, had taken an active part in the study of protein metabolism in seedlings, and after it had been shown in our laboratory that protein decomposition in seedlings is an enzymic process, Schulze came to the following general conclusions regarding the protein transformations in seedlings: Asparagin is formed in seedHngs at the expense of proteins and arises from the same material in etio- lated young green plants, and in young leaves and shoots; arginin also results in seedlings from direct decomposition of protein. Perhaps the individual amids arise in the leaf-buds in the propor- tions of their production from protein by hydrolysis with acid^ and other agcnts outside the organism, but probably with the differ- ence that, in the leaf-buds, neither aspartic acid nor glutamic acid is produced, the amids of these amino acids, viz., asparagin and glutamin, resulting instead. Amino acids, however, do not occur in plants in such proportions, since they are consumed in the plant metabolism, some more rapidly, it seems, than others. The accu- mulation of asparagin in seedlings is caused by the formation of this amide from other products (amino acids) of the trans formation of protein. One of the best arguments for this conception is the Observation, made by Schulze in his experiments and repeatedly em- phasized in his papers, that in many cases asparagin is produced abundantly even after the processes of protein decompositions in the plant have ceased. An admirable outcome of Schulze's investigations is his great compilation on the composition of cultivated plants, where he re- views briefly the methods of research and gives abundant data on the chemical constituents of these plants. The later years of Schulze's life were spent in close retirement because of a serious and long standing eye-disease that prevented him from appearing in public. He lived, at the end, only for his science and for his family. His colleagues often wondered how, with his weak eyes, he was able to do any experimental work what- ever. It was pathetic to see with what extreme care and patience. he had to tax himself in order to proceed with his work. When Schulze celebrated his seventieth birthday, two years ago, we all hoped that the twilight of his life might be long and happy, I9I2] Ernst IV int erst ein 5 but in vain, for pitiless death took him from iis. His pupils mourn, a beloved friend and guide; and science, a distinguished inves- tigator.^ Ernst Winterstein. Agriculturchemischen Laboratorium der Eidgenössischen Technischen Hochschule, Zürich. PUBLIICATIONEN VON PROF. DR. E. SCHULZE I. In den " Landwirtschaftlichen Versuchsstationen " Ueber die Elementarzusammensetzung der tierischen Fette, insbeson- dere der Fette vom Schaf, vom Rind und vom Schwein. E. Schulze und A. Reinecke. 9: 97-119 (1867). Ueber die sensiblen Stickstoff. Einnahmen und Ausgaben des voll- jährigen Schafes. E. Schulze und M. MÄRCKER. 11:201(1869). Ueber die Zusammensetzung und die Verdaulichkeit des im Wiesenheu enthaltenen Fettes. E. Schulze. 15: 81-90 (1872). Beiträge zur Kenntnis des Nährwerts und der Zusammensetzung der Rüben. E. Schulze. 15: 170-181 (1872). Zur Frage über die Verdauung des Heufetts. E. Schulze. 16 : 329-335 (1873)- Notiz über den Aspargingehalt von Lupinen Keimlingen. E. Schulze und W. Umlauft. 18: 1-3 (1875). Ueber die stickstoffhaltigen Bestandteile der Futter-Rüben. E. Schulze und A. Urich. 18: 296-324 (1875). Notiz betreffend das Vorkommen des Betains in den Futter-Rüben. E. Schulze und A. Urich. 18: 409 (1875). Ueber Schwefelsäurebildung in den Keimpflanzen, E. Schulze. 19 : 172-176 (1876). Einige Bemerkungen über die Sachsse-Kormannsche Methode zur Be- stimmung des in Amid-Form vorhandenen Stickstoffs. E. Schulze. 20: 1 17-123 (1877). Ueber die stickstoffhaltigen Bestandteile der Futterrüben. E. Schulze und A. Urich. 20: 194-245 (1877). Ueber den Gehalt der Kartoffelknollen an Eiweissstoffen und an Amiden. E. Schulze und J. Barbieri. 21: 63-92 (1878). * The foregoing biographical communication was translated from Prof. E. Winterstein's manuscript, in German, by Dr. Ernest D. Clark. Prof. Winter- stein's manuscript of the appended bibliography is reproduced verbatim. [Ed.] 6 Ernst Schulze [Sept Ueber ein neues Glukosid (Bestandteil von Lupinus luteus). E. Schulze und J. Barbieri. 24: i-ii (1880). Ueber das Vorkommen von Leucin und Tyrosin in den Kartoffelknol- len. E. Schulze und J. Barbieri. 24: 167-169 (1880). Ueber die Bestimmung der Eiweissstoffe und der nicht eiweissartigen Stickstoffverbindungen in den Pflanzen. E. Schulze. 24: 358- 365 (1880); 25: lyz--^?^ (1880). Zur Bestimmung der Eiweissstoffe und der nicht eiweissartigen Stick- stoffverbindungen in den Pflanzen. E. Schulze und J. Barbieri. 26:213-283 (1881). Neue Beiträge zur Kenntnis der stickstoffhaltigen Bestandteile der Kartoffelknollen. E. Schulze und E. Eugster. 27 : 357-373 (1882). Zur quantitativen Bestimmung der Eiweissstoffe und der nicht eiweiss- artigen Stickstoffverbindungen in den Pflanzen. E. Schulze. 27 : 449-465 (1882). Ueber das Vorkommen von Hypoxanthin im Kartoffelsaft. E. Schulze. 28: 111-115 (1883). Ueber das Glutamin. E. Schulze und E. Bosshard. 29: 295-307 (1883). Zur quantitativen Bestimmung des Asparagins, des Glutamins und des Ammoniaks in den Pflanzen. E. Schulze und E. Bosshard. 29 : 399-412 (1883). Zur Kenntnis der Methoden, welche zur Bestimmung der Amide in Pflanzenextrakten verwendbar sind. E. Schulze. 30 : 459-467 (1884). Ueber einige Bestandteile des Emmentaler Käses. B. Rose und E. Schulze. 31: 115-137 (1885). Ueber das Vorkommen von Glutamin in den Zuckerrüben und über das optische Verhalten desselben. E. Schulze und E. Bosshard. 32: 129-136 (1887). Untersuchungen über die stickstoffhaltigen Bestandteile einiger Rauh- futterstoffe. E. Schulze, E. Steiger und E. Bosshard. 33: 8^123 (1887). Ueber die Methoden, welche zur quantitativen Bestimmung der stick- stoffhaltigen Pflanzenbestandteile verwendbar sind. E. Schulze. 33: 124-145 (1887). Ueber das Vorkommen von Rohrzucker in unreifen Kartoffelknollen. E. Schulze und Th. Seliwanow. 34: 403 (1887). Ueber den Nachweis von Rohrzucker in vegetabilischen Substanzen. E. Schulze. 34: 408-413 (1887). I9I2] Ernst Winterstein 7 Ein Beitrag zur Erklärung der Veränderungen, welche die stickstoff- haltigen Bestandteile eingesäuerter Grünfutterstoffe erleiden. E. Schulze. 35: 195-208 (1888). Ueber die Zersetzung von Proteinstoffen in verdunkelten grünen Pflanzen. E. Schulze und E. Kisser. 36: 1-8 (1889). Ueber das Vorkommen eines unlöslichen, Schleimsäure gebenden Kohlenhydrats in Rotklee und Luzerne- Pflanzen. E. Schulze und E. Steiger. 36: 9-13 (1889). Untersuchungen über die stickstofffreien Reservestoffe der Samen von Lupinus luteus und über die Umwandlungen derselben während des Keimungsprozesses. E. Schulze und E. Steiger. 36 : 391- 476 (1889). Untersuchungen über die chemische Zusammensetzung einiger Legumi- nosen-Samen. E. Schulze, E. Steiger und W. Maxwell. 39 : 269 (1891). Ueber einige Bestandteile der Wurzelknollen von Stachys tuberifera. A. VON Planta und E. Schulze. 40: 277-298 (1892). Bestimmung des Stachyose-Gehalts der Wurzelknollen von Stachys tuberifera. A. von Planta und E. Schulze. 41 : 123-129 ( 1892) . Zur Kenntnis der in den Leguminosensamen enthaltenen Kohlenhy- drate. E. Schulze. 41:207-229 (1892). Ueber den Lecithingehalt einiger vegetabilischer Substanzen. E. Schulze und S. Frankfurt. 43: 307-318 (1894). Untersuchungen über die zur Klasse der stickstoffhaltigen organischen Basen gehörenden Bestandteile einiger landwirtschaftlich benutz- ter Samen, Oelkuchen und Wurzelknollen, sowie einiger Keim- pflanzen. E. Schulze in Verbindung mit S. Frankfurt und E. Winterstein. 46: 23-77 (1896). Zur Kenntnis der stickstoffhaltigen Bestandteile junger grüner Pflanzen von Vicia sativa. E. Schulze. 46: 383-397 (1896), Ueber das Vorkommen von Arginin in den Wurzeln und Knollen ein- iger Pflanzen. E. Schulze. 46: 451-458 (1896). Ueber die Verbreitung des Glutamins in den Pflanzen. E. Schulze. 48: 33-55 (1897). Ueber den Lecithingehalt einiger Pflanzensamen und einiger Oelkuchen. E. Schulze. 4g: 203-214 (1898). Die Notwendigkeit der Umgestaltung der jetzigen Futter- und Nahr- ungsmittel-Analyse. E.Schulze. 49:419-441 (1898). Ueber die Verbreitung des Glutamins in den Pflanzen. {Zweite Mitteilung.) E.Schulze. 49:442-446(1898). 8 Ernst Schulze [Sept. Ueber die Bestandteile der Samen von Pinus cemhra (Zierbeikiefer oder Arve). E. Schulze und N.Rongger. 51:189-204(1899). Ueber die Rückbildung der Eivveissstoffe aus deren Zerfallsprodukten in der Pflanze. E. Schulze. 55: 33-44 (1901). Ueber die Zusammensetzung einiger Koniferen-Samen. E. Schulze. 55:267-307 (1901). Können Leucin und Tyrosin den Pflanzen als Nährstoffe dienen? E. Schulze. 56:97-106(1902). Ein Nachtrag zu der Abhandlung über die Frage ob Leucin und Tyrosin den Pflanzen als Nährstoffe dienen können. E. Schulze. 56: 293-296 (1902). Zur Kenntnis der kristallisierten Stachyose. E. Schulze. 56 : 419- 423 (1902). Ueber das Vorkommen von Hexonbasen in den Knollen der Kartoffel {Solanum tuberosum) und der Dahlie {Dahlia variabilis). E. Schulze. 59: 331-343 (1904)- Ueber Methoden, die zur Darstellung organischer Basen aus Pflanzen- säften und Pflanzenextrakten verwendbar sind. E. Schulze. 59: 344-354 (1904). Zur Kenntnis des Glutamins. E. Schulze. 65: 237-246 (1906). Ueber die Bestandteile der Samen von Pinus cemhra. E. Schulze. 67: 57-104 (1907). Zur Kenntnis des Glutamins. (Zzveite Mitteilung.) E. Schulze und Gh. Godet. 67: 313-319 (1907). Ueber die chemische Zusammensetzung der Samen unserer Kultur- pflanzen. E.Schulze. 73:35-170(1910). Zur Kenntnis des Glutamins. (Dritte Mitteilung.) E. Schulze und G. Trier. 77: 1-12 (1912). 2. In den landwirtschaftlichen Jahrbüchern Untersuchungen über einige chemische Vorgänge bei der Keimung der gelben Lupine. E. Schulze, W. Umlauft und A. Urich. 5 : 821-862 (1876). Die stickstoffhaltigen Bestandteile der vegetabilischen Futtermittel und ihre quantitative Bestimmung. E.Schulze. 6:157-175(1877). Ueber die Prozesse, durch welche in der Natur freier Stickstoff in Stickstoffverbindungen übergeführt wird, E. Schulze. 6 : 695- 707 (1877). Ueber die Zersetzung und Neubildung von Eiweissstoffen in Lupinen- keimlingen. E.Schulze. 7:411-444(1878). jgi2] Ernst Winterstein 9 Ueber den Eiweissumsatz im Pflanzenorganismus. E. Schulze. 9: 689-748; 12: 909-920; 14: 713-729; 21: 105-130 (1880-1892). Untersuchungen über den Emmentaler Käse und über einige andere schweizerische Käsesorten. E. Benecke und E. Schulze. 16: 317-400 (1887). Ueber die Bildungsweise des Asparagins und über die Beziehungen der stickstofffreien Stoffe zum Eiweissumsatz im Pflanzenorganismus. E. Schulze. 17: 683-711 (1888). Ueber die stickstofffreien Bestandteile der vegetabilischen Futtermit- tel. E.Schulze, 21:79-103(1892). Zur Kenntnis der in den pflanzlichen Zellmembranen enthaltenen Kohlenhydrate. E. Schulze. 23: 1-26 (1894). Ueber die Bildungsweise des Asparagins in den Pflanzen. E. Schulze. 30: 287-297 (1901). Ueber den Abbau und den Aufbau der organischen Stickstoff Verbind- ungen in den Pflanzen. E. Schulze. 35: 621-666 (1906). 3. Im Journal für Landwirtschaft Welchen Einfluss haben die Zubereitung des Futters und die Futter- mischung auf den Nährwert des Futters? Mit welchen Futter- stoffen sind bei den gegenwärtigen Marktpreisen Futterrationen mit angemessenem Gehalt an Nährstoffen am billigsten herzu- stellen. E. Schulze, 17: 33-48 (1869). Untersuchungen über die sensiblen Stickstoff-Einnahmen und -Aus- gaben des volljährigen Schafs und die Ausnutzung einiger Futter- stoffe durch dasselbe. E. Schulze und M. Märcker. 18: 1-39; 19: 202-222, 285-326, 347-362; 20: 46-76 (1870-1872). Fütterungsversuche mit Schafen, E. Schulze und M. Märcker. 23: 141-174 (1875)- Ueber die Zusammensetzung einer pechschweissigen Schafwolle und des daraus gewonnenen Wollfetts. E. Schulze und J. Barbieri. 27: 125-144 (1879). Ueber die zur Gruppe der stickstofffreien Extraktstoffe gehörenden Pflanzenbestandteile. E. Schulze. 52: 1-30 (1904). Ueber die in den landwirtschaftlichen Kulturpflanzen enthaltenen, nicht proteinartigen Stickstoffverbindungen. E. Schulze. 52 : 305- 336 (1904). Ueber den Nährwert der in den Futtermitteln enthaltenen nichtprote- inartigen Stickstoffverbindungen. E.Schulze. 54:65-81(1906). 10 Ernst Schicke [Sept. 4. In dem Landwirtschaftlichen Jahrbuch der Schweiz Ueber die Entstehung der Salpetersäuren Salze im Boden. E. Schulze. 1890 : 109-121 ; 1891 : 82-86. Ueber die in den Futtermitteln enthaltenen Fettsubstanzen und über die Bedeutung derselben für die tierische Ernährung. E. Schulze. 1892 : 1-9. Ueber den Humus und seine Beziehung zum Leben der Pflanze. E. Schulze. 1901 : 1-13. Die Nährstoffnormen und die Beurteilung des Nährwertes der Futter- bestandteile nach ihrer Verbrennungswärme. E. Schulze. 1902 : 1-19. Ueber die chemische Zusammensetzung des Holzes und über einige aus demselben darstellbaren Produkte. E. Schulze. 1904: i-io. 5. In der Zeitschrift für physiologische Chemie Untersuchungen über die Amidosäuren, welche bei der Zersetzung der Eiweissstoffe durch Salzsäure und durch Barytwasser entstehen. E. Schulze, J. Barbieri und E. Bosshard. 9: 63-126, 253-259 (1885). Zur Kenntnis des Vorkommens von Allantoin, Asparagin, Hypoxanthin und Guanin in den Pflanzen. E. Schulze und E. Bosshard. 9: 420-444 (1885). Notiz betreffend die Bildung von Sulfaten in keimenden Erbsen. E. Schulze. 9: 616 (1885). Ueber einen neuen stickstoffhaltigen Pflanzenbestandteil. E. Schulze und E. Bosshard. ig: 80-89 (1886). Untersuchung über die Amidosäuren, welche bei der Zersetzung der Eiweissstoffe durch Salzsäure und durch Barytwasser entstehen. Zzveite Abhandlung. E. Schulze und E. Bosshard. 10:134-145 (1886). Ueber das Vorkommen von Vernin im Blütenstaub von Corylus avellana und von Pinus sylvestris. E. Schulze und A, von Planta, ig : 326-330 (1886). Ueber das Arginin. E. Schulze und E. Steiger, ii : 43-65 (1887). Zur Kenntnis der beim Eiweisszerfall entstehenden Phenylamidopro- pionsäure. E. Schulze und E. Nägeli. ii : 201-206 (1887). Ueber das Vorkommen von Cholin in Keimpflanzen. E. Schulze. 11:365-372 (1887). Ueber einige stickstoffhaltige Bestandteile der Keimlinge von Soja hispida. E.Schulze. 12:405-415 (1888). 1912] Ernst Winterstein ii Ueber den Lecithingehalt der Pflanzensamen. E, Schulze und E. Steiger. 13:365-384(1889). Zur Chemie der Pflanzenzellmembran. E. Schulze, E. Steiger und W. Maxwell. 14: 227-273 (1890). Bilden sich Cholesterine in Keimpflanzen, welche bei Lichtabschluss sich entwickeln? E. Schulze. 14: 491-521 (1890). Ueber die Farbenreaktion des Isocholesterins mit Essigsäureanhydrid und Schwefelsäure. E. Schulze. 14: 522-523 (1890). Ueber die basischen Stickstoffverbindungen aus den Samen von Vicia sativa und Pisum sativum. E. Schulze. 15: 140-160 (1891). Ueber das Lecithin der Pflanzensamen. E. Schulze und A. Likier- NiK. 15: 405-414 (1891). Zur Chemie der pflanzlichen Zellmembranen. (Zweite Abhandlung.) E. Schulze. 16: 387-438 (1892). Ueber einige stickstoffhaltige Bestandteile der Keimlinge von Vicia sativa. E. Schulze. 17: 193-216 (1893). Ueber die Konstitution des Leucins. E. Schulze und A. Likiernik. 17: 513-535 (1893). Zur Chemie der pflanzlichen Zellmembranen. (Dritte Abhandlung.) E. Schulze. 19: 38-69 (1894). Ueber die Bestimmung des Lecithingehaltes der Pflanzensamen. E. Schulze. 20:225-232(1895). Ueber das wechselnde Auftreten einiger krystallinischen Stickstoffver- bindungen in den Keimpflanzen und über den Nachweis derselben. E. Schulze. 20: 306-326 (1895). Ueber das Vorkommen von Glutamin in grünen Pflanzenteilen. E. Schulze. 20:327-334(1895). Ueber die Verbreitung des Rohrzuckers in den Pflanzen, über seine physiologische Rolle und über lösliche Kohlenhydrate, die ihn be- gleiten. E. Schulze und S. Frankfurt. 20: 511-555; 21: 108 (1895). Ueber die Zellwandbestandteile der Cotyledonen von Lupinus luteus und Lupinus angustifolius und über ihr Verhalten während des Keimungs Vorganges. E. Schulze. 21: 392-411 (1895). Ueber das Vorkommen von Nitraten in Keimpflanzen. E. Schulze. 22: 82-89 (1896). Ueber einen phosphorhaltigen Bestandteil der Pflanzensamen. E. Schulze und E. Winterstein. 22 : 90-94 (1896). Ueber das wechselnde Auftreten einiger krystallisierbaren Stickstoff- verbindungen in den Keimpflanzen. (Zzveite Abhandlung.) E. Schulze. 22:411-434(1896). ^ 12 Ernst Schuhe [Sept. Ueber die beim Umsatz der Proteinstoffe in den Keimpflanzen einiger Coniferenarten entstehenden Stickstoffverbindungen. E. Schulze. 22:435-448 (1896). Ueber den Umsatz der Eiweissstoft"e in der lebenden Pflanze. E. Schulze. 24: 18-114 (1898). Ueber die Spaltungsprodukte der aus den Coniferensamen darstell- baren Proteinstoffe. E. Schulze. 24 : 276-284 ; 25 : 360-362 (1898). Ueber die Bildung von Ornithin bei der Spaltung des Arginins und über die Konstitution dieser beiden Basen. E. Schulze und E. Winterstein. 26: 1-14 (1898). Ueber den Eiweissumsatz und die Bildungsweise des Asparagins und des Glutamins in den Pflanzen. E. Schulze. 26 : 41 1-426 ( 1899) . Ueber die Verbreitung des Rohrzuckers in den Pflanzen, über seine physiologische Rolle und über lösliche Kohlenhydrate, die ihn be- gleiten. (Zweite Abhandlung.) E.Schulze. 27:267-291(1899). Nachweis von Histidin und Lysin unter den Spaltungsprodukten der aus Coniferensamen dargestellten Proteinsubstanzen, E. Schulze und E. Winterstein. 28: 459-464 (1899). Ueber das Vorkommen von Histidin und Lysin in Keimpflanzen. E. Schulze. 28: 465-470 (1899). Einige Bemerkungen über das Arginin. E. Schulze. 29 : 329-333 (1900). Ueber den Umsatz der Eiweissstoffe in der lebenden Pflanze. (Zweite Abhandlung.) E. Schulze. 30: 241-312 (1900). Ueber die Ausbeute an Hexonbasen, die aus einigen pflanzlichen Ei- weissstoffen zu erhalten sind. E. Schulze und E. Winterstein. 33: 547-573 (1901)- Beiträge zur Kenntnis des Arginins und Ornithins. E. Schutlze und E, Winterstein. 34: 128-147 (1901). Ueber die Trennung des Phenylalanins von anderen Aminosäuren. E. Schulze und E. Winterstein. 35: 210-220 (1902). Beiträge zur Kenntnis einiger aus Pflanzen dargestellten Aminosäuren. E. Schulze und E. Winterstein. 35: 299-314 (1902). Beiträge zur Kenntnis der Hemicellulosen. E. Schulze und N. Castoro. 37: 40-53 (1902). Beiträge zur Kenntnis der Zusammensetzung und des Stoffwechsels der Keimpflanzen. E. Schulze und N. Castoro. 38: 200-258 (1903)- Beiträge zur Kenntnis der Hemicellulosen. E. Schulze und N. Castoro. 39: 318-328 (1903). igi2] Ernst Wintersfein 13 Zur Kenntnis der aus Pflanzen darstellbaren Lecithine. (Erste Mit- teilung.) E. Schulze und E. Winterstein. 40:101-119(1903). Ein Nachtrag zur Abhandlung über einen phosphorhaltigen Bestand- teil der Pflanzensamen. E. Schulze und E. Winterstein. 40 : 120-122 (1903). Beiträge zur Kenntnis der in ungekeimten Pflanzensamen enthaltenen Stickstoflfverbindungen. E. Schulze und N. Castoro. 41 : 455-473 (1904). Einige Notizen über das Lupeol. E. Schulze. 41: 474-476 (1904). Findet man in Pflanzensamen und in Keimpflanzen anorganische Phos- phate? E. Schulze und N. Castoro. 41: 477-484 (1904), Beiträge zur Kenntnis der Zusammensetzung und des Stoflfwechsels der Keimpflanzen. (Zzveite Mitteilung.) E. Schulze und N. Cas- toro. 43: 170-198 (1904). Ueber das Vorkommen von Ricinin in jungen Keimpflanzen. E. Schulze und E. Winterstein. 43: 211-221 (1904). Ueber das Verhalten des Cholesterins gegen das Licht. E. Schulze und E. Winterstein. 43: 316-319 (1904). Ueber die aus den Keimpflanzen von Vicia sativa und Lupinus albus darstellbaren Monoaminosäuren. E. Schulze und E. Winter- stein. 45: 38-60 (1905). Ueber das spezifische Drehungsvermögen einiger aus Pflanzen darge- stellten Tyrosinpräparate. E. Schulze und E. Winterstein. 45:79-83(1905)- Neue Beiträge zur Kenntnis der Zusammensetzung und des Stoff- wechsels der Keimpflanzen. E. Schulze. 47: 507-569 (1906). Ueber den Tyrosingehalt der Keimpflanzen von Lupinus albus. E. Schulze und N. Castoro. 48: 387-395 (1906). Bildet sich Homogentisinsäure beim Abbau des Tyrosins in den Keim- pflanzen? E. Schulze und N. Castoro. 48: 396-411 (1906). Ueber das Verhalten des Cholesterins gegen das Licht. (Zweite Mit- teilimg.) E. Schulze und E. Winterstein. 48:546-548(1906). Ist die bei Luftzutritt eintretende Dunkelfärbung des Rübensaftes durch einen Tyrosin- und Homogentisinsäuregehalt dieses Saftes bedingt? E. Schulze. 50: 508-524 (1907). Ueber den Phosphorgehalt einiger aus Pflanzen dargestellter Lecithin- präparate. E. Schulze. 52: 54-61 (1907). Zum Nachweis des Rohrzuckers in Pflanzensamen. E. Schulze. 52:404-411 (1907). Ueber die zur Darstellung von Lecithin und anderen Phosphatiden 14 Ernst Schulze [Sept. aus Pflanzensamen verwendbaren Methoden. E. Schulze. 55 : 338-351 (1908). Einige Bemerkungen zu den Arbeiten über den Nährwert der in den Pflanzen enthaltenen Amide. E.Schulze. 57:67-73(1908). Ueber den Calcium- und Magnesiumgehalt einiger Pflanzensamen. E. Schulze und Ch. Godet. 58: 156-161 (1908). Ueber das Stachydrin. E. Schulze und G. Trier. 59:233-235(1909). Ueber die zur Darstellung von Cholin, Betain und Trigonellin aus Pflanzen verwendbaren Methoden und über die quantitative Be- stimmung dieser Basen. E.Schulze. 60:155-179(1909). Untersuchungen über die in den Pflanzensamen enthaltenen Kohlen- hydrate. E. Schulze und Ch. Godet. 61: 279-350 (1909). Ueber das Vorkommen von Betain in den Knollen des Topinamburs (Helianthus tuberosus). E. Schulze. 65: 293-294 (1910). Studien über die Proteinbildung in reifenden Pflanzensamen. E. Schulze und E. Winterstein. 65: 431-476 (1910). Ein Beitrag zur Kenntnis des Vernins. E. Schulze. 66: 128-136 (1910). Ueber die in den Pflanzen vorkommenden Betaine. E. Schulze und G. Trier. 67: 46-58 (1910). Ueber das Stachydrin und über einige neben ihm in den Stachysknollen und in den Orangenblättern enthaltene Basen. E. Schulze und G. Trier. 67: 59-96 (1910). Ueber das Vorkommen von Hemicellulosen in den Samenhülsen von Pisum sativum und von Phaseolus vulgaris. E. Schulze und U. Pfenninger. 68: 93-108 (1910). Erwiderung auf R. Engelands Bemerkungen zu den Abhandlungen über die pflanzlichen Betaine und das Stachydrin. E. Schulze und G. Trier. 6g: 326-328 (1910). Ein Beitrag zur Kenntnis der in den Pflanzensamen enthaltenen Kohl- enhydrate. E. Schulze und U. Pfenninger. 69: 366-382(1910). Ueber die Identität des Vernins und des Guanosins, nebst einigen Be- merkungen über Vicin und Convicin. E. Schulze und G. Trier. 70: 143-151 (1910)- Studien über die Proteinbildung in reifenden Pflanzensamen. {Zweite Mitteilung.) E. Schulze. 71: 31-48 (1911). Untersuchung über die in den Pflanzen vorkommenden Betaine. "E. Schulze und U. Pfenninger. 71: 174-185 (1911). Zur Frage der Identität des aus Melasse dargestellten Guaninpentosids mit dem Vernin. E. Schulze und G. Trier, 76 : 145-147 (1912). I9I2] Ernst Winterstein 15 Untersuchungen über die in den Pflanzen vorkommenden Betaine. (Zzueite Mitteilung.) E. Schulze und G. Trier. 76: 258-290 (1912). Dasselbe. (Dritte Mitteilung.) E. Schulze und G. Trier. 79:235- 242 (1912). 6. Im Journal für praktische Chemie Ueber die Zusammensetzung der rohen Schafwolle. M. Märcker und E. Schulze. 108: 193-207 (1870). Ueber die Zusammensetzung des Wollfetts. E. Schulze. 7 (n. f.) : 1-16 (1873). Dasselbe. E. Schulze und A. Urich. 9: 321-339 (1874). Ueber die Eivveisszersetzung in Kürbiskeimlingen. E. Schulze und J. Barbieri. 20: 385-418 (1880). Ueber das Vorkommen von Allantoin und Asparagin in jungen Baum- blättern. E. Schulze und J. Barbieri, 25: 145-158 (1882). Zur Kenntnis der Cholesterine. E. Schulze und J. Barbieri. 25: 159-180 (1882). Ein Nachtrag zu der Abhandlung: "Zur Kenntnis der Cholesterine." E. Schulze. 25: 458-462 (1882). Ueber Phenylamidopropionsäure, Amidovaleriansäure und einige andere stickstoffhaltige Bestandteile der Keimlinge von Lupinns luteus. E. Schulze und J. Barbieri. 27: 337-362 (1883). Zur quantitativen Bestimmung des Asparagins und des Glutamins. E. Schulze. 31:234-246 (1885). Zur Kenntnis der stickstoffhaltigen Bestandteile der Kürbiskeimlinge. E. Schulze. 32: 433-460 (1886). 7. In den Berichten der Deutschen Chemischen Gesellschaft Über Maltose. E. Schulze. 7: 1047 (1874). Über die Zusammensetzung des Wollfetts. E.Schulze. 8:570(1875). Selenoidodiglykolsäure. A. Urich und E. Schulze. 8: yyT, (i875)- Die stickstoffhaltigen Bestandteile der Rüben. E. Schulze und A. Urich. 9:80 (1876). Keimung der Lupinensamen. E. Schulze und W. Umlauft. 9: 1314 (1876). Über die stickstoffhaltigen Bestandteile der Runkelrüben (Glutamin). E. Schulze und A. Urich. ig: 88 (1877). Über das Vorkommen eines Glutaminsäureamids in den Kürbiskeim- lingen. E. Schulze und J. Barbieri. ig: 199 (1877). Eiweisszersetzung in Keimpflanzen. E. Schulze, ii: 520 (1878). l6 Erfist Schuhe [Sept. Bildung von schwefelsauren Salzen bei der Eiweisszersetzung in Keim- pflanzen. E. Schulze, ii: 1234 (1878). Asparagin und Tyrosin in Kürbiskeimlingen. E. Schulze und J. Bar- BiERi. 12 : 710 (1879). Leucin aus Kürbiskeimlingen. E. Schulze und J. Barbieri. 12 : 1233 (1879). Über ein Glucosid aus Lupinus luteus. E. Schulze und J. Barbieri. 12: 2200 (1879). Über das spezifische Drehungsvermögen des Isocholesterins. E. Schulze. 13: 249 (1880). Über ein neues Glucosid. E. Schulze und J. Barbierl 13:681(1880), Amidosäuren in Lupinenkeimlingen. E. Schulze und J. Barbieri. 13: 1924 (1880). Über die Eiweisszersetzung in Kürbiskeimlingen. E. Schulze und J. Barbieri. 13: 2386 (1880). Über das Vorkommen von Allantoin im Pflanzenorganismus. E. Schulze und J. Barbieri. 14: 1602 (1881). Über das Vorkommen von Phenylamidopropionsäure unter den Zersetz- ungsprodukten der Eiweissstoffe. E. Schulze und J. Barbieri. 14: 1785 (1881). Zur Kenntnis des Cholesterins. E. Schulze und J. Barbieri. 15: 953 (1882). Über das Vorkommen von Allantoin und Asparagin in jungen Baum- blättern. E. Schulze und J. Barbieri. 15:955(1882). Beiträge zur Kenntnis der stickstoffhaltigen Bestandteile der Kartoffeln. E. Schulze und E. Eugster. 15: 1090 (1882). Über das optische Verhalten einiger Aminosäuren. E. Schulze und E. Bosshard. 17: 1610 (1884). Über die Bildung von Phenylamidopropionsäure beim Erhitzen von Eiweissstoffen mit Salzsäure und Zinnchlorür. E. Schulze und J. Barbieri. 17: 171 1 (1884). Über das optische Verhalten einiger Aminosäuren. E. Schulze und E. Bosshard. 18: 388 (1885). Über das Vorkommen von Glutamin in den Zuckerrüben und über das optische Verhalten desselben. E. Schulze und E. Bosshard. 18 : 390 (1885). Über einen neuen stickstoffhaltigen Bestandteil der Keimlinge von Lupinus luteus. E.Schulze. 19:1177(1886). Über Paragalactan. E. Schulze. 20: 290 (1887). Bilden sich Nitrate im Organismus lebender Pflanzen? E. Schulze. 20: 1500 (1887). I9I2] Ernst Winterstein 17 Über das Vorkommen von Cholin in Keimpflanzen. E. Schulze. 21 : 21 (1888). Über das Vorkommen von Rohrzucker in unreifen Kartoffeln. E. Schulze und Th. Seliwanow. 21 : 299 (1888). Über den Nachweis von Rohrzucker in vegetabilischen Substanzen. E. Schulze und Th. Seliwanow. 21 : 299 (1888). Ein Beitrag zur Veränderung, welche die stickstoffhaltigen Bestand- teile eingesäuerter Grünfutterstoffe erleiden. E. Schulze. 21 : 668 (1888). Über das Vorkommen eines unlöslichen Schleimsäure gebenden Kohlen- hydrats in Rotklee und Luzerne. E. Schulze und E. Steiger. 22:345 (1889). Über die Zersetzung der Proteinsubstanzen in verdunkelten grünen Pflanzen. E. Schulze und E. Kisser. 22:350(1889). Über einige stickstoffhaltige Bestandteile der Keimlinge von Soja his- pida. E. Schulze. 22: 599 (1889). Zur Kenntnis der chemischen Zusammensetzung der Pflanzenzellmem- branen. E. Schulze. 22: 1192 (1889). Betain und Cholin in den Samen von Vicia sativa. E. Schulze, 22 : 1827 (1889). Untersuchungen über die stickstofffreien Reservestoffe der Samen von Lupinus luteus und über die Umwandlung derselben während des Keimprozesses. E. Schulze und E. Steiger. 23: 405 (1890). Über ein Krystallisieren des Kohlenhydrats. A. v. Planta und E. Schulze. 23: 1692 (1890). Zur Kenntnis der chemischen Zusammensetzung der pflanzlichen Zell- membran. E.Schulze. 23:2579(1890). Darstellung von Lecithin aus Pflanzensamen. E. Schulze und A. LiKiERNiK. 24:71 (1891). Über den Lecithingehalt der Pflanzensamen. E. Schulze und E. Steiger. 24: 327 (1891). Zur Chemie der Pflanzenzellmembran. E. Schulze, E. Steiger und W. Maxwell. 24: 530 (1891). Über die Konstitution des Leucins. E. Schulze und A. Likiernik. 24: 669 (1891). Bilden sich Cholesterine in Keimpflanzen, welche bei Lichtabschluss sich entwickeln? E. Schulze. 24: 670 (1891). Über die Farbenreaktion des Isocholesterins mit Essigsäureanhydrid und Schwefelsäure. E. Schulze. 24: 671 (1891). Über die Bildung von stickstoffhaltigen Basen beim Eiweisszerfall im Pflanzenorganismus. E. Schulze. 24: 1098 (1891). i8 Ernst Schuhe [Sept. Zur Kenntnis der chemischen Zusammensetzung der pflanzlichen Zell- membran. E. Schulze. 24: 2277 (1891). Über die Bildung von Harnstoff bei der Spaltung des Arginins. E. Schulze und A. Likiernik. 24: 2701 (1891). Zur Kenntnis des Stachydrins, A. v. Planta und E. Schulze. 24: 2705 (1891). Über basische Stickstoffverbindungen in den Samen von Vicia sativa und Pisum sativum. E. Schulze. 25: 84 (1892). Über das Lecithin der Pflanzensamen. E. Schulze und A. Likiernik, 25:85 (1892). Zur Chemie der pflanzlichen Zellmembranen. E. Schulze. 25 : 434 (1892). Über das Vorkommen von Guanidin im Pflanzenorganismus. E. Schulze. 25:658(1892). Über einen stickstoffhaltigen Bestandteil der Keimlinge von Vicia sativa, E. Schulze. 25: 869 (1892). Zum Nachweis des Guanidins. E. Schulze. 25: 2213 (1892). Über das Vorkommen von Betain und Cholin in Malzkeimen und in den Keimen des Weizenkorns. E. Schulze und S. Frankfurt. 26: 2151 (1893). Über die Verbreitung des Rohrzuckers in Pflanzen. E. Schulze und S. Frankfurt. 27: 62 (1894). Über das Vorkommen von Raffinose im Keime des Weizenkorns. E. Schulze und S. Frankfurt. 27: 64 (1894). Über krystallisiertes Lävulin. E. Schulze und S. Frankfurt. 27: 65 (1894). Über das Vorkommen von Trigonellin in den Samen von Pisum sati- vum und Cannabis sativa. E. Schulze und S. Frankfurt. 27 : 769 (1894). Über ;8-Lävulin. E. Schulze und S. Frankfurt. 27: 3525 (1894). Vorkommen von Arginin in Knollen und Wurzeln einiger Pflanzen. E, Schulze. 29: 352 (1896). Verbreitung des Glutamins in den Pflanzen. E. Schulze. 29: 1882 (1896). Stickstoffhaltige Bestandteile der Keimpflanzen von Ricinus communis. E. Schulze. 30: 2197 (1897). Über die Spaltungsprodukte des Arginins. E. Schulze und E. Win- terstein. 30:2879(1897). Bestandteile des Wollfetts. E. Schulze. 31: 1200 (1898). Konstitution des Arginins. E. Schulze und E. Winterstein. 32 : 3191 (1899). I9I2] Ernst Winterstein 19 Über das spezifische Drehungsvermögen des Glutamins. E. Schulze. 39: 2932 (1906). Die Konstitution des Stachydrins, E. Schulze und G. Trier. 42 : 4654 (1909). 8. In verschiedenen Zeitschriften Ueber die Elementarzusammensetzung der tierischen Fette, insbeson- dere der Fette vom Schaf, Rind und Schwein. E. Schulze und A. Reinecke : Annalen der Chemie und Pharmacie, 142 ; 191-218 (1867). Untersuchungen über die Respiration des volljährigen Schafes bei Erhaltungsfutter. W. Henneberg, E. Schulze, M. Märcker und L. Busse : Zentralblatt für die medizinischen Wissenscliaften, 1870; 353-356; 369-370. Ueber Stickstoffausscheidung im Harn der Wiederkäuer. E. Schulze und M. Märcker: Zeitschrift für Biologie, 7; 49-62 (1871). Ueber die Bestimmung des aus Amiden abspaltbaren Ammoniaks in Pflanzenextrakten. E. Schulze. Zeitschrift für analytische Chemie: 21 : 1-26 (1882). Ueber das Stachydrin. E. Schulze: Archiv der Pharmacie, 231 ; 305 (1893). ^ Zur quantitativen Bestimmung der Kohlenhydrate. E. Schulze: Chemikerzeitung, 1894; 527. Ueber die Analyse der Pflanzensamen. E. Schulze: Ibid., 1894; No. 43- Ueber die Cellulose. E. Schulze: Ibid., 1895; No. 65. Inwieweit stimmen der Pflanzenkörper und der Tierkörper an ihrer chemischen Zusammensetzung überein und inwiefern gleicht der pflanzliche Stoffwechsel dem tierischen? E. Schulze: Viertel- jahr sschrift der Naturforschenden Gesellschaft in Zürich, 1894; 243- (A) Verbreitung des Glutamins in den Pflanzen. (B) Die in den Keimpflanzen der Coniferen enthaltenen Stickstoffverbindungen. E. Schulze. Verhandl. d. Schweiz. Naturf. Gesellsch., Zürich (1896), 126-127. Ueber die Zellwandbestandteile der Cotyledonen von Lupinus Intens und Lupinus angustifolius und über das Verhalten während der Keimungsvorgänge. E. Schulze : Berichte der Deutschen Bo- tanischen Gesellschaft, 14; 66-71 (1896). ^ Ueber den Eiweisszerfall und Eiweissbildung in der Pflanze. E. Schulze : Ibid., 18 ; 36-42 ( 1900) . 20 Ernst Schulze [Sept. Ueber Tyrosinbildiing in den keimenden Samen von Lupinus albus und über den Abbau primärer Eiweisszersetzungsprodukte in den Keimpflanzen. E. Schulze: Ibid., 21; 64-67 (1903). Ueber die Argininbildung in den Keimpflanzen von Lupinus luteus. E. Schulze: Ibid., 22 ; 381-384 (1904). 9. Dissertationen Ueber die Eiweisssubstanz der Kürbissamen und über die Zersetzungs- produkte, welche während des Keimprozesses aus derselben ent- stehen. J. Barbieri. 1878. Zur Kenntnis des Glutamins. Ueber Ammoniakbestimmung in Pflanz- ensäften und Pflanzenextracten. E. Bosshard. 1880. Ueber die chemische Zusammensetzung der Samen von Lupinus luteus und über ein in denselben enthaltenes dextrinartiges Kohlenhy- drat. E. Steiger. 1886. Ueber das pflanzliche Lecithin und über einige andere Bestandteile der Leguminosenchalen. A. Likiernik. 1891. Zur Kenntnis des pflanzlichen Amyloids und über einige andere Be- standteile der pflanzlichen Zellmembranen. E. Winterstein. 1892. Ueber die Zusammensetzung der Samen und etiolierten Keimpflanzen von Cannabis sativa und Helianthus annuus. S. Frankfurt. 1893. Ueber die Zusammensetzung der Samen und der etiolierten Keim- pflanzen von Lupinus angustifolius. Miron Merlis. 1897. Ueber die Bestandteile der Samen von Picea excelsa und über die Spaltungsprodukte der aus diesen Samen darstellbaren Protein- stoffe. N. Rongger. 1898. Versuche zur quantitativen Bestimmung der bei der Zersetzung der Eiweisskörper durch Säuren entstehenden Basen. O. Meyer. 1900. Versuche zur Bestimmung des Gehaltes einiger Pflanzen und Pflanzen- teile an Zellwandbestandteilen, Hemicellulosen und Cellulosen. A. Kleiber. 1900. Beiträge zur Kenntnis der Cholesterine und der Methoden, die zu ihrer Abscheidung aus den Fetten und zu ihrer quantitativen Bestim- mung verwendbar sind. E. Ritter. 1902. Beiträge zur Kenntnis der in den Pflanzensamen enthaltenen Kohlen- hydrate. Ch. Godet. 1909. Ein Beitrag zur Kenntnis der pflanzlichen Betaine und ihre Bedeutung. Das Stachydrin, seine Konstitution und seine Synthese. G. Trier. 1910. A RESUME OF THE LITERATURE ON INOSITE- PHOSPHORIC ACID, WITH SPECIAL REFER- ENCE TO THE RELATION OF THAT SUBSTANCE TO PLANTS ANTON RICHARD ROSE (Laboratory of Biological Chemistry of Columbia University, at the College of Physicians and Surgeons, New York) Contents. — Discovery: by the microscopist, 21; by the chemist, 22. Occur- rence, 2^; preparation, 25; properties, 26; Constitution, 31; terminology and Clas- sification, 35 ; analytical methods, 37 ; role in plants, 39. Bibliography, 46. DISCOVERY OF INOSITE-PHOSPHORIC ACID SALTS BY THE MICROSCOPIST In 1854 Hartig/ engaged in a microscopic study of the seeds o£ various plants, noted in all his sections small particles similar to the starch grains of the potato, which at that time were absorbing the interest of plant physiologists.^ These grains were obviously not starch, as they did not give the characteristic blue color with iodine in potassium iodid Solution. They are more commonly present in seeds than starch, the latter being frequently replaced by fat. Hartig considered them an essential reserve product designed to play an important part in the germination of the seed and the growth of the plant. He first called them " Klebermehl " but within a year renamed them " aleurone grains " f rom the Greek aXevpov (wheat fiour), a term still in use. Not only did he consider them significant for the plants in which they are found but also for the animals which eat them. The method which he employed for separating them from the other parts of the seed is the one usually followed by the investigators who have since worked with these ^ The papers ref erred to in the text and not accompanied by f ootnote ref er- ences are those which pertain specially to the literature of inosite-phosphoric acid and are given at the end of this review in the alphabetical order of the names of the authors. ^The starch grains were minutely studied by Nägeli and his coworkers. Their work (collected in Die Stärkekörner, 1858) probably afforded the Stimulus for the efforts which led to the discovery of inosite-phosphoric acid so early. 21 22 Literature on Inosite-Phosphoric Acid [Sept. grains, namely, extraction of the macerated seeds with ether and removal of the aleuron grains from the cellular debris by Sedimenta- tion in this medium. They are insoluble in both alcohol and ether, somewhat soluble in water, and quite soluble in dilute acids. In the years following Hartig's discovery several other botanists turned their attention to the aleuron grains and came to conflicting conclusions as to their nature. Von Holle considered them protein carriers and referred to them as " Proteinkörner." Both Sachs and Gris looked upon the particles as fat concentrates. By far the most important study in this field was the comprehensive work of Pfeffer in 1872. He differentiated the grains described by Hartig into three groups : (i) crystals of calcium Oxalate, (2) a protein sub- stance, and (3) a Compound giving no reactions for protein, fat, or inorganic salts. This last type was found in all of the one hundred different seeds which he examined. These particles he described as having rounded surfaces, assuming spheroidal shapes and fre- quently twinning, so as to present a convoluted appearance. Enough of the grains were obtained for a chemical examination, which was made for him by his colleague Brandon. The solubilities were the same as those reported by Hartig. Nitrogen could not be detected. Positive tests were obtained for calcium, magnesium, and phos- phorus. Organic matter was noted and the Suggestion made that the substance was a phosphate combined with a carbohydrate. These phosphorus-bearing spheroidal bodies occurring with or in the aleuron grains Pfeffer named "Globoid." DISCOVERY OF INOSITE-PHOSPHORIC ACID BY THE CHEMIST Palladin, while engaged on a study of the proteins of Sinapis niger in 1893, observed an unusual phenomenon. After extraction of the fat-free finely ground seeds with ten per cent. sodium chlorid Solution and heating the extract, he obtained a voluminous precipi- tate which partly dissolved on standing. A few trials showed that he had a substance soluble in cold but insoluble in hot water. By filtering off the permanent coagulum, reheating the filtrate and filtering while hot, he obtained a fairly pure product rieh in phos- phorus, containing calcium and magnesium, but no nitrogen. It proved non-reducing when tested with Fehling Solution, both before I9I2] Anton Richard Rose 23 and after hydrolysis with acids. It was soluble in water and aclds, and precipitated by the alkali earths and the heavy metals. Subsequently Schulze and Winterstein published a paper con- firming the observations of Palladin, and also noting that the phos- phorus was not precipitated by ammonium molybdate. These authors expressed the opinion that the Compound thus discovered by chemical procedure is identical with Pfeffer's " globoid." The fol- lovving year (1897), a more detailed paper was published by the junior author, in which the identity and properties of the substance were more fully revealed, and " inosite-phosphoric acid " was sug- gested as the proper name for the Compound, inasmuch as it yielded inosite and phosphoric acid on hydrolysis. The most complete study of this substance has been made by Posternak; his findings are embodied in eight papers and several applications for patents. He discarded the name suggested by Winterstein and proposed a structural formula which does not include the inosite ring. He gave to the substance the name "phytin" (from the Greek vT7]v) and under this trade name it is now placed on the market by a chemical firm in Basel. OCCURRENCE OF INOSITE-PHOSPHORIC ACID As already noted, phytin was first thought to be a storage product in seeds ; and this early Impression has been confirmed by subsequent investigation, no case having been reported of a seed in which it is completely lacking. The accompanying table (i) lists the plants specially mentioned in the literature in connection with the study of inosite-phosphoric acid. The relative data in the table are not in close accord, but no true comparison can as yet be drawn between the species, for most of the data were obtained at periods when adequate and uniform analytical methods were unavailable. The figures quoted in the table give an approximate idea of the quantitative signijficance of this important Compound, in relation to other forms of phosphorus available for seedling growth and the phosphorus requirement of man and beast. Posternak makes the Statement that seeds rieh in fat carry the largest amount of phosphorus, which is in harmony with the micros- copist's observations that the aleuron grains are particularly numer- 24 Literatur e on Inosite-Phosphoric Acid [Sept. TABLE I Recorded analytic data on the occurrence of inosite-phosphoric acid Plant. Total P Per Cent. Spruce fir 0.66 Pea (Pisum sativum).. 0.37 Pea, yellow Bean, white {Phaseolus vulgaris) 0.51 Bean, brown Hemp (Cannabis sativa) 1.46 0.76 Rice (Ory::a sativa)... 0.95 Rice flour Rice bran 2.22 Rice germ 6.20 Wheat ( Triticum sativa) 0.45 Wheat bran i.ii i.il Graham flour Sesame (Sesamum in- dicum) 0.77 Corn (Zea mays) 0.29 0.35 Oats (Avena sativa) . . 0.46 Barley (Hordium sati- vutn) 0.50 0.47 0.54 Barley bran Sunflower {Helianthus annuus) 0.83 Rye (Seeale cereale) . . 0.43 Ratio of the P in phyto- phosphate to Total P Per Cent. 91-5 70.8 19.0 81.6 58.0 91.4 15-0 45-9 69.0 74.1 89.2 29.9 84.0 52.0 29.0 16.3 54.0 48.9 48.0 87.4 Plant. 38.0 36.S 44.0 60.4 86.3 90.3 28.9 Total P Per Cent. Ratio of the P in phyto- phosphate to Total P Per Cent. 25.0 Rye flour Rape (Brassica napus olifera) 0.98 80.0 I-I9 44-5 0.54 38.0 Rape cake 49.5 Soy bean (Soja hispida) 0.57 58.0 Lentil (Lens esculenta) 0.30 82.6 0.30 9.3 Cocoanut (Cocus nuci- fera) 88.4 695 Cottonseed (Gossipium herhaceum) 93.6 Pine : Pinus cembra . . . 0.47 14.39 Pinus excelsa ... 0.63 21.6 Castor bean (Ricinus communis) 0.26 41.6 Millet (Panicum millia- ceum) 0.77 44-97 Vetch (Vicia faba mi- nor) 0.47 4.4 Red Clover (Hay) 0.24 70.0 Radish : Root (Rapha- nus vulgaris) 0.02 15.0 Turnip : Root (Bras- sica esculenta?) 0.02 15.0 Dahlia : Tuber (Dahlia variabilis) ..." Spheroids of phjrtin " Potato : Tuber (Alliuni cepa ) " Spheroids of phytin " The analytic results in the above table are those for seeds of the plants, except in the last five cases. They are compiled from a number of sources. Among the plants studied for their phyto-phosphate content, in which the rela- tive amount of this substance is not given, are the following: Beta vulgaris, Brassica campestris, Cucurbita pepo, Ervum lens, Lupinus albus, L. angustifolius, L. luteus, Pinus laricio, P. maritima, Sinapis nigra, Solanum tuberosum, and the tubers of AI Hunt cepa and Dahlia variabilis. In only two materials reported, namely, rutabaga root and alfalfa hay, could no phyto-phosphate be found. In several instances the total phosphorus was not reported. Where there is a close agreement between two or more results, only one figure is given above. 1912] Anton Richard Rose 25 ous in oily seeds. He also remarks that the smaller seeds such as cereals are the richest in " phytin." This Compound is not entirely confined to seeds, its presence having also been noted in the potato near the eye and as characteristic spheroids in the tubers of Allium and Dahlia. Roots functioning as storage organs, such as those of the Brassicae, contain small amounts. None was found by Totting- ham and Hart in the mature stems and leaves of the common fodder plants, but it occurs in clover leaves and in millet during the late flovvering period, and also in tender shoots. PREPARATION OF INOSITE-PHOSPHORIC ACID AND ITS SALTS To prepare phytin or its closely related Compounds from seeds, they should be finely ground and, if fat is present in large amounts, it should be removed by extraction with ether and alcohol. Most of the preparations reported in the literature have been obtained from cereals by leaching with 0.2 per cent. hydrochloric acid Solu- tion. Acetic acid has also been used in i per cent. Solution, and in a few cases acid Solutions of greater concentration have been employed. To remove the soluble proteins from the extract, Levene used picric acid; other investigators have coagulated them by heating and filtration after cooling; but when acidulated water is used the proteins do not seem to interfere appreciably with the preparation of pure phytin. The reserve proteins of the seeds are of the globulin type and are soluble only in the presence of salt in the extracting agent. Precipitation of inosite-phosphoric acid from its Solutions can be accomplished by several methods, such as the use of the acetates of the heavy metals, barium chlorid in ammoniacal Solution or magnesia mixture. In these cases the precipitated Com- pound is obviously in a form different from that in which it occurs in the original material. To obtain the salt more nearly in the form in which it is found in the seed, it may be precipitated by heating the Solution to almost boiling and filtering while hot; or better, by adding four volumes of ninety-five per cent. alcohol. In obtaining pure preparations of inosite-phosphoric acid or its salts, a number of reprecipitations are necessary. These have been made alternately with copper, lead, and barium. Salts of the first two are decom- posed by suspending in distilled water and bubbling hydrogen sul- 26 Literatlire on Inosite-Phosphoric Acid [Sept. phide through the liquid ; the third is removed by adding dilute sul- phuric acid. In all cases, the lead or copper salt is the last pre- cipitated in this manner of purification; and when the product is carefully washed, and the metal removed by hydrogen sulphide, the filtrate from the lead or copper sulphide is evaporated at a low tem- perature, leaving the inosite-phosphoric acid.^ The various salts which have been studied were made from this acid. In obtaining the acid for the preparation of pure Compounds, the greatest difficulty lies in removing the last traces of magnesium. Rising overcame this difficulty by taking up the syrupy acid with absolute alcohol, and adding ether until droplets of the acid formed. He then filtered off the acid magnesium inosite-phosphate and again evaporated. The commonest impurity in phytin is inorganic ortho- phosphate which, however, is easily removed. Starkenstein uses the calcium salts of the mixed acids and washes with glacial acetic acid, which dissolves the inorganic part but not the organic phosphorus Compound. Forbes precipitates with magnesia mixture, removes the excess of this reagent by washing with ammonia water, washing again with alcohol, and extracting with 95 per cent. alcohol con- taining 0.2 per cent. mineral acid, which also dissolves all the in- organic phosphorus and none of the " phytin." Attempts to prepare these salts synthetically will be referred to in a later section. PROPERTIES OF INOSITE-PHOSPHORIC ACID AND ITS SALTS The substance widely known as "phytin," and described in the middle of the last Century by the microscopists as " spheroid bodies," frequently assumes the globular shape when forced out of Solution, In most cases, the precipitate comes down as a flocculent amorphous mass. Inosite-phosphoric acid has not as yet been obtained in crys- talline form. At room temperature, it is a syrup of light straw color, which becomes very viscid on cooling to — 20° C, and darkens on heating to 100° C. Vorbrodt found that this coloration could not be prevented by replacing the air with an inert gas during the heating, and from this concludes that the change is not due to oxidation. If the heat is allowed to reach 125° C, an insoluble dark char is produced (cf. Posternak). Inosite-phosphoric acid may 'For details of the method of preparing the acid see Hart and Patten (page48). 1912] Anton Richard Rose ^y form neutral salts, acid salts, double salts, or acid double salts. The acid, neutralized with alkali and evaporated to dryness, gives a brownish horny mass; but if an alkali earth is also present, double salts are formed, which crystallize in fine needles with eight mole- cules of water. The magnesium salts crystallize in small and uni- form spherules, while the copper salts form large and irregulär spherules. Twin forms are frequently produced in the copper pre- cipitates, resembling the globoids of which drawings appear in Pfeffer's paper. These spheroid masses may be Clusters of needles of approximately equal lengths, as is suggested by the regularly pitted surfaces sometimes seen, and the term spherocrystal can accordingly be applied to them. The copper Compounds are green ; the others, as far as reported, are white. Occasionally a faint pinkish cast has been noticed in pure preparations. The acid is miscible in all proportions with water. It is soluble in alcohol but not in the other common lipoid solvents. Ether added to an alcoholic Solution precipitates the acid in droplets. According to Posternak, the acid-alkali and acid-magnesium salts are soluble in alcohol and water. The double salts are soluble in water, forming opalescent Solutions from which they are precipitated by chlorid and acetate of potassium, redissolving if these are added in excess. The decrease in solubility of the salts of inosite-phosphoric acid is in the following Order : alkali, alkali earth and heavy metal. The magne- sium Compounds are more soluble than the calcium salts and the latter more soluble than those of barium or Strontium. The same Order of solubility also holds for acid salts, double salts and normal salts. These phytophosphates are more soluble in cold than in hot water, and heating frequently precipitates them, even in the presence of dilute acetic acid. This precipitation is largely influenced by other Compounds in Solution, halogens and sulphates inhibiting, and phosphates facilitating the reaction. Posternak noted that the precipitates thus formed by heating were not always completely dis- solved on cooling; also that the phytophosphates not readily soluble in cold water were changed to more soluble forms by dissolving in dilute acid and precipitating with alcohol. Dilute mineral acids are solvents for all of these Compounds. Acetic acid does not dissolve the salts of inosite-phosphoric acid with the heavy metals, barium. 28 Literature on Inosite-PJwsphoric Acid [Sept. calcium and Strontium ; but the magnesium and alkali salts, and the double salts, are very soluble in this reagent. Posternak says that the alkali salts of this acid are solvents for the Compounds with alkali earths, and that on standing, crystals of double salts form in these Solutions, tending to arrange themselves in rosettes — a further Suggestion as to the mode of formation of the characteristic sphero- crystals mentioned above. Inosite-phosphoric acid Solutions do not polarize light, and pass through semi-permeable membranes com- paratively slowly. All the salts of inosite-phosphoric acid, except the alkali salts and the acid magnesium salts, are precipitated f rom aqueous Solution by four volumes of alcohol. The acid and its salts in alcoholic Solu- tion are precipitated by ether. The addition of neutral Solutions of silver, lead, copper, cadmium, iron, uranium, Strontium, barium and calcium precipitates the acid f rom its Solutions ; so also do magnesia mixture and albumin. According to Posternak, precipitation with copper is prevented by the presence of fat. The copper salts are soluble in ammonium hydroxid Solution. Ammonium molybdate in nitric acid does not cause precipitation in dilute Solutions of inosite phosphates, but in concentrated Solutions white needles are formed on long Standing which are insoluble in nitric acid and soluble in water. Preparations f rom seeds retain persistently small amounts of magnesium, several reprecipitations being necessary to get a salt containing a single metal. It is equally difficult to get a preparation free from the hydrogen ion, and it may be said in general that the property of forming acid salts and double salts is very characteristic of inosite-phosphoric acid. The most important contribution to our knowledge of the nature and properties of its salts has been made recently by Anderson. From acid purified by means of barium pre- cipitation and the method described by Hart and Patten, the follow- ing Compounds have been prepared : tri-barium, penta-barium, penta- barium di-ammonium, penta-magnesium, penta-magnesium di-am- monium, tetra-cupric di-calcium, tetra-calcium, penta-calcium, hexa- cupric, octa-silver, and hepta-silver salts. Most of them are white amorphous powders, but the tri-barium and tetra-calcium salts can be reprecipitated in irregulär crystalline form. Pure preparations have been made and analyzed by several other investigators. The results of their work are given in Table 2. I9I2] Anton Richard Rose 29 TABLE 2 Analytic data pertaining to inosite-phosphoric acid (Compüed from results reported in the literature of the subject) Name of Author Anderson. Contardi. Hart, Patten. Hart. Tottingham. Horner. Levene. Elements Carbon Hydro- gen Phosphorus Barium Magne- sium Calcium Other Metals Ratios' C=6 P=x P=6 M'=x (Prepared from the purified commercial product.)^ 6.42 4-59 10.56 10.76 1-44 1-15 3-21 3.22 37-21 48.87 46.99 14-13 42-9 13-03 22.46 17.66 14.69 16.87 13.46 14.07 21.29 26.37 26.16 19.07 20.62 21.75 22.53 16.88 11.94 13-02 (Synthesized by means of inosite and ortho-phosphoric acid.) 6.42(K) 33-54(Cu) 55-98(Ag) 52.43(Ag) 6 6 6 6 6 6 6 6 6 6 6 6 6 14.24 14-23 9.16 9.60 ( 12.62 8.17 3-45 3-61 1.64 1.68 24.09 24.31 16.34 16.05 35-57 34-48 4 4 4 4 (Synthesized by means of inosite and pyro-phosphoric acid.) 3-24 1.58 (acid) 26.51 16-55 ( 26.08 21.08 35-90 (Prepared from rice bran.) 9.0 13-8 10.89 10.63 17-30 3-00 3-40 3-63 28.10 12.50 15.60 21.40 (Preparations from wheat bran.) 25-98 56.2 23.2 8.9 13-5 22.1 (Cu) 16.38 26.08 5-8 I-13 2.6 (K) 6 2 (Similar preparations.) (The commercial preparation.)- 20.32 1 I 1.45 I 11-96 I 9-84 5.66 17.90 7-70 1.47 3-57 23-00 13-93 13-16 11-95 (Preparations from hemp.) 44-50 40.14 5-5 6 1-5 2 6 10 12 12 12 10 8 IG 12 6 4 (Synthesized by means of inosite and ortho-phosphoric acid.) 12 12 12 12 63 - 1 7 8.5 4 9-5 ^ The empirical formulas have been calculated from the analytic results, and the relations between the carbon and phosphorus, and the phosphorus and base (M'), are given in these two columns. The carbon and the phosphorus are, in each case, assumed to be six atoms per molecule. ^ Placed on the market by the Gesellschaft für Chemische Industrie in Basel. ^ A 0.2 per cent. HCl extract of wheat bran several times reprecipitated from weakly acid Solution by alcohol. * The first substance; obtained by extracting with sodium chlorid Solution and, after removing protein with picric acid, precipitating with copper and removing the copper. 30 Literature on Inosite-Phosphoric Acid [Sept. TABLE 2 (coniinued) Name of Author Elements Carbon Hydro- gen Phosphorus Barium Magne- Calcium Other Metals Ratiosl C=6 P=x P=6 M'=x Posternak. Rising. Suzuki, Yoshimura, Takaishi. Vorbrodt. Winterstein. Winterstein, Schulze. (A large number of preparations were raade in which several kinds of seeds were used.) 9-97 4-79 7-25 7-44 6.51 26.08 25.89 12.70 19-42 19-73 6 6 6 6 3-70 I.OO 1-34 1.49 50-45 8.41 i9.02(Na) 5-37 1 1-03 I (Preparations from barley bran.) 13-08 I I I l52.6s(Ag)| (Preparations from rice bran.) 1.21 I 23.48 I I S-81 I 17-48 I (Preparations from corn.) 15.18 I42.62 I I (Preparations from black mustard.) 18.42 I I 7-8 I I 9.65 I 2.83 I 15-20 I I I 5-5 I - I 3.7 II 4 12 I 7 1 6 I 10.6 8 6.5 — ?* The alkali salts are hygroscopic, but the others do not change in weight under ordinary conditions. Posternak assigns to the double alkali salts of the alkali earths eight molecules of water. The barium salt prepared by Vorbrodt lost 9.33 per cent. of its weight in the presence of phosphorus pentoxid, but regained it when exposed to a moist atmosphere. At 110° C, 11.5 per cent. was lost. Ander- son reports for his tri-barium salt five molecules of water, for his tetra-calcium salt twelve, and for his penta-magnesium salt twenty- four. Inosite-phosphoric acid is easily decomposed by heating with strong acids in sealed tubes but does not spontaneously break down into its cleavage p'roducts. Mendel and Underhill kept a Solution of the acid for many months and found at the end of the time no apparent change. Posternak states that heating phytin in alkaline Solution to 100° C. causes no decomposition, but Winterstein found that if a twenty per cent. Solution of alkali were used (sodium hydroxid) and the temperature raised to 230° C, cleavage occurred. Contardi states that cleavage does not occur when these salts are * Extracted with sodium chlorid Solution, precipitated by heating, and fil- tered hot. I9I2] 'Anton Richard Rose 31 heated in water to 200° C. under pressure. According tc Giacosa it is more readily hydrolyzed than lecithin. From the fact tbat the products of hydrolysis are inosite and phosphate, Winterstein came to the conclusion that the Compound is inosite-phosphoric acid. CONSTITUTION OF INOSITE-PHOSPHORIC ACID Posternak, who has done more work on this substance than any other one investigator, did not agree with Winterstein in the con- clusion set forth above. From his analyses he first constructed the following formula: HC(OH)OP .(OH) (X) I HC(OH)0 P^ (OH) but, as benzoyl chlorid gave no positive test for the hydroxyl group, a second formula was proposed (anhydro-oxymethylene-diphos- phoric acid) : CH,-0-P(' / ^(OH), («) o: \ /^ \cHj-o— ?<;■ %(0H), He was of the opinion that inosite is synthesized from the products of hydrolysis when the "phytin" is heated under pressure with mineral acids. A number of chemists have expressed doubt concern- ing the probability of such a formation of inosite, either from this organic group or any part of it that might result from the action of the acid thereon. In 1907 Suzuki and his co-workers obtained inosite from "phytin" by the action of an enzyme, from which they concluded that inosite is an integral part of the "phytin" molecule and constructed the following formula to represent their view : 32 Litcrature on Inosite-Phosphoric Acid [Sept. HO-^P— O— Q Ho/ H -C— 0-P' /^ -OH \0H (3) P_0— CH HC— 0— Pf HO O^ =0 v\0H sOH hq// ;P— O— c- h -C— O— P= h =0 \\0H HO/ \0H M. W., 660; C = 10.91%; P = 28.18%. Neuberg came to similar conclusions the following year when he obtained inosite and furfurol on mixing " phytin " with phosphoric acid and distilling under reduced pressure, and also showed that furfurol can be obtained from inosite. He proposed the following f ormula : H -c- (4) HO. H0\ H HO>P-0-(; HO^. HO-7P— O— CH HC— O HO/ H /OH O— ^<0H /OH -P.^OH \0H t -CH HO— P\ /P— OH HO/ ^0^ \0H M. W., 714;C = 10.085% ; P = 26.05%. Levene, working with a preparation from hempseed, was led to believe that the "phytin" of this grain contained in its molecule phosphate, inosite and a carbohydrate of the pentose group. His work was criticized by Neuberg, who claimed that there were impurities in the preparation. In view of the known intimate asso- ciation of the phytin with protein and carbohydrate in the aleuron grain, and the possible occurrence of a chemical combination of both phyto-phosphate and carbohydrate with protein, it is conceiv- able that Levene had a product holding pentose as an integral part igi2] 'Anton Riclmrd Rose 33 and not as an impurity, though in view of all the available evidence Neuberg's criticism seems at the present time somewhat justifiable. Starkenstein also refused to accept the simple formula proposed by Posternak and offers the f ollowing : HO-^P— OH.HO— C-C— OH.HO— P^OH ^\ j i /^ HO— P— OH. HO— C C— OH.HO— P^H o/ 11 \) (5) HO— C— C— OH HO OH 0=P P=( /\/\ OH O OH M. W., 714 ; C = 10.985^ ; P = 26.05 He argues that the phosphoric acid is in the pyro-form^ from the fact that its silver salt is the same color as silver pyro-phosphate, and that its behavior when titrated with Standard uranium acetate Solution is also like that of pyro-phosphoric acid. That it is not combined in the usual form of an ester but held loosely in a complex "addition form," he maintains from the fact that an increase of inosite and inorganic phosphate resulted from heating some of the calcium salt for an hour at ioo° C. These arguments are not alto- gether convincing. Anderson has prepared a silver salt of the ortho- tetra-phosphoric acid ester with inosite and reports it as being white like the pyro-phosphoric acid salt. In the quantitative titration of pyrophosphoric acid with a uran- ium acetate Solution, standardized by ortho-phosphate and using fer- rocyanide as indicator, only one half of the phosphorus value is obtained. Starkenstein explains this phenomenon by the assump- tion that one half of the more reactive ions of the phosphoric acid have been removed in the dehydration. Now if two phosphoric- acid groups had formed esters with one polyalcohol, analogous con- ditions would have resulted as far as the ions are concerned, and bivalent ions would be expected to connect the two phosphoric acid * That phosphorus occurs in plants in the pyro form may seem stränge to many, but this is not the first time that such an occurrence has been suggested. In 1892 Hardin (5". C. Exp. Sta. Bull., N. S., No. 8) reported his finding both pyro- and meta- phosphate in cottonseed meal, when he sought, in this feeding material, a substance toxic to cattle. 34 Literature on Inosite-Phosphoric Acid [Sept. radicles. This may be illustrated by these two graphic represen- tations : .HO: OH^^^* \ /OH«-^ \ / 0=:P OH 0=P— OH ^ -f— ua. The idea of this type of reaction for ortho and pyro-forms is in harmony with the fact that when inosite-phosphoric acid is precipitated in acid Solutions by divalent metals, the tri-metal salt is the more readily formed. The activity of the hydrogen ions is relatively greater in the inosite-phosphoric acid than the sum total of the ions of the ortho-forms would be, probably due to the influ- ence of increased negative electric charges in the many phosphorus atoms held in one molecule, so that, altho six having been eliminated in the assumed ester formation, the very reactive ions are eight in number. The last column in Table 2 is interesting in this con- nection. Finally, the Statement that the inosite-phosphoric acid is decomposed by dry heat has been shown to be erroneous by both Anderson and the writer. That phytin is a salt of inosite-phosphoric acid seems to be con- clusively demonstrated by the synthetic work of Contardi, whose preparations from rice bran gave analyses identical with a synthetic preparation obtained by heating anhydrous inosite with ortho- phosphoric acid (sp. g. 1.7). Other workers have attempted to substantiate this result, but so far without success. Carre could obtain only a mixture of the two chemicals ; Anderson was able to produce tetra-ortho and di-pyro-phosphoric acid esters. It does not follow that these preparations of inosite-phosphoric acid are identical in form with the organic phosphorus Compound occurring in plants. The writer calls attention elsewhere to a differ- ence of behavior between the phytophosphate in seeds and in prepa- rations.^ Certain data, not as yet published, as well as differences in the products described in the literature, fall in with those suspicions.^ What inosite-phosphoric acid is, in terms of a definite chemical 'Rose: Technical Bulletin 20 of the New York Agricultural Experiment Station. ' Cf . Preparations analyzed by Patten and Hart, Winterstein and Schulze, and Levene, in table 2, p. 29. I9I2] Anton Richard Rose 35 structure, is an open question. It is probably an ester of phosphoric acid with inosite, in which six phosphoric acid groups are united with each inosite molecule. This ratio, Cß : Pg, is indicated for the vast majority of the pure preparations analyzed; exceptions are the preparations by Levene, Vorbrodt and Rising (Table 2), these authors giving the ratio Cq : P5.5. Anderson has pointed out that bis and Rising's silver salts are probably identical, Rising's analysis being equally well adapted to the formula C6Hi7027P6Ag7. It seems probable that the molecular weight when accurately deter- mined will be reported as 714 or will differ from this by the molec- ular weight of three molecules of water, The molecule seems to contain twelve hydrogen atoms readily separated in ionization, six of which are exceedingly reactive ; the remaining hydrogen atoms gradually diminish in reactivity by twos, the last four being slow to enter into an exchange with bases. The most readily formed salts are therefore those corresponding to an octavalent acid and the other common ones are in six and tenvalent combinations. TERMINOLOGY AND CLASSIFICATION The investigations of which this paper is a brief review have brought to the biological chemist and plant physiologist a type of phosphorus Compound from the plant world which is relatively new and probably of prime importance. The phosphorus which occurs in acid and water extracts was formerly considered inor- ganic phosphate, and awakened no especial interest, and the mention of organic phosphorus immediately brought to mind nucleoproteins and lecithins. In the organic laboratories combinations of phos- phorus with various organic radicals have been made and recently- prepared phosphoric acid esters'^ resemble " phytin " in some respects, and so may be considered of special concern to the biological chemist, as possibly bearing on problems in his field. A cleavage product of inosinic acid, c?-arabinose phosphoric acid,^ is significant as showing that carbohydrate esters are not confined to those pro- duced by the synthetic Operations of the laboratory. Even more 'v. Lebedew: Biochem. Zeit., 1909, 20, 114; Neuberg and his coworkers : Ber., 43, 2060; Biochem. Zeit., 23, 515; 26, 115 and 529; 36, 5; Langheld, Ber., 44, 2076. 'Levene and Jacobs, Ber., 191 1, 44, 746. 36 Literatur e on Inosite-Phosphoric Acid [Sept. striking are Iwanow's experiments® in which, when yeast was allowed to ferment sugar in the presence of sodium phosphate, there was noted a disappearance of the inorganic phosphorus, amounting to f rom eighty to ninety-three per cent. ; and in the liquors there was foiind an organic phosphorus Compound optically active and giving reactions for aldehydes and ketones. Biochemical syntheses of this class have also been successfully made by other investigators.^*' Of special interest are the inosite esters with ortho- and pyrophos- phoric acid prepared by Anderson. Mention may also be made of the spherocrystals discovered by Hansen" in the parenchyma cells of the Euphorbia caput medusae, which he describes as amorphous masses of calcium and magnesium phosphate, but which Belzug^^ later has shown to be salts of a new organic acid, phosphomalic acid. We may reasonably expect that additional phosphorus-bearing sub- stances of this kind will be discovered in nature by the phyto- chemist for which a rational System of nomenclature will be required. Rising, in his paper on inosite-phosphoric acid, refers to soluble phosphorus Compounds obtained from grains, which he promises to discuss in later contributions. These substances he considers closely related to "phytin," and proposes classifying them as a single group with the generic name " phyto-phosphoric acid." This term we may profitably adopt to indicate the acid radicals of those organic phos- phorus Compounds which may be found in the water and dilute acid extracts of plant materials. In this group will be included the gly- cerophosphates, phosphomalates, such hexose and pentose phos- phates as may be discovered in plants, and the phytin-like substances. The term " phytin " as used at present seems to designate that substance which is extracted from seeds by leaching with dilute acids, reacting positively in the tests for calcium, magnesium, and, after hydrolysis, for phosphoric acid and inosite. The multiplica- tion of trade names for definite chemical Compounds is not desirable. There are many students and other workers who must of necessity 'Iwanow: Zeit, für physiol. Chein., 1107, 50, 281-288. "Young and Hardin : Biochem. Zeit., 191 1, 32, 173-188; Proc. Chem. Soc, 21, 23, 24; Proc. Roy. Soc., London, 77, 80, 81, 82; Euler and Ohlsen: Biochem. Zeit., 191 1, 37, 313. " Hansen : Arbeit, des bot. Inst., Würzburg, 1888, 92-122. "Beizug: Jour. de bot., 1893, 7, 211-229. I9I2] Anton Richard Rose 37 carry in memory more names of organic Compounds than they can reasonably be expected to define in terms of chemical formulae, if the common names do not in themselves off er suggestions of chem- ical structure. Unsystematic naming is contrary to the modern spirit of chemical nomenclature. Winterstein's " inosite-phosphoric acid" has priority over Posternak's "phytin" and the further ad- vantage of being a chemically descriptive term. The preference of several authors for this latter designation is evidenced by the fact that the name phytin is not adhered to or is given in parenthesis after the name " inosite-phosphoric acid." In this particular case the probability of confusion is very miich increased by the fact that the term " phytine " is already applied to Chlorophyll preparations whose chemical composition we cannot hope to know for some time and for convenience must perforce carry a non-chemically descrip- tive appellation. The word " phytin " seems to have all the Psycho- logie requirements of a really good trade name and the substance which it designates in the market is widely advertised in the Euro- pean medical Journals for its therapeutic properties, which are more than likely of questionable character, and the term will undoubtedly persist. It can be readily conceived that this may not be the only inosite- phosphoric acid in plants and we should look for other combinations in which the phosphorus may be in the ortho or pyro form — even the meta phosphate — and be present as the hexa, tetra, di, or mono phosphoric acid. Various incidents have suggested to the writer that some of these forms occur in preparations from seeds when certain treatment other than those described above is used. ANALYTICAL METHODS^ The quantitative estimation of phytin phosphorus has been effected only by determining the difference between the total soluble phosphorus and the inorganic phosphorus. Phytin research in animal and plant metabolism is therefore very largely dependent upon the accuracy of the determination of inorganic phosphorus. " The analytical methods are here treated very briefly, for their development is as yet imperfect and the literature conflicting. Many papers have not been mentioned and the reader is referred for these to the bibliography on page 46. A more complete Statement with experimental data will be published later. 38 Literature on Inosite-Phosphoric Acid [Sept The term soluble phosphorus above and elsewhere means of course the phosphorus Compounds which dissolve in cold acidulated water ; the amount is obtained by evaporating the extract and destroying the organic matter with sulphuric and nitric acids according to the method of Neumann,^^ after which the phosphorus is determined by the usual ammonium molybdate and magnesia mixture method as described by Sonnenschein and later modified by Woy. As ex- tracting agents both acetic acid and hydrochloric acid have been used. The first method to approximate an accurate determination of inorganic phosphorus in the presence of soluble organic phosphorus was that used by Hart and Andrews in 1903. Their extracting agent was 0.2 per cent. hydrochloric acid Solution, a solvent which has since been used by most investigators. Hart and Andrews noted that ammonium molybdate did not precipitate the phytin phosphorus, and used this fact to devise a method for separating the two kinds of phosphorus combination in Solution. They had some apprehension lest the strong acid in the usual molybdate Solutions would hydrolyze some of the organic phosphorus Com- pounds and thus yield high results for the inorganic portion. They determined the minimum amount of nitric acid necessary to give a rapid, complete, and crystalline Separation of the yellow precipitate (2 c.c. of nitric acid, specific gravity 1.2, in each 250 c.c. of Solu- tion) and added to the liquid of this acidity neutral ammonium molybdate Solution. Vorbrodt, in his excellent monograph on " phytin," developed a method which is based on a triple precipitation of the inorganic phosphorus, first precipitating in general by means of magnesia mixture and dissolving the precipitate in the least amount of nitric acid. This is diluted to 50 c.c, heated to 100° C, and treated with an equal volume of ammonium molybdate Solution. The yellow precipitate is dissolved in ammonia water, 25 c.c. of 5 per cent. barium chlorid are added,^^ and the precipitate after being washed and dried is weighed; or the phosphorus may be precipi- tated with magnesia mixture and weighed as magnesium pyro- phosphate. "Neumann: Zeit, für physiol. Chetn., 1902, 37, 115. "Riegler: Zeit. Anal. Chem., 1902, 41, 675. 1912] 'Anton Richard Rose 39 Stutzer in Germany and Forbes in this country, working inde- pendently, introduced a new idea in the determination of inorganic phosphorus, namely, the use of acid alcohol. Forbes and bis asso- ciates make an acidulated water extract and precipitate with mag- nesia mixture; the precipitate is then washed successively with ammonia water and alcohol, and the inorganic phosphorus separated f rom the phytins by digesting in cold 95 per cent. alcohol containing 0.2 per cent. of nitric acid. This alcoholic Solution is finally filtered, the filtrate evaporated, and phosphorus determined in the residue in the usual way. Starkenstein has studied in some detail the application of titra- tion methods to this problem, and his results point to the possibility of determining quantitatively these different forms of phosphorus in the same Solution. He found that titration of a Solution con- taining ortho-phosphate, pyro-phosphate and inosite-phosphate with uranyl acetate standardized by ortho-phosphate, using cochineal as an indicator, gave in each case true values for total phosphorus; that with ferrocyanide as an indicator, the total phosphorus was equivalent to all of the phosphorus as ortho-phosphate, one half of that as pyro-phosphate and inosite-phosphate, the glycero-phosphate not entering into the reaction at all. Anderson notes that pyro- phosphoric acid can be converted into the ortho form by heating with dilute acids, while the inosite-phosphoric acid is not affected by this treatment. With these facts in mind a Volumetrie process may readily be devised. THE ROLE OF INOSITE-PHOSPHORIC ACID AND ITS SALTS IN PLANTS The literature on phosphorus metabolism in animals has become voluminous, but the botanists have published comparatively little on the changes of these Compounds and their probable significance in a plant's life history. That cell functioning is impossible in the absence of phosphorus is again emphasized in the recent work of Frouin,^^ which shows its absolute necessity in the growth of micro-organisms. The study of the role of phytin in plant life in- volves an investigation of the changes and distribution of all the ^'Frouin: Compt. Rend. Soc. Biol., 1910, 68, 801-803. 40 Literature on Inosite-Phosphoric Acid [Sept. pliosphorus Compounds and of inosite in the several stages of plant development. Since the methods of differentiating between the various combinations of phosphorus in plant substances are becom- ing highly perfected, we may expect rapid developments in our knowledge of their functions in plant processes. The universal presence of phytin in propagating and growing parts must be highly significant. This constant occurrence led Starkenstein to assume that "phytin" plays a specific role in the mechanism of growth of both plants and animals. If this be so, its biochemical reactions must be closely linked with carbohydrate and protein formation, and its occurrence with these substances in the aleuron grain must be more than a mere coincidence. In this connection it may be well to review briefly the literature regarding the aleuron grains. The best summary was found in Vines's text book of plant physiology (1886) but this is too brief to be satisfactory. From Pfeffer's comprehensive description, it appears that these grains form in the vacuoles during the ripening and desiccation of the seed; that the forms assumed are globular, which are less distorted and attain a larger size in the more fatty seeds. They consist morphologically of three parts: the large pro- tein particle, Pfeffer's globoid, and a membrane. Crystals of cal- cium Oxalate are sometimes present. Weyl^'^ isolated the grains from the " Paranuss " employing the method of all the previous inves- tigators, and made an extensive study of their proteins. This was in the days of the vegetable vitellins (globulins), and the chief protein of the aleuron grain having been shown to belong to this group, Weyl thought that the membrane was a modified form of the same protein, an albuminate. Three or four years later, Vines^^ under- took a study of these proteins and from his observations on mate- rial from a large variety of seeds, grouped them into five classes: vegetable peptone (water soluble), vegetable myosin, crystalloid, vit ellin (all three soluble in sodium chlorid Solution), albuminate (soluble in sodium carbonate Solution). These are described as plastic proteins, in part transported to the cells of the seed from other portions of the plant. According to Posternak, these pro- ^ Weyl : Zeit, für physiol. Chem., 1S77, i, 84-96. "Vines: Proc. Roy. Soc. London, 28, 218; 30, 387; 31, 59, 62; see also Lundtke : Jahrb. wiss. Bot., 1890, 21, 62. I9I2] Anton Richard Rose 41 teins constitute from fifty to seventy-five per cent. of the aleuron grain. It is doubtful whether they are simple proteins; more likely they contain both phosphorus and bases in their molecules. Besides protein, Posternak found carbohydrates which were not free, but combined with some other substances of the grain; also ash, to the extent of twenty-five to fifty per cent. The following analytic data were recorded : Per Cent. Per Cent. Phosphorus O.11-3.83 Magnesium 0.28-1.27 Sulphur 0.64-0.81 Calcium 0.11-0.37 Silicon 0.01-0.36 Iron 0.03-0.09 Potassium 2.29-2.71 Manganese Trace These results were obtained from aleuron grains of sunflower, white lupin, hemp, and red fir. The author calls attention to the interesting fact that all the elements essential to plant growth are present in these bodies. These results are notable when com- pared with Bernardini's analyses of rice embryo: P2O5, 0.95; SO3 (not given); SiOs, 0.25; K2O, 1.691; MgO, 1.389; CaO, 0.279; FesOg, 0.06; Mn, trace; NagO, trace. One would like to know whether the Silicon in these two substances is present in organic combination. The globoid or "phytin" is a calcium-magnesium salt of inosite-phosphoric acid. Phyto-phosphate is also combined in the protein granules, possibly in the form of the potassium salt, as Posternak believed, inasmuch as he could not separate the potassium salt from the globulin, although it is soluble in alcohol and globulin is not soluble in this liquor. He concluded that it is chemically attached to the protein. In germination, the aleuron grains swell up, forming a granulär viscid mass; both globoid and crystalloid go into Solution, enzymatic action sets in, and both phytin and pro- tein are hydrolyzed. The presence of an enzyme having the power to decompose phy- tin into inosite and an inorganic phosphate was first demonstrated by Suzuki and his associates in the bran of rice. It has also been found by Vorbrodt in other small grain, including wheat, rye, and barley ; likewise in larger seeds, as vetch and lentils. An extract of the kemel of indian corn gave no evidence of the presence of a phy- tase, but it was shown to develop during the germination of the grain. 42 Literatlire on Inosite-Phosphoric Acid [Sept. In the earlier analyses of seeds, the inorganic phosphorus was not given a very prominent place and was usually reported as that por- tion obtained by subtracting the sum of the protein and lecithin phos- phorus from the total phosphorus; but as this dement began to re- ceive special attention its direct determination was attempted, and consequently the amount of inorganic phosphorus reported was lessened. Thus Umikoff^^ estimates the inorganic portion as fully half of the total phosphorus, but the more recent workers report it in very small percentages. With the germination of the seed, the inorganic phosphorus gradually increases at the expense of the organic form. This was at first attributed to the breaking up of the phosphorus-bearing proteins and lecithins. Tammann,^'' one of the earliest investigators to make direct determinations of inorganic phosphorus in seeds and their sprouts, found during germination an increase of this form which, in terms of P2O5, was from 0.324 per Cent, to 0.443 P^^ cent. in a period of only twelve days. Ac- cording to the work of Prianischnikow^^ and Merlis,^^ lecithin de- creased one half during fifteen days' germination of Vicia sativa and Lupinus angustifolius. Iwanow found that the inorganic phos- phorus increased from a very small amount to 93.7 per cent. of the total phosphorus in germinating seeds of Vicia faba. He held that lecithin is the most stable of the organic forms and is altered very little. Phytin, owing to the presence of phytase, is practically all changed by this process. Vorbrodt has shown that the phosphorus Compounds, especially inosite-phosphoric acid, are particularly abundant in the germ. When the seed begins to sprout, this supply is increased by trans- portation of phosphorus from other parts of the grain, as is indi- cated by Zalesky's^^ Observation that in the sprouts of Lupinus angustifolius the total phosphorus increased in twenty-five days from 0.302 gram to 0.514 gram; the inorganic phosphorus doubled in amount, while the protein and lecithin phosphorus remained prac- "Umikoff: Russian Dissertation, 1895. (Cited by Zalesky: Ber. bot. Ges., 1902, 20, 426-433.) ^Tammann: Zeit, für physiol. Chem., 1885, 9, 416-418. " Prianischnikow, 1895. (Cited by Zalesky. See footnote 19.) ^'Merlis: Landw. Vers. Stat., 1897, 18. (Cited by Zalesky. See footnote 19.) ^Zalesky: Ber. bot. Ges., 1902, 20, 426-433. I9I2] Anton Richard Rose 43 tically unchanged. Bernardini found that the phytin decreased also in the germination of wheat, but the lecithins increased. In the early stages of plant development subsequent to germina- tion, inorganic exceeds organic phosphorus. Staniszkis could find no trace of inosite-phosphoric acid in millet during this period. The phosphorus is now drawn f rom the soil and its increase in the plant is proportional to the increase in dry matter. The organic forms of phosphorus are synthesized from this supply, according to Stan- iszkis, very slowly until the heads are formed. Hart and Totting- ham found no phytin phosphorus in the dried forage plants. Balicka-Iwanowska, working with barley, found the phosphorus Compounds at the end of the fourth week present in the same pro- portion as that in the seed ; thereafter there was a constant decrease in the inorganic phosphorus. In the seventh week the protein phos- phorus had doubled and the inosite-phosphoric acid had increased to seven times the amount present in the fourth week. As the barley seeds began to form, in the ninth week, the increase of organic phosphorus occurred mostly through the synthesis of nucleoproteins. The small increase which Staniszkis reports was more in the form of lecithins and protein phosphorus Compounds than of inosite-phos- phoric acid. At the period of flowering, the lecithins reach their maximum, which may be, according to Stoklasa, 71.6 per cent. of the total phosphorus. During the formation of millet seed, the syn- thesis of phytin goes on energetically at the expense of both inor- ganic and protein phosphorus. In the barley, Balika-Iwanowska found an increase of inosite-phosphoric acid and also one of nucleo- protein which was even greater than that of the phytophosphate. In the ripening of the seeds of millet the formation of inosite phos- phates and nucleoprotein ran parallel, but in barley the inosite phos- phate increased at the expense of some of the protein phosphorus. As the panicle grew and matured in both of these plants, there was a transportation of both the phosphorus and the protein to this part of the plant. The mobilisation of phosphorus and changes in its form in Vicia faha and other plants were also studied by Iwanow. The most interesting part of his contribution is the relation between sunlight and changes in the form of phosphorus. The plants which remained in a dark room contained more inorganic phosphorus than 44 Literature on Inosite-Phosphoric Acid [Sept. those which had snnlight. Opaque shields on the leaves produced the same results in the protected part of the leaf, hence the change of inorganic into organic phosphorus may be attributed in part if not altogether to photosynthesis. Stoklasa and his pupils^'* consider phosphorus an integral part of Chlorophyll, existing in a form which does not give the HPO4 ion. Schimper^^ in his rather comprehen- sive study of the assimilation of the ash constituents in plants also noted the decrease of the inorganic phosphorus through the action of light. Posternak, attempting to account for the formation of "phytin," which he then thought to be anhydro-oxymethylene-di- phosphoric acid, assumed that it was formed simultaneously with the reduction of carbon dioxide by a direct combination of the orod- ucts of the photo-chemical action and inorganic phosphates, an hypothesis suggested by Schimper's experiments. If Posternak's assumption is true, even in part, phosphorus may play a very signi- ficant röle in carbohydrate anabolism. Several authors have ex- pressed doubt about this explanation of "phytin" synthesis, advanc- ing the argument that inosite-phosphoric acid is not found in the early stages of growth, and when formed later is not uniformly produced, as would be expected if it were due entirely to the action of the chloroplasts. There is still the possibility that it is so syn- thesized and instantly broken down in the formation of other Com- pounds. Soave could find no inosite in dormant seeds unless they were first boiled in strong acid, but after they began to sprout its pres- ence could be easily demonstrated until the reserve material of the cotyledons was almost exhausted. It was also present in unripe seeds, indicating that the inosite-phosphoric acid is formed in the seed by the combination of the inorganic phosphorus, abundantly present at this stage, with the inosite. The occurrence of inosite in the unripe seed and the green parts of the mature plant, and the later disappearance of this substance as inosite-phosphoric-acid -forms, indicate that phytin is probably produced by the reversible action of an intracellular phytase, and that Posternak's explanation is in- correct. Rising suggests that inosite-phosphoric acid may be an interme- ** Stoklasa, Brdlika and Ernest : Ber. d. deut. bot. Ges., 1910, 27, i, "Schimper: Flora, 1890, 23, 207-261; Bot. Zeifg., 1888, 46, 81. igi2] 'Anton Richard Rose 45 diary product in the formation and destruction of the lecithins, and in this way may play an exceedingly important part in the life cf the plant. The possible relation between the two Compounds is shown in the following graphic representation : HjOjPOHC— CHOPO3H, — CHOPOjHj HjOgPOHg (JHOPOjH, ^ CHOPOjH, HjOjPOHC— CHOPOjHj — CHOPO,H, 2 The most striking Suggestion as to the functions of inosite-phos- phoric acid is contributed by Starkenstein, who thinks that the inosite is in itself inert and incidental and functions only in its com- bination with phosphorus. He has demonstrated that, in the body, inosite yields lactic acid, an interesting fact in view of its possible significance in carbohydrate formation. He assigns to inosite- phosphoric acid, as its special function, some part in the process of growth, basing his view on his experiments with animals. In har- mony with this view is the distribution of phosphorus in the seed, the greater part being localized in the germ; according to Bernardini over eighty per cent. of the total phosphorus in the rice germ is in the form of inosite-phosphoric acid. It is interesting to note in this connection the Observation of Iwanow that there is a tendency to concentration of phosphorus in the parts of the plant where growth is most active, and also that when the phosphorus supply of the Sub- strate is insufficient, the phosphorus of the other parts of the plant is rapidly transported to the growing shoots. As previously stated the phosphorus of the seed is in the form of the calcium-magnesium Salt of inosite-phosphoric acid, but, according to Posternak, the phosphorus, in transportation, is in the potassium salt of this acid. Phyto-phosphoric acids, whether they are inosite esters or other Compounds, undoubtedly play very significant roles in all higher plants, but as their specific functions have not as yet been ascertained, even the chemical structures being as yet uncertain, nearly all State- ments on the subject must be pure conjectures. The chief sug- gestions from experimental work are that these acids are concerned in the process of photo-synthesis or in the changes of the photo-syn- thetic products, for example, the formation of carbohydrates and fats; that it is an intermediary step in the synthesis of phospho-pro- teins and lipoids; and that it acts as a specific Controlling factor in 4^ Literature on Inusite-Phosphoric Acid [Sept. growth. The varioiis functions as thus outlined are probably over- estimated, but those who have worked in this field seem to be strongly of the opinion that inosite-phosphoric acid is more than a reserve material. It is attracting considerable attention and as the necessary analytical methods are perfected, we may expect to see, in increasing number, valuable contributions that will eluci- date in detail the part which this interesting Compound plays in nature. BIBLIOGRAPHY OF INOSITE-PHOSPHORIC ACID ("PHYTIN") Anderson. N. Y. Agr. Exp. Sta., Geneva, N. Y. ; A''. Y. Agr. Exp. Sta. Tech. Bull, ig, 21, 1912; /. Biol. Chem., 1912, 11, 471-487; 12, 97-113, and 447-464. Important contribution on the salts of inosite-phosphoric acid, also impor- tant synthetic work. Aso and Yoshida. Imp. Univ., Tokio; /. Coli. Agr., Imp. Univ., Tokio, 1909, i, 153-168. Compares the value of " phytin " with other forms of phosphorus as a fertilizing material. Balika-Iwanowska. Agr. Chem. Inst., Krakow; Ras. Akad. Univ., 1906, 2° ser, 6, B, 24. (From Maly's Jahrsb., 1907, 36, 741.) Gives the influence of the HPO4 ion on plant growth; also the changes in form and distribution of phosphorus at the various stages of development of barley plants. Bernardini. Chemica Agr. Scuola, Portici ; Atti. Acc. Lincei, 1912, 21, 1°, 283-289. Bernardini and Morelli. Atti. Acc. Lincei, 1912, 21, 1°, 357-362. Boorsma. Batavia ; Von Bemmelen Festschrift, 210-213 ; (see Chem. Centr., 19H (I), 296). Preparation and properties of the inosite-phosphoric acid from rice bran. Carte. Bull. soc. chim., 1901, (4), 9, 195-199. (From Chem. Zentr., 1911 (I), 1196.) Repeats the work of Contardi and reports negative results. Collison. Ohio Agr. Exp. Sta., Wooster, O. ; /. Biol. Chem., 1912, 12, 65-73; /. Ind. and Eng. Chem., 1912, 4, 606-608. Modified method of determining inorganic phosphorus in the presence of phyto-phosphates. Cf. Forbes. Contardi. Lab. di chim. org. della Reale Scuola Agr., Milan; Atti. Acc. Lincei, 1909, 5 ser., 18 (1° sem.), 64-67; 1910, 5 ser., 19 (1° sem.), 823-827. Prepa- ration of inosite-phosphoric acid from rice bran, analyses of the pure prepa- ration, synthesis of phytin by heating inosite with phosphoric acid. Syn- thetic product gave same analytic data as the preparation from rice bran. Important papers. Cook. U. S. Dept. Agr., Washington, D. C; Bur. of Chem., U. S. Dept. Agr., Bull. 123, 1909. Feeding experiments on rabbits. Dox and Golden. la. Agr. Exp. Sta., Ames, la. ; /. Biol. Chem., 191 1, 10, 183- 186. Demonstration of the presence of phytase in certain common fungi. Donath. Wien. klin. Woch., 191 1, 24, 1192-1197. Therapeutic. Fingerling and Hecking. Versuchsstation, Hohenhain; Biochem. Zeit., 1912, 37. 452. Contains a note on Stutzer's method. Forbes, Lehmann, Collison and Whittier. Ohio Agr. Exp. Sta., Wooster, 1912] Anton Richard Rose 47 Ohio; Ohio Agr. Exp. Sta. Bull., 215, 1910. Gives a good method for the determination of inorganic phosphorus. See Collison. Fürst. Centr. Kinderheilk., 1904, 409. Therapeutic. Giacosa. Giorn. della Real. Accad. di med. di Torina, 1905, 68, 369-374; 1907, 70, 290-295. (From Maly's Jahrsb., 1906, 35, 124 ; 1908, 37, 473 ; also Biochem. Centr., 6, 573.) Pharmacological study on man. Gilbert and Posternak. L'Oeuvre Medico-chirurgical, 1903, No, 36. (From Maly's Jahrsb., 1904, 34, 729. Therapeutic. Gilbert and Lippmann. La Presse Medicale, 1904, Aug. 2y and Sept. 10. Phar- macological studies on rabbit and guinea pig. Hardin. S. C. Exp. Sta., Fort Hill, S. C. ; 5". C Exper. Sta. Bull., n. s. 8, 1892. Pyrophosphoric acid in cottonseed meal; probably associated with phytin. Cf. Crawford, /. Pharm, and Exp. Ther., 1910, i, 519. Hart, McCoUum and Humphrey. Wis. Agr. Exp. Sta., Madison, Wis. ; Wis. Agr. Exp. Sta. Research Bull., 5, 1909; Am. J. Physiol., 1909, 23, 86-102; 24, 246-277. Feeding experiment with the cow. Cf. Jordan. Hart and Andrews. N. Y. Agr. Exp. Sta., Geneva, N. Y. ; A''. F. Agr. Exp. Sta. Bull., 238, 1903; Am. Chem. Jour., 1903, 30, 470-486. Gives the first approximately reliable method of determining inorganic phosphorus in the presence of phytin and other organic forms of phosphorus. Hart and Tottingham. Univ. of Wis., Madison, Wis.; /. Biol. Chem., 1909, 6, 431-444. Determination of the amounts of phytin in certain feeding stuffs. Hartig. Braunschweig; Bot. Ztg., 1855, 13, 881-882; 1856, 14, 257-355. Micro- scopic study of seeds; discovery of the substance later known as phytin. See also "Lehrbuch der Anatomie und Physiologie der Pflanzen," 1891, 48. Horner. Pathol. Inst., Univ. of Berlin ; Biochem. Zeit., 1906, 2, 428-434. Phar- macological studies on dog and rabbit. Iljin. Russ. Wratsch., 1906, No. 13, from Maly's Jahrsb., 1907, 36, 54. Com- pares the properties of phytin, lecithin and nucleoprotein. Iwanoff. Jahrb. wiss. Bot., 1901, 36, 355-379; /. Exp. Agr. (Russian), 1902, i (cited by Zaleski) ; Ber. bot. Gesell., 1902, 20, 366-372; Zeit, physiol. Chem., 1907, 50, 281-288. Studies on the changes of the forms of phosphorus in germinating vetch seeds and in the growing plant. Production of phyto- phosphates by yeast-fermentation of sugar in the presence of di-sodium phosphate. Jegorow. Landw. Inst. Petrowskoje-Rasumowskoje, Moskow; Biochem. Zeit., igi2, 42, 432-439. Study on stability of inosite-phosphoric acid and disput- ing the existence of phytase. Jordan, Hart and Patten. N. Y. Agr. Exp. Sta., Geneva, N. Y. ; N. Y. Agr. Exp. Sta. Tech. Bull, i, 1903; Am. J. Physiol., 1906, 16, 268-313. Feeding experiments with the milk cow. Cf. Hart. Korolev. Moscow. Izv. Moscov. Selsk. Khoz. Inst., 1910, 16, 1-98. (From Chemical Abstracts, 191 1, 1962.) Studies on organic phosphorus in the soil. LeClerc and Cook. U. S. Dept. Agr., Washington, D. C. ; /. Biol. Chem., 1906, 2, 203-217. Feeding experiments on rabbits. Levene. Rockefeller Inst., N. Y. ; Biochem. Zeit., 1909, 16, 399-405- Studies of phytin preparations made from hemp. Obtained a Compound consisting of three groups : inosite, pentosan and phosphate. Maestro. Lo Spermentale, 1904, 59, 456-458. (From Maly's Jahrsb., 1905, 35, 91.) Pharmacologic. 48 Literature on Inosite-Phosphoric Acid [Sept. McCoUum and Hart. Univ. of Wis.. Madison, Wis. ; /. Biol. Chem., 1908, 4, 497-500. Showing the presence of phytase in animal tissues. Mendel and Underhill. Yale University; Am. J. PhysioL, 1906, 17, 75-88. In- fluence of phytin on bacteria; pharmacological studies on dog and rabbit. Nagaoka. Imp. Univ., Tokio; Bull. Coli. Agr., Tokio, 1906, 6, No. 3. Value of inosite-phosphoric acid and other phosphorus Compounds in plant waste products as fertilizers, as compared with animal wastes, showing that the latter are the more efficient. Neuberg. Pathol. Inst., Univ. of Berlin; Biochem. Zeit., 1908, 9, 557-560; 1909, 16, 405-410. Analyses of several preparations of phytin. Concludes that the substance is inosite-phosphoric acid and does not contain a carbohy- drate group. Novi. Pharm. Lab. Bologna; Mem.r. accad. sei. Bologna, igii,^,s^r. 6. (From Zentr. Biochem. u. Biophys., 1911,11,871.) Influence of phytin and glycero- phosphates on muscle reaction. Palladin. Zürich Polytechnicum ; Zeit. Biol., 1894, 31, 191-203. Discovery of inosite-phosphoric acid by chemical procedure. Patten and Hart. N. Y. Agr. Exp. Sta., Geneva, N. Y. ; A''. Y. Agr. Exp. Sta. Bull., 250, 1904; Am. Chem. Jour., 1904, 31, 564-672. An important contri- bution on the properties and composition of inosite-phosphoric acid. Peters. Allg. med. Centralzeitg., 1908, No. 9. (From Centr. Nervenheilk. u. Psychiatrie, 1908, 31, 1081.) Therapeutic. Pfeffer. Marburg; Jahrb. wiss. Bot., 1872, 8, 429-574. Comprehensive study of aleuron grains, identification of "globoid," and approximation of its chem- ical nature. Polacci. Royal Bot. Inst., Univ. of Pavia; Malphigia, 1894, 8, 361-379. Study of phosphorus in the aleuron grain. Posternak. Zürich Polytechnicum; Pasteur Inst.; Basel; Compt. Rend., 1905, 140, 322-324; 1903, 137, 202-203; 2>37-2>29; 439-441; Bul. soc. chim., 1904, 33» 116; Rev. Gen. Bot., 1900, 12, 5-24; 65-73; Compt. Rend. Soc. Biol., 1903, 55, 1190-1192. German Patents, Kl. 12, Nos. 155798, 159749, 160470, 164298; Münch. med. Woch., 1907, p. 827. The first and most comprehensive study of the physical and chemical properties of phytin, giving methods of prepa- ration, results of analyses of pure products, and speculations on the Consti- tution and biological function of this product. Rising. St. Albonvorstadt, Basel; Svensk Kern. Tidskrift, 191 1, 22, 143-150. Study of organic phosphorus Compounds in food materials ; the name phyto- phosphoric acid is suggested, methods are given for analysis of the various forms of phosphorus, and analyses are reported for a preparation of the silver salt of inosite-phosphoric acid. Rogosinski. Anz. Akad. Wiss., Krakow, 1910, 260-310. (From Chem. Centr., 1910 (II), 1549; Chem. Abstr., 1911, 5, 1476.) Feeding experiment with the dog. Rose. N. Y. Agr. Exp. Sta., Geneva, N. Y. ; N. Y. Agr. Exp. Sta. Tech. Bull., 20, 1912. Feeding experiment with the milk cow; Dept. Biol. Chem., Colum- bia Univ., N. Y., BiocHEMiCAL Bulletin, 1912, i, 428-438. Influence of phytin on the growth of lupin seedlings. Sechert. These de Paris, 1904. (From Maly's Jahrsb., 1904, 34, 729.) Therapeutic. Schulze and Castoro. Zürich Polytechnikum; Zeit, physiol. Chem., 1904, 41, I9I2] Anton Richard Rose 49 477-484. Presents a method of analysis, and data on content of inosJte- phosphoric acid in various seeds. Schulze and Winterstein. Zürich Polytechnikum; Zeit, physiol. Chem., 1903, 40, 120-122. Phytin prepared and analyses of it made. Scofne. Pharm. Inst., Turin; dorn, della Real. Acc. di med. dt Torino, 190S, 56, 630. (From Biochem. Zentr., 1905.) Fate of inosite-phosphoric acid in the animal organism and paths of elimination. Soave. Sias, spernt. agrar. Ital., 1906, 39, 413-427, 434-438. (From Chem. Zentr., 1906, 1726.) Ann. R. Accad. di Agr. di Torino, 1906, 49, p. i et seq. (From Centr. Physiol., 1906, 772.) Shows the relation between inosite and phytin in seeds. Staniskis. Jagel Univ., Krakow ; Ans. Akad. Wiss., Krakow, 1909, 95-123. (From Chem. Zentr., 1909 (II), 114.) Determination of the distribution of the forms of phosphorus in millet at various stages of development. Starkenstein. Pharm. Inst., Univ. of Prague; Zeit. exp. Path. u. Therapie, 1908, 5, 378-389; Biochem. Zeit., 1910, 30, 56-98; 191 1, 32, 234-265. A study of inosite and its relation to animal and plant life; the relation between inosite and inosite-phosphoric acid — chemical, biochemical and biological ; the nature and Constitution of phytin; its significance in animal and plant growth ; toxicity ; reaction to common indicators ; and estimation by Volu- metrie methods. A noteworthy contribution. Streffer. Zentr. ges. Therapie, 1908, 25, 135. Therapeutic. Stutzer. Biochem. Zeit., 1908, 7, 471-487. Methods of analysis. Suzuki, Yoshimura and Takaishi. Imp. Univ., Tokio; Bull. Coli. Agri., Tokio, 1907, 7, 495-502, 503-512. Preparation of inosite-phosphoric acid from rice bran; discovery of phytase; Suggestion of a new formula, containing the inosite nucleus. Tsuda. Imp. Univ., Tokio; /. Coli. Agri., Tokio, 1909, i, 167-168. Study of the forms of phosphorus in vegetable wastes. Tyshnjenko. Therap. klin. militarmed. Akad., St. Petersburg; Dissertation (Russian), 1909, p. 117. (From Maly's Jahrsh., 1909, 39, 589.) Feeding experiments on man. Vorbrodt. Univ. Krakow; Bull, de l'Acad. des Sei. de Cracovie, 1910, ser. A, 414-511. A study of the general reactions of phytin; comparison of methods of analysis, and Suggestion of a desirable modification; the phytin content of a number of seeds reported; comparative study of phytases; ultimate composition of phytin and a proposed empirical formula. An excellent paper. Weismann. Therap. Monatshefte, 1908, 22, 470. Therapeutic. Windisch. Jahrb. Vers. u. Lehrs. f. Brauerei, Berlin, 1907, 10, 56-58. (See also Wochschr. Brauw., 1906, 23, 516; Chemical Abstracts, 1907, i, 81.) Showing that the inosite-phosphoric acid of barley does not pass into beer but disappears in the malting process. Winterstein. Zürich Polytechnikum ; Ber., 1897, 30, 2299-2302. The first inten- tional preparation of phytin; analyses of the material; cleavage products; introduction of the name inosite-phosphoric acid. Zeit, physiol. Chem., 1908, 58, 118-121. Discusses the Constitution of phytin. A NEW TYPE OF ARTIFICIAL CELL SUITABLE FOR PERMEABILITY AND OTHER BIOCHEMICAL STUDIES E. NEWTON HARVEY (Physiological Lahoratory, Princeton University) Research on the permeability of membranes has been largely confined to a study of the properties of what may be termed macro- scopic membranes; composed of parchment, collodion, rubber, or silk impregnated with various substances. The best example of membranes of a type and size comparable to the surface film of cells and yet available for permeabiHty studies are the precipitation membranes of Traube, investigated by Waiden, Tamann, and Meerburg. Protein membranes of exceeding fineness are formed at the surface of various non-miscible fluids shaken with protein Solutions, such as the surface film of oil globules in protein-oil emulsions, or the films formed on Chloroform or benzol when shaken with albu- men Solutions.^ Such membranes are useless for permeability studies so long as they Surround fluids that do not mix with water. However, it is an easy matter to replace the fluid within the mem- brane by a watery Solution, provided the former fluid is readily volatile and slightly soluble in water. Chloroform conforms to these conditions. When Chloroform is shaken with egg albumen Solutions, the globules, in the course of 10-15 minutes, shrink in size and their membranes become crumpled, due to the passage of Chloroform from water to air and from globule to water. Lecithin, if previously dissolved in the Chloroform, will take up water as the Chloroform passes out. In the course of one to two hours, in an open vessel, all the Chloroform disappears and we obtain, instead of a Chloroform Solution of lecithin, a water Solution of lecithin enclosed in a fine protein membrane, the whole of a size comparable with cell sizes. The diameter of the droplets may be varied at will ^ Robertson : Journal of Biological Chemistry, 4, p. i, 1908. 50 igi2] E. Newton Harvey 5^ according to the degree of shaking. The role of the lecithin is to hold the water as the water replaces the Chloroform. The protein membrane is impermeable to lecithin. These artificial lecithin cells are stable, persisting until destroyed by bacteria. In many ways — in shape, in general appearance and in consistency — they resemble, to a very remarkable degree, sea-tirchin or star-fish eggs. Some of their properties have been described in Science (n. s.), V'ol. 36, p. 564, 1912. The point I wish to emphasize here, however, is not that we can prepare artificial cells closely resembling real cells, but that a Solution of lecithin may be obtained within a protein membrane, the whole of known composition and of a size comparable with cell sizes. Much can be inferred concerning the living cell from a knowledge of the properties of such artificial cells where compo- sition is definitely known. As Chloroform is exchanged for water, some of the lecithin separates in the form of granules, most of which agglutinate in a dense clump. The cell as a whole, but more particularly these granules, take up neutral red from dilute Solution, becoming red in color. (Chloroform alone takes up only the yellow base of neu- tral red. When lecithin is dissolved in Chloroform it unites with the yellow base, forming a red salt.) If the permeability for alkalies of such red-stained cells is studied, a marked difference from that of living cells is noted. Both ammonium hydroxid and sodium hydroxid in w/2000 con- centration enter rapidly and at the same rate. It will be remem- bered that all living cells are very easily permeable to ammonium hydroxid, but very slightly so to sodium hydroxid.^ The surface membrane of living cells is evidently of quite different composition from the protein film which condenses on Chloroform droplets. Living cells behave toward alkalies as though they were sur- rounded by a layer of a fat solvent, as postulated by Overton. Lipoid-soluble alkalies (ammonium hydroxid) penetrate readily, lipoid-insoluble alkalies (sodium hydroxid) do not. The lipoid solubility of ammonium hydroxid can be readily demonstrated by means of a benzol-lecithin Solution shaken with egg albumen solu- ^ Harvey, E. N. : Journal of Experiniental Zoology, 10, p. 507, 1911 and BiocHEMicAL Bulletin, i, p. 227, 1911. 52 New Type of Artificial Cell [Sept. tion. The same type of protein-film is formed on these globules but they differ from chloroform-lecithin globules in that the benzol is not replaced by water. If the benzol-lecithin globule is stained in neutral red Solution and placed in 7?/iooo ammonium hydroxid Solution, the color change from red to yellow takes place almost instantly. But it is only after 15-20 minutes that sodium hydroxid in relatively high (;i/io) concentrations can enter. Ammonium hydroxid is readily soluble in the benzol droplet, while sodium hydroxid is not; and in this fact lies the explanation of the differ- ence in penetrability. When stained in neutral red Solution, prac- tically all living cells behave as though they were protected from alkali by a benzol-lecithin surface layer. It is a simple matter to introduce various substances into these cells by dissolving or suspending the material in the chloroform- lecithin Solution before it is shaken with the protein Solution. Thus, oil may be dissolved by Chloroform and will separate in the cell in several large droplets much like those in a Nereis egg. Or cholesterol, starch grains and finely divided protein particles can be likewise included ; or substances to be used as indicators in study- ing the permeability of the protein film. Such cells, regarded as complex Systems of biological sub- stances, offer exceptional advantages for interpreting phenomena observed in living cells under special conditions; for example, dur- ing the passage of an electric current. Movements and disintegra- tions take place which I have as yet only partially investigated. In the near future I intend to describe these phenomena and shall give more complete data upon the permeability of the film which sur- rounds the cells. ON A NEW FUNCTION OF THE CATALYZER CALLED " PEROXIDASE " AND ON THE BIOCHEMICAL TRANSFORMATION OF ORCIN TO ORCEIN^ JULES WOLFF In a recent publication I have described the influence which peroxidase exerts on certain phenols in the presence of various salts and alkalies.^ When dissolved in a weak sodium carbonate Solu- tion freely exposed to the air, orcin, for example, fixes from four to five times more oxygen in the presence of peroxidase than in its absence. In this note I wish to call attention to the fact that perox- idase has other powers than the fixation of atmospheric oxygen. In determining the combined influence of ammonia and perox- idase upon aqueous Solutions of orcin, I have studied conditions which favor the transformation of orcin^ into orcein, the beautiful coloring matter which is one of the principal constituents of com- mercial orseille. My observations are interesting from many points of view. They show that (i) if a 2 per cent. Solution of orcin is exposed to the air in a thin layer and subjected to the influence of different proportions of ammonia, orcein is not formed even after a month under such conditions, but, instead, there is produced a substance which imparts a brownish-red color to the liquid. (2) If, how- ever, a portion of the same Solution is put in a narrow tube, so that the reaction takes place in a deeper layer, and the surface of con- tact with air is limited (other conditions being equal), one observes a very slow but regulär formation of orcein. (3) If (everything eise being equal) one repeats the first experiment (i), but adds to the ammoniacal Solution of orcin a suitable quantity of peroxidase, orcein fails to appear, just as in the first experiment. (4) If, how- * Translated from the author's manuscript, in French, by Dr. J. J. Bronfen- brenner. [Ed.] ''Wolff: Comptes rendus, 1912, clv, p. 618. 'Orcin has been the subject of interesting work by Robiquet, Dumas, Liebig, and Laurent and Gerhardt. 53 54 A New Function of " Peroxidase" ever (all other conditions being eqiial), one repeats the second ex- periment (2), but adds to the ammoniacal Solution of orcin a siiitable amount of peroxidase, the transformation into orcein takcs place very rapidly and is quite advanced in four or five days^ Compar- ing the coloration intensities of products 2 and 4, one sees that in five days 4 contains more than twice as mtich orcein as 2. This gain is due to the action of the peroxidase. By boiling the perox- idase Solution for f rom 5 to 6 minutes, before adding it to the ammo- niacal Solution of orcin in experiment 4, there is no acceleration in the formation of orcein, evidently because the peroxidase, as the active agent in the transformation, is thus destroyed. In Order to determine the rate of oxidation in the different ex- periments, I measured the volumes of absorbed oxygen. In experi- ment 2, eight to nine times more oxygen was absorbed than in i, in the course of 48 hours. In experiments 3 and 4 there was a similar difference but, because of the presence of peroxidase, the proportions of absorbed oxygen were larger. Without discussing the nature of the combined action of am- monia, oxygen, and peroxidase upon orcin, we may conclude that in dilute Solutions of orcin, slozv oxidation by ammonia is the pri- mary condition for the formation of orcein. If, however, to this condition is added the accelerating influence of peroxidase, the action is directed toward formation of coloring matter rather than toward increased absorption of oxygen. These facts may possibly be helpful in the commercial preparation of orseille. Paris, France. * My first and third experiments could easily be performed in flasks with flat bottoms. A small test tube would be satisfactory for experiments 2 and 4. The proportions of materials indicated below are well adapted for the purposes of the experiments : ^ , ( 2 c.c. of 2.8 per Cent. Solution of ) tnm x j -.i For I and 2 < , , , ^j^ > Diluted with water to 3.5 c.c. ( orcm and 50 mg. of NH3 ) 2 c.c. of 2.8 per Cent. Solution of For 3 and 4 ■{ orcin, 50 mg. of NH3 and \- Diluted with water to 3.5 c.c. I c.c. of active peroxidase Solution STUDIES OF DIFFUSION THROUGH RUBBER MEMBRANES I. Preliminary observations on the diffusibility of lipins and lipin-soluble substances WILLIAM J. GIES (Biochemical Laboratory of Columbia University, at the College of Physicians and Surgeons, New York) CONTENTS I. Introduction c :- IL On the diffusibility of biological substances through rubber cg III. A demonstration of osmotic pressure exerted by fat 50 IV. A demonstration of the diffusion of pigments from fat through rubber into fat 60 V. Comparative dialysis experiments, with demonstrations 61 VI. Experiments on the diffusibility of alkaloids through rubber 62 I. INTRODUCTION When I proposed to my biochemical associates in Cancer re- search, in 1909, that we undertake a study of intracellular chem- istry/ I realized that new analytic methods and unconventional experimental procedures were prerequisites for material progress in this as in any other chemical relation. The greatest obstacle in the path of progress in intracellular chemistry is the evident lability of the essential intracellular constituents. Our best chemical methods increase this predicament because each is essentially anti-biological in character. Biochemical discoördinations are enforced whenever any of our present chemical processes is efifectively applied to proto- plasmic material. In reflecting on the properties and possible coördinations of intracellular lipins, it seemed probable that such lipins might be separated from protoplasmic material with the least chemical vio- * Gies : Studies in cancer and allied subjects, conducted under the auspices of the George Crocker Special Research Fund; Volume III, Department of Bio- logical Chemistry, Introduction (in press). 55 56 SUidies of Diffusion through Rubber Membrancs [Sept. lence, and isolated with the least possible alteration of their qualities, if they cotild be removed by dialysis.^ When this idea first came to mind, however, execution of its essential feature appeared to be im- possible. I believed that the diffusion of a solute depends very largely on chemical affinity between the separating membrane and the solvents on both sides of the partition. In that view, it seemed highly improbable that any of the ordinary membranes, except possibly collodion, could be of Service in the dialysis of lipins under any circumstances. Collodion appeared to possess favorable quali- ties because of its solubility in common lipin solvents and its pos- sible affinity for the latter under conditions of dialysis.^ Collodion is the only one of the available membranes which, while soluble in ether-alcohol Solutions, freely permits the passage of salins, extractives, carbohydrates, and proteins from aqueous Solutions to water, or to aqueous Solutions, outside, and vice versa, At first thought this suggested special availability of collodion for the work in mind. On the other hand lipins could not be expected to dialyze through collodion in the presence of much water and, as preliminary dehydration seemed an inevitable necessity for the dialysis of lipins from cellular matter, the permeability of collodion membranes to zoater-soluble substances did not appear, after all, to imply any practical advantages for the diffusion of lipins. I also recalled the fact that, in some experiments in another relation, we found that collodion was occasionally rendered defective by ether when the latter was used as a preservative of aqueous Solutions undergoing dialysis.^ Continuing actively to consider these matters from one view- point and then another, I thought of rubber as a possible choice of membrane. Recalling the well-known fact that rubber swells very markedly in ether and even in ether vapor, I assumed that the rub- ber expands in ether under such conditions because ether dissolves in the rubber or combines with it. This was but the prelude to the ' After preliminary desiccation by treatment with anhydrous sodium sulfate or other suitable process. ° Collodion is a serviceable membrane for such purposes. See page 70. * In some experiments which Professor Welker has conducted at my request, we have found that the disintegrative effect of ether on collodion membranes may be due to contained alcohol and other impurities. (See page 70.) igi2] William J. Gies 57 deduction that if ether dissolves in or combines with rubber, ether would also carry dissolved lipins with it into a rubber membrane; and if ether were on the opposite side of such a membrane, to work inwardly under such conditions, ether currents would develop; and lipins would pass from the Solution o£ higher concentration to that of the lower, and there accumulate until an equilibrium was estab- lished. This conception was so attractive that I proceeded at once to State it to Dr. Rosenbloom and, with his Cooperation, immediately tested it. The solid residue from an evaporated ether extract of egg yolk offered the greatest advantages for a preliminary test. We accordingly made an ether Solution of such a yellow residue, transferred the deep yellow Solution to a rubber condom, immersed and supported the latter in ether in a stoppered bottle, and almost immediately observed diffusion currents as well as the rapid egress of lipochrome. Fat and cholesterol were easily detected in the diffusate. Assuming that this prompt positive result might be due to defects in the rubber, we made many tests to satisfy ourselves that the ob- servations were or were not what they appeared to be. Dr. Rosen- bloom gave very earnest attention to this phase of the matter for some time and established the fact that we were dealing, except in a few cases of obviously imperfect membranes, with true diffusion phenomena. The original experimental observations were made on March i, 1910. At that time I was ignorant of similar results of previous work with rubber membranes, although I recalled rather vaguely the fact that Kahlenberg had made use of such membranes in another connection. The references to Kahlenberg's work which are given in the Chemisches Zentralblatt [1906 (2), pp. 1391 and 1772], the only ones we could find on this subject at that time, satisfied us that if we extended these experiments, the observations of a previous observer would not be repeated.^ A month or two after the work "The references to which I allude gave the substance of a paper in the Transactions of the Wisconsin Academy of Sciences, Arts, and Letters, 1905, xv (i), pp. 209-272, entitled: " On the nature of the process of osmosis and osmotic pressure, with observations concerning dialysis." The results with which our own could be directly compared were the f ollowing ones : Copper oleate was $8 Stiidies of Diffusion through Rubber Membranes [Sept. was inaugiirated we also saw a late reference to the well-known fact, regarding the swelling of rubber in lipin solvents, on which our work was based.*^ Ten months later we demonstrated these findings at a meeting of the American Society of Biological Chemists (see page 64). The succeeding sections of this paper present reprinted prelim- inary reports on various portions of the studies which thus far have developed from the observations described above. It is my Inten- tion to discuss in detail each seotion of the work, and additional ex- periments, at the earliest opportunity, when I hope to dwell more particularly on the significance of such results for the Student of the functions of cell membranes, and for the investigator of the co- ordinations and equilibria in intracellular affairs. IL ON THE DIFFUSIBILITY OF BIOLOGICAL SUBSTANCES THROUGH RUBBER' The writer and his associates have f ound that many ether-soluble substances of biological origin, such as fat and cholesterol, pass readily from ether Solutions through rubber membranes into ether when the mechanical conditions for such diffusions are favorable. Lecithans appear to be wholly indiffusible. Many substances which are soluble in fatty oils, Chloroform, al- cohol, acetone, ethyl acetate, and other solvents of similar powers, or in mixtures of such solvents, promptly diffuse through rubber under suitable conditions. Collodion is one of the products which appears to be indiffusible under such circumstances. When an ordi- nary ethereal Solution of collodion (containing 24 per cent. of alco- hol) is dialyzed in a rubber Condom against ether in a closed vessel, the alcohol rapidly passes to the exterior and the collodion gradually gelatinizes. Liquid accumulates in the bag under these conditions. Various inorganic substances diffuse through rubber under the found to diffuse from benzene through a rubber membrane into benzene; and camphor diffused from pyridin, alcohol, and toluol through rubber membranes into the same solvents, respectively. A recent study of Kahlenberg's paper in the original makes it evident that our results may be explained on the theory of diffusion which Kahlenberg has done much to render convincing. 'Flack and Hill: Journal of Physiology, 1910, xl, p. xxxiii. ' Gies : Proceedings of the Biological Section of the American Chemical Society: Science, 1911, xxxiv, p. 223; Biochemical Bulletin, 1911, i, p. 125. igi2] William J. Gies 59 conditions mentioned above. Ferric sulphocyanate readily passes from ether Solution through rubber into ether. The writer inaugurated these studies, with Dr. Rosenbloom's Cooperation,^ in the hope of devising improvements in the methods. for the isolation of Hpins. The work is progressing along several lines, especially with reference to methods of isolation and purifi- cation, and to osmosis (see page 64). III. A DEMONSTRATION OF OSMOTIC PRESSURE EXERTED BY FAT' In the first of two demonstrations, a cylindrical rubher bag (Con- dom), 13^ inches in diameter and 8 inches long, was lowered into an oiled miislin bag of aboiit the same dimensions. The rubber bag was then filled to overflowing with olive oil. The rubber bag ex- panded, as the oil filled it, to the füll length and width of the muslin sheath. The sheath prevented further extension of the rubber bag and imparted rigidity to the Osmometer that was ultimately con- structed. The double bag, füll of oil and with its mouth wide open, was then raised so as to inclose about an inch of the lower end of a long glass tube which was firmly supported vertically above the demonstration table. The glass tube was 5 feet long and its bore was 4 mm. in diameter. Ligatures were tightly secured around the neck of the double bag against the immersed lower end of the verti- cal tube. The bag then hung directly from the end of the tube. The bag and its sheath were in a tightly distended condition and a stationary column of oil an inch high in the tube was visible above the protruding edge of the sheath. The tube and bag were then lowered into a salt-mouth liter bottle on the table until the bag almost touched the bottom of the bottle. The height of the bottle and the length of the bag were nearly equal. The tube was then marked with a label on the plane of the oil meniscus just above the neck of the bag, and enough ether was poured into the bottle to pro- vide Immersion for the bag to the depth of an inch. For a mo- ment no change in the volume of oil was apparent, and the lateral ^Rosenbloom and Gies: Proceedings of the American Society of Biological Chemists, 191 1, ii, p. 8; Journal of Biological Chemistry, 191 1, ix, p. xiv. •Rosenbloom and Gies: Proceedings of the Society for Experimental Biology and Mediane, 191 1, viii, p. 71. 6o Studies of Diffusion through Rubber Memhranes [Sept. pressure of the ether was obviously without mechanical effect. But in a miniite or two downward diffusion currents were visible along the surface of the bag and oil rose rapidly in the tube. After the initial effects of the ether had been shown, the bottle was filled with ether containing Sudan III, and a 5-foot vertical extension of the same bore was added to the upright glass tube. In a moment the upward movement of the liquid was markedly accelerated. The demonstration was started at about 9 p. m. At 10 p. m. the osmotic pressure had carried the column of oily fluid to the top of the lo-foot tube, and liquid continued to run rapidly from the Upper orifice until the apparatus was dismantled after the adjourn- ment of the meeting, at about 11.30 p. m. During the progress of the demonstration, Sudan III diffused rapidly from the exterior, through the rubber, to the very top of the rising column of fluid, before any of the liquid passed out of the Upper opening. Oil diffused rapidly through the rubber into the ether. The second demonstration was essentially the same in principle and technic as the first. Instead of a lo-foot upright tube, however, the authors substituted an L tube with an inside diameter of 6 mm. The vertical extension of the tube was 17 inches, the horizontal ex- tension was only 3 inches. The latter extension was drawn out to a narrow bore in an inclined plane, to facilitate direct delivery of any liquid that might pass through that end of the tube. When partial immersion of the bag first occurred there was no visible response, but, in a minute or two, oil began to rise in the tube. The bag was then completely covered with ether. The up- ward movement proceeded rapidly ; and in about an hour nearly 200 c.c. of liquid passed through the upper orifice into a graduated cylin- der which was supported underneath the outlet to catch the overflow. IV. A DEMONSTRATION OF THE DIFFUSION OF PIGMENTS FROM FAT THROUGH RUBBER INTO FAT»" The writer has found that many fat-soluble pigments, such as Sudan III and Scarlet R, diffuse readily from solid and liquid fats " Gies : Proceedings of the Society for Experimental Biology and Mediane, 191 1, viii, p. 73. 1912] William J. Gies 6i through rubber into various solid and liquid media, among them both solid fat and oil. Thus, when Sudan III is dissolved in melted lard, the red liquid poured into a rubber bag, the bag supported in melted lard in a bottle, and the apparatus promptly immersed in ice water — the fatty matter will congeal before any sign of pigmentary diffusion occurs but, in a few hours, a reddish tinge will develop outside of the bag, and on each successive day for several weeks further extension of the pigmented matter may be witnes'sed, until the whole of the external lard is deeply suffused with red. This process takes place quite rapidly when the lard and apparatus are kept in a thermostat at 40° C. The demonstrations were intended to exhibit a few instances of such pigmentary diffusions as occur speedily enough at room tem- No. Contents of the Rubber Bag Nature of the Liquid in which the Bag was Suspended Oil Pigment I 2 3 4 S Olive oil Cocoanut oil Lard oil Paraffin oil Olive oil Scarlet R Scarlet R Sudan III Sudan III Sudan III Olive oil Cocoanut oil Lard oil .Paraffin oil Ether Visible diffusion of the pigment oc- curred promptly Visible diffusion of the pigment oc- curred promptly Visible diffusion of the pigment oc- curred promptly Visible diffusion of the pigment oc- curred promptly Visible diffusion of the pigment oc- curred almost immediately perature to yield positive results within an hour. The accompany- ing summary indicates briefly the precise nature and results of the demonstrations (including two control tests — 4 and 5), which were made with thin rubber bags in ordinary glass bottles. The bags were securely supported in the bottles, and the mix- tures were shaken occasionally during the demonstration. The bags were found, after the adjournment of the meeting, to be with- out defects. V. COMPARATIVE DIALYSIS EXPERIMENTS, WITH DEMONSTRATIONS" When dry bags of rubber, gold-beater's skin, parchment, and collodion, each containing olive oil and Sudan III, are separately " Goodridge and Gies : Proceedings of the Society for Experimental Biology and Mediane, 191 1, viii, p. 74. 62 Studies of Diffusion through Rubber Membranes [Sept. immersed in olive oil, and the remaining conditions of the environ- ment are uniform, diffusion of the pigment promptly occurs through rubber, but does not take place at all through any of the other three membranes. When the bags are lifted from the oil, washed ex- ternally with ether, and then immersed in ether,^^ the pigment quickly passes through the rubber, but diffuses very slowly if at all through the remaining membranes. Successive immersions of nioist impermeable membranes (gold- beater's skin and parchment) in alcohol and ether, for different periods of time, fail to render the treated membranes more perme- able to Sudan III than before. The authors demonstrated the general facts in this connection pertaining to rubber and gold-beater's skin. VI. EXPERIMENTS ON THE DIFFUSIBILITY OF ALKALOIDS THROUGH RUBBER^" Various ether-soluble substances, when dissolved in ether and placed in rubber bags immersed in ether, readily pass through the rubber membranes thus imposed (I-V). We have found that various alkaloids and some related substances readily diffuse through rubber under such conditions. Our experiments were conducted as follows : A moderate quan- tity of the pure ether-soluble substance was mixed with 15 to 25 c.c. of ether.^^ This mixture was poured through a funnel into a new air-tight rubber condom in such a way as to preclude the possibility of overflow upon the external surface. The bag was then immersed in about 50 c.c. of ether in a narrow salt-mouth bottle 7 inches high. With the bag suspended at füll extension in this position, its mouth was about an inch above the opening in the bottle. The protrud- "In experiments which the senior author has been conducting with Prof. Welker's Cooperation, it has been found that collodion bags are disintegrated by ether containing more than about 1.5 per cent. of alcohol. Pure ether does not dissolve or in any way disorganize collodion membranes. A collodion bag con- taining pure ether may be immersed for a week or more in pure ether without undergoing any appreciable deterioration. (See page 70.) ^ Sidbury and Gies : Proceedings of the Society for Experimental Biology and Medicine, 191 1, viii, p. 104. " Substances which did not dissolve readily were triturated with ether in a mortar. igi2] William J. Gies 63 ing Condom was supported in the neck of the bottle by a tightly fitting cork Stopper, which also served to keep the bag closed. After a diffusion period of convenient length (sometimes 2 to 5 days)/^ the Condom was cautiously removed from the bottle, the ether diffus- ate was poured into a porcelain dish, and the ether completely re- moved by evaporation on a steam bath. At least one appropriate test was then applied to the residue.^^ Meanwhile, the ether Solution in the Condom was removed. A large volume of water was then poured into the suspended bag, which, during its distention by the water, was carefully examined for signs of leakage. In a few instances defective membranes tem- porarily rendered the outcome doubtful. All results with such bags were ignored, of course. Each of the tests, even after reliable pos- itive responses, was repeated at least once with a nezv rubber bag. The substances named below (the complete list of those already tested in this connection) are readily diffusible under the conditions of these experiments : — A. Apomorphin, atropin, brucin, caffein, Cocain, codein, col- chicin, coniin, morphin, narcein, narcotin, nicotin, physostigmin, quinin, strychnin, veratrin. B. Acetanilid, antipyrin, phenacetin, picric acid, picrotoxin, pyramidon, salicylic acid. Experiments with other solvents, and with additional substances of alkaloidal type, will be added to this series. " Some of the alkaloids pass through rubber almost immediately under the conditions of these experiments. *' In the experiments with nicotin, the " tobacco odor " of the concentrated liquids was very pronounced. STUDIES OF DIFFUSION THROUGH RUBBER MEMBRANES 2. Diffusibility of lipins from ether through rubber membranes into ether JACOB ROSENBLOOM (Biochemical Laboratory of Columbia University, at the College of Physicians and Surgeons, New York) I. INTRODUCTION Many experiments, in completion of the diffusion work I have been doing in collaboration with Dr. Gies/ have been performed to determine the diffusibihty or non-diffusibihty of hpins and similar substances (page 57). Such data must obviously be obtained in detail, if any attempt to devise methods for the isolation and purifi- cation of hpins by dialysis through rubber can be successful. I present here briefly the essential resuks of the work aheady completed in this connection. II. DIFFUSION EXPERIMENTS Methods. In the experiments described below, ordinary rubber Condoms were used as diffusion membranes.^ Various kinds of "sheet rubber," known as "pure Mexican plantation rubber," and fumed with carbon-disulfid, were found to be good membranes for this kind of work, but besides allowing fats, fatty acids, soaps, cho- lesterol and hpochrome to diffuse through it, this sheet rubber also permits the passage of lecithans under the conditions to be described, although the lecithans pass through the sheet rubber very slowly compared with other lipins such as fat and cholesterol. Condoms ^ Rosenbloom and Gies : Proceedings of the American Society of Biological Chemists, 191 1, ii, p. 8; Journal of Biological Chemistry, 191 1, ix, p. xiv. ° Bef ore the Condoms were used for this purpose, they were placed in f resh portions of ether daily for several days, to free them from the powder adherent to them. This is especially important when one proposes to test the dialysates for phosphorus, since the adherent powder has been found to contain phosphorus. 64 I9I2] Jacoh Rosenbloom 65 do not permit the diffuslon of lecithans under the conditions of the tests to be described, and they were preferred for this work for that reason. The cause of the observed difference in permeability is unknown to us, but will soon be made the subject of special inquiry. The substances to be tested in the diffusion experiments were dis- solved in 100 to 200 c.c. of ether (anhydrous and distilled over sodium), the concentrations of the Solutions varying from 0.5 to 5 per cent, The Solution or Suspension was carefully poured through a funnel into a new air-tight rubber condom in such a way as to preclude the possibility of overflow upon the external surface. The bag was then immersed in from 100 to 200 c.c. of pure ether in a wide-mouthed bottle of convenient size, and suspended loosely by a thin cord held securely between the stopper and neck of the tightly stoppered bottle. The bottle was kept well stoppered throughout the whole of each test to prevent egress of ether and ingress of dust and other extraneous matter. 1. Ether extract of egg yolk. Within five minutes after ether extract of egg yolk is subjected to the diffusion treatment described above, the lipochrome appears in the dialysate, diffusion currents being visible about the same time. The following substances can be detected in the dialysate after short periods of dialysis: fat, fatty acid, cholesterol, and lipochrome. The lecithans do not pass through the Condoms, even during prolonged periods of dialysis. We tested for lecithans in the dialysate by analyzing the evapora- tion residue for phosphorus by the fusion and Neumann methods. and by seeking an " acetone precipitate " in the concentrated ether Solution after the addition of electrolyte (sodium chlorid). Some- times a positive phosphorus test was obtained from a dialysate which did not yield an " acetone precipitate." In such cases, it was found that this result was due to the presence of glycerophosphoric acid in the dialysate. If to a Solution of lecithans, which, after dialysis in a condom, does not yield a phosphorus Compound to the dialysate, one adds some glycerophosphoric acid, and then dialyzes this Solution through the same rubber condom, glycerophosphoric acid appears in the diffusate. 2. Ether extract of brain. The dialysate from ether extracts of brain contained fat, fatty acid, and cholesterol. Lecithans failed to dialyze. 66 Studies of Diffusion through Rubber Membranes [Sept. 3. Ether extract of heart muscle (ox). Fat, fatty acid, lipo- chrome, and cholesterol were detected in the dialysate from ether extracts of the heart muscle of oxen. Lecithans did not diffuse. 4. Ether extract of kidney and liver (dog). Fat, fatty acid, lipochrome, and cholesterol appeared in the diffusates from ether extracts of dog kidneys and livers. Lecithans did not dialyze. 5. Ether extract of blood (dog). Fat, fatty acid, lipochrome, and cholesterol occurred in the dialysates from ether extracts of dog blood. No lecithans dialyzed. 6. Ether extract of carrots. The coloring matter dialyzed very rapidly from ether extracts of carrots. A small amount of fat was also present in the dialysate. 7. Ether extract of Xanthoma (skin). The yellow coloring matter dialyzed very quickly from an ether extract of xanthomatous skin, but it faded in twelve hours. 8. Ether extract of cerumen. Cholesterol, fat, and fatty acid were present in the dialysates from ether extracts of cerumen. Neither the coloring matter nor the lecithans diffused. g. Ether extract of yeast. The dialysates from ether extracts of yeast exhibited a peculiar opalescence, even at the end of six weeks' dialysis. A small amount of fat dialyzed, but lecithans did not diffuse. 10. Ether Solutions of individual substances er special prod- ucts. The following substances or special products, when subjected to diffusion by the method described above, were found to be diffusible : Acetic acid Ethyl butyrate Palmitic acid Acetone Formic acid Potassium palmitate* Beta-hydroxy-butyric acid GlyceroP Potassium stearate* Butter (fresh and rancid) Lactic acid Propionic acid Butyric acid Lead oleate Sodium palmitate* Cholesterol-acetate Mutton tallow Sodium stearate* ' When ether-alcohol Solutions of glj'cerol are dialyzed against ether-alcohol, and alcohol Solutions of glycerol are dialyzed against ether, the dialysates contain glycerol. * Treated with water, then with alcohol to the point of precipitation, then with ether until a precipitate was produced. The filtrate was dialyzed against water, alcohol, and ether in identical proportions. 912] Jacob Rosenhloom 6y Cholesterol-benzoate Oleic acid Stearic acid Cholesterol (from brain, Olive oil Sudan III egg yolk, and gall-stones) Olive oil stained with Urochrome' Ethyl acetate Sudan III Valerianic acid In some special experiments we found^ that cholesterol benzoate, cholesterol stearate, cholesterol oleate and cholesterol palmitate, when dissolved in ether, readily diffuse through rubber into ether. Cholesterol stearate with a molecular weight of 652.61 diffuses, whereas the various lecithans, with molecular weights considered to be 770 to 785, do not. If we assume that the diffusion of a sub- stance depends on the size of its molecules, the above facts strengthen Hiestand's conclusion that the molecular weight of ^gg- yolk lecithin is 1446, which figure he obtained by a molecular weight determination, II. Indiffusible substances. The following substances, when subjected to diffusion by the method described above, were found to be indiffusible.'^ Sodium chlorid Lecithans from yeast Lecithans from brain Lecithans from wheat embryo Lecithans from egg yolk Kephalin from brain Lecithans from heart muscle Cuorin from heart muscle Lecithans from pig testicle Compound of lecithin with platinic chlorid Lecithans from liver and kidney Koch^ has lately described the preparation of various Compounds with lecithans, but it is uncertain whether these Compounds are colloidal adsorptions, mechanical mixtures, or true chemical Com- pounds. It seemed of interest to study the behavior of these sub- stances in ether Solution, when subjected to dialysis in rubber bags suspended in ether. The preparations used in these experiments were made accord- ' Ether-alcohol Solution (equal amounts) dialyzed against ether-alcohol. ' Boas and Rosenbloom : Proceedings of the Society for Experimental Biology and Medicine, 191 1, viii, p. 132. ''We have found that lecithans prepared by the Zuelzer, Bergeil, or Roaf and Edie method, when dialyzed, always yield traces of cholesterol to the dialysate; and often fat. * Koch and collaborators : Journal of Pharmacology and Experimental Therapeiitics, 1910, xii, 239-269. 68 Studies of Diffusion through Rubber Membranes [Sept. ing to the method described by Koch. For the dialysis tests the Solutions of the lecithan Compounds were evaporated to dryness at 38° and the residues triturated with ether. The extracts were fil- tered, and the filtrates placed inside of rubber bags and dialyzed against ether for thirty-seven days. The dialysates were tested weekly to see if the substance combined with the lecithan had diffused. Compounds of lecithin with glucose, lactic acid, strychnin, digi- tonin, salicin, urea, creatin, Creatinin, and caffein were prepared. It was found that the glucose and lactic acid dialyzed completely, the strychnin, digitonin, and salicin dialyzed partially, while urea, creatin, Creatinin, and caffein did not dialyze at all.^ It was thought that some of the various substances which did not diffuse might do so in the presence of a considerable amount of dif- fusible material, but on dialyzing various mixtures of the above- named indiffusible substances with varying amounts (up to 15 grams), of neutral fat, fatty acid, cholesterol, or olive oil, no diffu- sion of lecithan occurred. When Solutions of lecithans are subjected to dialysis by the method described above, they take up a great deal of ether, and the volume of liquid in the bag is greatly increased. We have demon- strated that lipins exert strong osmotic pressure. ( See page 59. ) We have also placed ether Solutions of lecithans with cholesterol and fat in closed rubber bags suspended in Soxhlet extractors. Soxhlet extraction in the usual way failed to remove lecithan from the bag under these conditions. These findings favor the develop- ment of a method for the thorough removal of impurities from lecithan Solutions. It is perhaps superfluous to add that the results already mentioned may be obtained by placing the Solution to be tested outside the rubber bag and allowing dialysis to take place into pure ether con- tained in the bag. III. SUMMARY OF GENERAL CONCLUSIONS I. Most lipins, chief among them, fat, fatty acid, soaps, cho- lesterol, cholesterol-esters, lipochrome, and various other ether-sol- • Boas and Rosenbloom : Loc. cit. I9I2] Jacob Rosenhloom 69 üble substances, diffuse from ether Solution through rubber membranes into ether, 2. Sodium Chlorid, lecithans prepared from various sources, kephalin, cuorin, and the Compound of platinum with lecithin, do not diffuse under such conditions. 3. One or more of the diffusible substances in these experiments may be dialyzed from Solutions containing them, together with one or more of the indiffusible ones, without inducing any of the latter to pass through the membrane. STUDIES OF DIFFUSION THROUGH RUBBER MEMBRANES 3. Diffusibility of protein through rubber membranes, with a note on the disintegration o£ coUodion membranes by common ethyl ether and other solvents WILLIAM H. WELKER (Biochemical Laboratory of Columbia University, at the College of Physicians and Surgeons, New York) l. INTRODUCTION In the course of our studies of proteins, under the auspices of the George Crocker Special Research Fund, we obtained a protein product which is soluble in a mixture of equal parts of absolute alcohol and absolute ether, and which responds to the biuret, xantho- proteic, Millon and Hopkins-Cole tests. The material was prepared by the following method : 25 grams of Witte peptone were dissolved in 500 c.c. of 0.2 per cent. hydrochloric acid Solution. The liquid was evaporated to a thick syrup on a water bath. This syrup was thoroughly extracted with absolute alcohol and the resultant yellow liquid filtered. The filtrate was treated with an equal volume of absolute ether, which produced a white flocculent precipitate. After the Sedimentation of the precipitate, the supernatant liquid was de- canted and filtered. When five volumes of absolute ether were added to this filtrate, a white flocculent precipitate was produced. This product was isolated by filtration, washed with absolute ether, and exposed to the air in a thin layer on a watch glass, where it solidified as yellowish and somewhat hygroscopic granulär material, which could easily be pulverized. The product dissolved promptly in absolute alcohol. From concentrated alcoholic Solution the prod- uct may be precipitated by the addition of an equal volume of abso- lute ether. Whether the product is peptone or a much simpler Poly- peptid has not yet been determined. Dr. Gies and his collaborators have lately given much attention to 70 1912] William H. Welker 71 the diffusibility of lipins and other substances throtigh rubber mem- branes. The solubility of the above mentioned product in alcohol- ether Solution led Dr. Gies to propose a study of the comparative diffusibility of the protein through membranes of rubber, parchment and collodion. Such an investigation was accordingly conducted by the diffusion process described on page 55. The data in the accompanying tables indicate the conditions and the results of the tests in this connection.^ IL COMPARATIVE DIFFUSION EXPERIMENTS Experiments with rubber membranes. The results of the tests in the first four series show collectively [Table i (1-15)] that biuret-reacting matter appeared in the diffusates; that the rubber itself did not yield such substance; and that the occurrence of biuret- TABLE I Results of experiments with rubber membranes A. Data showing the dißusibility of biuret-reacting material First Series. With Rubber Condoms. Results of Results of the Duration of Liquid outside the biuret test for leaks at Exp. No. the experi- Contents of the bag of the bag test in the the end of the ment, days diffusate experiment I 4 r 30 c.c. ether-alc. sol. \ 10 c.c. abs. ether Abs. ether + + + Small hole in the bag 2 4 f 20 c.c. ether-alc. sol. \ 20 c.c. abs. ether Abs. ether + + No leak 3 4 f 10 c.c. ether-alc. sol. l 30 c.c. abs. ether Abs. ether + No leak Second Series. With Rubber Condoms. 4 5 / 30 c.c. ether-alc. sol. \ 10 c.c. abs. ether Abs. ether + + + Small hole very high up in the bag 5 5 f 20 c.c. ether-alc. sol. \ 20 c.c. abs. ether Abs. ether + + No leak 6 5 f 10 c.c. ether-alc. sol. \ 30 c.c. abs. ether Abs. ether + No leak * In the tables, " ether-alc. sol." indicates the protein Solution as it was made available by the above mentioned process before final precipitation with five volumes of ether. Such precipitation was efifected only when the solid was desired for special reasons. See the data pertaining to the eighth series of tests, Table i. 72 Studies of Diffusion through Rubber Membranes [Sept. TABLE I (continued) Third Series. With Rubber Condoms. Exp. No. Duration of the experi- ment, days Contents of the bag Liquid outside of the bag Results of the biuret test in the difTusate Results of the test for leaks at the end of the experiment 7 S / 30 c.c. ether-alc. sol. 1 lo c.c. abs. ether Abs. ether + + + No leak 8 5 f 20 c.c. ether-alc. sol. \ 20 c.c. abs. ether Abs. ether + + No leak 9 5 f lo c.c. ether-alc. sol. \ 30 c.c. abs. ether Abs. ether + No leak lO« 5 Abs. ether ("control") Abs. ether — No leak II» 5 Abs. ether ("control") Abs. ether — FouRTH Series. With Bags of Sheet Rubber.* 12 30 / 60 c.c. ether-alc. sol. \ 20 c.c. abs. ether Abs. ether + + + No leak 13 30 f 40 c.c. ether-alc. sol. 1 40 c.c. abs. ether Abs. ether + + No leak 14 30 / 20 c.c. ether-alc. sol. L 60 c.c. abs. ether Abs. ether — No leak IS 30 Abs. ether ("control") Abs. ether — No leak B. Data showing that the diffusible biuret-reacting material ii-15) was true protein Fifth Series. With Rubber Condoms. Duration of the experi- ment, days Contents of the bag Liquid outside of the bag Results of the tests for protein in the diffusate Results of the test for Exp. No. Biuret Hop- kins- Cole Xan- thopro- teic leaks at the end of the experiment 16 17 3 3 / 30 c.c. ether-alc. sol. \ 10 c.c. abs. ether 30 c.c. ether-alc. sol. Abs. ether Abs. ether + + + + + + No leak No leak SixTH Series. With Bags of Sheet Rubber.* 18 3 / 60 c.c. ether-alc. sol. \ 20 c.c. abs. ether Abs. ether + + + No leak 19 3 r 60 c.c. ether-alc. sol. \ 20 c.c. abs. ether Abs. ether + + + No leak Seventh Series. With Rubber Condoms. 20 31 3 3 40 c.c. ether-alc. sol. 40 c.c. ether-alc. sol. Abs. ether Abs. ether + + + + + + No leak No leak •This control experiment (10) was carried out in duplicate, with negative results in each case. *In this experiment (11) a new, clean, empty condom, with the bottom removed, was suspended in absolute ether, for " control " purposes. * " Pure Mexican plantation rubber." I9I2] William H. Welker 73 TABLE I (continued) C. Data showing the eßect of water on the diffusion phenomena (.r-21) EiGHTH Series. With Rubber Condoms.' Exp. No. Duration of the experi- ment, days Contents of the bag Liquid outside of the bag Results of the biurei test in the diffusate Results of the test for leaks at the end of the experiment 3 C.C. abs. alc. sol. 6 c.c. abs. alc. 22 5 ' 9 c.c. abs. ether 6 c.c. H2O 3 c.c. abs. alc. 3 c.c. abs. alc. sol. 18 c.c. abs. ether . 1.2 c.c. H2O 9 c.c. abs. alc. •" No leak 23 5 21 c.c. abs. ether I c.c. H2O 6 c.c. abs alc. 21 c.c. abs. ether I c.c. HsO — No leak 24 5 \ r 3 c.c. abs. alc. sol. l 3 c.c. abs. ether Equal volumes of abs. ether + No leak and abs. al- cohol D. Data showing the effect of fat on the diffusion phenomena {1-24) NiNTH Series. With Rubber Condoms. 25 10 Olive oil and Witte Pep- tone (solid) Olive oil _e 26 10 Olive oil, Witte peptone (solid) and H20^ Olive oil ^" Tenth Series. With Rubber Condoms. 27 28 30 30 Olive oil and Witte pep- tone (solid) Olive oil, Witte peptone and HjO^ Olive oil Olive oil Eleventh Series. With Rubber Condoms. 29 10 f Ether-alc. sol. iLard Abs. ether _ 30 10 f Ether-alc. sol. "iLard Abs. ether — Twelfth Series, With Rubber Condoms. 31 1 / Ether-alc. sol. LLard Abs. ether + No leak 32 I / Ether-alc. sol. iLard Abs. ether + No leak Thirteenth Series. With Rubber Condoms. 33 2 f 30 C.C. ether-alc. sol. ILard 30 c.c. abs. ether + No leak 34 3 / 30 C.C. ether-alc. sol. ILard 30 c.c. abs. ether + No leak 'For this series of tests, we used 0.2 gram of the protein material dissolved in 10 c.c. of absolute alcohol. • On the water-soluble extract of the oil. ^ Water sufficient to make a paste of the Witte peptone was used. The paste was triturated into the oil. 74 SUidies of Diffusion throngh Rubber Membranes [Sept. reacting material in the diffusate was not due to perforations of the bags. The diffusion of biuret-reacting material was always greatest in degree through the bags containing the largest proportion of protein. The results of tests 1-15 show that biuret-reacting material dif- fiised through the rubber membranes under the conditions imposed. In Order to determine more definitely, however, whether protein dif- fused through the rubber, we repeated the essential features of tests 1-15, but applied additional tests to the diffusates [Table i (16- 21)]. The results of tests 16-21 (Table i) confirm the findings of tests 1-15, and also show definitely that the dififusible biuret-reacting ma- terial was triie protein. The data of tests 1-2 1 suggest that osmosis depends upon affin- ities between the membrane, and the solvent or solute, or both. We made a direct test of this mattter in a preliminary way by adding water to the solvent and thus disturbing its affinities with the mem- brane without decreasing the solubility of the solute. The findings are given in the summary pertaining to the eighth series (Table i ). The results of tests 22-24 show that water exerted a disturbing osmotic influence and that diffusion of the protein was entirely pre- vented by the water. We extended these experiments to determina- tions of the influence of associated, readily diffusible lipins, in the presence or absence of water. The results are given in tests 25-34. In the tenth series the oil in the diffusates was emulsified with a little soap Solution and then repeatedly extracted with ether until all the fat was removed. The water containing the soap, and the aqueous extract of the oil, were evaporated to dryness and the biuret test applied to a concentrated Solution of the residue. Experiments with parchment-paper bags. The foregoing re- sults with rubber membranes naturally increased our desire to make comparative observations with bags of parchment and collodion. The results of the tests with parchment are given in tests 35-42, Table 2. That osmosis depends upon accord between the solvent and the membrane is obvious from these results also, for the protein substance, which is readily diffusible through parchment from aqueous Solution, does not dialyze through such a membrane from an alcohol-ether Solution. I9I2] William H. Welker n TABLE 2 Results of experiments with bags of parchment paper FOURTEENTH SeRIES. Duration of the Results of the Exp. No. experiment, Contents of the bag Liquid outside of biuret lest in days the bag the diffusate 35 I f 10 c.c. ether-alc. sol. \ 30 c.c. abs. ether Abs. ether 36 2 / 3 c.c. ether-alc. sol. \ 3 c.c. abs. ether Abs. ether — FiFTEENTH SERIES.« 37 10 / 30 c.c. ether-alc. sol. \ 10 c.c. abs. ether Abs. ether ^ 38 10 f 20 c.c. ether-alc. sol. 1 20 c.c. abs. ether Abs. ether — 39 IG f 10 c.c. ether-alc. sol. \ 30 c.c. abs. ether Abs. ether — SiXTEENTH SERIES.» 40 10 f 30 c.c. ether-alc. sol. \ 10 c.c. abs. ether Abs. ether _ 41 10 f 20 c.c. ether-alc. sol. \ 20 c.c. abs. ether Abs. ether -" 42 10 / 10 c.c. ether-alc. sol. \ 30 c.c. abs. ether Abs. ether — III. ON THE UTILITY OF COLLODION.BAGS IN EXPERIMENTS OF THE KIND DESCRIBED IN THE FOREGOING SECTIONS Experiments like those in the sixteenth series (Table 2) were attempted with bags made of collodion, but in each case the bags were perforated and in part dissolved, by the Contents, before the experiment could be fairly started. Several years ago, Dr. Gies observed, in some dialysis experi- ments with collodion membranes, that ethyl ether could be kept on the aqueous Contents of collodion bags, for preservative purposes in such tests, without inducing distintegration of the bags. In repeti- tions of the experiments a year or two later, however, it was found that ether under such circumstances often caused deterioration of *The results in this series, while apparently negative in each case, were somewhat doubtful owing to the fact that the paper contained some soluble material, which rendered the biuret test more or less uncertain. See the results of the tests in the sixteenth series. *The parchment paper was washed free from soluble matter before the beginning of the tests. 76 Studies of Diffusion through Rubber Membranes [Sept. the membrane, but that occasionally it did not. The reason for such variations in the action of the ether could not be conveniently ascer- tained at the time. The prompt Perforation of the collodion bags in our several attempts, as stated above, to determine the diffusibihty through col- lodion of the alcohol-ether soluble protein, recalled Dr. Gies' pre- vious experiences and led him to suspect that the alcohol, in the Solu- tions employed by us, was responsible for the observed destructive effects on the collodion membrane in these experiments. He be- lieved, also, that the previous variations in the action of ether on collodion in dialysis experiments, as already related, were due to differences in the degrees of purity of the ether employed. At Dr. Gies' request, therefore, I made direct tests of the solvent action of ether containing alcohol, and various other substances related in one way or another to alcohol and ether. Collodion bags were made, in test tubes, from U. S. P. col- lodion.^*^ It was found that such bags were not perforated by abso- lute ether when it was poured into them 10 minutes after their re- moval from the tubes, i. e., after fairly complete evaporation of the residual alcohol. The time required for the evaporation of the residual alcohol is dependent on the prevailing temperature. At low temperatures the alcohol disappears from the collodion membrane very slowly. Common ether (Merck's 0.720 sp. gr.), however, when poured into such bags, passed through them almost immedi- ately, with general Solution of the collodion, even after 2 hours of preliminary exposure of the bag outside the mould. In the first tests of the effects of alcohol it was found that absolute ether con- taining 1.5 per Cent, or more of added absolute alcohol promptly penetrated the bags. In a series of more careful tests of absolute ether containing various percentages of added absolute alcohol, it was found that the bags were penetrated promptly by ether containing more than 1,25 per Cent, of alcohol, but that the mixture containing 1.25 per cent. of alcohol acted more slowly. Ether containing less than 1.25 ^r cent. of alcohol exhibited no destructive action. Qualitative tests showed that acetone, acetaldehyde, ethyl acetate, methyl alcohol and glacial acetic acid attack and penetrate collodion "An ether Solution containing alcohol. igi2] William H. Welker 77 bags^^ immediately, toluol slowly, whereas formic acid, formalde- hyde (40 per cent.), Chloroform, petroleum ether, carbon tetra- chlorid, carbon bisulfid and paraffin oil were without distinguishable solvent action, even after long periods of contact. Acetone (5 per cent.) in absolute ether attacks collodion bags slowly, while a 10 per cent. Solution acts rapidly. Acetaldehyde (4 per cent.) in absolute ether attacks the bags slowly, but a 5 per cent. Solution acts rapidly. Methyl alcohol (3 per cent.) in absolute ether dissolves the bags, but a 2 per cent. Solution does not. Glacial acetic acid (2 per cent.) in absolute ether attacks the bags slowly, a 3 per cent. Solution acts rapidly but a i per cent. Solution appears to be inert. Five per cent. Solutions of Chloroform, toluol, petroleum ether, carbon tetra- chlorid, carbon bisulfid, benzol, ethyl acetate, and paraffin oil, in absolute ether, were without visible effect on collodion bags. Five per cent. Solutions of formic acid (sp. gr. 1.2) and formalde- hyde (40 per cent.), in absolute ether, immediately attacked and penetrated collodion bags. Further work along these lines is in progress. My cordial thanks are due Dr. Gies for his kind direction and assistance in these experiments. "Bags practically free from residual alcohol were used. STUDIES OF DIFFUSION THROUGH RUBBER MEMBRANES 4. The comparative diffusibility of various pigments in different solvents GEORGE D. BEAL and GEORGE A. GEIGER {Biochemical Laboratory of Columbia University, at the College of Physicians and Surgeons, New York) I. INTRODUCTION Dr. Gies and his associates have shown that many biological substances diffuse through rubber under suitable conditions (page 55). Inorganic as well as organic substances exhibit this capacity and various colloids share it with crystalloids. Lipochrome and ferric sulfocyanate are among the colored substances which, in the early experiments, were found to be diffusible from ether Solution through rubber membranes into ether. At Dr. Gies' Suggestion we undertook a similar study of the diffusibility of common pigments, especially " food colors." Fol- lowing his advice we also sought data which might be of service in devising methods for the purification of pigments, and for their Separation and detection under various circumstances. Our diffusion tests were conducted by the following general method: A moderate quantity of the pigment was mixed with 15- 25 c.c. of the solvent. The Solution, or Suspension, was carefully poured into a rubber Condom in such a way as to preclude the pos- sibility of overflow upon the external surface. The bag was then immersed in about 50 c.c. of solvent in a narrow salt-mouth bottle 7 inches high. With the bag suspended at füll extension in this Posi- tion, its mouth was about an inch above the opening in the bottle. The protruding condom was supported in the neck of the bottle by a tightly fitting cork stopper, which served to keep both the bag and the bottle closed. The diffusion periods varied from a few minutes \o a week or more, according to the obvious requirements in each case for a definite conclusion regarding diffusibility. 78 I9I2] George D. Beul and George A.. Geiger 79 Most of the original tests were made with new Condoms. Many tests were repeated with Condoms which had previously been em- ployed by us in pigment-diffusion experiments but which, prior to being used again, had been thoroughly washed with portions of the solvent to which they were soon to be subjected in the new diffusion tests. Defects in the rubber could easily be detected. All doubtful results were ignored. Numerous repetitions prevented erroneous deductions. In the accompanying summary we present an outline of the various tests and the main results of each. For the sake of con- venience we use in the summary the following abbreviations : D, diffusion; Di, pigment appears in the diffusate within lo minutes; D2, pigment does not diffuse within 10 minutes, but appears in the diffusate within 30 minutes; D3, pigment does not diffuse within 30 minutes, but appears in the diffusate within i hour; D4, pigment does not diffuse within i hour, but appears in the diffusate before the lapse of 2 hours ; D5, pigment cannot be seen in the diffusate before the third hour of diffusion, but appears before the fourth hour; D6, pigment cannot be seen in the diffusate before the sixth hour of diffusion, but appears before the eighth hour; D7, pigment cannot be seen in the diffusate before the tenth hour of diffusion, but appears before the twelfth hour; D8, pigment appears in diffusate in about 24 hours; O, no visible diffusion at any time within a week. IL SUMMARY OF DIFFUSION DATA Inside Outside solvent Result Solvent Pigment Remarks I Ether. . . Sudan III Ether Ether + alco- hol (25%). Ether + alco- hol (50%). Ether -f alco- hol (75%). Alcohol (100%)... Chloroform . . Di Di Di Di Di Di D very rapid. 2 Ether. . . Sudan III 3 Ether . . . 4 Ether. . . 5 Ether. . . 6 Ether . . . Sudan III D rapid though slower than i. Sudan III D slower than 2. Sudan III D slower than 3. Sudan III Ether was withdrawn, leaving a concentrated Solution of Sudan III. Currents very distinct. CoUection of color near top very marked. Color zone on top. No diffusion currents downward. 8o Studios of Diffusion through Rubber Membranes [Sept. II. SUMMARY OF DIFFUSION DATA {continued) Inside Solvent 7 Ether. 8 Ether . . . 9 Ether. . . 10 Ether . . . 11 Alcohol. . 12 Chloro- form . . . 13 Alcohol . . 14 Ethyl acetate . . 15 Acetone.. 16 Gl. acetic acid .... 17 Ether. . . 18 Ether. . . 19 Ethyl acetate . . 30 Acetone.. 21 Alcohol. . 27 Ether. . . 23 Ether. . . 24 Ether . . . 25 Ether. Pigment 26 Ether . . 27 Ether. . 28 Ether. . 29 Ether. . 30 Ether . . 31 Ether. . 32 Ether. . 33 Ether. . 34 Ether . . 35 Ether. . 36 Ethyl acetate . 37 Ethyl acetate . Sudan III Sudan III Sudan III Sudan III Sudan III Sudan III Sudan III Sudan III Sudan III Sudan III Picric acid Hematoxylin Methyl violet Methyl violet Methyl violet Methyl violet Magenta Naphthol yellow . . . Methyl violet and Sudan III Outside solvent Methyl alco- hol Acetone . Petroleum ether. . . . Gl. acetic acid Ether Chloroform . Alcohol . . . . Ethyl acetate Acetone .... Gl. acetic acid Ether Ether Ethyl acetate Acetone . Alcohol . Ether. . . Ether. . . Ether... Resnlt Ether. Chlorophyll Ether. Annatto Alcannin Metanil yellow. . Martius yellow. . Scarlet R Malachite green. Brazil wood . . . . Chrysoldin Turmeric 'Ether. Ether. Ether. Ether. Ether. Ether. Ether. Ether. Ether. Annatto . Chlorophyl . Ethyl acetate Ethyl acetate Di Di Di Di Di Di Di Di Di Di Di D3 DS D6 O D5 Ds O Di Di Di Di O Di Di D2 Di D2 Di D2 Di Remarks Ether was withdrawn, leaving layer of dye inside. Ether withdrawn. Solution of pigment in bag concentrated. Moderate diffusion. Diffusion slow. Rapid diffusion. Diffusion slow. Slow diffusion. Moderate diffusion. Slow diffusion. Rubber yellow; color not re- moved by ether. Denser Solution in bag than outside. (See 36.) Rapid diffusion of the Sudan III. Ether changed three times in 2 hours after which practically all Sudan III had been removed, leaving the methyl violet in the bag. Vary slight diffusion. Very slight diffusion. Very rapid diffusion. Rapid diffusion. Very rapid diffusion. Bag colored green. Slow diffusion. Very slow diffusion. Rapid diffusion. Pigment Solution inside concen- trated.« Pigment Solution inside concen- trated. • " Fat-soluble " chlorophyl was used in all the chlorophyl tests. * In these cases (36-44) the solvent diffused more rapidly than the solute. 1912] II. George D. Beal and George A. Geiger SUMMARY OF DIFFUSION DATA (continued) 8i Inside Outside solvent Result T< ATVl f l^e Solvent Pigment JxCularKa 38 Ethyl acetate . . Alcannin Ethyl acetate Di Pigment Solution inside concen- trated. 39 Ethyl acetate . . Martius yellow .... Ethyl acetate Di Pigment Solution inside concen- trated. 40 Ethyl acetate . . Scarlet R Ethyl acetate Di Pigment Solution inside concen- trated. 41 Ethyl acetate . . Brazil wood Ethyl acetate Di Pigment Solution inside concen- trated. 42 Ethyl acetate . . Chrysoidin Ethyl acetate D3 Pigment Solution inside concen- trated. 43 Ethyl acetate . . Turmeric Ethyl acetate D3 Pigment Solution inside concen- ^-'O trated. 44 Ethyl acetate. . Metanil yellow Ethyl acetate D3 Pigment Solution inside concen- trated. 45 Ethyl acetate . . Malachite green. . . . Ethyl acetate D6 46 Ethyl acetate . . Naphthol yellow . . . Ethyl acetate 47 Ethyl acetate . . Sudan I Ethyl acetate Di Rapid diffusion. 48 Ethyl acetate . . Sudan G Ethyl acetate Di Moderate diffusion. 49 Ethyl acetate . . Rhodamin Ethyl acetate D in about 2 days. 50 Ethyl acetate . . Fast red A Ethyl acetate D3 Diffusion very slight in each of 51 Ethyl tests 50-56 inclusive. acetate . . Rose bengal Ethyl acetate D2 52 Ethyl acetate. . Erythrosin Ethyl acetate D2 53 Ethyl acetate . . Methylene violet . . . Ethyl acetate D5 54 Ethyl acetate. . Phloxin red Ethyl acetate D3 55 Ethyl acetate . . Auramine Ethyl acetate D5 56 Ethyl acetate . . Orange G Ethyl acetate D in about 5 hours. 57 Methyl alcohol. . Gold orange Methyl alcohol .... D8 Color of diffusate very slight i week later. 58 Methyl alcohol. . Naphthol yellow . . . Methyl alcohol. . . . No appearance of color in 8 hours. 59 Meth alcohol. . Carmosin B Methyl alcohol .... D8 Color of diffusate very slight i week later. 82 Studics of Diffusion through Ruhher Memhranes [Sept II. SUMMARY OF DIFFUSION DATA (continued) Inside Solvent 60 Methyl alcohol . 61 Methyl alcohol . 62 Methyl alcohol . 63 Methyl alcohol . 64 Amyl alcohol . 65 Amyl alcohol . 66 Amyl alcohol . 67 Amyl alcohol . 68 Amyl alcohol . . 69 Amyl alcohol. . 70 Amyl alcohol. . 71 Amyl alcohol. . 72 Amyl alcohol . , 73 Amyl alcohol. , 74 Amyl alcohol . . 75 Amyl alcohol. , 76 Amyl alcohol . , 77 Acetone. 78 Acetone. 79 Acetone. 80 Acetone. 81 Acetone. 82 Acetone. 83 Acetone. 84 Acetone. 85 Acetone. 86 Acetone. 87 Acetone. 88 Acetone. 89 Acetone. Pigment Ponceau, G. A.. Ponceau, 2 R. . Naphthol red S. Curcumin S . . . Fast red A . Safranin . . . Eosin A. Phloxin . Rose bengal . Rhodamin . . Erythrosin . . Chrysoldin Sudan I . . . Sudan G. . . Sudan III. Alcannin. . Chlorophyl . Alcannin . . , Auramine. . Barwood . . , Chlorophyl . Chrysoldin . Fast red A . Methylene violet. . Malachite green. . . Martins yellow. . . . Metanil yellow. . . . Naphthol yellow S. Picric acid Rhodamin Outside solvent Result Methyl alcohol. . . . Methyl alcohol. . . . Methyl alcohol. . . . Methyl alcohol. . . . Amyl alcohol Amyl alcohol Amyl alcohol Amyl alcohol Amyl alcohol Amyl alcohol Amyl alcohol Amyl alcohol Amyl alcohol Amyl alcohol Amyl alcohol Amyl alcohol Amyl alcohol Acetone. . . Acetone. . . Acetone . . . Acetone . . . Acetone . . . Acetone. . . Acetone . . . Acetone. . . Acetone . . . Acetone . . . Acetone. . . Acetone. . . Acetone. . . O O O O D7 D7 O O D7 D8 D3 D2 D2 D2 D3 D3 Di Di Di D3 D5 D4 D4 D5 D3 D8 O Dl D7 Remaxks Color of diffusate very slight I week later. D in about 3 days. Color of diffusate very shght i week later. Color of diffusate very slight I week later. Very rapid diffusion. Color of diffusate not very strong I week later. I9I2] George D. Beal and George A. Geiger 83 II SUMMARY OF DIFFUSION DATA {continued) Inside Outside solvent ^Result Remarks Solvent Pigment 90 Acetone.. Sudan I Acetone Acetone Acetone Acetone Dl Di D8 91 Acetone.. Sudan G 92 Acetone.. Fustic 93 Acetone.. Rose bengal Color of dif?usate very slight i week later. 94 Acetone . . Phloxin Acetone . D4 Color of diffusate very slight I week later. A ^^^ V.. W^^ 4 Jk^h^ • • ■ • a 95 Acetone.. Eosin W. gelblich . . . Acetone D7 Color of diffusate very slight I week later. 96 Acetone.. 97 Acetone.. Eosin A Acetone Acetone D4 Cape aloes 98 Gl. acetic acid .... Sudan G Gl. acetic acid Di Slow diffusion. 99 Gl. acetic fc..^**.^ TT *.A**Ä 1.4V^ A^..r AA # acid .... Sudan III Gl. acetic acid Dl Rapid diffusion. 100 Gl. acetic acid. . . . Sudan I Gl. acetic acid Di Slow diffusion. loi Gl. acetic acid. . . . Alcannin Gl. acetic acid D3 102 Gl. acetic acid .... Chlorophyl Gl. acetic acid D6 103 Gl. acetic acid .... Rose bengal Gl. acetic acid DB 104 Gl. acetic acid .... Phloxin Gl. acetic acid D in about 2 days. 105 Gl. acetic acid. . . . Malachite green. . . . Gl. acetic acid 106 Gl. acetic acid .... Methyl violet Gl. acetic acid D8 107 Gl. acetic acid. . . . Scarlet R Gl. acetic acid D2 108 Gl. acetic acid .... Methylene violet . . . Gl. acetic acid D in about 10 days. 109 Gl. acetic acid .... Martius yellow Gl. acetic acid D8 iio Gl. acetic acid .... Biebrich Scarlet. . . . Gl. acetic acid D8 III Gl. acetic acid. . . . Erythrosin Gl. acetic acid D in about 4 days. 112 Gl. acetic acid .... Oranee G Gl. acetic acid 113 Gl. acetic ^.—1' ^ v***^^ ^- ^-* ••••»• •«• ■ ^t^H« * ■ fc*^-v* W^V^ ^.l>VrA^.J acid. . . . Tropeolin OO Gl. acetic acid Very slight color after 2 days, which did not increase after Standing about four days. 114 Gl. acetic acid. . . . Aurainine Gl. acetic acid 115 Gl. acetic acid. . Rhodamin Gl. acetic acid D8 116 Gl. acetic ^^.AA V.' X.A%4* AA * A^ ■«•■*«*«■ acid .... Eosin A. gelblich . . . Gl. acetic acid D8 117 Gl. acetic acid .... Chrysoidin Gl. acetic acid D in about s days. 118 Gl. acetic acid .... Eosin W Gl. acetic acid D in about 4 days, which did not increase during the suc- M rf\J\^i**-* »■• •••••■••• ceeding 3 days. 84 Stndics of Diffusion through Rubber Membranes [Sept. II. SUMMARY OF DIFFUSION DATA (continued) Inside Solvent 119 Gl. acetic acid. . . . 120 Gl. acetic acid .... acetic acetic acetic 121 Gl. acetic acid 122 Gl acid . . 123 Gl acid. 124 Gl acid. . . . 125 Gl. acetic acid .... 126 Alcohol. . 127 Alcohol. . 128 Alcohol. . 129 Alcohol. . 130 Alcohol. . 131 Alcohol. . 132 Alcohol. . 133 Alcohol. . 134 Alcohol. . 135 Alcohol. . 136 Alcohol. . 137 Alcohol. . 138 Alcohol. . 139 Alcohol. . 140 Alcohol. . 141 Alcohol. . 142 Alcohol. . 143 Alcohol. . 144 Alcohol. . 145 Alcohol. . 146 Alcohol. . 147 Alcohol. . 148 Alcohol.. 149 Alcohol. . 150 Alcohol. . 151 Alcohol. . 152 Alcohol. . 153 Alcohol. . 154 Alcohol. . 15s Alcohol. . Pigment Fast Red A Safranin Turmeric Metanil yellow Barwood Annatto Picric acid Auramine ChrysoTdin. .._..... Eosin A Eosin W ' Fast Red Methyl violet Methylene violet. . . Malachite green. . . . Martins yellow Metanil yellow Rose bengal Rhodamin Sudan G Sudan I Bismarck brown . . . Benzopurpurin Tropeolin OO Phloxin Safranin Naphthol yellow . . . Alcannin Chlorophyl Barwood Fustic Turmeric Cape aloes Curcumin S Naphthol green. . . . Orange G Carmosin B Outside solvent Gl. acetic acid Gl. acetic acid Gl. acetic acid Gl. acetic acid Gl. acetic acid Gl. acetic acid Gl. acetic acid Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Alcohol Result D3 O D7 O D7 D7 O D7 D8 D7 DB D7 D7 O O D2 D2 O O D7 O DS D7 O O O O O Remarks D in about 4 days, which did not increase during the suc- ceeding 3 days. D in about 2 days. D in about 6 days. D in about 3 days. Color of diffusate very slight I week later. Color of diffusate very slight I week later. Color of diffusate very slight I week later. Color of diffusate very slight I week later. D in about 3 days. D in about 5 days. Slight I week later. Color of diffusate very slight I week later. D in about 2 days. D in about 2 days. I week later. D in about 2 days. I week later. Very slight Very slight I9I2] George D. Beal and George A. Geiger 85 III. ATTEMPTS TO SEPARATE PIGMENTS BY DIALYSIS The outcome of Test 25 encouraged us to ascertain whether two dissimilar pigments like scarlet R and malachite green might be wholly separated from each other by dialysis thru rubber in a suit- able solvent, e. g., ethyl acetate (see tests 40 and 45). A mixture of the two pigments dissolved in ethyl acetate was accordingly sub- jected to the usual mechanical treatment, but the diffusate was re- peatedly replaced with fresh solvent. The results are indicated in the f ollowing summary : Continuous differential diffusion of scarlet R and malachite green. March 28 — ist diffusate ii-i p.m. Bright red. March 28 — 2nd diffusate 4 p.m. Bright red. March 28 — 3rd diffusate 11 p.m. Deep red. March 29 — 4ith diffusate 12.30 a.m. Deep purplish red. March 29 — 5th diffusate i a.m. Light purplish red. March 29 — 6th diffusate 10.45 ^■^- Deep red with decidedly bluish tinge. March 29 — 7th diffusate 12.30 p.m. Light blue. March 29 — 8th diffusate 9.15 p.m. Blue green. March 30 — 9th diffusate 11.50 a.m. Blue green. March 31 — loth diffusate 9.15 a.m. Blue green. April I — iith diffusate i p.m. Blue green. April 2 — I2th diffusate 11.50 p.m. Light green. April 3 — I3th diffusate 11.50 p.m. Light green. Altho scarlet R and malachite green showed widely different rates of diffusion when they were treated separately, the results detailed above made it evident that it would be difficult if not im- possible to obtain all the scarlet R from mixtures like the one em- ployed without removing some of the malachite green with the red pigment. By subjecting Solutions of scarlet R and malachite green of simi- lar concentrations independently to diffusion in the usual way, we duplicated the blue and green effects with malachite green and the red effects with scarlet R, but the purplish colorations could not he ohtained under such circumstances. That these purplish effects were due to early diffusion of the malachite green with scarlet R, and that the red pigment facilitated the passage of the green one, are clearly indicated by the results. 86 Studies of Diffusion through Rubber Membranes [Sept Repeated removals of the diffusate in the independent scarlet R experiment, and replacements with fresh solvent for a period of about a weck, led to the Separation from the original pigment- product of all its red diffusible matter. The bag, at that stage of the treatment, contained considerable brownish-red, indifTusible material, which evidently was not scarlet R. This result, and simi- lar observations with other pigments, emphasized Dr. Gies' opinion that it might be possible to purify pigment preparations in this way and that their value as coloration agents, for histological staining especially, might thus be considerably enhanced. It will be noted that those pigments which diffused most rapidly were the so-called " fat colors," i. e., those soluble in, or staining, the common fats and oils. Again, with these pigments the dif- fusion is the most rapid, and therefore the most satisfactory, when the solvents are those which, in the State of vapor, soften rubber. It will thus be seen that apparently the membrane, as well as the solvent, exert selective action. This is true to a far greater extent in experiments of this kind than in the ordinary dialyses in aqueous media. When we arrived at this point in these experiments, to which we could give but a few hours weekly, our period of residence at Columbia University was about to close and, after completing some repetitions of previous observations in this connection, we were obliged to discontinue the work. It is Dr. Gies' intention to pro- ceed along lines suggested by the results already obtained in this preliminary investigation. THE COLLOIDAL NITROGEN IN THE URINE FROM A DOG WITH A TUMOR OF THE BREAST MAX KAHN AND JACOB ROSENBLOOM (Biochetnical Laboratory of Columbia University, at the College of Physicians and Surgeons, New York) In 1892 Töpfer^ found that the urine of patients suffering from Cancer contained a very large amount of "extractive substance." This " extractive substance " was calculated by first determining the total nitrogen, and then subtracting from this amount the sum of the nitrogen values for the urea, uric acid, and ammonia contained in the same urine. Bondzynski and Gottlieb,^ five years later, reported that the nitrogen in oxyproteic acid was 2 to 3 per cent, of the total urinary nitrogen. Salkowski,^ and Hess and Saxl,* using different procedures in their researches, came to the conclusion that the oxy- proteic acid or the alcohol-precipitable substances are increased in the urine of human beings suffering from Carcinoma. Salkowski and Kojo,^ in a preliminary communication, recently suggested several methods for the determination of colloidal nitro- gen in the urine. A year later Kojo published the results of a com- parative study of the various procedures suggested in this connec- tion.^ Einhorn, Kahn and Rosenbloom^ studied the zinc sulfate- precipitable, colloidal, nitrogenous material from the urine of nor- mal subjects as well as from the urine of carcinomatous patients, and came to the conclusion that the amount of colloidal nitrogen was invariably increased in subjects with carcinomatous growths. * Töpfer: Wiener klin. Wochenschrift, 1892, v, p. 49. 'Bondzynski and Gottlieb: Zentralbl. f. d. med. Wissenschaften, 1897, xxxv, P- 577- * Salkowski : Berliner klin. Woch., 1910, xlvii, p. 1746. *Hess and Saxl: Beiträge zur Carcinomforschung, 1910, Part II. 'Salkowski and Kojo: Berl. klin. Woch., 1910, xlvii, p. 2297. 'Kojo: Zeitschr. f. physiol. Chem., 191 1, Ixxiii, p. 416. ^Einhorn, Kahn and Rosenbloom: Amer. Journ. of Gastro-enterology, 1911, i, p. 2; and Archiv f. Verdauungs-Krankheiten, 191 1, xvii, p. 557. 87 88 Colloidal Nitrogen in Urinc from a Dog [Sept. The writers lately embraced an opportunity to study the colloidal nitrogen Output in the urine of a dog with a large tumor. The dog upon which this study was made had a hard calcified growth about the size of an orange in one of the breasts. The tumor involved the nipple and the breast tissue for some distance around the nipple. Several metastatic deposits were present along the "breast lines." Microscopic examination of sections of the original growth and of the metastatic infiltrations, according to several pathologists who examined them, indicated that the tumor was a chondroma which had undergone carcinomatous degenera- tion. Other pathologists, on the contrary, believed the growth to be of a benign nature, with the histological structure of a chon- droma. For the determination of colloidal nitrogen the alcoholic pre- cipitation method of Salkowski was used, with modifications, as follows :^ The total nitrogen was determlned in 5 c.c. of the urine by the Kjeldahl process. Two portions of 100 c.c. each of the urine were evaporated in a porcelain dish over a gently steaming water bath tili they were of the consistency of thin syrup. The residues were then taken up in 100 c.c. of alcohol (98.5 per cent.) and thoroughly stirred. The alcoholic extracts were then filtered through ashless filter papers, and the precipitates washed with alcohol. We determined the effect of dialysis upon this alcohol-precip- itable, so-called "colloidal," nitrogenous material. Most colloidal substances fail to dialyze through the very best grade of parchment paper. Only that fraction of the alcoholic precipitate which would remain indiffusible under suitable conditions of dialysis could be called "colloidal," at the present stage of our knowledge of the subject. Accordingly, the two precipitates on the ashless filter paper were treated as follows : The precipitate on one filter paper, together with the filter, was placed in a Kjeldahl flask, digested with sulfuric acid, and the nitrogen determined in the usual way. The second precipitate and *Before subjection to analysis the urine was first tested for protein, which, if found, was removed by means of heat coagulation aided by the addition of a few drops of dilute acetic acid Solution. I9I2] Max Kahn and Jacob Rosenbloom 89 filter paper were placed with water in a bag of the finest grade of parchment paper and dialyzed for forty-eight hours. The liquid in the bag was then analyzed quantitatively for nitrogen. The appended summaries present the results obtained for urine from the dog with the breast tumor and also for urine f rom several normal dogs. In the Salkowski method for the determination of " colloidal " nitrogen (as the results in the summary show), diffusible nitrog- enous substance is precipitated as "colloidal" nitrogen. It has not yet been shown that such diffusible nitrogenous matter in the colloidal precipitate is true colloidal material. A. Data pertaining to the urine of normal dogs ßpecimen No. Total Nitrogen in 100 c.c. of Urine Colloidal Nitro- gen in 100 o.e. of Urine Percentage of Total Nitrogen as Colloidal Nitrogen Indiffusible Colloidal Nitrogen Percentage of Total Nitrogen as Indif- fusible Colloidal Nitrogen I 2 3 4 Grams 2.304s 3-2051 0.8590 1.6436 Gram 0.0437 0.0314 0.0172 0.0214 1.85 0.98 2.00 1.28 Gram 0.02775 0.01634 0.01202 0.01841 1.2 O.S 1.4 i.l B. Date pertaining to the urine of the dog with a tumor of the breast Sa. 4.0088 0-3392 8.40 0.0939 2.3 5 b. 6.3034 0-3897 6.10 0.2293 3.6 SC. 4-4591 0.3210 7.10 0.0767 1-7 5d. 3.6862 0.3294 8.10 0.0817 2.2 Se. 3-1414 0.0958 3-04 0.0867 2.7 5 f. 3.9642 0.3566 8.90 0.1175 2.8 5 g- 2.5139 0.4617 13.10 0.1342 3.6 The results demonstrate that the " colloidal " nitrogen, both before and after dialysis, was greater in amount in the urine of the dog with the tumor than that in the urines from normal dogs. It is desirable to study the effect of dialysis upon the " colloidal " nitrogenous substances in the urine of Cancer patients. GENERAL ASPECTS OF FASTING^ PAUL E. HOWE (Department of Physiological Chemistry, University of Illinois, Urbana, III.) Fasting (starvation or inanition) is a State in which the dietary elements are withheld, either wholly or in part, so that the organism is compelled to draw upon its own resources to maintain its exist- ence. In discussing this subject it is my purpose to make a rather general survey of the changes which take place as the result of fast- ing; to show briefly how such results have been used to elucidate other scientific problems; and, also, to touch upon the therapeutic value of fasting, with relation to man. A distinction is made between physiological and experimental fasting. The first form is illustrated by the hibernation of mam- malia (hedge hog and bear), and cold blooded animals (frog), by the normal condition of the salmon during the spawning season and by the period of metamorphosis of the insects, these being natural phenomena for which the organism has made suitable preparation. In experimental fasting the animal is forced to live without sus- tenance, of one kind or another. Under this last State we may con- sider pathological fasting as a special case in which the organism is forced to fast as a result of impairment of some organ or of a gen- eral diseased condition. These forms of inanition present certain differences as evidenced in the effect upon the organism; yet it is quite probable that they are chiefly phylogenetic and we can conceive that any of the animals which do not experience these periodical physiological fasts might do so under the proper adverse circum- stances. In our discussion we will consider only the phenomena which take place as the result of experimental fasting. Here, too, we must distinguish between a number of forms of fast; such as the com- ^ A lecture delivered at the College of Physicians and Surgeons, New York, May I, 1912, under the auspices of the Columbia University Biochemical Asso- ciation. 90 I9I2] Paul E. Howe 91 plete fast in which there is total abstinence from both food and water; a modification of this, in which the subject is permitted to take water "ad libitum" or caused to ingest a uniform quantity from day to day ; and the incomplete fast, in which one or more of the food principles or chemical elements contained therein is with- held, such as a diet lacking in protein, fat, carbohydrate, water, salt or certain amino acids. There is not a marked distinction between complete fasting and fasting with water taken "ad libitum," for under the latter con- ditions the quantity of water taken decreases as the fast progresses until finally there is a natural abstinence from water. Some hold that the desire for water returns just before death. The ingestion of water causes a lengthening of the life of the animal and the severity of the fast is lessened. If at any time the quantity of water given is increased there will be for a time an increase in the metab- olism (14). This condition also holds for the well nourished animal (8), i. e., under all conditions when the water ingestion of the animal is sufficiently increased the general metabolic processes of the organism are stimulated. The length of a fast which would result in death depends upon the size, the species, the age, the nutritive condition, the external surroundings (e. g., temperature, humidity, etc.) and the intrinsic rate of metabolism. In general, we may say that the smaller the subject the shorter will be the time it can live without food; but this does not hold in all cases, for certain of the lower animals can fast much longer than the higher forms, e. g., the Salamander, which is about 3-4 inches long, has been fasted for more than 125 days (19). Adult organisms can fast longer than the young of the same species. Thus, a young pup can fast but a few days, while a füll grown dog will fast from 20 to 60 days. Of the fasts on man and other warm blooded mammals, the longest on record is one of 117 days (15). This experiment was conducted in our laboratory, a Scotch collie dog being the subject. Subsequent to this long fasting interval the dog was fed, and it returned to its normal condition. A comparison of the results obtained by various investigators shows that death does not ensue until there is a loss of between 40- 50 per Cent, of the original body weight. The real cause of death 92 General Aspects of Fasting [Sept. from fasting has not been determined. The probable reason is the failure of some organ or life process (27) and not the depletion of all possible nutritive material. From our experiments (10) it would appear that a certain definite minimal proportion of nitrogen- holding substance must be present in the body for life to exist. Fasts have been reported upon men covering periods of from 2-50 days, upon dogs as long as 117 days and Salamanders for 125 days. In each of the extreme cases, the subjects were subsequently fed and they returned to normal. The influenae of repeated fasting upon the resistance of the animal to subsequent fasts is a phenome- non which appears to be intimately associated with hibernation. As has been shown by Russian investigators (20), and more recently in our laboratory (10), repeated fasting decreases the rate of metab- olism in each succeeding fast. A French investigator (21) has shown that repeated fasting, in which the subjects were alternately fasted and fed during equal periods of about a week each, resulted in the ultimate death of the animals. From the experimental data Et band it seems that where the animal is permitted to recover completely from a fast before it is subjected to another, there will be an increased resistance to the ravages of the succeeding fast. The number of men who have made a study of the changes which take place as the result of fasting is so great that it is difficult to name those who have made the most important contributions upon this subject without doing an injustice to others. The inves- tigations of Cathcart (4) in England and of Benedict (2) in this country, upon men, are the most extensive that have been con- ducted with the more refined methods of analysis which we possess today. The names of Succi, Cetti and Breithaupt stand out in the literature as the subjects of important experimental fasts. What changes take place in an organism as the result of a fast? Outwardly the subject becomes emaciated, his body weight de- creases, he becomes weak and apathetic and, should the fast proceed long enough, he would probably die in a State of coma. In man it has been demonstrated that the brain retains its activities unimpaired during a fast and that hunger is evident only at the beginning of the ordeal. These facts are substantiated in the populär writings upon fasting and also by an experiment made by us (11) in which the I9I2] Paul E. Howe 93 subject prepared for the preliminary examination for the degree of Doctor of Philosophy during a seven-day fast. The fasting State is indicated in the body by certain changes; such as a general decrease in the body metaboHsm, represented by variations in the nitrogen excretion and the respiratory exchange, a decrease in the fat and glycogen Stores, a decrease in the volume of muscle and in the size and weight of certain organs. The tempera- ture remains normal, for a time at least, but shows a tendency to decrease toward the end of the fast. The decrease in the general metaboHsm is well Illustrated by the data obtained from the respiration calorimeter experiments. It has been shown by the earlier investigators and more recently by Bene- dict (2) that, in a well fed man, the quantities of protein and fat which were utiHzed, and the energy change (calories) per day, decreased very gradually and tended toward a constant minimum. In addition to the excreted carbon dioxide, Benedict determined the amount of oxygen consumed. From these data it was shown that the glycogen consumption, which is most rapid on the first day, decreases as the fast progresses. It is probable that the glycogen Store is never depleted and that even in fasting there is a resynthesis of glycogen from the protein material present in the body. Decreased metaboHsm in fasting is also shown by the quantities of nitrogen-containing substances eliminated in the excreta. We are particularly concerned with the losses of nitrogen, for it is the protein material which is the most fundamental nutritive substance and which the body strives to protect. In fasting, the nitrogen- containing substances in the urine or feces arise from the tissues and hence the total nitrogen excretion is a measure of the quantity of muscular or organ tissue catabolized. The excretion of total nitro- gen in the urine decreases rapidly at the beginning of the fast and soon reaches a minimum, which is maintained for some time. This minimum of nitrogen excretion, representing a minimum protein disintegration, is Held to represent the " maintenance " metaboHsm of the individual, i. e., that amount of protein substance which if supplied, with sufficient fats or carbohydrates, in the form of food would sustain life. This minimum has variously been shown to be greater or less than the metaboHsm as represented by fasting ex- periments. 94 General Aspects of Fasting [Sept The muscular disintegration is influenced by the factors already mentioned; the nutritive condition and the experience of previous fasts, or repeated fasting. The diet just before the fast influences the nitrogen excretion for a number of days. This has been demon- strated in the classical experiments of Voit (26), in which he fed varying amounts of meat and bread to a dog and showed that, when fasted the rate of nitrogen excretion varied, but that in each case the animal came to the same level of catabolism on about the seventh day of fasting. The fat available in the body exerts a marked efTect upon the protein metabolism and the Hfe of an animal. So long as there is sufficient fat in the organism to supply the energy requirements, the protein metabolism will remain at a minimum. When, how- ever, the fat deposits are depleted, the body is forced to use protein to furnish the necessary energy. The result is a more rapid protein consumption and an earlier death. This increased protein con- sumption, is, of course, accompanied by an increased nitrogen excre- tion, which has been designated as the " premortal rise." The feed- ing of carbohydrate or fat sufficient to supply the energy require- ment of the body would prevent this increased consumption of pro- tein and thus lengthen the life. Repeated fasting will also modify the rate of metabolism. This point is well illustrated by the results obtained on a subject in a repeated fast (10), in which there was a rapid and increasing con- sumption of the protein reserves of the body during the first fast, but a more gradual and uniform consumption during the second fast. The total body weight and nitrogen losses were practically identical in the two fasts and the data f rom the intermediate feeding period would indicate that an increased fat störe was not the cause of the more gradual utilization of the body resources. A study of the differential distribution of the nitrogen in the urine serves to bring out certain points with regard to the protein metabolism of fasting animals. The percentage of total nitrogen occurring as urea-nitrogen decreases in man and is accompanied by an increased ammonia-nitrogen excretion. This has been explained as due to the condition of acidosis, which may result, at least in part, from the accelerated utilization of the fat deposits and the decreased oxidative powers of the animal. I9I2] Paul E. Howe 95 In the case of dogs there is a difference o£ opinion as to the relation between the urea-nitrogen and the total nitrogen. Schön- dorf (22) and others hold that the percentage of urea-nitrogen decreases, while in all of our experiments it has remained nearly constant, which fact, coupled with the failure to find marked quan- tities of organic acids in the urine, would show that dogs are better able to utilize their body Stores. This may be due to the fact that the dog is naturally a "high-protein" animal. The daily Creatinine excretion, which is a constant for any indi- vidual under normal conditions of feeding and is generally believed to be a function of the muscular metabolism, decreases gradually as the fast progresses and in correspondence with the decreasing amounit of protoplasm. Creatine, which does not occur in normal urines, or is found only in cases associated with muscular disintegration, appears dur- ing fasting and ordinarily becomes a constant constituent. It has recently been shown that the feeding of carbohydrate causes the excretion of creatine to stop (5, 17) ; while ingested fats may even cause an increase in the excretion of this form of nitrogen. In one of our dog experiments (15) there was a disappearance of urinary creatine from the iQth to the 59th fasting days. This phe- nomenon might be explained on the above basis. It is improbable, however, that the body could synthesize sufficient glycogen at this stage of the fast to cause the disappearance of the creatine. The real explanation is therefore not apparent. In connection with the repeated fast, previously mentioned, it is interesting to note that the excretions of creatine as well as of total nitrogen were practically the same during each of the two fasts, notwithstanding the fact that the second fast was twice as long as the first. • This would indicate an intimate relation between the total-nitrogen excretion and the quantity of creatine excreted. When the data representing the creatine and Creatinine excretions of a fasting animal are examined, it is seen that there is generally a progressive increase in the creatine Output and an accompanying decrease in the Creatinine elimination, until the output of creatine exceeds that of the Creatinine. In other words, when expressed graphically, the curve representing the course of the creatine excre- 96 General Aspects of Fasting [Sept. tion crosses that representing the Creatinine Output. This phenome- non has been termed by us the "creatine crossing" and is believed to be very significant. It occurs with great uniformity a few days previous to the decrease in the total nitrogen excretion that precedes the pre-mortal rise of excreted nitrogen. By means of the " creatine crossing" the length of the subsequent Hfe tenure of the animal may be quite closely estimated. Certain pathological constituents, other than creatine, may ap- pear in the urine as the result of fasting, such as acetone, diacetic acid, lactic acid, bile pigments, albumin, etc. The processes in the large intestine during fasting have received but httle attention. Various authorities contend that it is difficult to make a Separation (2, 18) of fasting feces. Müller (19a) has shown that indican, which is now considered as an index of intes- tinal putrefaction, disappeared upon the third day of fasting. We have been able to make an undoubted Separation of fasting feces and have found indican present during the whole of a seven day fast on man {22,), Fasting feces are distinct from those of the normal individual in that they are of a peculiar brown color and are pasty in consistency. The percentage of nitrogen present is higher than in normal feces. The bacterial content of feces has received but little attention and only recently have results upon the bacterial content of fasting feces been determined. The results indicate a lovver percentage content of bacteria (3). There is not an equal wasting of all of the organs and tissues of the body, those organs most necessary for the maintenance of life show only a slight decrease in size and weight, while others are reduced to but a fraction of their original proportions. Thus the heart, lungs, and nervous System exhibit but little change while the muscles and fatty tissues exhibit a marked reduction of both volume and weight. The organs of regeneration are also resistant to the ravages of a fast. This fact is of especial significance for it demon- strates the tendency of nature to preserve the species. A histological examination of the tissues and organs of fasting animals shows a decrease in the volume of the cells as a whole and of the nuclei. MorguHs (19) has shown that the decrease in the volume of the cells of the Salamander is greater than that of the igi2] Paul E. Howe 97 nuclei and further that the nuclei become elongated. In the case of the liver, the cell walls finally begin to disappear and the small masses of pigment to clump together. Such a condition does not necessarily result in death, for Salamanders of the same size have been caused to fast for even a longer time than those whose tissues demonstrated these changes ; and, af ter feeding, it was f ound that the cell walls again appeared and the liver returned to a normal condition. We will not take up the question of the localization of the degenerative changes, i. e., as to whether they occur in an organ as a whole or in localized portions. It is an interesting fact, however, that even when the organism is undergoing the degenerative effects of a fast, there are still evidences of mitotic division of the nuclei. What changes take place which enable one organ to waste away while another retains its normal condition? The explanation most generally accepted is that of the nourishment of the more vital Organs by transference of the nutritive material from the less im- portant tissues. Thus the less resistant tissues gradually give up their stores of fat and protein to the blood stream which in turn furnishes them to the actively functioning organs. This idea has received further proof from the researches of Hottes.^ This botanist (9) worked with beans and has shown that upon removing the cotyledons, and thus the food supply, from seed- lings, the meristematic tissue which would normally go to produce lateral roots is transferred to the tip of the root (meristem) and there used for growth ; and that at the end of from three to f our weeks all the cells in the Upper part of the root have lost the major portion of their protoplasm and the only actively functioning cells are those at the tip. Hottes has also shown that the decrease in size of the root is due rather to a reduction in the number of the cells than to a mere decrease in size. This is in Opposition to the findings of certain zoölogists who hold that the reduction in the weight and volume of the organs is due more to reduction in size than in number, altho they admit a small decrease in the number of contained cells. 'I am indebted to Professor Hottes of the University of Illinois for this Information which was taken from some of his unpublished work. 98 General Aspects of Fasting [Sept The blood of fasting subjects which are ingesting water shows in general a decrease in the number of erythrocytes and leucocytes, and of the percentage of hemoglobin. The differential distribution of leucocytes varies with the species. In the dog (12) there is a decrease in the percentage of polymorphonuclear leucocytes and a corresponding increase in the small lymphocytes. The changes in the other forms of cells are but nominal. Fasting studies have been of great importance in the study of the minimum of food necessary to maintain life and upon which to base the calculation of dietary Standards. Such studies have also been utilized in the explanation of phenomena occurring in patho- logical States and of metabolism in general. Underhill and Rand (25) have explained certain anomalies in the urinary changes which occur in pernicious vomiting of preg- nancy from their knowledge of fasting metabolism. Two agriculturalists have recently made use of the results of fasting studies to elucidate problems of importance to both the scientist and the farmer. McCollum (16), fed a nitrogen-free diet to pigs and studied the efficiency of individual grains as feeding stuffs, as well as the nature of the repair processes in protein metab- olism. He shows that the difference in the nutritive values of the wheat, oat and corn kerneis is not so great as would be expected from the difference in the chemical composition; and further, that the repair processes of the cell are of a different character from those of growth, and that the cellular catabolism and repair do not involve the destruction and resynthesis of entire protein molecules. This last Statement is not in entire accord with the most widely accepted theories of metabolism. Certain zoölogists have also shown that the changes of regeneration are unlike those which occur in growth. Dietrich^ shows (7) that fasting so reduces the plane of metab- olism that the quantity of food which was insufficient for main- tenance before fasting was afterward sufficient, not only for main- tenance, but to produce a positive nitrogen balance ; in other words, the animals were more efficient machines after fasting. 'I wish to thank Professor Dietrich of the University of Illinois for per- mission to refer to his unpublished data. I9I2] Paul E. Howe 99 Aron (i), in his studies upon nutrition and growth, subjected dogs to incomplete fasts. The results showed that a growing animal, receiving only enough food to provide for little or no in- crease in weight, " is in a condition of severe starvation." Under such conditions the skeleton grows at the expense of the flesh, the Organs retain their weight and the brain reaches its normal size. The fat and protein of the muscles are largely used up, altho this loss of material is balanced by gain of water and by the growth of the skeleton. The biologists have made use of the fasting subject in the study of the Problems of degeneration, of regeneration, and of growth, The work of Morgulis and of Hottes already considered was of this nature. The therapeutic value of fasting is realized in the preliminary treatment of some digestive disorders and in the partial fasts of obesity eures. These latter eures consist in supplying only the protein requirements of the body and thus forcing the individual to utilize the surplus fat deposits to make good the energy require- ments. The increasing populär literature upon fasting and the tendency to fast on the part of certain people, especially the pronounced physical culturists, and their general good health, would seem to indicate that there are some beneficial results to be obtained from fasting. The various books upon fasting, of which the superficial, yet interesting book of some six hundred pages by Carrington (6) upon " Vitality, Fasting and Nutrition " is the most complete, lend strength to the idea that fasting as a therapeutic measure is impor- tant. The chief contention of this fasting cult is that by depriving the body of food the digestive organs are given a chance to recu- perate and the body is enabled to rid itself more effectively of the waste products and toxic substances. Fasting for short and widely-separated periods may be a bene- ficial procedure in some individuals. This conclusion is supported by the observed effects on dogs, which acquire increased resistance from repeated fasting. This view is strengthened, also, by the foregoing data pertaining to pigs as well as by Seeland's (24) results on pigeons and chickens, which show that repeated fasts. loo General Aspects of Fasting [Sept. for periods of f rom one to two days, were foUowed by better growth and greater strength. It is probable, then, that fasting under proper conditions may be advantageous. Long fasts, however, seem to be devoid of benefit and may endanger health. BIBLIOGRAPHY 1. Aron. The Philippine Journal of Science, 6, i, 1911, 2. Benedict. Carnegie Publications, 77, 1907. 3. Blatherwick, Sherwin and Hawk. Proc. Amer. Soc. of Biological Chem- ists, 2, 42, 191 1. 4. Cathcart. Biochemische Zeitschrift, 6, 109, 1907. 5. Cathcart. Journal of Physiology, 39, 311, 1909. 6. Carrington. Vitality, Fasting and Nutrition. Rebman Co., N. Y. 1908. 7. Dietrich. Unpublished data. Illinois Agricultural Experiment Station. 8. FowLER and Hawk. Journal of Experimental Medicine, 12, 388, 1910. 9. HoTTEs. Publication of the Carnegie Institution. (In press.) IG. HowE AND Hawk. Journal of the American Chemical Society, 33, 215, 1911. 11. HowE AND Hawk. Proc. Amer. Soc. of Biological Chemists, 2, 65, 191 1. 12. HowE AND Hawk. American Journal of Physiology, 30, 174, 1912. 13. HowE, Mattill and Hawk. Journal of the American Chemical Society, 33, 568, 191 1. 14. Howe, Mattill and Hawk. Journal of Biological Chemistry, 10, 417, 1911. 15. Howe, Mattill and Hawk. Journal of Biological Chemistry, 11, 103, 1912. 16. McCoLLUM. American Journal of Physiology, 29, 215, 191 1. 17. Mendel and Rose. Journal of Biological Chemistry, 10, 213, 191 1. 18. Mendel and Fine. Journal of Biological Chemistry, 11, 5, 1912. 19. MoRGULis. Archiv für Entwicklungsmechanik der Organismen, 32, 169, 1911. 20. Quoted by Pashutin. Pathological Physiology, 1902. 21. Richet. Comptes rendus de la Societe de Biologie, 61, 546, 1906. 22. Schöndorf. Archiv für die gesammte Physiologie, 117, 257, 1907. 23. Sherwin and Hawk. Journal of Biological Chemistry, 11, 169, 1912. 24. V. Seeland. Biologisches Centralblatt, 7, 145, 1887. 25. Underhill and Rand. Archives of Internal Medicine, 5, 61, 1910. 26. Voit. Zeitschrift für Biologie, 2, 307, 1866. 27. Voit, E. Zeitschrift für Biologie, 41, 188, 1901. THE PHYSICO-CHEMICAL BASIS OF STRIATED MUSCLE CONTRACTIONi 2. Surface tension WILLIAM N. BERG (WITH PLATE l) If the physico-chemical basis of muscle contraction is ever to be understood or explained, it is almost certain that it will be brought about thru speculation and experiment of a quantitative, rather than of a qualitative nature. The mere Statement that muscle con- traction is caused by surface tension, or thru osmotic action, etc., unless accompanied by quantitative data of an experimental or theoretical nature, can add little toward the Solution of the problem of the transformation of energy by muscle. It is, perhaps, regret- able that so many of the " theories of muscle contraction " which have appeared in the recent literature belong to the qualitative class. Occasionally someone attempts to treat the subject quantitatively. From this point of view the works of Bernstein,^ and of Zuntz^ are particularly meritorious, even if the problem has not yet been solved by them. Among the latest qualitative contributions to the theory of muscle contraction, is that of Strietman and Fischer.^ They studied the contraction and relaxation of catgut strings immersed in various Solutions. By attaching the strings to the usual arrangement of iever and recording drum, they found that when a catgut string is immersed in water or physiological salt Solution, even for some time, no changes in length take place (p. 66). But if the string be immersed in Solutions of hydrochloric or lactic acids (w/8o to ^ Berg, W. N. : Biochemical Bulletin, 1912, i, 535. ^ Bernstein, J. : Arch. f. d. ges. Physiol., 1901, 85, 271-312. ^Zuntz, N. : Die Kraftleistung des Tierkörpers. Festrede; Berlin, 1908. * Strietman, W. H., and Fischer, M. H. : Ztschr. f. Chemie und Industrie der Kolloide, 19 12, 10, 65-77. lOI I02 Physico-Chemical Basis of Striatcd Musclc Contraction [Sept n/20) it contracts. On replacing the acid Solution by water, the string relaxes. The relaxation is faster, however, when the acid is replaced, not by water, but by a Solution of some salt such as sodium bicarbonate, which can neutralize the acid. From their diagrams it would seem that a minute or more may be required for a Single contraction or relaxation, depending upon the strength of the acid, etc., etc. These observations are, no doubt, interesting in themselves. But before connecting them with muscle contraction, might it not be well to consider whether the conditions under which a catgut string can contract and relax are at all similar to those existing in muscle ? Strietman and Fischer State that because lactic acid is formed in a working muscle and because a catgut string will contract when immersed in a lactic acid Solution and will relax when the acid is removed, therefore, in the working muscle, the contraction is brought about by the formation of lactic acid. They quote several other investigators who have stated their belief in the same idea of the connection between lactic acid formation and contractility with- out, however, making any of the simple calculations that would naturally suggest themselves. Their theory is open to the following objections: (i) It is not likely that there is any free lactic acid in the working muscle, it is probably neutralized at once by the phosphates present in lymph. At least this would be inferred from the work of Henderson^ who showed that the mixture of phosphates and other substances in blood and various tissue fluids was such as to enable them to maintain an absolute neutrality in spite of the formation of even considerable quantities of acid or of alkali. This point was not overlooked by Zuntz (p. 20, 1. c.) when calculating the amount of energy made available by the transformation of dextrose into lactic acid. The heat of neutralization of the lactic acid by sodium, as well as the heat required to separate the sodium from its presumable combina- tion with protein, are given due consideration by Zuntz, who cal- culated that the heat liberated in the formation of lactic acid from dextrose is equivalent to 3.4 per cent. of the heat of combustion of dextrose. * Henderson, L. J. : Ergebnisse d. Physiologie, 1909, 8, 254-325. I9I2] William N. Berg 103 A repetition of the experiments of Strietman and Fischer, in which catgut strings would be immersed in Solutions comparable with lymph containing lactic acid (not exceeding the maximal amount possible if all of the muscle glycogen were changed at once to lactic acid), would probably give results more decisive than those in which free acids were used. (2) And even if free lactic acid existed in muscle, or if com- bined lactic acid could induce proteins to swell, one such Observa- tion is only one of very many that are needed for a rational theory of muscle contraction. The Statement that lactic acid swells protein adds very little to our knowledge of the mechanism in muscle by which the potential energy of the food is transformed into the kinetic energy of the moving muscle and its load. It is to be regretted that the work of Brod^ on the swelling of fibrin in acid Solutions has received practically no attention in the recent literature. The paper can be profitably studied by those con- templating studies on protein swelling. A brief resume of Brod's results is given by Berg."^ A good example of a quantitative theory of muscle contraction is the calculation of Bernstein^ on the possible changes in the sur- face energy resident on the muscle fibrils. The method of making the calculations is, perhaps, unnecessarily complicated and, in one or two instances, the mathematical equations are of doubtful correct- ness. Bernstein finds that in order that a muscle may lift an ordi- nary load, the surface tension between fibril and sarkoplasm must have an improbably great magnitude. He nevertheless concludes that the principle, that energy is transformed in muscle thru changes in surface energy, is correct. There are several reasons why, to the Student at least, a proper understanding of some of the recent applications of physical chem- istry to biology should be so difficult, if not altogether impossible. First: The indefiniteness of certain Statements that the writer has ' Brod : Beiträge zu der Lehre von der Eiweissverdauung. Dissertation, Würzburg, 1892. "Berg, W. N. : Amer. Jour. Physiol., 1909, 23, 427. Brod's method has recently been used by Tracy and Gies, Biochemical Bulletin, 1912, i, 468. * Bernstein, J. : Arch. f. d. ges. Physiol, 1901, 85, 271-312. 104 Physico-Chcniical Basis of Striated Muscle Contraction [Sept. frequently seen in the literature. This is an example taken from Freundlich's Kapillarchemie, p. 4: Stirface energy = surface tension X area of surface. A similar Statement is made by Michaelis,^ and others. Nothing further was stated that would enable the reader to use such a formula in making calculations were it desired. Expressing the surface tension in dynes per centimeter and the area of the surface in cm.2, what is the surface energy? The answer is very simple after one has taken the time to look the matter up. After a formula such as the above, a numerical example ought to be given, so that it means more than so many words to the average reader. Suppose it is desired to calculate the amount of energy required to form a water-surface (in contact with air) of i sq. cm. area? Or, what is the same thing, how much energy is liberated or is available for external work when the above water-surface diminishes by i sq. cm.? According to Michaelis (1. c, p. 14), this will require (or liberate) 70 ergs or 7 X lo"'^ kilogram-meters. The method of using the formula to obtain this result, simple as it is, was not given by Michaelis, altho at least one example of the use of a formula is desirable because it will enable the reader to make many other cal- culations. Following is a numerical example of the kind mentioned above. How much energy is required to form a water-surface (against air) of I sq. cm.? In the formula it is assumed that the surface tension remains constant during the change in area : surface energy required ^surface tension X increase in area, or surface energy liberated = surface tension X decrease in area, (ergs) = (dynes per cm.) X (cm.^). Since the surface tension of water-air is about 70 dynes per cm., it is evident that-^^ X i cm.2^70 ergs = the amount cm. of energy required. The erg is a unit of work (or energy) and is the work done when a mass is moved i cm. by a force of i dyne. ' Michaelis : Dynamik der Oberflächen, p. 13. Dresden, 1909. I9I2] William N. Berg 105 The element of time does not enter into the definition of the erg. The vvork done (ergs) is equal to the product of the force (dynes) times the distance (cm.) thru which the force acts. Of course, other Units may be used. The surface tension may be expressed in grams per cm., and the area in Square cm. The work then is ex- pressed in gram cm. But on account of the unfortunate use of the Word ' gram ' to designate a certain mass or quantity of matter and also to designate a force, it is better, for the present, to use the erg and the dyne, and later to convert ergs into kilogram-meters, or any other of the customary units for expressing muscular work. It is, of course, absolutely necessary that the terms used in such calculations be consistent. Here is the second reason why some so-called appHcations of physical chemistry to biology are not easily followed. An equation will sometimes be given that is not correct in its dimensions. To State that 2 sq. cm. = 2 cubic cm. is obviously incorrect. Such an inconsistency is to be found in one of Bern- stein's^" equations : 'Wir werden daher in dem Falle des isometri- schen Tetanus, in welchem alle chemische Energie als Wärme er- scheint, dp — ar = c. Wp setzen können, wenn Wp die in einer Zeit- einheit erzeugte Wärmemenge, c eine Constante und «p und oLt die Oberflächenspannung im Tetanus und in der Ruhe bedeuten. Da wir nun oben (S. 296) gesehen haben, dass ctr gegen CLp verhältniss- mässig sehr klein ist, so können wir annähernd ap=:c. Wp an- nehmen. ' Here are two equations in which surface tension is equated with work (or heat). It makes no difference what units are used, on one side there is a force (surface tension expressible in dynes per cm.) and on the other a quantity of energy or work (ergs, or dynes X cm.). The constant above referred to is probably meant to be the mechanical equivalent of heat. These equations are interesting for another reason. It is true that in isometric tetanus, a muscle does work in the physio- logical sense of the word. But not in the physical sense. In physics (or mechanics) work is defined as a product of force times distance thru which the force has acted. If either factor is zero, the product, work, is zero. The columns that support ^^ Bernstein, J. : Loc. cii., p. 307. io6 Physico-Chemical Basis of Striatcd Muscle Contraction [Sept. a biiilding do no work in the physical sense, n'or would a man who took the place of one of them; altho physiologically he would do a great deal of work. In this case the distance thru which the force acts (the weight supported or the upward thrust of the man's Shoulders) is zero and hence the work is zero. The above equa- tions of Bernstein could be made consistent if there were two factors on the left-hand side; one, the surface tension or change in surface tension (expressed in dynes per cm.), the other, the area or change in area (expressed in cm.^). The product (ergs) could be calculated to calories (gram-degrees C.) by dividing by 4.2 X 10''^, since i small calorie (gram-degree C.) is equivalent to 4.2 X 10'^ ergs. It is difficult to see what the other factor (omitted by Bern- stein) can be. In an isometric tetanus the muscle does not change its length. In what way can an internal diminution in area take place? If the contractu units — whatever their shape may be — do not change in length, how do their areas diminish? This difficulty does not arise in the case of the ordinary (isotonic) contraction. Here one can assume a decrease in the areas of contact between contractil unit and sarkoplasm caused by an increase in the surface tension between the same surfaces. The product of these two quantities, according to the theory, should be an amount of work sufficient to account for the external work done and perhaps also for the heat liberated at the same time. We have stated before that Bernstein's calculations on the magnitude of the surface energy changes in muscle are probably unnecessarily complex, involving, as they do, several pages of cal- culus. The same result is obtained in the following calculations, in which two simple quantities are calculated and then compared : (i) the amount of energy liberated in a working muscle thru in- crease of surface tension times diminution of area of contractil Units; and (2) the external work done in lifting a weight a known distance. Assume that in i c.c. of muscle a right section contains (as Zuntz assumes, 1. c, p. 24) 62 million rods, and that there are 800 such layers, making a total of very nearly 5 X 10^^ rods in i c.c. of muscle. Assume the general structure of muscle to be that de- scribed by Hürthle (see diagram), and that the muscle rod is the LU I- < _l Q. o > I- UJ CD _l < Ü LU I o g m s I i I ^1 «- W6 ü ' ni H 9 ^• O ^ ■< 1) o c o c 'oidO-CJ.QS'IUir w b hJ fci u «+H cn P 1^ ^ o -Ci, w H ^ < 1 — 1 ■^ Pi — I CO < < u (LI O j= 's; S öl -*-t u W u I o u I— I CO >^ Ph o -^ J9fi-VJ 07doJ}0S-IUV- ü 2 P3 O igi2] William N. Berg 107 CONTRACTIL UNIT (Hürthle, p. 157). From the dimensions 011 the accompanying diagram, it is evident that the lateral area of a rod diminishes from 4.8 /a^ when relaxed, to 2.8 /^^ when contracted. (We omit the simple geometrical calculation.) The base areas are increased, but the energy apparently required for this work will for the present be disregarded — it is an additional load on a probably overloaded theory. Since the total area of the relaxed rod (5.2 /a^) is greater than that of the contracted rod (3.8 fi^), it follows that there is an increase in the surface tension immediately preceding the contraction, according to the requirements of the theory. To calculate the energy liberated as being equivalent to the diminution in area times the surface tension of water is probably incorrect, for without an increase in surface tension there seems to be no reason why the rods should contract against the external resistance — the downward pull of the weight lifted. The rod contracts presum- ably, because in the relaxed State the surface tension on the rod surface is low. How low can it be ? It may be as low, perhaps, as 23 dynes/cm. if we assume the rod to be covered with a layer of pure acetic acid, and that the acid has the same surface tension in con- tact with the water that it has when in contact with air. Other fatty acids also give low values for the surface tension of their Solutions, and they have still lower surface tensions in the pure State. Pres- ently, the surface tension is raised, presumably by the removal of the fatty acid or other agent causing low surface tension, by instan- taneous combustion, let us say. How high can the surface tension be raised? It might be raised" to 85 dynes/cm. if we imagine the rod now to be covered with a layer of saturated sodium chlorid Solu- tion. The surface tensions of aqueous Solutions of salts cannot be raised^^ very muchbeyond that of pure water, which varies between y2 to y6 dynes/cm. (at 18° C), according to the method of measure- ment. The upper limit for any Solution that possibly could exist in the muscle might be assumed, then, to be the value for saturated sodium chlorid Solution, or any other concentrated salt Solution that might probably occur in living muscle. Of course, the existence of the films of pure acetic acid and of strong salt Solution over the " Freundlich, H. : Kapillarchemie, p. 27 and 62. Leipzig, 1909. *^ Heyd Weiler, A. : Ann. Physik., 1910, 33, 145-185. io8 Physico-Chemical Basis of Striatcd Musclc C ontraction [Sept. muscle rods is purely hypothetical. The surface tension theory requires that changes in surface energy take place, and from what follows it is apparent that these changes must be great — greater, in fact, than the probable actual change on the rod surface. For it seems hardly possible that such great changes in concentration and in surface tension could take place. The values for the surface tensions of pure acetic acid and concentrated salt Solution have been taken from the literature ; whether such limiting values are ever reached in living muscle is, for the present, purely hypothetical. If it be assumed that, during the chemical changes taking place in a working muscle, the inorganic ions in the rod-surface film rapidly change their concentrations, the film might be regarded as an electrical double layer or Helmholtz double layer. Without a doubt, changes in surface tension would result from the changes in ion concentration. The more ions in one of these layers covering a rod, the more they repel one another and the lower is the surface tension, and vice-versa. But as has been pointed out before,^^ it is not certain that such a double layer really exists between living particles and their surrounding medium. And even if there were such a layer, the total change in surface tension in such a layer is hardly significant for the present purpose. The small Variation in surface tension when the Variation is caused only by ions was prob- ably overlooked by Robertson^"* and others who advocated a capil- lary electric theory of muscle contraction. It is really a special case of surface tension in which the variations in surface tension are caused by the mutual repulsion of the ions in each of the layers. But insofar as small amounts of certain organic substances, such as fatty acids, can affect (depress) the surface tension of water very much more than even improbably large amounts of inorganic salts, the surface tension theory is given the benefit of the greatest possibilities by assuming the changes in concentration from pure acetic acid (23 dynes/cm.) to saturated sodium chlorid Solution (85 dynes/cm.). This is as large a difference as can be assumed from the experimental data on the surface tensions of Solutions. "Berg, W. N. : New York Med. Journal, 1907, July 20 and 27; and Ion, 1910, 2, 161-188. "Robertson, T. Brailsford: Trans. Royal Soc. South Australia, 1905, 29; and Quarterly Jottr. Exper. PhysioL, 1909, 2, 303-316. I9I2] William N. Berg 109 If in I c.c. of muscle there are 5 X lo^*' rods, the lateral area of each of which diminishes from 4.8 /a^ to 2.8 /^^ when the muscle con- tracts, the total reduction in area is 5 X 10^" X 2 /x,2__io^i fx^ = lO'^ cm. 2 (i ju. = 0.001 mm.). The calculations will be simplified if it be assumed that the increase in surface tension is instantaneous, giving the contracting muscle the largest surface tension during the entire contraction phase. Then since surface energy liberated = diminution in area X surface tension, (e.,s) (c..^) {^) the energy liberated is 1000 X 85 ergs. Let it be assumed that all of this is transformed into external work — lifting a weight — and that the resultant heat arises from the activity of a different mechanism ; in short, that the muscle is an engine having an efficiency of 100 per Cent. How great a weight will this i c.c. of muscle lift? Since there are 800 layers of rods, and each layer shortens by 3 /-i during the contraction (see Plate i herewith), the muscle shortens by 2400 /a or 2.4 mm., lifting a mass of W grams 2.4 mm. The energy (ergs) expended in lifting a mass of W grams thru the distance D (cm.) is PF X ■C' X 981 ergs, since gravity = 98i dynes. Therefore the 8s,ooo 85,000 ergs will hft ^^3^ = 361 grams. According to Zuntz (1. c, p. 23) i gram of muscle substance can do 0.002 kilogram-meter of work in one contraction under favorable conditions. If this muscle shortened 0.24 cm. as the above muscle did, it would lift a trifle more than 800 grams. Bern- stein^^ mentions 600 grams at least, as the pull of i cm.^ of frog muscle in an isometric contraction. Insofar as i cm.^ of many kinds of muscle can support without lengthening (but not lift) several kilograms — about 6 kilograms for human, and probably more for certain types of insect muscle — the above figure of 361 grams, as the weight a muscle could lift, is small, especially when it is borne in mind that it is an improbable maximum. The foregoing discussion may be summarized as f oUows : I. Too often there is a general lack of definiteness in the mathe- " Bernstein, J. : Arch. f. d. ges. PhysioL, 1905, 109, 326. HO Physico-Chemical Basis of Striated Muscle Contraction [Sept. matical treatment of a biological problem. Formulae are stated vvith no information as to their use or application to the problem under disciission. 2. Bernstein's calculations on the surface energy changes in working muscle are criticized. A much simpler method of calcula- tion is used with a result similar to Bernstein's, namely, the energy expended by a working muscle is much greater than the probable changes in surface energy can furnish, Of course, future investi- gations may bring to light sources of surface energy within muscle as yet unknown. Washington, D. C. A STUDY OF SOME PROTEIN COMPOUNDS WALTER H. EDDY (Biochemical Laboratory of Columbia University, at the College of Physicians and Surgeons, New York) Contents. (A) Morphin mucoid, 112; (B) strychnin mucoid, 114; (C) conin mucoid, 115; (D) piperidin mucoid, 115 ; (E) anilin mucoid, 115; (F) morphin nucleoprotein, 115; (G) morphin caseinogen, 116, strychnin caseinogen, 116, calcium caseinogen, 116; (H) strychnin ovo-mucoid, 117; (I) histon mucoid, 118; (J) histon nucleoprotein, 121; (K) histon ovo-mucoid, 121. I. INTRODUCTION When I began Ph.D. work in this laboratory, six years ago, Dr. Gies was actively engaged in studies of the properties of various protein Compounds which he had prepared as early as 1904.^ He inaugurated that work from the Standpoint of his interest in the chemical composition of protoplasm, and the nature of the struc- tural and dynamic relationships of cell constituents and products. He believed that the knowledge gained from studies of artificial protein Compounds would pave the way for successful inquiry into the nature of the protein correlations in the cells — relationships of the most fundamental biological character. At his Suggestion, and in furtherance of this object, I have conducted the experiments de- scribed in this paper.^ The general plan of the research was: (A) The production of protein salts by combining organic hases, such as strychnin, mor- phin, conin, piperidin, etc., with acid-reacting proteins, such as tendo-mucoid, ovo-mucoid, yeast nucleoprotein, etc.; and {B) the production of protein salts by combining hasic proteins, such as ^Gies: Proc. See. Path. and Physiol., Amer. Med. Assn., 1906, p. 121. ''The detailed results of this work have been described in the writer's dissertation, On the synthesis of some protein salts, Columbia University, 1909 (pp. 61). A preliminary report was published by Eddy and Gies in the Pro- ceedings of the Society for Experimental Biology and Medicine, 1907, iv, pp. I4S-6. III 112 Some Protein Compounds [Sept. histons and protamins, with the acid-reacting proteins eiiumerated above. In developing the latter part of this plan certain anomalies arose in connection with the preparation of thymus histon, which led to a collateral investigation of histons. The results of the latter studies will be embodied in a future paper. (See page 169.) IL EXPERIMENTAL I. Salts of various proteins with organic bases. 'Ä. Mor- phin MUCOiD. Purification of the materials. The first step in the preparation of a typical product was the removal of free alkali f rom the base — and free acid from the protein. Chemically pure, pul- verized, morphin was washed with distilled water until the wash- ings were entirely neutral to litmus. Tendo-mucoid was prepared after the manner of Cutter and Gies^ but dehydration with alcohol and ether was omitted. The dry scales w^ere soaked in distilled water until they softened. The protein was then washed with dis- tilled water until the washings were entirely neutral to litmus. Union of base and protein. The base and the protein were then triturated together in a mortar, a very little water being added to ensure an intimate mixture. A mechanical excess of the base was used in every case. Evidence of chemical action was seen in the peculiarly viscid, smeary character of the mixture. Mucoid and w-ater give a thick, milky mixture, but it is not viscid or smeary. The mixture w^as finally treated with sufficient water in excess to dissolve the product. The viscid liquid was filtered through a wet, fluted, hardened, filter paper, but the first portions of iiltrate were returned to the paper until a clear opalescent liquid appeared. This filtrate was neutral to litmus. Purification of the product. The filtrate, preserved with tol- uene, was subjected to continuous dialysis in a parchment bag, im- mersed in frequently renewed distilled water, until the dialysate, even when concentrated to a very small volume at 40° C, gave no test for the base (morphin). The contents of the bag were then evaporated to dryness at 40° C, toluene being used and frequently renewed during the process. The resultant dry produot was then ' Cutter and Gies : Amer. Journ. PhysioL, 1902, vi, pp. 155-6. I9I2] Walter H. Eddy . 113 pulverized in a mortar and extracted three times with a large excess of ether for the removal of traces of admixed free base (morphin). Isolation of the product. The powder was next dissolved in a small amount of water and this Solution poured into a mixture of Yz ether and Yz alcohol. A copious precipitate resulted. The pre- cipitate was gelatinous and dissolved easily in water. After dissolv- ing the precipitate in water and filtering the Solution, the filtrate was precipitated with alcohol-ether. This process was repeated sev- eral times. The final product was dehydrated in the usual manner with alcohol and ether. Special difficulties in the preparation of morphin mucoid. The first Solutions of the Compound filtered very slowly. It was found that this was due to excess of mucoid. When a large excess of morphin was used there was less insoluble mucoid residue and filtra- tion became correspondingly more rapid. Precipitation of the purified product with alcohol became in- creasingly difficult with the increasing purity of the product. Am- monium Sulfate, in excess, precipitated the product from its aqueous Solution, but long dialysis was required to remove the salt. The purified product failed to respond to the iodic acid test for morphin^ This fact was carefully investigated. The results showed that the failure was not due to the quality of the iodic acid used nor to interference with the test by the mucoid. In the puri- fication of the product there seemed to be continuous loss of mor- phin. This was presumably due to hydrolytic dissociation. Evidence of the compound-naturc of the product. The product was water-soluble, demonstrating that it was neither mucoid nor morphin, nor a mechanical mixture of the two. The aqueous Solu- tion of the product frothed strongly on shaking and gave a good biuret test, indicating its protein character. Addition of a few drops of 0.2 per cent. hydrochloric acid Solution yielded a flocculent precipitate of mucoid. Conclusions regarding morphin mucoid. Morphin and mucoid *For the detection of morphin, the iodic acid test was applied as follows: I c.c. of the Solution to be tested was added to an equal volume of dilute sulfuric acid Solution. To this was added a few c.c. of iodic acid Solution and finally a little Chloroform. After vigorous shaking, the presence or absence of a violet coloration served to indicate the presence or absence of morphin. 114 Some Protein Compounds [Sept. react to form a water-soliible protein Compound which, in aqneous Solution, yields mucoid on treatment with 0.2 per cent. hydrochloric acid. The morphin enters into combination in a proportion so small as to be incapable of responding to the iodic test, or is united in such a way as to fail to respond to the test. B. Strychnin mucoid. In view of the extreme delicacy of the dichromate test for strychnin, this base was selected for the second series of preparations. Care was taken to insure purity of the original materials as in the preparation of the morphin-mucoid products. Preparation. The method of preparation was identical with that for morphin mucoid (page 112) except in the following details: The final water-solution of the product was again evaporated to dry- ness at 40° C. and the dry matter, after pulverization, was extracted with Chloroform. Twenty-six voluminous washings were neces- sary to free the powder from admixed strychnin and to obtain a strychnin-free washing. In view of the insolubility of strychnin in water and its ready solubility in ether this result seemed difficult to explain on any other basis than partial dissociation by the Chloroform. Evidence of chemical combination. The protein character of the Compound was established by the following results : The water- solution gave a strong biuret test; was precipitable by Saturation with ammonium sulfate or magnesium sulfate; gave a flocculent precipitate with 0.2 per cent. hydrochloric acid and 4 per cent. acetic acid Solutions ; and f rothed strongly on shaking. The aqueous Solu- tion of the product was neutral to litmus. The presence of strychnin was shown by the intense bitter taste and by strong "dichromate tests." Filtrates from precipitates formed by addition of 0.2 per cent. hydrochloric acid Solution yielded, in every case, strong "di-chromate tests " for strychnin. Four physiological tests were also made to establish the presence of the strychnin. The results and methods follow : The lethal dose of strychnin sulfate is about 2.5 mg. per kilo of weight for frogs and 7.6 mg. per kilo of weight for dogs. Vol- umes of aqueous Solution of strychnin mucoid (0.575 ^S- P^^* ^■^•) containing quantities equal to the lethal dose of strychnin sulfate were injected subcutaneously in frogs and dogs. I9I2] Walter H. Eddy ' 115 In the first of two experiments on frogs, the initial dose failed to produce any strychnin effects. An effect followed the second injection of an equal dose, but it required three doses to produce Opisthotonus. Recovery was complete. In the second frog, each of two doses injected successively produced Opisthotonus. The frog recovered. For the first dog a double dose was required to produce hyperes- thesia and tetanus. The results with the second dog duplicated those with the first. In all these physiological tests the strychnin appeared to be liberated slowly in the animal, the effects Coming on gradually and extending over a period of 3-5 hours, with complete recovery. Concliisions regarding strychnin mucoid. The results seemed to leave no doubt regarding the compound-nature of this product. Whether it is a true salt or an adsorption Compound can not be de- cided from the available data, but its neutrality, its water-solubility, and its power to yield both strychnin and mucoid, strongly suggest the production of a salt by a process directly comparable to the neutralization of base by acid. The physiological tests show that the Compound evidently con- tains a much smaller proportion of strychnin than that in the com- mon Sulfate. The quantitative examinations have not yet been completed. C. Conin mucoid. Conin combines with mucoid very rapidly and yields a Solution which filters easily. The product, af ter purifica- tion by dialysis and alcohol precipitation, is water-soluble and biuret- reacting. As in the case of morphin mucoid, however, it was im- possible to demonstrate the presence of the alkaloid. All tests were negative with the potassio-mercuric iodid and phospho-tung- stic acid reagents. D. PiPERiDiN MUCOID. The purified piperidin product gave the protein tests and also a test for piperidin with platinic chlorid. E. Anilin mucoid(?). A water-soluble product of mucoid and anilin was obtained but the anilin disappeared early in the puri- fication process. F. Morphin nucleoprotein. Two attempts were made to produce a Compound of morphin with yeast nucleoprotein. The ii6 Sonic Protein Compounds [Sept. method of preparation was similar to that described on page 112. Neither attempt was successful in establishing the presence of mor- phin in the final product. A water-soluble protein of different char- acter from the nucleoprotein resulted in each case. G. Morphin caseinogen, strychnin caseinogen, and cal- cium CASEiNOGEN. Studies were made of the effects of morphin, strychnin and calcium hydroxid on caseinogen. In each case water- soluble, biuret-reacting products were obtained. Rigorous purifi- cation was not attempted. Salts of ovomucoid. Neumeister^ investigated a glucoprotein in eggs which he named " pseudopeptone." This Compound was studied by Salkowski,^ Mörner/ and Eichholz,^ and called by them " ovo-mucoid." As a " cell-protein," this substance seemed to offer good material for our experiments. A pure product was prepared by Mörner's''' well-known process. Preparation of ovo-mucoid. (From eggs.) With increasing purity, precipitation with alcohol became correspondingly difficult. Alcohol-ether did not remove this difficulty but the addition of a few drops of sodium chlorid Solution brought about precipitation in every case. The final water-solution was freed from chlorid by dialysis in a parchment bag in the presence of toluene. The Solu- tion, which then was acid to litmus, was evaporated to dryness at 40° C, yielding yellow flakes which were ground to a white powder. Properties of the ovo-mucoid product. This ovo-mucoid was readily soluble in water and gave a good biuret test. The water- solution frothed on shaking, but was not viscid. Phosphotungstic acid, 0.2 per cent. hydrochloric acid, 4 per cent. acetic acid and tannic acid Solutions precipitated the aqueous Solution, which was acid to litmus. (From shad roe.) The roe was ground in a mortar with sand and this mixture poured into boiling, slightly acidulated, water. The remaining Steps were identical with those for the preparation of ovo-mucoid from eggs and the product responded to the same tests. These two products were used in the following studies. ' Neumeister : Zeitschrift für Biologie, 1890, xxvii, p. 331, 'Salkowski: Centralhlatt f. d. med. Wissensch., 1893, xxxi, pp. 513 and 706. ^ Mörner : Zeitschr. f. physiol. Chem., 1893, xviii, p. 525. ' Eichholz : Journ. Physiol., 1898, xxiii, p. 163. igi2] Walter H. Eddy 117 H. Strychnin ovo-mucoid (egg). The method of prepara- tion followed the lines of the morphin-mucoid process (page 112) with the following abbreviation : After dialysis the Solution was at once precipitated with absolute alcohol. No other methods of puri- fication were used. The filtrate f rom the original mixture of ovo-mucoid and strych- nin was turbid and acid to litmus, but became neutral on standing, in the presence of toluene. On dialysis, and consequent dilution with water, the Solution clarified. The dialysate on the other band became turbid but failed to give a protein or strychnin test, When the dialyzed liquid was treated with absolute alcohol, in excess, a mixed, cheesy and gelatinous precipitate was produced. The alcoholic filtrate from this precipitate was acid and gave a strychnin test, suggesting dissociation. The precipitate dissolved readily in water and the Solution was then filtered. It was nozv acid in reaction and gave no strychnin test. Precipitated again with alcohol, the solid product failed to give the strychnin test, was acid and resembled in every way the original ovo-mucoid. A portion of this precipitate was dissolved in water and the So- lution evaporated to dryness at 40° C. A new trituration with strychnin was made with this product. The results were the same as with the first preparation, viz., a turbin Solution that cleared on dilution with water by dialysis and gave in this condition both strychnin and protein tests. Alcohol again dissociated it into strychnin and ovo-mucoid (?). From the above results it was deemed desirable to make a care- ful study of the reactions of the product and a second preparation was conducted for this purpose. The turbid filtrate obtained from the initial mixture of strychnin and ovo-mucoid was found to be actually amphoteric to litmus, though acid to Phenolphthalein. Its alkalinity to litmus was not increased by retriturating it with strych- nin. Dilution with water resulted again in a clear Solution, giving both strychnin and protein tests. On standing for a considerable time in a parchment bag, in the presence of toluene, the amphoteric reac- tion gradually disappeared and the Solution became distinctly acid to litmus. It also finally yielded a precipitate in the bag and lost its power to respond to the strychnin test. The turbid dialysate grad- ii8 Sonic Protein Compounds [Sept. ually acquired protein material, but the frequent renewals of water and large voliime made it impossible to determine the presence of strychnin. Apparently complete dissociation resulted, but neither the character of the dissociation products nor the manner in which the strychnin separated was determined. Strychnin ovo-niucoid (roe). The results with ovo-mucoid from shad roe were identical with those in the case of tgg ovo-mu- coid except that the disappearance of the strychnin on dialysis was much slower. It was ten days before the contents of the bag failed to give the strychnin test. Concentration of the dialysates in this case before applying the strychnin test failed to make its detection possible. Evidence of the Compound natnre of the ovo-mucoid products. The clear amphoteric Solution, with its response to strychnin and protein tests, indicates a chemical combination, especially in view of the water-insolubility of strychnin. The dissociability in alcohol of the shad roe product, and the results of dialysis, indicate that it is more stable than the strychnin product with &gg ovo-mucoid. Again the question of whether we are here dealing with a true chemical Compound or with an adsorption product remains open for further investigation. 2. Protein-protein Compounds. The foregoing experiments were preliminary to attempts to bring about combinations between acid-reacting and basic-reacting proteins, such as protamins and histons. /. HiSTON MucoiD. Preparation of histon hydrochlorid. His- ton was prepared by the method of Huiskamp.^ Thymus glands from freshly killed calves were freed from fat with a knife and minced in a meat chopper. The hash was then placed in a large bottle and extracted in an ordinary ice box for 24-48 hours with distilled water. About 300 c.c. of water were used with each 100 grams of thymus. The extract was filtered through wet fluted filter papers. Nucleohiston was precipitated from the filtrate with 5 c.c. of IG per Cent, calcium chlorid Solution per 100 c.c. of extract. The precipitate was then filtered off and redissolved in water to which a little ammonia had . been added. This Solution was filtered and reprecipitated with calcium chlorid Solution in the usual way. The * Huiskamp : Zeitscjir. f. physiol. Chemie, 1901, xxxii, p. 145. igi2] Walter H. Eddy 119 precipitate was then extracted with 0.8 per cent. hydrochloric acid for the production of the hydrochlorid. This extract of histon hydrochlorid was finally dialyzed in a parchment bag against dis- tilled water until neutral to litmus. This Solution of histon hydro- chlorid was used for the preparation described below. Preparation of potassium mucoid. Acid-free mucoid was dis- solved in 0.3 per cent. potassium hydroxid Solution and the liquid filtered. The filtrate was then dialyzed in a parchment bag against distilled water (in the presence of toluene) until neutral to litmus. The product in this neutral Solution was presumably potassium mucoid. Preparation of histon mucoid. Histon hydrochlorid Solution was added drop by drop to the potassium mucoid Solution. A pre- cipitate formed immediately, and sedimented quickly beneath the clear supernatant liquid. Excess of the histon hydrochlorid Solu- tion dissolved the precipitate. The product was then filtered off and washed with water until the washings no longer gave precipi- tates with ten per cent. ammonium hydroxid or 0.2 per cent. hydro- chloric acid Solution. Evidence of the Compound nature of the histon mucoid product. A portion of the precipitate was triturated with 0.05 per cent. so- dium carbonate Solution. A colloidal Solution was obtained. Its filtrate gave a heavy precipitate with 0.2 per cent. hydrochloric acid Solution and a distinct precipitate with ammonium hydroxid Solution. These results did not determine whether the sodium carbonate merely dissolved the histon mucoid, or dissociated it into a histon So- lution and a sodium mucoid Solution. To ascertain these points the following tests were made : (a) A portion of the sodium carbonate Solution was poured into 95 per cent. alcohol. It failed to precipitate at once or on Standing. (b) A portion of the sodium carbonate Solution was poured into 95 per cent. alcohol, to which one drop of 10 per cent. sodium chlorid Solution had been added. A precipitate appeared on Standing. (c) Alcohol-ether failed to precipitate the Solution but with the addition of a drop of salt Solution a precipitate appeared. 120 Some Protein Compounds [Sept. {d) The precipitates obtained in {h) and (c) failed, in this first set of tests, to dissolve in water and the washings gave no hydro- chloric acid or ammonia precipitate. The precipitates dissolved in 0.05 per Cent, sodium carbonate Solution and the filtrates gave both the ammonia and hydrochloric acid tests. {e) Histon hydrochlorid Solution was not precipitated by alcohol even when sodium chlorid was present. Alcohol also failed to pre- cipitate potassium mucoid Solution but did so in the presence of a trace of sodium chlorid. These results Warrant the inference that the sodium carbonate acted as a solvent rather than as a dissociant. They also indicate that precipitation by alcohol in the presence of salt served to dif- ferentiate the histon mucoid from the histon hydrochlorid, and that our product was a Compound and not a mixture. A Solution of histon hydrochlorid was tested with an excess of alcohol in the absence of salt. A similar Solution of potassium mucoid was made. When these two clear Solutions were mixed a precipitate of histon mucoid formed at once. This histon mucoid was then dissolved in 0.05 per cent. sodium carbonate Solution and the filtered Solution precipitated with alcohol in the presence of a little salt. This precipitate, unlike that above (d), dissolved readily in zvater. The water-solution gave both the ammonia precipitate and the hydrochloric acid precipitate. This and similar results indi- cated the formation of a soluble histon mucoid Compound. Finally a new histon mucoid product w^as made by the original method. This product was washed free from excesses of both his- ton hydrochlorid and potassium mucoid, as before, and then treated as f ollows : A portion was macerated in a mortar with o.i per cent. hydro- chloric acid Solution and a second portion in another mortar with 0.1 per cent. potassium hydroxid Solution. These liquids were fil- tered. The acid filtrate gave a precipitate with ammonia but not with hydrochloric acid. The alkali filtrate gave a heavy precipitate with hydrochloric acid but none with ammonia. These results suggest that mixtures of (a) histon hydrochlorid and potassium mucoid Solutions yield a precipitate of histon mucoid; (&) pure histon mucoid and o.i per cent. hydrochloric acid I9I2] Walter H. Eddy 121 Solutions yield histon hydrochlorid and insoluble mucoid; (c) pure histon mucoid and o.i per cent. potassium hydroxid Solutions yield a potassium mucoid histon complex. The failure to get a histon precipitate with ammonia in the potassium hydroxid extract may have been due to the small amount of resultant histon mucoid or to the formation of an insoluble form o£ histon, such as ammonia produces. Whatever the explanation of this failure, there seemed to be no doubt of the power of histon to combine with mucoid to form a Compound different in proper- ties from either histon hydrochlorid or potassium mucoid. /. Histon nucleoprotein (yeast). Neutral potassium nu- cleoprotein (obtained by dissolving yeast nucleoprotein in o.i per cent. potassium hydroxid Solution and dialyzing free from hydroxyl ions) combines with histon hydrochlorid in the same way as potas- sium mucoid (page 119). Much more of the Solution of histon is necessary for the production of the salt. The product was similar to histon mucoid in being insoluble in water; in dissolving readily in 0.05 per cent. sodium carbonate Solution but incompletely in 0.5 per cent. sodium carbonate Solution ; and in forming, with sodium carbonate, a water-soluble sodium-histon nucleoprotein complex. K. Histon ovo-mucoid. Preparation. The ovo-mucoid {tgg) was purified to such a degree as to be practically soluble in salt-free alcohol (page 116). A similarly pure Solution of histon hydro- chlorid was used. When the water Solutions of these two substances were com- bined, a precipitate formed slowly. A slight excess of the histon Solution dissolved the precipitate. The precipitate dissolved to a turbid Solution in 0.05 per cent. sodium carbonate Solution. This turbid fluid was filtered and divided into two portions. One por- tion was poured into 95 per cent. alcohol to which 3 drops of 10 per cent. sodium chlorid Solution had been added. The other por- tion was saturated with ammonium sulfate. Both portions gave heavy precipitates, which were soluble in water; the Solutions were precipitated in part by ammonia. When purification of the ammo- nium sulfate precipitate by dialysis was attempted, the Compound broke down. The "alcohol precipitate" was hydrolyzed with hydrochloric acid. The resultant liquid, neutralized with potassium 122 Some Protein Compounds [Sept. hydroxid, rediiced the Fehling-Benedict reagent. This result, with the precipitation by ammonia, seemed to show the presence of both glucoprotein and histon in the precipitate. When alcohol Solutions of ovo-mucoid and histon hydrochlorid were poured together, a precipitate formed at once that gave both the ammonia test and the reduction test. The latter process is the simplest and quiekest method of obtaining this product. The results of these researches have shown that the methods for the preparation of histon, as outlined in the literature, are in serious need of revision. In fact, the results suggest that so-called histon is a protein salt rather than a simple protein. In a future paper will be presented the findings in regard to histon preparation. EFFECTS OF INTRAPERITONEAL INJECTIONS OF EPINEPHRIN ON THE PARTITION OF NITRO- GEN IN URINE FROM A DOG JACOB ROSENBLOOM and WILLIAM WEINBERGER (Biochemical Laboratory of Columbia University, at the College of Physicians and Surgeons, New York) I. INTRODUCTION The action of epinephrin on nitrogenous metabolism has been the object of investigation by several authors. The experimental results of Kraus and Hirsch/ and Quest,^ indicate that intravenous or subcutaneous injections of epinephrin exert very Httle influenae on the nitrogenous metaboHsm of healthy dogs, the insignificant increase of eliminated nitrogen being caused both by the glycosuria and (after subcutaneous injections) by skin necrosis. Fasting ani- mals seem to be differently affected. Falta and Rudinger,^ and Underhill and Closson* were able to show an accelerating influ- enae, on protein metaboHsm, of subcutaneous and intravenous injec- tions of epinephrin. Underhill and Closson have shown that the subcutaneous injec- tion of " adrenalin chlorid " Solutions into dogs is not attended by any significant change in the proportions of the urea-, ammonia- and creatinin-nitrogen of the urine, in partial disagreement with Paton,^ who also found that, although on a sufficient diet, the catabolism of proteins is not interfered with, there is a markedly increased produc- tion of ammonia. In all the above mentioned experiments the epinephrin was in- jected into veins or into subcutaneous tissues. The intraperi- toneal way has not been utilized by previous observers in this con- ^ Kraus and Hirsch. Cited by Kraus and Friedenthal : Berl. klin. Woch., 1908, xlv, p. 1709. ^Quest: Zeit. f. exp. Path., 1908, v, p. 43. ' Falta and Rudinger : Central, f. klin. Med., 1908, Ixvi, p. i. * Underhill and Closson : Anier. Journ. Physiol., 1906, xvii, p. 42. ' Paten : English Journ. of Physiol., 1903, xxix, p. 286 ; 1904, xxxii, p. 59. 123 124 Epinephrin Effects on Metabolism [Sept. nection, althongh it is possible that this mode of administering epinephrin has a different effect on nitrogenous metabolism, as is the case in carbohydrate metabolism (Löwi).® IL DESCRIPTION OF THE EXPERIMENTS This investigation consisted of two metabolism experiments. One animal was used for both experiments. An intermediate pe- riod served to allow the animal to recuperate from the effects of the first experiment before the second one was begun. The metabolism work was conducted by the general methods in use in this labo- ratory.'^ We determined the nitrogen content in the several ingredients of the food. Urinary nitrogen, in the leading forms, was deter- mined as f ollows : ammonia, each day ; total, urea, creatin and Crea- tinin (as Creatinin), every third day; purins, at the end of each period. The urine was preserved with thymol. Total nitrogen was determined by the Kjeldahl process ; ammonia and Creatinin by the Polin methods f iirea^ by Benedict's method ;^^ purin nitrogen by a combination of the Arnstein" and Salkowski^^ methods. The authors used two specimens of the colorless "adrenalin Chlorid" (i:i,ooo) of Parke, Davis and Co. They were pur- chased in the open market. Each was tested for its pressor action at the conclusion of the corresponding injection experiments and was then found to be practically as active as ever. Varying amounts and concentrations of "adrenalin chlorid" were injected into the peritoneal cavity ; in the first experiment the concentration was 1 : 10,000 — in the second, i : i,ooo. In one injection period of *Löwi: Von Noorden's Metabolism and practica! medicine, 1907, iii, p. 1181. ' Mead and Gies : Anier. Journ. Physiol., 1901, v, p. 106 ; also Gies and collab- orators: Biochemical Researches, 1903, i, Reprint No. 21; Gies: Amer. Journ. of Physiol., 1905, xiv, p. 403; Gies: Amer. Journ. of Physiol., 1901, v, p. 235; also Gies and collaborators : Biochemical Researches, 1903, i, Reprint No. i ; Gies : Proc. Amer. Physiol. Soc, Amer. Journ. of Physiol., 1904, x, p. 22; Hawk and Gies: Amer. Journ. of Physiol., 1904, xi, p. 177. * Polin : Amer. Journ. of Physiol., 190S, xiii, p. 45. " No glycosuria occurred. Examinations were made repeatedly. ^^ Benedict : Journ. of Biol. Chem., 1910, viii, p. 405. " Arnstein : Zeit. f. physiol. Chem., 1897, xxiii, p. 417. " Salkowski : Salkowski's Manual of physiol. chem. and path., 1904; Arch. d. ges. Physiol., 1897-98, Ixix, p. 268. I9I2] Jacoh Rosenhloom and William Weinherger 125 eighteen days, a total of 62 c.c. o£ i : 10,000 Solution was given intraperitoneally ; in another injection period of six days, a total of 29 c.c. of 1 : 1000 Solution was administered. The accompanying tables contain the metabolic data obtained in this study. TABLE I. FIRST METABOLISM EXPERIMENT (jUNE I2-JULY l8, I9I2) A. Daily Records I. Fore Period. Normal Condition Number of the day I 2 3 4 5 6 7 8 9 10 Body weight (kilos) 6.3 270 1,017 6.33 210 1.025 6.3 270 1,020 6.3 190 1,024 6.31 192 1,024 6.3 212 1,021 6.28 275 1,020 6.27 240 1,018 6.3 188 1,021 6.33 Urine, volume (c.c.) 170 1,026 TJrine. so. er II. Dosage Period. Intraperitoneal Injections Number of the day Body weight (kilos) . , Urine, volume (c.c.) . , Urine, sp.gr , Adrenalin Solution (i 10,000) c.c. 6.34 135 1,027 S 6.27 157 1,031 5 6.28 80 1,042 5 6.26 100 1,040 5 6.35 165 1.030 8 6.25 224 1,020 6.33 160 1.030 5 8 6.26 245 1,020 6.31 180 I,02S 5 Number of the day Body weight (kilos) . . Urine, volume (c.c.) . . Urine, sp. gr Adrenalin Solution (i 10,000) c.c. 10 II 12 13 14 IS 16 17 6.26 6.33 6.41 6.40 6.42 6.37 6.35 6.34 200 105 153 175 255 230 205 270 1.023 1.037 1.025 1,023 1,019 1,017 1,020 1,020 2 3 3 — 4 — 4 4 18 6.43 167 1,023 4 III. After Period. Normal Conditions Number of the day I 2 3 4 5 6 7 8 Body weight (kilos) Urine, volume (c.c.) Urine. so. sx 6.34 135 1,027 6.40 145 1,020 6.44 200 1,022 6.40 245 1,020 6.44 220 1,018 6.41 185 1,022 6.40 214 1,019 6.46 214 1,020 B. Analytical Totais and Daily Averages of Urinary Data for Each Period 3 > - 4) IS Nitrogen UreaN Ammonia N Creatin and Creatinin N Purin N Period 2S H So Daily average, grams 2 S E-i dt Daily average, grams H bo Daily average, grams Total, grams Daily average, gram 15 H Daily average, gram Fore (10 days). Dosage (18 days). After (18 days). 2,217 3.206 1.558 221 178 195 41-594 74.225 33-077 4-159 4.123 4-134 36.85 63-58 28.63 3.685 3-532 3.578 I.5II 2.945 I-I95 0.1511 0.1636 0.1493 1.046 I.714 I.IOI 0.1046 0.0952 0.1376 0.067 0.147 0.081 0.0067 0.0081 O.OIOI 126 Epinephrin Effects on Metaholism [Sept, TABLE 2. SECOND METABOLISM EXPERIMENT (jULY 2I-AUGUST 9,. I912) A. Daily Records I. Fore Period. Normal Conditions Number of the day Body weight (kilos) Urine, volume (c.c.) Urine, sp.gr 6.52 190 1,020 6.53 190 1,023 6.53 230 1,019 6.54 200 1,020 6.57 193 1,021 IL Dosage Period. Intraperitoneal Injections Number of the day I 2 3 4 5 6 Body weight (kilos) . . . Urine, volume (c.c). . . Urine. so. er 6.54 ISO 1.028 4 6.69 134 1,028 5 6.68 215 1,019 5 6.63 275 1,018 5 6.70 185 1,020 5 6.71 240 1,016 Adrenalin Solution (i : 1,000) c.c 5 III. After Period. Normal Conditions Number of the day Body weight (kilos) Urine, volume (c.c.) Urine, sp.gr I 2 3 4 5 6 7 8 6.68 245 1,019 6.72 232 1,018 6.72 230 1,018 6.76 200 1,019 6.78 202 1,017 6.77 240 1,018 6.78 200 1,019 6.83 223 1,017 6.80 23s 1,019 B. Analytical Totais and Daily Averages of Urinary Data for Each Period E 3 "o > >> > > " Nitrogen UreaN Ammonia N Creatin and Creatinin N Purin N Period — B E — « 2 E rt r: n E ü E V := t« E S E « H So CS u cd >" f^ Fore (5 days) . . . Dosage (6 days) After (9 days) . . 1,003 1,199 2,007 201 199 223 20.997 24.678 34-937 4.199 4-II3 3.882 18.22 21.46 29-39 3-643 3-5766 3.266 0.767 0.967 1.499 0.1533 0.1611 0.1664 0.671 0.7314 1.266 0.1342 O.I2I9 0.1407 0.039 0.039 0.059 0.0076 0.006s 0.0066 TABLE 3. PARTITION OF THE URINARY NITROGEN Period I. Fore period Injection period .... Post injection period II. Fore period Injection period .... Post injection period Urea N, per Cent. 88.5 85-7 86.5 86.7 87.0 84.2 Ammonia N, per Cent. 3-64 3-75 3-61 3-65 3-92 4.29 Creatin and Creatinin N, per Cent. 2.51 2.50 3-33 3-20 3-73 3-62 Purin N, per Cent. 0.16 0.20 0.24 0.19 0.16 0.17 Undeter- mined N, per Cent. 5-19 7.86 6.32 6.26 5-19 7.72 I9I2] Jacob Rosenhloom and William Weinherger 127 III. CONCLUSIONS The results of these experiments show conclusively that intra- peritoneal injections of "adrenalin chlorid" Solutions were without appreciable effect on the proportions of nitrogen (in the forms of Urea, ammonia, creatin and Creatinin, purins, and undetermined sub- stances) in the urine of a healthy dog. /THE BIOCHEMICAL SOCIETY, ENGLAND In a previous note, which appeared in the Biochemical Bul- letin (i: 484), it was stated that the recently founded Bio- chemical Club would probably develop into a society with a Journal of its own. This is now an accomplished fact, and the Biochemical Society of England has been launched into being. It has been instituted for the purpose of facilitating intercourse between those biologists and chemists who are interested in problems common to both, such as the chemical questions connected with agriculture, brewing, animal and vegetable physiology and pathology, etc. Meetings are held at different centers throughout the country for the communication of papers and demonstrations. The Honorary Secretary is Dr. R. H. A. Flimmer, University College, London, W. C, from whom further Information can be obtained. The Bio-Chemical Journal, which has hitherto been under the editorship of Professor Moore, F.R.S., of Liverpool, will in the fu- ture be conducted by the Biochemical Society, and will be issued by the Cambridge University Press, Fetter Lane, London, E. C. The editors are Professor W. M. Bayliss, F.R.S., University College, London, W. C, and Professor A. Harden, F.R.S., Lister Institute, Chelsea Gardens, London, S. W. The first issue of the Journal under these editors is expected in January next. The price is £1.1.0 ($5) per volume. One f€els sure that our American confreres will heartily support the new enterprise. W. D. Halliburton King's College, London. 128 MEETINGS OF THE SECTION (II) ON DIETETIC HYGIENE AND HYGIENIC PHYSIOLOGY OF THE FIFTEENTH INTERNATIONAL CONGRESS ON HYGIENE AND DEMOGRAPHY, WITH AB- STRACTS OF SOME OF THE PAPERS Proceedings reported by THE Secretary, LAFAYETTE B. MENDEL The meeting o£ the Congress was noteworthy for the unusual opportunity which it afforded to American men of science to meet some of their foreign colleagues, particularly those from the Con- tinent, in a personal way. It can scarcely be said that the proceed- ings of the Section on Dietetic Hygiene and Hygienic Physiology were unique in any way; nor could they be expected to attract the Chief interest where so many important disciplines and conflicting or overlapping scientific fields were involved. The Symposium on the specific dynamic action of foodstuffs deserves special comment, however, both on account of the new views which were forcefully presented there for the first time, and the preeminent part played by all of the referees in the development of this field of study. I. OFFICIAL LIST OF PRESIDENTS AND VICE-PRESIDENTS OF THE SECTION Honorary Presidents : Dr. Max Rubner, Professor of Phys- iology and Director of the Physiological Institute, Berlin, Germany ; Dr. Artur Schattenfroh, Professor of Hygiene in the Univer- sity of Vienna, Austria ; Dr. Axel Holst, Professor of Hygiene, University of Christiania, Norway ; Dr. A. B. Macallum, Profes- sor of Biochemistry, University of Toronto, Canada. President: Dr. Russell H. Chittenden, Professor of Phys- iological Chemistry, Sheffield Scientific School of Yale University, New Haven, Conn. 129 130 Biochcmical Proceedings, Hygienic Congrcss [Sept. Vice-presidents : Dr. Graham Lusk, Professor of Physiology, Cornell University Medical College, New York City; Dr. David L. Edsall, Professor of Clinical Medicine, Harvard Medical School, Boston, Mass. II. OFFICIAL PROGRAMM All the meetings of the section were held on Sept. 23 to Sept. 27, inclusive, in Washington, D. C, at the new National Museum, Room 376. 1. Monday afternoon, September 23. The physiological SIGNIFICANCE OF SOME SUBSTANCES USED IN THE PRESERVATION OF FOOD : Dr. John H. Long, professor of chemistry, Northwestern Uni- versity Medical School, Chicago, 111. (page 132) ; Dr. Artur Schat- tenfroh, professor of hygiene in the University of Vienna, Austria. 2. Tuesday morning, September 24. The specific dynamic ACTiON OF FOODSTUFFS : Dr. Max Ruhner, professor of physiology and director of the Physiological Institute, Berlin, Germany. {A) The work of digestion and specific dynamic action : Dr. N. Zuntz, Direktor des tierphysiologischen Laboratoriums der landwirtschaft- lichen Hochschule, Berlin, Germany {presented by Prof. F. G. Bene- dict). — (5) The influence of the Ingestion of food upon metabolism: Dr. Francis G. Benedict, director of the Nütrition Laboratory of the Carnegie Institution of Washington, Boston, Mass. (page 134). — (C) The influence of foodstuffs and their cleavage products upon heat production : Dr. Graham Lusk, professor of physiology, Cornell University Medical College, New York City (page 135). 3. Tuesday afternoon, September 24. Nutrition and GROWTH. (A) An anatomical analysis of growth : Dr. Henry H. Donaldson, The Wistar Institute of Anatomy, Philadelphia, Pa. — (B) Nutrition of the embryo: Dr. John R. Murlin, assistant pro- fessor of physiology, Cornell University Medical College, New York City. — (C) The nutrition and growth of bone: Dr. Francis H. Mc- Crudden, chemist at the Hospital of the Rockefeiler Institute for Medical Research, New York City (page 137). — {D) The role of proteins in growth : Dr. Lofayette B. Mendel, professor of physi- ological chemistry, Shefiield Scientific School of Yale University, New Haven, Conn. (page 138). — (E) The influence of the quantity * Abstracts of the papers appear on the pages indicated by the numerals in parenthesis. I9I2] Lafayette B. Mendel 131 and quality of food upon the growing organism : Dr. Hans Aron, director of the scientific laboratory of the University Children's Clinic, Breslau, Germany {presented by Prof. Lafayette B. Men- del). — (F) Direct calorimetry of infants, with a comparison of the results obtained by this and other methods: Dr. John Howland, Professor of pediatrics, Johns Hopkins University, Baltimore, Md. (page 139). 4. Wednesday morning, September 25. The röle of in- ORGANIC SUBSTANCES IN THE NUTRITION OF MAN. {A) The antag- onistic action of salts : Dr. Jacques Loeb, head of the department of experimental biology, Rocke feller Institute for Medical Research, New York City. — (B) The distribution of soluble salts in living cells and the forces Controlling it: Dr. Archibald B. Macalliim, pro- fessor of biochemistry, University of Toronto, Canada (page 140). — (C) The röle which common salt and water assume in the nutri- tion of man : Dr. Hermann Strauss, professor of clinical medicine, University of Berlin, Germany (page 141). 5. Thursday morning, September 26. Practical dietetics, {A) Cost and nutritive value of f oods : Dr. C. F. Langworthy, ex- pert in charge of nutrition investigations, U. S. Department of Agriculture, Washington, D. C. — {B) The influence of the prepara- tion of food on its nutritive value : Dr. Max Riibner, professor of physiology and director of the Physiological Institute, Berlin, Germany. — (C) The choice of foods, with regard to disease: Dr. Carl von Noorden, professor of internal medicine and director of the First Medical Clinic, Vienna, Austria (page 143). — {D) Diet in relation to disease, chiefly in relation to some forms of partial un- derfeeding (beriberi and scurvy) : Dr. Axel Holst, professor of hy- giene, University of Christiania, Norway. — {E) Diet and metabo- lism in fever : Dr. Warren Coleman, Cornell University Medical College, New York City (page 145). 6. Thursday afternoon, September 26. Ventilation in its HYGiENic ASPECTS, {A) Organic matter in the expired air: Dr. Milton J. Rosenmi, professor of preventive medicine, Harvard Med- ical School, Boston, Mass. — (B) A consideration of the unknown fac- tors in the ill-effects of bad Ventilation: Dr. Yandell Henderson, professor of physiology, Yale Medical School, New Haven, Conn. (page 146). — (C) The hygienic physiology of work in compressed 132 Biochcmical Proceedings, Hygienic Congress [Sept. air: Dr. J. J. R. Macleod, professor of physiology, Western Re- serve Medical School, Cleveland, Ohio (page 147). 7. Friday morning, September 27. The hygienic physiol- ogy OF EXERCISE. {A) The influence of exercise on the nervous System : Dr. Leon Asher, a. o. professor of physiology, Bern, Swit- zerland {presented by Prof. L. B. Mendel). — {B) The influence of exercise on the heart : Dr. R. Taif McKenzie, director of physical education, University of Pennsylvania, Philadelphia, Pa. — (C) Certain aspects of the influence of muscular exercise upon the respi- ratory System : Dr. Theodore Hough, professor of physiology, Uni- versity of Virginia, Charlottesville, Va. (page 148). — {D) Physical training in the United States Naval Service: Dr. J. A. Murphy, surgeon, U. S. N., U. S. Naval Academy, Annapolis, Md. Additional papers. The prevention of arteriosclerosis and heart disease in otherwise healthy individuals past middle life: Dr. Louis F. Bishop, New York City, — Tuberculosis and metabolism: Dr. Diesing, chief physician, Recreation and Convalescent Home, Gross-Hansdorf, Hamburg, Germany. — On the nature and impor- tance of the diet as the most important factor of causal therapy in severe diseases of the stomach and intestines, in nervous and mental diseases, and in disorders of the circulation and of the metabolism : Dr. W. Plönies, Hanover, Germany. — Public baths: Dr. Simon Baruch, president, American Association for Promoting Hygiene and Public Baths, New York City. — The significance of hydrother- apy for hygiene, therapeutics and medical Instruction: Prof. Dr. L. Brieger, Hydrotherapeutische Universitäts-Anstalt, Berlin, Ger- many. — The importance of the nutritive salts for healthy and sick people : Dr. R. Peters, Hanover, Germany. III. ABSTRACTS OF SOME OF THE PAPERS = The physiological significance of some substances used in the preservation of food JOHN H. long This paper dealt with the action on the human organism of a number of substances employed as food preservatives, or otherwise, in the preparation of food. * Reprinted f rom the official pamphlet containing " abstracts of papers to be read at the congress," Sept. 23-28, 1912 (pp. 11-28). igi2] Lafayette B. Mendel 133 Something of the history of food preservatives was recited, and it was shown that a considerable number of substances are added to food largely because of their preservative properties, rather than because of flavors they may impart. Some of the so-called " na- tural " preservatives come under this head. Modern conditions of living and modern scientific advances have called for the introduc- tion of more efficient substances, the so-called " chemical " or " arti- ficial " preservatives. Many of these substances have been con- demned, and perhaps properly, but frequently the condemnation is solely on the ground of their origin. This basis of condemnation has no justification in fact, as all preservatives are as truly chem- ical as are those of recent introduction made by industrial processes. The active principles in cloves, cinnamon, allspice, etc., are true chemical Compounds, and in their action on the body and final dis- position are much like benzoic acid, now made largely by laboratory processes. A number of important investigations on the physiological action of sodium benzoate have been carried out in the last few years, and the results of these were discussed. The effects of large and small amounts of benzoic acid are known, and it has been clearly shown that the use of the small quantities employed in the ordinary pro- tection of the condimental foods is quite unobjectionable. Such small amounts are normally disposed of in the human body without ill effects. The use of copper salts in coloring vegetables was next discussed. There is an enormous literature on the subject, especially from France and Germany, where copper has long been used in the can- ning Industries. Several commissions have pronounced in favor of permitting the use of copper salts, although others have opposed it. But all authorities have come to agree that the toxicity of these salts is much less than was at one time assumed. This toxicity depends somewhat on the combinations in which the salts are in- gested. The effects of copper as used in young peas or string beans are far less marked than are those of its inorganic salts. It is, there- fore, not quite justifiable to draw conclusions as to the behavior of copper from experiments with copper sulfate alone. If only very young and fresh vegetables, with plenty of chlorophyl, were treated 134 Biochemical Proccedings, Hygienic Congress [Sept. with copper, and if the amount were strictly limited, there might be but little fault foiind. But with older vegetables the combination is far less stable and the effects approach those of the inorganic salts. The amounts of copper taken up by the liver and other Organs from inorganic salts may be considerable, and such absorp- tion cannot be held free from danger. The use of these salts serves no real good purpose and should be condemned. The paper touched also on the employment of sulphurous oxide and sulphites in certain food Industries. The influence of the ingestion of food upon metabolism FRANCIS G. BENEDICT Three interpretations of the increase in metabolism following the ingestion of food are current : first, the theory in which the mechanical work of the digestive processes plays the most prom- inent röle; second, the less sharply defined theory in which the con- ception of the development of free heat unavailable to the cells is the dominant note, and, finally, the opinion expressed by Friedrich Müller, that there is absorbed out of the food certain substances w'hich are carried by the blood to the cells and there stimulate the cells to a greater metabolic activity. The evidence used for the evaluation of these views in this paper is based almost exclusively upon experiments made upon men in our laboratory. It was found that although the ingestion of sodium sulfate produced a powerful peristalsis, no measurable increase in the me- tabolism as measured by the oxygen consumption was noticed. Similarly, the ingestion of large amounts of agar-agar produced very voluminous, bulky stools, but did not increase metabolism measurably. As subsidiary evidence, in unpublished experiments on dogs with deficient pancreatic secretion, it was found that al- though the decreased assimilation of protein and fat resulted in large, bulky, fatty stools, there was not an increase in the carbon dioxide production. The evidence points strongly to the fact that the ingestion of meat by depancreatized dogs, accompanied as it is with large, voluminous, bulky stools, results in absolutely a smaller increase in metabolism than is experienced with the ingestion of meat by normal dogs. I9I2] Lafayette B. Mendel 135 Both of these pieces of evidence, therefore, can be taken as strongly contrary to the work of digestion as an explanation of any considerable proportion of the increased metabolism generally noted after the ingestion of food. Experiments both on dogs and on men show that foUowing the ingestion of food there is an increased muscle tonus as indicated by the pulse rate, and frequently by the respiration rate, showing that the animal is living on a higher metaboHc plane than formerly. The increased heat is thus a product of cell action, and the question as to its economic value acquires a new significance. A man asleep, with lowest heat production, is of little value to the world ; awake, with no external muscular activity, he has increased internal activity and is capable of intellectnal life. The ingestion of protein alone stimulates metabolism with the possibility of some differences in the kinds of protein. Carbohy- drates show rapid effects, not so great as protein, and different car- bohydrates give different results. The metabolism 12 hours after the last meal of a carbohydrate-free, fat-rich diet, with moderate amounts of protein, is much greater than the metabolism under sim- ilar time-conditions after a mixed diet with the same amount of protein. Diabetics with varying degrees of intensity of the disease show marked differences in the total metabolism 12 hours after the last meal. A high acidosis is coincident with a high metabolism. A carbohydrate-free, fat-rich diet, eaten by a normal individual, is accompanied by the presence of an acidosis and an increased metabolism. The evidence suggests that coincidental with what is commonly termed a " State of acidosis " there is present in the blood a substance or substances, probably of an acid nature, that stimulate the cells to a greater metabolism. ö' The influenae of foodstuffs and their cleavage products upon heat production GRAHAM LUSK If meat in large quantity be given to a dog, the heat production rises in the second hour almost to its maximum, reaches its maxi- mum in the third hour and continues at this level through the tenth 13Ö Biochemical Proceedings, Hygienic Congress [Sept. hour when it beglns to fall. In one instance the heat production during a morning hour was 22.3 calories, and after the ingestion of 1,200 grams of meat it had risen in the second hour to 36 calories, reaching 40 calories in the third hour at which level it remained through the tenth hour, after which it gradually feil to 25 calories in the twenty-first hour. During the second hour the nitrogen elim- ination was one third the maximal nitrogen Output as evenly main- tained between the third and tenth hours. The second hour also showed that the calculated non-protein respiratory quotient ranged between 90 and 99, which indicated that that part of the metabolism which was not due to protein, as calculated from urinary nitrogen, originated largely from carbohydrate. During the later hours, the increased heat production is proportional to the nitrogen in the urine. During a period of 15 hours, protein carbon was retained in the or- ganism and when the oxygen absorption as computed on the basis of such retention in the form of dextrose is compared with the actual oxygen absorption, the two agree within 0.9 per cent., whereas computed on the basis of carbon retained as fat, there is a discrepancy of 10 per cent. between the calculated and actual value. Administration of 50 grams of dextrose in 150 c.c. of water to a dog causes a rise of heat production from 16.2 to 20 calories, at which level it is maintained during the second, third and fourth hours, falling nearly to the basal level in the fifth hour. The skin temperature rises to a greater extent than the rectal temperature. The absorption from the intestine is completed in the fourth hour. The urine is scanty until the fourth hour when 100 c.c. are suddenly eliminated. The sugar content of the blood in per cent. rises in the first hour but becomes normal after that. After the first hour the percentage of hemoglobin in the blood falls but returns to nor- mal subsequent to the fourth hour. Hence, after sugar ingestion, osmotic phenomena cause an increased volume of blood. When the absorption is complete, the glycogenic function removes the dex- trose from the blood, and the blood returns to its normal composi- tion through the elimination of water by the kidney. Water alone or a Solution of salt or of urea have no effect on the metabolism, hence the increase in metabolism is probably due to the increased number of molecules of dextrose carried to the cells and not to I9I2] Lafayette B. Mendel 137 changes due to osmosis. Liebig's extract of beef is without influ- ence on the metabolism. Fifty grams of olive oil cause a consider- able increase in heat production. GlycocoU causes a very great in- crease in heat production, alanin also acts powerfully, leucin and ty- rosin less so and glutamic acid not at all. It is concluded that the heat production may be increased by increasing the quantity of sugar and fat reaching the cells, or it may be increased through the direct Stimulation of the cells by amino- acids, notably glycocoll and alanin. Nutrition and bone growth FRANCIS H. MCCRUDDEN The question of the nature of bone metabolism in health and disease is one that until recently can hardly be said to have been attacked experimentally. The pathologists, with one or two excep- tions, have generally considered bone as a dead tissue not undergo- ing metabolism, once it is laid down. This opinion has, of course, colored their views regarding the nature of the process in various bone diseases. In osteomalacia, for example, a disease in which there is a decrease in the mineral content of the bone, it has been supposed that the process is due to the action of an acid which dis- solves out the mineral constituents. Numerous investigations, chemical, histological and clinical, dur- ing the last few years have shown that these views regarding the nature of the process in osteomalacia and the nature of normal bone metabolism cannot be correct. Bone, like the other tissues, under- goes metabolism throughout life. Old bone is continuously being resorbed and new bone laid down. If the new bone laid down is not qualitatively of the right composition, the result may be rickets, osteomalacia, osteoporosis, or Osteitis deformans, depending on the age of the patient and other factors. The bones act as a störe of lime salts to be called on in time of need just as the subcutaneous tissue acts as a störe of fat and the liver as a störe of glycogen; and a flux of calcium from the bones started by a growing fetus, a hardening callus, metastatic bone formation, etc., may, under cer- tain circumstances, lead to decalcification enough to result in osteo- 138 Biochemical Proceedings, Hygienic Congress [Sept. malacia and similar conditions. An important factor is the degree to which overproduction takes place, a factor involved also in im- munity and, in fact, in all tissue repair. Other disturbances which may be said to involve quantitative dis- turbance in bone growth, — the rate of growth, — rather than dis- turbances in the qualitative character of the bone produced, are the various types of dwarfism. In some of these the failure to grow seems to depend on an absence of the " growing tendency " on the part of the bones; in others, some disturbance in the supply of lime salts available for bone growth seems to be at fault. The role of proteins in growth LAFAYETTE B. MENDEL Some of the views held in the past regarding the interrelation of the food supply and growth are no longer tenable. Growth has often been associated in a causal way with the relative abundance of protein in diet. The parallelism between the protein content of the milk of various species and rate of growth may, in the familiär cases be an example of correlation rather than of causation. Recent in- vestigations have shown that the assumed association of growth with high protein intake is not confirmed by the evidence at band. Growth is a function of the cells. This inherent capacity ap- parently cannot be exaggerated by feeding ; but growth can be held in abeyance by various conditions. These include inadequacy of the food supply in respect to both quantity and quality of the nutrients. Attention must be directed to the chemical as well as the energetic aspects of the problems involved. In the past physiologists have largely disregarded the relative values of the individual members of different groups of food substances in nutrition, owing to an igno- rance of the chemical characteristics of the individuals. In considering the uses of protein in the organism, the distinc- tion between the requirement for maintenance and that for growth must be clearly kept in mind. The development of a successful method of investigation by Osborne and Mendel has made it easy to approach some of the problems experimentally. The method was explained in detail. Normal rate of growth has been induced in I9I2] Lafayette B. Mendel i39 rats with dietaries containing various Single purified proteins. But not all proteins suffice to promote growth under otherwise favorable conditions. Some suffice for maintenance without growth, whereby a prolonged period of stunting, or suppression of growth, can be in- duced ; still other proteins are alone insufficient for the maintenance requirement. The capacity to grow is not lost even after comparatively long periods of dwarfing and a subsequent normal unimpaired rate of growth may be attained with a suitable protein dietary. Aside from the apparent nutritive inequalities of the different proteins, other incidental findings, such as the synthetic features in growth, and diverse questions raised thereby, present a multitude of view- points which may serve to direct further research in this field. Direct calorimetry of infants, with a comparison of the results obtained by this and other methods JOHN HOWLAND A discussion of the various methods for determining the ex- change of energy in infants with their advantages and disadvantages and their limitations. The results of direct calorimetry obtained with four children by means of a modified Atwater-Rosa-Benedict calorimeter. The carbon dioxide excretion, oxygen consumption and heat production of two essentially normal children. The effect of the Ingestion of food, and especially of an excess of protein. The effect of eighteen hours' fasting. The heat production and respira- tory exchange of two extremely emaciated children. Difficulty of comparing results with those obtained by other methods on account of the different conditions under which the experiments have been conducted, the different Information that has been supplied and also on account of the unsatis facto ry formulas for determining the surface areas of infants, which give errors of 20 per Cent, and more. A new, more accurate and simple formula for determining this. 140 Biochcmical Proceedings, Hygienic Congress [Sept. The distribution of soluble salts in living cells and the force^ Controlling it ARCHIBALD B, MACALLUM 1. The distribution of salts in living matter is held to be due to the forces that make the distribution of salts uniform in an ordinary Solution. These forces are the same as those which determine the distribution of the molecules of a gas in an enclosed space. Conse- quently, in a living cell the salts are supposed to be uniformly dis- tributed throughout the fluid of the cytoplasm, that is, the osmotic force, or the pressure exercised by the molecules and ions through- out the fluid, is due to this uniform distribution of the solute throughout the System. The quantity of a soluble salt present in living matter is, therefore, a measure of the osmotic pressure therein and hence exchange between the salts within and those without would, in all cases, if the cell membrane were permeable, develop so as to ad just the pressure equally within and without the cells. 2. This conception leaves wholly out of account the action of surface tension. Every particle of the colloid of which living mat- ter is composed presents to the fluid in which it is suspended an In- terface where the surface tension of the fluid is lower than such a fluid has at its free surface. In consequence the Gibbs-Thomson principle comes into Operation and there results a condensation of the molecules and ions of the solutes on the Interfaces of all par- ticles. As the united interfacial surfaces must, in relation to the total volume of the Solution or fluid, present a very great area, a very large proportion of each of the solutes must be so Condensed, and the general concentration is, accordingly, greatly reduced or brought to the vanishing point. This would reduce the osmotic pressure due to such solutes to a very low value or even to nil. 3. The degree to which concentration on surfaces or on In- terfaces obtains depends on the degree of diminution of the tension of the fluid at an Interface, but it also depends on the nature of the solute, for the concentration in case of certain salts greatly exceeds the value demanded by the Gibbs equation, while in other salts the ascertained value approximates the theoretical value. 4. Such surface condensation of the salts of living matter can. I9I2] Lafayette B. Mendel 141 in a number of cases, be demonstrated microchemically. In the case of potassium salts this Is especially feasible. They are in this way found Condensed in interfaces inside of living cells, and also on sur- faces in tissues, i. e., on the external surfaces of nerve cells, of renal, pancreatic and salivary tubules, and thereby relations are established which determine the processes of excretion and secretion of these salts. In such cases the potassium salts in the tissue fluids elsewhere than at the surfaces or interfaces are scarcely detectible microchem- ically. 5. Surface tension is, therefore, an all-important factor in de- termining the distribution of salts in living matter. The role which common salt and water assume in the nutrition of man HERMANN STRAUSS Common salt plays an important part as a regulator of the osmotic processes in the human organism, whereby the latter with greatest tenacity holds fast the percentage concentration of its fluids. Man can get along on relatively small quantities ( jE^ gm. ) of " salt required by the tissues." But the majority of civilized men con- sume much greater quantities of common salt, and the principal quantity taken in food plays the part of a " seasoning salt." There- fore, the reduction, in the diet, of common salt has its limits, since dis- turbances may ensue f rom too great a reduction. Where the supply is too abundant, the excess is excreted. As a result of reduction of ingested common salt, a diminution in the secretion of gastric juice has been noted in dogs. In diseases of the stomach in men, it has been proposed, in the case of lack of hydrochloric acid in the gastric juice, to introduce copious quantities of common salt ; in the case of increase in the secretion of gastric juice, to decrease the quantity of common salt in the food. But, in practice, with such a proce- dure, it has been possible to obtain only inconstant results. I myself have pointed out, that an excretory insufficiency of the renal function may be traced to a retention of common salt. Through the retention of water, this condition favors the develop- ment of dropsy, since the principal amount of the retained salt finds 142 Biochemical Proceedings, Hygienic Congress [Sept. lodgement in the organism in the form of a " seroretention," while only a small part is deposited in the form of a " historetention." In consideration of these established opinions, for a decade, I have recommended a limitation of the supply of salt in the food, and a medicinal Stimulation of salt-elimination, in the prevention and treatment of hydronephrosis. The Situation, with regard to uncomplicated diseases of the heart (as well as incipient compensation disturbances in subjects of heart disease) is different from that in cases of parenchymatous nephritis. Also, in inflammatory discharges, and in ascites resulting from cir- rhosis of the liver, the circumstances are otherwise. In these con- ditions, the results of a deprivation of chlorin are very inconstant. For alimentation in diabetes insipidus, there have been established certain correct requirements similar to those laid down for parenchy- matous nephritics with an inclination toward dropsy. The signifi- cance of a limitation of salt as a means of lessening the thirst, in all cases in which there is a question of a decrease of fluid in the aliment, is now more highly appreciated than formerly. The question of dry retention of chlorin is now not wholly clear. At present, exact investigations as to the salt-content of the skin are lacking. Also, the relation of salt-retention to the development of uremia has not yet been fully explained. I should be inclined at this time to State only that, ccoteris parihus, uremia occurs more readily in the nephritic organism which is poor in water, than in one where water is abundant, and I may also State that, in the vom- ited matter of uremics, an extraordinary quantity of common salt is found. Through recent researches, a marked relationship between bro- min and chlorin has also been brought to light. Bromid poisoning may be success fully treated by means of an abundant supply of common salt. In complete deprivation of salt, and, likewise, in thorough lim- itation of fluids, an increase in the disintegration of protein may be noted. On the other band, an increase in the combustion of fat cannot be shown. As a rule, salt-equilibrium is restored in 24-48 hours. On the contrary, following a previously sharp decrease in the supply of salt, it requires several days for the restoration of salt- 1912] Lafayette B. Mendel 143 equilibrium ; and, in extreme retention of salt, increased elimination may be checked for many days. It cannot be denied that many healthy persons consume too great quantities of common salt. Moderate amounts are not injurious. A certain quantity of salt, as seasoning, is permissible for civilized individuals accustomed to substances which stimulate the sense of taste. The choice of foods, with regard to disease CARL VON NOORDEN The discussion pertained to the lessons dietetlc therapy holds for US in its connection with various diseases and disease groups, and to the foods that are serviceable or a hindrance to the attainment of the end desired. Only the major groups of food substances, such as proteins, fats, carbohydrates, spices and salts were considered. 1. Obesity. Principle : Decrease in the caloric value of the food. This is best attained through a decrease or total exclusion of the supply of fat. Carbohydrates, where relatively plentiful in the diet, should be barred out. In anti-fat treatments, the amount of con- tained protein should, where possible, amount to not less than 100 grams. The supply of water must be curtailed only if the obesity is accompanied by disturbances of the circulation. 2. Forced alimentation. Principle : Increase of the caloric sup- ply over the diet for maintenance. Theoretically, it is all the same, whether the center of gravity rests upon a large supply of carbohy- drates or fat. In reality, 250 gm. of carbohydrate is seldom ex- ceeded, because most carbohydrate foods possess a very great vol- ume. The supply of protein may not ordinarily be increased be- yond 100-120 gm. By means of these, approximately 1,300 calo- ries, no satisfactory alimentative results may be obtained. The practical results depend always upon the increase in the supply of fat. In most cases, the latter may be increased to 250 or 300 gm. daily, and then increases in weight of about 2 kilos per week may be gained. 3. Gout and uric acid diatheses. Principle : Decrease in animal foods; eventually total exclusion of the same. It is useful, in the 144 Biochemical Proceedings, Hygienic Congress [Sept. case of every gouty patient, to undertake a separate "test of tolera- tion," and, from the result of this test, to establish the patient's diet 4. Diabetes mellitus. Principle : Avoidance of such foods as in- cite the organ of sugar-production, the liver cells, to increased for- mation of sugar. Every undue Stimulation of the sugar-forming organ has, as its result, not only an immediate lavish production of sugar, but also increases f or the f uture its morbid excitability ; while systematic care of the organ renders its recovery possible. There- fore, decrease, and, under some circumstances, total exclusion of the carbohydrates. Moreover, decrease of protein substances. It is essential, in every case of diabetes, to exactly determine under what dietetic regime and manner of living the least amount of superfluous sugar is formed. That order of diet is best under which the patient continues free from superfluous sugar. 5. Feverish diseases and morbus Basedowii. Principle: In both these diseased conditions there occurs an abnormal increase in caloric production. Simultaneously ensues a heightened sensibility in re- lation to the specific dynamic influence of proteins. In order to limit as far as possible the caloric production and the loss in weight, practical empiricism and theory likewise call for a scanty protein supply, while weight is gained by an ample provision of carbohy- drates. 6. Diseases of the digestive organs. Principle : A food supply which is sufficiently nourishing, while imposing as little tax as pos- sible upon the diseased organs. A discussion of the injurious effect of certain combinations of foods. Report upon enterotoxic neuritis. Attack by means of protracted pure milk diet. 7. Kidney diseases. Principle : As much rest as possible for the kidneys. The amount of the intake of those nutrient media whose products of metabolism leave the body through the kidneys, should be reduced. The proteins conie first in this regard. But this limitation should not be carried too far, since patients with chronic kidney diseases become anemic and weak if strict curtail- ment of the proteins is too long continued. Many spices irritate the kidneys, and indulgence therein must be limited ; the same is the case with regard to alcohol. Common salt and water severely tax the kidneys. I9I2] Lafayette B. Mendel i45 Final words : Warning against schematic employment of dietary precepts. An effort must be made, on the one band, to hold fast to the basic rules of nutrition-therapy, but, on the other, to duly take into account the individuality of the patient. Diet and metabolism in fever WARREN COLEMAN Empiricism has been a signal failure as a basis for the fever diet. Through studies of metabolism the Solution of this problem appears to be at band. With diets containing large amounts of carbohy- drate it is possible to bring typhoid fever patients into nitrogen equilibrium, or nearly so. The apparently excessive quantities of food required for the purpose are almost completely absorbed. The heat-production in typhoid fever, as determined by indirect calorimetry, averages about 35 calories per kilogram at absolute rest. Diets furnishing only sufficient energy to cover the heat-pro- duction do not Protect the body against nitrogen or weight loss. The explanation of this discrepancy has not yet been found. Respiratory quotients in typhoid fever below 0.65 to 0.70 appear to be due to errors of technique. The lowest quotient we obtained in the fasting state, during the febrile period, was 0.70. During the same period, patients on a füll diet gave quotients varying from 0.75 to 0.95 at short intervals after food. The quotient rises dur- ing the later stages of the fever and reaches i.o to 1.15 early in con- valescence. During a relapse, a quotient of 1.04 was obtained while the patient had a temperature of 102° F. (37.7° C). The oxygen consumption during the febrile stage varies between 4 and 6 c.c. per kilogram a minute. Compared with the amount used in the fasting stage, the oxygen consumption is not greatly increased by the quantity of food administered. The body burns carbohydrate by preference during fever as long as it is available. As indicated by a falling quotient, from 100 to 120 grams of lactose is, for the most part, consumed, or deposited in the glycogen depöts, after 4 to 5 hours. The optimum amount of carbohydrate in the fever must be determined for each patient in- dividually, but is always large. The optimum amounts of fat and protein have not yet been determined. 146 Biochcmical Proceedings, Hygienic Congress [Sept. A consideration of the unknown factors in the ill-effects of bad Ventilation YANDELL HENDERSON The facts regarding Ventilation present an extraordinary contra- diction. Fresh air, sunlight and dry cool climates exert a decidedly beneficial effect upon health. Ill-ventilated dwellings decrease vitality. In some persons under certain conditions even a few min- utes in a crowded room may produce acute ill-effects. As to how these effects are produced physiology has up to the present time afforded no satisfactory explanation. The evidence is almost en- tirely negative. The ill-effects of bad Ventilation can not be due to lack of oxygen. It is probable that they are not due in any con- siderable degree to excess of CO2. The idea that they are due to some poisonous substance contained in the expired air has in recent years been regarded as untenable. Recently this conception has been revived in a novel form by the brilliant work of Rosenau. Even Rosenau's investigations do not appear, however, to afford the Solu- tion of this problem. The recent investigation of Hill in England and of Flügge and his pupils in Germany makes it highly probable that the effects of fresh or vitiated air are brought about not by a direct action upon the lungs but indirectly through the skin. It appears probable that the temperature and moisture of the air sur- rounding the body are the essential Clements. According to the explanation to be suggested in this paper the condition of the skin exerts a potent influence upon the lungs. This may be in part a vaso-motor reflex acting upon the pulmonary circu- lation. More probably it is a chemical or hormone influence upon certain pulmonary processes. The evidence accumulated during recent years indicates that the lungs are not mere passive organs through which gases diffuse as through non-living membranes. The investigations of Bohr, of Haidane and his co-workers and of the recent Pikes Peak expedition all tend to indicate that the lungs are the seat of vital activities of great importance to health. Thus under certain conditions the lungs secrete oxygen into the blood, and it appears that considerable oxidation may take place in the blood during its passage through the pulmonary vessels. The evidence I9I2] Lafayette B. Mendel 147 available, although still far from complete, suggests that these pul- monary activities are indirectly but powerfiilly influenced through conditions affecting the skin, and that it is in this manner that Venti- lation influences health. The hygienic physiology of work in compressed air J. J. R. MACLEOD Although it is now a well-established fact that the Symptoms of Caisson disease and diver's palsy are due to the sudden liberation o£ bubbles of nitrogen in the blood and tissue fluids, on account of too sudden decompression, there are several peculiarities regarding the conditions which influence the safety of decompression about which there is still a certain degree of uncertainty. This is the case more particularly with regard to : ( i ) Whether the decompression should be uniform or in stages; (2) how long it should take in proportion to the time of the shift and the pressure employed; (3) the degree to which the breathing of oxygen increases the safety of decompres- sion. Although, as insisted on by Haidane and others, it is no doubt the case that " the absolute air pressure can always be reduced to half the absolute pressure at which the tissues are saturated without risk " yet, in practice, it has not been found that the method is in any way superior to that of gradual decompression. The time that should be taken in decompression depends on the length of the shift in the caisson, because the Saturation of the re- moter parts of the body with nitrogen continues for a long time after this has been attained in the blood and the more accessible tis- sues. Tables indicating what time should be allowed have been prepared by Haidane and by Japp. The advantages of breathing oxygen are not only that it accel- erates the diffusion of nitrogen out of the lungs and, therefore, out of the blood; but, if Symptoms have already appeared, it supplies enough oxygen to keep life going when the circulation is danger- ously obstructed by nitrogen bubbles. In using oxygen at higher pressures, its toxic action must however be kept in mind. Recompression, either by placing the caisson worker in a pres- sure Chamber or by having the diver descend again to a certain depth 148 Biochemical Proceedings, Hygienic Congress [Sept. whenever the first Symptoms appear, is by far the most efficient treat- ment, as both experiment and the experience of engineers testify. On account of the heat and the high relative humidity of com- pressed air, the worker in a caisson is under conditions which tend to lower his efficiency. Not only this, but his appetite is likely to suf- fer and his general condition after some time to deteriorate so that he becomes liable to infections if not to caisson disease itself. The Caissons should therefore be well ventilated and the wet bulb ther- mometer kept as low as possible. Means for doing this were dis- ciissed. In the choice of men for caisson work attention should be paid to age, body weight and f atness and while engaged in the work the men should be kept in good training. Certain aspects of the influenae of muscular exercise upon the respiratory System THEODORE HOUGH Muscular activity increases the respiratory exchange from three- to tenfold, thus making demands on the System comparable only with those of the more severe forms of dyspnea. In meeting the respiratory needs of the tissues there are secondary effects of hy- gienic importance, such as the increased aspiration of the thora'x upon the return of venous blood to the heart and also upon the flow of lymph in the larger lymphatics ; this increased lymph flow is feit in the interstitial Spaces of every organ in the body, thus favorably influencing the environment of every cell. The introduction with more vigorous exercise of physiological strain makes it important to inquire into the exact condition of the organism revealed by the accompanying respiratory phenomena. Of the conditions known to increase the work of the respiratory Center Geppert and Zuntz have excluded, as exciting causes of the in- creased breathing movements of muscular activity, afferent Impulses from the working muscles and deficiency of oxygen in the arterial blood; their work also shows a decrease of the total {i. c, free and combined) carbon dioxid of the arterial blood; it does not establish a diminished tension of this gas in the respiratory center, and it is possible (Haidane) that there may be increased tension of this gas in igi2] Lafayette B. Mendel I49 the Center along with a fall of the total amount in the blood. No direct determinations of the condition of the blood in this respect have been made. Determinations of the alveolar tensions of oxygen and carbon dioxid during and at varying periods after muscular activity show that with increasing intensity of work there is first a rise of CO2 tension, then a fall to and below normal. In the latter case the CO2 tension sinks still further after the cessation of the exercise and may remain subnormal for over half an hour or even an hour. Review of the evidence as a whole leads to the conclusion that during more vigorous exercise the main cause of the increase of breathing movements is some catabolite (other than CO2) of the working muscle. During moderate exercise the increase of CO2 tension of the blood is probably an adequate explanation ; the respi- ratory condition of the organism would thus differ not only in degree but also in kind with moderate and with more vigorous exercise. Theory that the distress which is relieved by " second wind " is due to excessive CO2 tension not well established by the evidence at band, but worthy of further study. Bearing upon this question is the effect of previous Inhalation of oxygen in lessening the distress of maximal effort. Should not the measurement of respiratory power in physical examination be extended so as to include not only the anatomic features of ehest expansion and vital capacity, but also the ability of the respiratory System to meet successfully the conditions of the more vigorous forms of muscular activity? Yale University, New Haven, Conn. MEETINGS OF THE SECTION ON BIOCHEM- ISTRY, INCLUDING PHARMACOLOGY (VIII, D), OF THE EIGHTH INTERNATIONAL CON- GRESS OF APPLIED CHEMISTRY Proceedings reported by THE Secretary, JOHN A. MANDEL I. LIST OF OFFICERS OF THE BIOCHEMICAL SECTION President, John J. Abel; Vice President, William J. Gies; Secre- tary, John A. Mandel; Executive Committee: Reid Hunt, Thomas B. Osborne and the officers. IL SECTIONAL PROGRAM^ The meetings of the Section were held on September 6 to Sep- tember 12, inclusive, at Columbia University, in Room 301 of Havemeyer Hall. The morning sessions were opened at 10 o'clock and the afternoon sessions at i o'clock. Friday morning, September 6. In the chair: The Vice President. Julius Stoklasa: Ueber die photochemische Synthese der Kohlenhydrate unter Einwirkung der ultravioletten Strahlen. — L. Marchlezvski: The present State of our knowledge of the relation- ship of the chemistry of the blood coloring matter and Chlorophyll. — L. Marchlezvski and C. A. Jacobson: On the quality of Chlorophyll and the variable ratio of the two constituents, and on methods for determining this ratio. — *Guido M. Piccinini: II manganese del punto di vista delle funzioni enzimatiche. — ^ Jules Wolff: Sur la re- sistance de la Peroxydase ä l'ammoniaque et sur son activation par contact avec l'alcali. — *Jides Wolff: Sur une nouvelle fonction du catalyseur dit " Peroxydase" et sur le transformation biochemique de l'orcine en orceine (page 53). — Walter Jones: Some new phases *The asterisks indicate the papers which were actually presented. Some of the titles were received after the official program had been printed and are included here informally. Abstracts of most of the papers were published in Volume 19 of the preliminary report of the proceedings of the Congress. 150 1912] ■ John A. Mandel 151 of the nuclein fermentation. — Carl Voegtlin: Further studies in biologic oxidations. — "^Walter R. Bloor: Fatty acid esters of glucose. — C. C. Guthrie: A comparative study of the action of Solu- tions on the preservation of the vitality of tissues. — *R. Delaimay and O. Bailly: Les pepsines fluides etude du sediment qui se produit dans certaines d'entre elles. — William J. Gies: Modified collodion membranes, with demonstrations. Saturday morning, September 7. In the chair: Prof. Mauthner, of Vienna. ^Gabriel Bertrand and F. Medigreceanu: Sur la presence normale du manganese chez les animaux. — *M, Lin- det: Sur les elements mineraux de la caseine du lait. — *P. Malvezin: La question de l'acide sulfureux dans les vins blancs. — *Z. Mimu- roto: Ueber das Vorkommen von Adenin und Asparaginsäure in Maulbeerblättern. — U. Suzuki and 5". Matsunaga: Ueber das Vor- kommen von Nikotinsäure (m-Pyridinkarbonsäure) in der Reis- kleie. — Zozo Sakaguchi: Ueber den Fettgehalt des normalen und pathologischen Harns. — W. N. Berg: Effect of sodium chlorid and cold storage upon the activities of proteolytic enzymes. — *Thonias B. Aldrich: The iodine content of the small, the medium and the large thyroid glands of beef, sheep and hogs. — *Lezvis W. Fetzer: The chemical changes taking place in milk under pathological con- ditions. — *Ma.Y Kahn: A study of the chemistry of renal calculi, Monday morning, September 9. In the chair : The Pres- ident. *M. Nicloux: Moyen de caracteriser de petites quantites d'alcool methylique dans le sang et dans les tissus. — Zennoshin Hatta: Zur Kritik der Zuckerbestimmungsmethode nach Ivar Bang. — Munemichi Taniura: Zur Prüfung der Kumagawa-Sutoschen Fett- bestimmungsmethode in Bezug auf die Oxydation der fettsäuren und unverseif baren Substanzen im Verlaufe des Verfahrens. — Yuji Sueyoski: Eine neue approximative Eiweissbestimmungsmethode bei Albuminurie. — "^ Franz Herles: Schnelles Verfahren zur Bestim- mung der Harnsäure im Harn. — W. Worth Haie and Atherton Seidell: The comparative estimation of epinephrin in suprarenal glands and in its Solutions, physiologically and by color tests. — Lyman B. Stookey: The Cammidge reaction. — ^Herbert H. Bunzel: Oxidase determinations. — /. P. Atkinson: On the Separation of cer- tain alkaloids from nerve tissue. — *W. H. Schidtz and Atherton 152 Biochcmical Procccdings, Chemical Congrcss [Sept. Scidcll: The determination of thymol in dog feces. — "^Shiro Tashiro: A new apparatus for the detection and estimation of exceedingly minute quantities of carbon dioxide in biological materials. — "^Shiro Tashiro: Carbon dioxide production in the nerve fibre during an excitation. Its apphcation for detection of life in protoplasm. — *F. Klein: Die selenige Säure — ihr Verhalten gegen Eiweiss und tierische Haut. — ^Gabriel Bertrand and H. Agulhon: Sur la presence normale du bore chez les animaux. Monday afternoon, September 9. In the chair : The Pres- ident. "^ Felix Ehrlich: Ueber einige chemische Reaktionen der Mikroorganismen und ihre Bedeutung für chemische und biologische Probleme. — ^Gilbert T. Morgan and E. Ashley Cooper: The influ- ence of the chemical Constitution of certain organic hydroxyl and aminic derivatives on their germicidal power. — Takaoki Sasaki: Ueber den Abbau einiger Polypeptide durch Bakterien. II. Unter- suchungen mit nicht verflüssigenden Bakterien. — Naganiichi Shi- bata: Zur Frage der Fettzersetzung durch einige Saprophyten. — *A. Trillat: Influence des impuretes gazeuses de l'air sur la vitalite des microbes. — *M. Javillier: Influence exercee par le zinc sur Vas- pergilliis niger au point de vue de l'utilisative par la plante. — *Car/ L. Aisberg and O. F. Black: Biochemical and toxicological studies upon Penicillium stolonifernm. Tuesday morning, September 10. In the chair : The Pres- ident. *M. Mane: Relations de la plante avec les Clements fertili- sants; loi du minimum et loi des rapports physiologiques. — *M. Gerber: Etüde comparee des pressures des Vamanite phalloide et de Vamadoiivier. — *i?. Dubois: Sur l'atmolyse et sur l'atmolyseur. — */?. Dubois: Recherches sur les vacuolides de la purpurase. — *i?. Dubois: La biophotogenese reduite a une action zymasique. — Oszvald Schreiner: The physiological röle of organic constituents in plant metabolism. — *C F. Langworthy : The study of problems of vegetable physiology by means of the respiration calorimeter; a progress report. — ^Hozvard S. Reed: The enzyme activities in- volved in certain plant diseases. — ^Ernest D. Clark: Origin and sig- nificance of starch. — William J. Gies: Studies of diffusion, with demonstrations. Tuesday afternoon, September 10. In the chair.- The jgi2] John A. Mandel 153 President. *P. Carles: Les phosphates et le son de froment dans ralimentation animale. — *P. Carles: Entretien du tissu dentaire par une alimentation appropriee. — Minoru Maeda: Versuche über die Ausnutzung von " Konnyak " (einer japanischen Speise). — *PanlE. Hozve and Philip B. Hawk: The utilization of various protein foods by man after repeated fasting. — *L. F. F oster and Philip B. Hawk: A study of the utilization of ingested food when undermas- ticated ("bolted") and overmasticated ("fletcherized"). — S. P. Beebe: The influence of the thyroid on the excretion of ammonia. — ^Andrew Hunter and Maurice H. Givens: Purin metaboHsm in the monkey. — William Salant and /. B. Rieger: The influence of alcohol on protein metabolism. — Jacob Rosenbloom: Chemical and pharma- cological studies of human duodenal contents. Wednesday morning, September 11. In the chair: The President. *M, Sauton: Nutrition minerale du bacille tubercu- leux. — * Walter J. Dilling: Charts of spectra representing visible and invisible bands of various hemoglobin derivatives, with explana- tory booklet. — *G. O. Higley: Some notes on the form of the curve of carbon dioxide excretion resulting from muscular work follow- ing forced breathing. — *G. 0. Higley: The influence of barometric pressure on the carbon dioxide excretion in man. — *Joseph L. Miller and Dean D. I.ezvis: Physiological action of the various anatomical components of the hypophysis. — Isaac Levin: Immunity and specific therapy in experimental Cancer. — Lafayette B. Mendel: The physio- logical behavior of lipoid-soluble dyes. — *5. B. Crohn: Experiences with duodenal and stool ferments in health and disease. — Hertnan M. Adler: Experimental production of lesions resembling pellagra. — William J. des: Studies of edema, with demonstrations. Wednesday afternoon, September 11. In the chair: The President. George W. Crile: Neuro-cytological changes resulting from the administration of certain drugs. — *G. A. Menge: Some new Compounds of the cholin type. — Reid Hunt: Physiological action of some new Compounds of the cholin type. — Arthur S. Loev- enhart: Further observations on the action of oxidizing substances. — W. H. Schultz: Pharmacological action of proteins and some of their derivatives. — */^. H. Schultz and Atherton Seidell: Subcu- taneous absorption of thymol from oils. 154 Biochemical Proceedings, Chemical Congress [Sept. Thursday morning, September 12. In the chair: The President. */. M. Fortesciie-Brickdale: The arylarsonates : their pharmacology considered from the experimental and practical stand- points. — *//. A. D. Jowett, V. F. L. Pyman and V. F. G. P. Remfry: The relation between chemical Constitution and physiological action, as exempHfied by the glyoxahnes, isoquinolines and acid amides. — Walther Straub: Pharmakologische Bedeutung der Zellmembranen. — Charles Baskerville: Inhalation anesthetics. — "^Thomas B. Al- drich: On feeding young white rats the anterior and posterior parts of the pituitary gland. — /. A. E. Eyster: The relation of calcium to the inhibitory mechanism of the heart. — Clyde Brooks: On the action of alcohol on the circulation. Thursday afternoon, September 12. In the chair: The President. Giovanni Bufalini: Reazioni caratteristiche del veleno del rospo (Bufo vulgaris.) — Giovanni Bufalini: Meccanismo dell' azione narcotici del chloridrine. — *E. Fonrneau and V. K. Ochslin: Chlorure de l'acide dichloroarsinobenzoique ; ethers des acides benz- arsineux et benzarsinique. — *C R. Marshall: The pharmacological action of brom-strychnins. — *C R. Marshall: The influence of hy- droxyl and carboxyl groups on the pharmacological action of nitric esters. — Isaac Adler: Studies on chronic adrenalin, lead and nicotin intoxications. — *Ivo Novi: II calcio e il magnesio del cervello in varie condizioni fiziologiche e farmacologiche. — *L. Launoy: Action de quelques amines, en particulier du chlorure et de l'hydrate de tetra- methylammonium sur la secretion pancreatique. — *R. Delaunay and O. Bailly: Examen critique des conditions d'essai des pancreatines medicinales. III. ATTENDANCE Among the many in attendance at one or more sectional meet- ings, the Secretary noted the presence of the colleagues named below : John J. Abel, T. B. Aldrich, C. L. Aisberg, J. P. Atkinson, W. N. Berg, G. Bertrand (Paris), Samuel Bookman, Harold C. Bradley, H. H. Bunzel, Ernest D. Clark, F. C. Cook, F. Ehrlich (Breslau), Frank R. Eider, B. G. Feinberg, Lewis W. Fetzer, M. S. Fine, Harry L. Fisher, A. O. Gettler, Wm. J. Gies, A. J. Goldfarb, R. A. Gortner, Isidor Greenwald, M. L. Hamlin, G. A. Hanford, 1912] John A. Mandel i55 Robert A. Hatcher, Philip B. Hawk, G. O. Higley, B. Horowitz, E. M. Houghton, Paul E. Howe, Reid Hunt, Max Kahn, F. Klein, P. A. Kober, W. M. Kraus, P. A. Levene, Isaac Levin, Alfred P. Loth- rop, Wm. G. Lyle, John A. Mandel, Samuel Matthews, T. Mauthner (Vienna), F. Medigreceanu, G. M. Meyer, Jas. L, Miller, G. T. Morgan (Dublin), Max Morse, Victor C. Myers, W. A. Pearson, F. B. Power (London), Howard S. Reed, A. I. Ringer, C. J. Rob- inson, Anton R. Rose, Jacob Rosenbloom, William Salant, Emily C. Seaman, Atherton Seidell, B. Setlik (Prague), H. C. Sherman, Torald Sollman, Matthew Steel, M. X. Sullivan, Shiro Tashiro, Rodney H. True, H, Vieth (Ludwigshavn), Charles Weisman, Louis E. Wise. University and Bellevue Hospital Medical College, New York City. SIXTH SCIENTIFIC MEETING OF THE COLUMBIA UNIVERSITY BIOCHEMICAL ASSOCIATION, AT THE COLLEGE OF PHYSICIANS AND SUR- GEONS, NEW YORK, JUNE 3, 1912 PrOCEEDINGS RePORTED BY THE Secretary, ALFRED P. LOTHROP The sixth scientific session (third "annual" meeting) of the Columbia University Biochemical Association was held at the Co- lumbia Medical School on the evening of June 3, 191 2. The execu- tive proceedings of this session were published on pages 570-573 of Volume I of the Biochemical Bulletin (June number). The scientific proceedings consisted of research Communications by members of the Association. Abstracts of the papers are pre- sented here (pages 158-187) in two groups: (I) Abstracts of papers on research by non-resident members^ and (II) abstracts of papers from the Columbia Biochemical Department and affiliated laboratories. The appended summary will faciliate reference tp the abstracts (1-44). A SUMMARY OF THE NAMES OF THE AUTHORS AND OF THE TITLES OF THE SUCCEEDING ABSTRACTS I Allan C. Eustis. On the physio- WiLLiAM N. Berg. The physico- logical action of some of the amins Chemical basis of striated-muscle produced by intestinal putrefaction. contraction. (i) (s) William N. Berg, with L. A. Rogers, Allan C. Eustis. Solubilities and C. R. Potteiger and B. J. Davis. action of ^-imidazolylethylamin and Factors influencing the flavors of the relation to asthma and anaphy- storage butter. (2) laxis. (6) Isabel Bevier, for Anna W. Wil- A. J. Goldfarb. On the production of liams. A study of ropy bread. (3) grafted multiple embryos. (7) Allan C. Eustis. On the toxicity of Max Morse. Non-toxicity of inor- guinea pig urine and its relation to ganic colloid Solutions upon protozoa. anaphylaxis. .(4) (8) ^ Members of the Association who were not officially connected with the Columbia biochemical department when the research was conducted. 156 I9I2] Alfred P. Lothrop 157 Max Morse, for L. B. Ripley. Larvae of Lepidoptera obtained with sul- furic acid. (9) Anton Richard Rose. A study of the metabolism and physiological effects of certain phosphorus Com- pounds in milk cows. (10) II David Alperin. Contribution to the knowledge of nucleoprotein metab- olism, with special reference to uri- colysis and to the properties of uricase. (11) George D. Beal and George A. Geiger. The comparative diffusibility of vari- ous pigments in different solvents. (12) Stanley R. Benedict. The occur- rence and estimation of Creatinin in urine. (13) Louis E. Bisch. An endeavor to pre- pare Phrenosin from protagon. (14) Louis E. Bisch. Mucoid-silver prod- ucts. (15) Sidney Born. Protein-copper prod- ucts. (16) J. J. Bronfenbrenner and Hideyo NoGUCHi. A biochemical study of the phenomena known as comple- ment Splitting. (17) Ernest D. Clark. Notes on the chemical natura of Lloyd's " tannin mass." (18) Walter H. Eddy. A study of some protein Compounds. (19) Walter H. Eddy. The preparation of thymus histon. (20) Frank R. Elder and William J. Gies. The influence of proteases on the swelling of collagen and fibrin par- ticles in alkalin and acid media con- taining a biological electrolyte. (21) William J. Gies. A convenient form of apparatus for demonstrations of osmotic pressure exerted by lipins. (22) William J. Gies. Some interesting properties of thymol. (23) William J. Gies. A convenient method of preparing starch that swells rapidly in water. (24) R. f. Hare. A study of the carbo- hydrates of the prickly pear and its fruits. (25) Henry H. Janeway and William H. Welker. The relation of acapnia to shock. (26) Max Kahn. Biochemical studies of sulfocyanate. (27) Max Kahn. The chemical Constitu- tion of renal calculi. (28) Max Kahn and Jacob Rosenbloom. The colloidal nitrogen in urine from a dog with a tumor of the breast. (29) Max Kahn and Frederic G. Good- ridge. A non-protein, colloidal, ni- trogenous substance in milk. (30) John L. Kantor. A biochemical test for free acid, with a review of the methods for estimating the various factors in gastric acidity. (31) Marguerite T. Lee. A study of modi- fications of the biuret reagent. (32) Alfred P. Lothrop. A chemical study of salivary mucin. (33) C. A. Mathewson. A study of some of the more important biochemical tests. (34) Jacob Rosenbloom. A quantitative study of the lipins of bile obtained from a patient with a biliary fistula. (35) Jacob Rosenbloom and William Weinberger. Effects of intraperi- toneal injectionsof epinephrin on the partition of nitrogen in urine from a dog. (36) Oscar M. Schloss. A case of allergy to common foods. (37) Carl A. Schwarze. The comparative enzyme content of green and varie- gated leaves of Tradescantia. (38) 158 Proceedings CGlumhia Biochcmical Association [Sept. Emily C. SE.^MAN. Biochemical studies of beryllium sulfate. (39) Clayton S. Smith. Chemical changes in fish during long periods of cold storage (40) William Weinberger. An attempt to sharpen the end point in Benedict's method for the quantitative deter- mination of sugar in urine. (41) William H. Welker. Diffusibility of protein through rubber merhbranes, with a note on the disintegration of collodion membranes by common ethyl ether and other solvents. (42) Charles Weisman. A further study of the Bardach test for protein. (43) Harold E. Woodward. A study of the surface tension of dog blood-serum by the drop-weight method. (44) I. ABSTRACTS OF PAPERS ON RESEARCH BY NON- RESIDENT MEMBERS^ 1. The physico-chemical basis of striated-muscle contrac- tion. William N. Berg. {Washington, D. C). Part I was published in the June issue of the Biochemical Bulletin; part II is presentcd in this issue} 2. Factors influencing the flavors of storage butter. Wil- liam N. Berg, with L. A. Rogers, C. R. Potteiger, and B, J. Davis. {Dairy Division Research Laboratories, Bureau of Aninial Industry, Washington, D. C.) The official government bulletin on this subject is in press. 3. A study of ropy bread. Isabel Bevier, for Anna W. Williams. {Research Laboratory, Department of Household Sci- ence, University of Illinois, Urbana, III.) Published in fidl in the June issue'^ of the Biochemical Bulletin. 4. On the toxicity of guinea pig urine and its relation to anaphylaxis. Allan C. Eustis. {Laboratory of Clinical Med- iane, Department of Nutrition, Tidane University, New Orleans, La.) The nrine of guinea pigs, fed on Kohlrabi or cabbage, con- tains a great excess of indican, which readily oxidizes to indigo. Such urine also contains excess of putrefactive amins. Tests for /8-imidazolylethylamin, as well as efforts to isolate it, have been negative. Experiments on fifteen guinea pigs weighing 300 grams each, with different specimens of guinea pig urine, indicate that 1.5 c.c. constitutes a lethal dose when injected intravenously. In these ^ Members of the Association who were not officially connected with the Columbia biochemical department when the research was conducted. ^Berg: Biochemical Bulletin, 1912, i, pp. 535-7; ii, pp. loi-io. * Williams : Biochemical Bulletin, 1912, i, pp. 529-534. I9I2] Alfred P. Lothrop i59 animals, the Symptoms were identical with those observed after in- jections of /8-imidazolylethylamin. There was no delay in coagu- lation of the blood, but there was marked lowering of blood pres- sure and lowering of body temperature. After intravenous injections of filtered giiinea pig iirine into three dogs, Symptoms resembUng those of anaphylactic shock were exhibited, but the fall in blood pressure was not constant as it is after anaphylactic shock, and there was no delay in the coagulation of the blood. There is evidently some relation between the occur- rence of putrefactive amins and anaphylactic shock, but the writer's results do not bear out Pfeiffer's opinion regarding that relation. 5. On the physiological action of some of the amins produced by intestinal putref action. Allan C. Eustis. (Laboratory of Clinical Medicine, Department of Nutrition, Tulane University, New Orleans, La.) Putrescin (tetramethylendiamin) and cada- verin (pentamethylendiamin), in doses as small as o.i mg., are in- stantly fatal when injected intravenously into guinea pigs. Non- fatal doses produce marked lowering of blood pressure, dyspnea from edem.a of the lungs, salivation and prostration. The pulse is quickened. Phenylethylamin is immediately fatal to a guinea pig weighing 300 gm. when 0.05 gram is injected intravenously; 0.03 gram was fatal in two minutes when injected intravenously into a 300 gram guinea pig, with immediate prostration and paralysis of the respira- tory center ; 0.02 gm. produced a distinct chill in a 300 gram guinea pig, followed by prostration but with ultimate recovery. ß-imidasolylethylamin, in doses of o.oi gram intravenously, caused death in three minutes with typical anaphylactic Symptoms, the animals dying in attacks of forcible inspiratory effort, the heart continuing to beat after the respiration had ceased. Parahydroxyethylamin, as well as isoamylamin, produced marked rise in blood pressure. 6. Solubilities and action of /?-imidazolylethylamin and the relation to asthma and anaphylaxis. Allan C. Eustis. (Lab- oratory of Clinical Medicine, Department of Nutrition, Tiüane University, New Orleans, La.) I. A specimen of chemically pure )S-imidazolylethylamin, obtained through the courtesy of Dr. Dale i6o Proccedings Columbia Biochemical Association [Sept of the research laboratory of Burroughs, Welcome & Co., was in- soliible in cold Chloroform, benzene, tolnene, amyl alcohol, but slightly soluble in xylol, easily soluble in methyl alcohol, and soluble in cold carbon disulfide and hot amyl alcohol. Aqneous Solutions were tested with several reagents, to dis- cover if possible some means of detecting the presence of /?-imida- zolylethylamin in the tissties or blood, as follows: Bromine water, no precipitate, no coloration; copper sulfate, negative; potassium ferrocyanid, negative; Paidy's reagent, cherry red coloration; pic- ric acid, yellow precipitate insoluble in water, alcohol, ether, xylol and toluene, but which gave the positive Pauly reaction; phospho- tungstic acid, gray-blue precipitate, soluble in barium chloride Solu- tion, and in barium hydroxid Solution, which gave a positive Pauly reaction; sodiiim nitrite, negative; magnesium sulfate, negative; niercuric chlorid, negative ; gold chlorid, negative. Efforts to detect, by microchemical means, the presence of ß-\m\- dazolylethylamin in the bronchioles of guinea pigs dying from ana- phylactic shock, were without results. IL Tests of the physiological action of /J-imidazolylethylamin were conducted upon rabbits, guinea pigs and dogs by intravenous, subcutaneous and intraperitoneal injections. Intravenous injections of 0.5 mg. in guinea pigs caused immediate respiratory embarräss- ment, lowered blood pressure and diminished body heat, the animal dying in six minutes from suffocation due to complete occlusion of the bronchioles ; it being impossible to either f orce air into the lungs or to withdraw air, after the contraction had become complete. The Symptoms were typical of anaphylactic shock, and the post- mortem examination revealed the presence of enormous emphysema, the heart continuing to beat long after respiration had ceased. In dogs and rabbits there was also a lowering of the blood pressure and some respiratory embarrassment, but the occlusion of the bron- chioles was not as complete as in guinea pigs. Stibctttaneons and intraperitoneal injections were much less toxic and, in some instances, were entirely negative, suggesting that the tissues are able to utilize /?-imidazolylethylamin. The writer has seen many cases of asthma relieved entirely along dietetic lines by a " low protein " diet, and empirically has I9I2] 'Alfred P. Lothrop i6i found that red meats predispose to asthmatic attacks. )8-imidazo- lylethylamin is produced in the putrefactioii of histidin, and hemo- globin yields a large percentage of histidin on decomposition. It is possible, therefore, that /3-imidazolylethylamin causes asthma. Un- like clinical asthma, however, experimental asthma produced by iß-imidazolylethylamin is not relieved by injections of epinephrin ("adrenaHn chlorid"). 7. On the production of grafted multiple embryos. A. J, GoLDFARB. (Marine Biological Lahoratory, Woods Hole, Mass., and the Department of Natural History, College of the City of New York.) Grafted multiple embryos were first successfully produced in considerable numbers by Driesch, with the eggs of either of two genera of echinoderms, namely, Echiniis and Sphaere- chinus. Though several investigators have endeavored to repeat these experiments with American echinoderms they have failed completely. By slightly modifying the Herbst-Driesch method as described below, an unusually large number of grafted multiple embryos and larvae were produced from the eggs of Arbacia punc- tidata. After removing the fertilization membranes, the eggs were placed either directly into a sodium hydroxid Solution, or first placed in calcium-free sea water, then in an alkaline liquid of the following composition: 4 to 20 drops of 0.5 per cent. sodium hy- droxid Solution in 200 c.c. of sea water. This treatment sufficed in Driesch's experiments with Echinus and Sphaerechinus, giving rise to about 4 per cent. of agglutinated and fused embryos. For Arbacia eggs it was necessary to Supplement this treat- ment by centrifuging the eggs in tubes with very narrow bores, so that the eggs whose outer surfaces had previously been gelatinized were compressed against one another. These eggs gave rise to about 40 per cent. of agglutinated and fused embryos and larvae. The multiple embryos of Arbacia, so produced, were of the same general character as those described by Driesch, such as true twins, incomplete fusions, and complete fusions of the respective embryos. 8. Non-toxicity of inorganic colloid Solutions upon pro- tozoa. Max Morse. (Boardman Laboratories, Trinity College, Hartford, Conn.) Colloidal platinum prepared by the Bredig 102 Proceedings Columbia Biochemical Association [Sept. method, in which the house current of iio volts was reduced to 70 volts by lamps in parallel and passed through glass-distilled water by means of platinum electrodes, was used as a medium in which cultures of Paramecium and other protozoa were permitted to rest. Drop-ctilture slides were also made of these cultures in hanging drops of the platinum black. In all cases there was no augmenta- tion of division-frequence or size of the organism, nor any evi- dence of toxicity. Attempts with a Solution of mastic in ether and alcohol, which gave beautiful pictures under the Dunkelf eldtheleuch- tung of Zeiss, were not clear in their results. The colloidal Solu- tion was dialyzed for seven days in a fish-bladder, which freed it from the ether and alcohol, leaving a colloidal mass with excellent brownian movement. However, there is good reason to believe that this is not toxic in any way on protozoa. No attempt was made to " ultra-filter " the colloidal Solutions, in order to study the effects of small and larger colloidal particles upon protozoa, because of the apparent indifference of the organisms to the mixed Solution. 9. Larvae of Lepidoptera obtained with sulfuric acid. Max Morse, für L. B. Ripley. (Boardjnan Laboratories, Trinity Col- lege, Hartford, Conn.) Larvae were obtained from unfertilized eggs of the moth, Cecropia, by painting them with Baker's conc. sulfuric acid (sp. g. 1.84) for from 3 to 6 seconds and immediafely washing in pure water until entirely free from the acid. They were then left to dry and to develop. Checks were made by treating one- half of the batch from a given female with the acid and leaving the other half untouched. The females had been raised and isolated, from cocoons. The typical blueing of the developing eggs could be observed in the early stages of the eggs treated with acid while the control eggs remained white. The larvae emerged sev- eral days later in the case of the artificially fertilized eggs than in those normally fertilized. The percentage of errors was low. The larvae after emerging from the eggs were fed upon wild cherry, but thus far they have not been carried to the adult stage. This is now being tried. Petrunkevitch, Tichomorow and others have succeeded in obtaining larvae from silk-worm eggs by artificial means, but Cecropia has thus far failed to yield larvae under arti- ficial conditions. Short exposure and thorough washing may be the key to the success obtained in the present case. I9I2] 'Alfred P. Lothrop 163 10. A study of the metabolism and physiological effects o£ certain phosphorus Compounds in milk cows. Anton Richard Rose. (New York Agricultural Experiment Station, Geneva, N. Y.) The phosphorus requirement of a cow, aside from the milk phosphorus, would seem from the results of this experiment to be about 26 mg. per kilo of body weight. When the phosphorus supply is less than this amount, the physiological functions are con- tinued at the expense of the phosphorus previously stored in the tissues. Storage takes place when a greater amount than that indi- cated above is ingested. When the ingested insoluble phosphorus did not exceed 14 grams per day, there was approximate regularity in the phosphorus elimination in the feces independent of the In- gestion, suggesting that all the forms of phosphorus were digested, with liberation of phosphates; also that the fixed phosphorus of the feces was entirely due to the cellular matter from the mucosa and the intestinal flora. The soluble organic phosphorus in the feces was relatively slight in quantity, even in the periods when " phytin " was fed in liberal amounts. The calcium phytate added to the washed-bran ration was not utilized as economically as the " phytin " of the whole bran, and the "phytin" of the partially washed bran also gave a lower digestion coefficient. The addition of "phytin" to the "low phosphorus" ration in- creased the potassium Output in both feces and urine. The fecal potassium dropped in quantity when the "phytin" was withheld, but the urinary potassium did not. The amount of fecal mag- nesium was constant through the several periods except in the fourth, when it seems to have been influenced by the increased in- take of calcium phytate. At the beginning of the experiment the magnesium in the urine was equal to half that in the feces, but con- tinually decreased until the mobile magnesium of the body had been largely eliminated. The calcium in the urine increased remarkably when the phosphorus intake decreased. In the calcium phytate period, the calcium increase in the feces was approximately equiva- lent to the calcium increase in the rations. In all cases the addition of organic phosphorus to the " low phos- phorus " ration was followed by a decrease in the milk flow, and the withdrawal of this phosphorus from the ration was followed by a 164 Proceedings Coliunbia Biochemical Association [Sept. larger yield of milk. The percentage of fat in the milk fluctuated regularly with the changing amount of phosphorus ingested. The response was immediate, but the quantities of milk-fat bear no con- stant ratio to the amount of phosphorus in the rations. Aside from those pertaining to the fat, there were practically no changes in the composition of the milk, not even in the percentage of phosphorus in the fat-free solid matter. The moisture relations in the problem seem significant, though the intake and outgo of water could not be accurately measured in this experiment. The margin, after allowing for the influence of temperature, leads one to suspect a large retention of water in the last two periods. Up to the sixtieth day there was no outward sign of any physio- logical disturbance, but about that time the appetite began to wane. On the seventy-seventh day the milk-flow declined rapidly and serious trouble developed. A few days later the cow was placed in a box-stall and fed alfalfa, silage and vvheat bran, which caused all signs of malnutrition to disappear in the course of a week and also increased the milk-flow. IL ABSTRACTS OF PAPERS FROM THE COLUMBIA BIOCHEMICAL DEPARTMENT AND AFFILIATED LABORATORIES 11. Contribution to the knowledge of nucleoprotein metab- olism, with special reference to uricolysis and to the properties of uricase.^ David Alperin. The author studied the relative efficiency of the Wiener, Rosell, Croftan, Wiener and Wiechowski, and Galeotti methods for the preparation of uricase, and indicated the properties of the products. Wiener and Wiechowski have sug- gested that the subcutaneous or intravenous administration of uricase preparations is an effective procedure for the eure of gout and allied diseases. The author concludes that "practical demon- stration of the efficiency of this method of treatment has not been made." 12. The comparative diffusibility of various pigments in different solvents. George D. Beal and George A. Geiger. (Piiblishcd in füll in this issue of the Biochemical Bulletin.)^ "Alperin: Dissertation, Columbia University, 1912. 'Beal and Geiger: Biochemical Bulletin, 1912, ii, pp. ydr-^. 1912] Alfred P. Lothrop 165 13. The occurrence and estimation of Creatinin in urine/ Stanley R. Benedict, l'he work contemplates a thoroiigh inves- tigation of the question as to whether the Jaffe reaction in urine is due entirely, as is usually assumed, to the form of Creatinin which is ordinarily isolated from urine, or whether other substances may not be partially responsible for the reaction. The results indicate that there are two (or more) forms of Creatinin in urine, both of which yield the Jaffe reaction and also a zinc chlorid Compound, but which differ from each other in certain specific properties. A change in the ratio between these two forms of Creatinin in the urine has been observed in certain abnormal conditions. The most marked change was noted in inanition. There is probably a third substance contributing to the Creatinin reaction of urine which is in no wise related to Creatinin, but appears to be a weak acid. The study is in progress. 14. An endeavor to prepare phrenosin from protagon.^ Louis E. Bisch. Thudichum's method^ of isolating phrenosin has apparently never been reviewed. It was assumed that this method could be applied with success directly to protagon. The author was unable to do so, however. Repetitions of each of the numerous Steps in the process, with as much as 1450 grams of protagon at a time (in faithful accord with Thudichum's description), failed to yield sufficient material with which to complete the directions. It is possible that losses, which seem to have occurred at all stages of the process, totally consumed any phrenosin that existed in the orig- inal protagon. It is Dr. Gies' Intention to study this possibility further. 15. Mucoid-silver products.^^ Louis E. Bisch. Mo'ist, a-cid- free tendomucoid, triturated with a moderate amount of moist, alkali-free silver oxid, yields a brown to black mixture which be- comes very viscid when a small volume of ammonium hydroxid So- lution is stirred into it. A mechanical excess of 10 per cent. ammonium hydroxid Solution converts the viscid mass into a brown ^ Under the auspices of the George Crocker Special Research Fund. * Bisch : Dissertation (Part I), Columbia University, 1912. * Thudichum : A treatise on the chemical Constitution of the hrain, 1884, pp. 136-8. "Bisch: Dissertation (Part II), Columbia University, 1912. i66 Proceedings Columbia Biochemical Association [Sept. to black Solution, from which free alkali and free silver can be removed by dialysis. The neutral Solution thus prepared appears to contain argent-ammonium-mucoid, which may be obtained by precipitation with alcohol or by direct desiccation. The aqueous Solutions of these products are similar to those of argyrol in many respects yet appear to keep indefinitely. The purified material is antiseptic, and retards the growth of plants, but is seemingly non- irritant to the Cornea or other animal tissues. Fairly large quan- tities fail to induce toxic effects when injected subcutaneously or intravenously into dogs. The product in aqueous Solution is decom- posed by acidification. The purified material yields about i6 per cent. of ash. The silver content will be given special attention in the near future. i6. Protein-copper products. Sidney Born. Concentrated aqueous Solutions of various indiffusible proteins, when rendered slightly alkalin with sodium hydroxid Solution and treated with a moderate quantity of copper sulfate Solution, exhibit the typical biuret reaction in marked degree, but the excesses of alkali and copper may be removed by dialysis and, as the process continues (although no color may appear in the diffusate), the deep "biuret color" slowly changes until finally a blue or green persists. The resultant protein-copper product was isolated by precipitation of such a Solution with alcohol or by its direct desiccation. The Pro- portion of copper in six products made from edestin, gelatin, and serum protein ranged from 4.2 to 6.3 per cent. Injected subcuta- neously into frogs, the edestin and gelatin products (1.3 c.c. of concentrated aqueous Solution in each case) caused death in three hours. The properties of the dialyzed Solutions and the products therefrom will be described in some detail later. 17. A biochemical study of the phenomena known as com- plement Splitting. J. J. Bronfenbrenner and Hideyo Nogu- CHi.^^ A. It is generally accepted that complement may be split into a mid-piece and an end-piece. The mid-piece is thought to be in the globulin fraction, and the end-piece in the albumin fraction. 11 Bronfenbrenner: Dissertation, Columbia, 1912; Bronfenbrenner and Noguchi : Journal of Experimental Mediane, 1912, xv, 598-643. Most of the work was conducted at the Rockefeller Institute for Medical Research. I9I2] 'Alfred P. Lothrop 167 The restoration of complement activity by putting together the albu- min and globulin fractions does not prove, however, that each fraction contained a part of the complement, for the albumin fraction can be reactivated in the absence of the globulin fraction. Complement-splitting as brought about by hydrochloric acid, carbon dioxid, and dialysis, is really an inactivation of the whole complement by certain acids or alkalis, either added in the free State to the serum, or liberated as a result of the dissociation of certain electrolytes. That the whole complement, and not a part only, is present in the albumin fraction of the serum can be demonstrated by the re- moval of the inhibitory action of the acid or alkali. This can be effected by the addition, not only of alkali or acid, but also of any amphoteric substance. When hydrochloric acid, carbon dioxid, or dialysis are employed to produce the phenomenon known as com- plement Splitting, the complement is merely inactivated, not split. B. Thus far, most investigators have made but little distinction between the Splitting phenomenon obtained by chemical interference and that which takes place in the biological phenomenon known as complement fixation. In this study we have shown that these two sets of phenomena exhibit certain fundamental differences and that the so-called complement Splitting by physical conditions leading to chemical interaction, or directly by chemical means, is not a real Splitting of the complement, but an inactivation of the active prin- ciple of complement through an alteration in the reaction of the medium caused by an excess of either anions or cations. The modi- fication of the reaction of the medium may cause a more or less definite combination of the complement with the free ions, but the latter can readily be removed by an appropriate number of opposite ions, and render the complement active once more. The fluids that have hitherto been regarded as containing the end-piece of com- plement, contain, as a matter of fact, the whole complement tem- porarily deprived of its activity by certain ions derived either from the Salt constituents of the serum itself under a modified physical condition (dialysis against water or dilution with water) or intro- duced in the form of dissociable electrolytes. On the other hand, the Splitting of complement in the fixation i68 Proccedings Columbia Biochemical Association [Sept. reaction seems far more complicated than that caused by the phys- ical or chemical procedures. The supernatant fluid from the fixa- tion test differs from all the other end-pieces prepared by chemical methods in being active upoii persensitized sheep corpuscles only (not upon human corpuscles). The addition of various mid-pieces, obtained by different methods, to sensitized sheep corpuscles does not render the Wassermann supernatant fluid active. It is quite remarkable that the persensitized sheep corpuscles are, on the other hand, easily attacked, not only by the supernatant fluids of fixation tests, but also equally well by the other end-pieces. It is not at all improbable that in the fixation reaction, where so many factors come into play, there is a most complicated physical as well as chemical interaction leading to such an entangled mixture of factors that a substance carrying one set of ions alone cannot reverse the activity of complement, and hence the reversion takes place only when cer- tain electrolytes with both ions are employed. At all events there seems to be no doubt that the inactivation of complement is far more complicated in the Wassermann reaction or the Bordet-Gen- gou phenomenon than in the inactivation by physical or chemical means. Nevertheless, no one has as yet proved conclusively that the supernatant fluid of a fixation test necessarily contains the end- piece of complement. i8. Notes on the chemical nature of Lloyd's " tannin mass." Ernest D. Clark. Chemical studies were made upon "tannin masses" prepared by Lloyd from the fruit of the persim- mon. The original material dissolved in alkalies to form a purple jelly-like Solution. In dilute mineral acid Solutions the "masses" turned bright red in color and no swelling was observed. Upon hydrolysis with 0.2 per cent. and 2.0 per cent. hydrochloric acid Solutions, cherry-red colorations were obtained. Such Solutions contained both tannin and phloroglucin in considerable proportions. The presence of phenolic substances like vanillin was also indicated. An insoluble gelatinous substance was removed, by filtration, from the hydrolyzed acid mixture and seemed to be cellulose or a related material. Hydrolysis with 0.5 per cent. and 5.0 per cent. sodium hydroxid Solutions gave thick, dark-colored liquids and large amounts of insoluble gelatinous residue. Alkaline hydrolysis pro- igi2] 'Alfred P. Lothrop 169 duced the same kinds of materials as those that resulted from acid hydrolysis. The "tannin masses" seem to be combinations of tannin and phloroglucin associated with cellulose-like substances. With ferric chlorid, phloroglucin gives a dark blue product but not the blackish precipitate characteristic of the tannin-ferric chlorid reaction. Theref ore, " iron reagents " do not detect tannin in the presence of phloroglucin. 19. A study of some protein Compounds. Walter H. Eddy. (Published in füll in this issue of the Biochemical Bulletin. )^2 20. The preparation of thymus histon. Walter H. Eddy. As outlined by Bang, the properties of histon may be summarized as follows : Water-soluble, non-coagulable by heat, precipitated by am- monia in the presence of salts, precipitated from neutral Solution by "alkaloidal reagents," produces precipitates of several soluble pro- teins from their aqueous Solutions. The current method of preparing thymus histon, as recom- mended in Standard handbooks such as Abderhalden's and Oppen- heimer's, may be summarized as follows : Extraction of the minced glands with water. Precipitation of the water extract by acid or calcium chlorid, and extraction of this precipitate with 0.8 per cent. hydrochloric acid Solution. Precipitation of the hydrochloric acid extract with ammonium hydroxid Solution, either before or after removing free hydrochloric acid by dialysis. Washing the "am- monia precipitate " free from ammonia with alcohol and ether. Kossei, who discovered histon in goose blood, obtained it by saturating the hydrochloric acid extract with sodium chlorid. He alone calls attention to the anomaly noted in our experiments, viz., that treatment with ammonium hydroxid Solution results invariably in the precipitation of a substance that is practically insoluble in water. In a series of many preparations, extending in time over a period of two years and involving materials obtained from many calves, we have come to the conclusion that the " ammonia-pre- cipitation" of a hydrochloric acid Solution of thymus histon results invariably in a water-insoluble product. Furthermore, our experiments show that of two fractions of the same hydrochloric acid Solution, the fraction saturated with sodium chlorid invariably ^^Eddy: Biochemical Bulletin, 1912, ii, p. 111-22. I/o Proceedings Columbia Biochemical Association [Sept. yields a prodtict which (when free from sodium chlorid) is water- soluble, gives all the qualitative histon tests, and contains less nitrogen than the "ammonia precipitate" from the other fraction; the " ammonia precipitate " being water-insohtble and appar- ently a very different stibstance. Finally, when the " sodium chlorid precipitate " of histon is dissolved in vvater, and the aqueous Solution is treated with a few drops of ammonium hydroxid Solu- tion, a precipitate is produced which is insoluble in water. Quanti- tative studies now under way show marked differences in the nitro- gen content of the two products. The results suggest that " histon " as commonly prepared is an adsorption product or a salt, rather than a simple protein. The following method is suggested as a means of obtaining water-soluble histon from thymus : Mince f resh thymus glands and extract the hash with distilled water for 24 hours (best in the cold), Precipitate the aqueous extract with acetic acid Solution and ex- tract the precipitate with 0.8 per cent. hydrochloric acid Solution (after Lilienfeld) ; or add sufficient calcium chlorid to the aqueous extract to make its content of that substance 0.2 per cent. and extract the precipitate with 0.8 per cent. hydrochloric acid Solution (after Huiskamp) ; or add sufficient hydrochloric acid to the aqueous extract to make its content of the acid 0.8 per cent. and let stand 24 hours (after Kossei and Kutscher). Filter off the hydrochloric acid extract, and either remove the free acid or precipitate the histon directly by Saturation with sodium chlorid. Remove ad- mixed sodium chlorid by dialysis. Filter the resultant salt-free Solution and evaporate it to dryness at 45° C. This material, ground to a powder, may be heated to 105° C. without loss of water-solubility. 21. The influenae of proteases on the swelling of Collagen and fibrin particles in alkalin and acid media containing a bio- logical electrolyte. Frank R. Elder and William J. Gies. (Published in full in the June issue of the Biochemical Bulletin. )^^ 22. A convenient form of apparatus for demonstrations of osmotic pressure exerted by lipins. William J. Gies. The " Eider and Gies : Biochemical Bulletin, 1912, i, pp. 540-545. I9I2] 'Alfred P. Lothrop 171 writer repeated the demonstration described on page 59.^^ Instead, however, of using a thin rubber bag in a muslin sheath, he employed a i2-inch section of ordinary bunsen-burner tubing. The rubber tube had been swollen to its maximum extension by immersion in ether for about an hour previous to its use. It was then closed at one end by the Insertion of a short, tightly fitting, section of a thick glass rod, which was fastened by a ligature. After the swollen tube had been filled with olive oil and a narrow glass tube about 10 feet in length (in two sections) had been tied into the open end and held upright, the rubber-oil portion of the vertical tubulär appa- ratus was completely immersed in ether in a tall, narrow cylinder. The oil began to rise in the tube almost immediately, and rapidly proceeded upward until the liquid emerged from the open top. 23. Some interesting properties of thymol. William J. GiES. During the course of recent experiments on enzymes as pos- sible factors in the development of edema/^ we had occasion to study the effect of trypsin on elastin in ammonium hydroxid Solu- tions containing a biological electrolyte (NaCl). To our surprise we not only failed to obtain the swelling results which we had pre- viously observed under similar conditions/^ but the elastin particles in use gradually became green, ultimately blue. With repeated shaking, the elastin particles were more deeply colored, and the supernatant liquid slowly became green; finally, bluish green. The color of the particles slowly diminished in intensity as the pigment accumulated in the liquid. Unlike the elastin used in the previous experiments, this product had been prepared about 10 years before. The fresh-ligament hash had been put in water and preserved there with considerable alcoholic thymol Solution; later, had been put in alcohol; ultimately, had been dried and bottled. The main supply of the dry elastin smelled strongly of thymol. Some of the above-mentioned green and blue ammoniacal liquids, when shaken with ether or toluene, were quickly transformed into purplish, then reddish mixtures. The ether layer on the quiescent liquid was bright red — all green and blue had disappeared from the " Gies : Biochemical Bulletin, 1912, ii, p. 55. " Eider and Gies : Ibid., 1912, i, p. 540. *' Tracy and Gies : Ibid., 1912, i, p. 472. 1/2 Proceedings Columbia Biochemical Association [Sept. alkalin liquid imderneath, which was colorless. By spontaneous evaporation, the ether extract yielded a purplish-red oily product, vvith a pronounced thymol odor. When a small quantity of thymol (Kahlbaum) was mixed with lO per Cent, ammonium hydroxid Solution, the liquid became green- ish in about 2 hours ; then gradually turned blue. Alcohol appeared to accelerate the transformation. Shaken with ether, the blue was wholly removed and a beautiful, red, ether-layer obtained. Such ether extracts yielded, by spontaneous evaporation, a purplish-red oily product, which dissolved readily in ether, toluene and alcohol, the Solutions being bright red. In some cases the oily product be- came crystalline, due apparently to the presence of unchanged thy- mol ( ?). The red alcoholic Solution was turned deeply bluish by a drop of n/io sodium hydroxid Solution; the red was restored by a drop of w/io hydrochloric acid Solution. These transformations could be elicited repeatedly in the same Solution. The changes were so sharp that the material may prove to be a valuable indicator for use in the titration of alcoholic liquids. Concentrated alcoholic Solu- tions yielded reddish white precipitates when they were diluted with water — a ready means of isolating the substance. The reddish white precipitate dissolved promptly in alcohol, ether and toluene, and formed a red Solution in each case. An excess of thymol, added to a green or blue ammoniacal Solu- tion in its original condition, completely changed the green or blue to red, and wholly dissolved the red material, behaving, in this respect, like toluene and ether. These phenomena did not appear to be due to impurities in the thymol. A general survey of thymol literature has not revealed the explanation of these results, although certain inferences are sug- gested by several color reactions of thymol. The chemical nature of the colored substances derived from thymol in these preliminary experiments, the possible Utility of the products — their probable antiseptic, pharmacologic and other rela- tionships, suggest numerous interesting biochemical inquiries which will be undertaken in the near future. 24. A convenient method of preparing starch that swells rapidly in water. William J. Gies. For the purpose of study- I9I2] ^Alfred P. Lothrop 173 ing the effects of amylases on the power of starch to imbibe water (prior to hydrolytic cleavage), the writer prepared markedly hydro- phylic starch in the following way: A very thick starch paste was speedily prepared by rapidly pouring a thicl<: potato-starch Suspen- sion through musHn into boiling water while the latter was being vigorously stirred. The vessel containing the paste was inimersed in ice water immediately after the last portion of starch Suspension had been added. By constant stirring of both liquids, and by the maintenance of a low external temperature, the paste was speedily cooled,^'^ when it was poured into, and thoroughly stirred in, a large excess of 95 per cent. alcohol. After the Sedimentation of the prod- uct, and the decantation of the alcoholic liquid, the snow white ma- terial was treated with fresh portions of alcohol until its viscidity disappeared and it became firmly granulär. After several washings with ether, to remove alcohol, the product was rapidly freed from ether in a current of air from an electric fan. Although somewhat hygroscopic, the material formed hard, snow-white masses which could be granulated easily in an ordinary pulverizer. Placed in water, the particles swell very rapidly into bloated glassy forms. " Starch paste " may be made almost instantly from the product. The powder can easily be freed from its soluble car- bohydrate impurities by dialysis. The material promises to be of special Service in many connections. Mr. Nathan Rosenthal has undertaken a study of the effects of amylases on the swelling of material of this kind in various anti-hydrophylic media, such as dilute alcohol. 25. A study of the carbohydrates of the prickly pear and its fruits. R. F. Hare.^^ The difficulties encountered in the practical laboratory Separation of the sugars from the mineral matter, muci- lages, gums and dextrinoid substances have been numerous, and the Operations time-consuming. Many attempts to obtain the sugars free and in crystalline form have usually resulted unsuccessfully; so that it became necessary to make the individual tests not on the sugar crystals, but on the syrups previously purified as much as pos- sible by different methods. " The Operations were conducted rapidly in order to prevent undue hydrol- ysis. It is probable that satisfactory results can be obtained by pouring the hot paste directly into alcohol. "Hare: Dissertation, Columbia University, 191 1. 174 Proceedings Columbia Biochemical Association [Sept. The Juice of the ripe fruit contains 1.57 per cent. of pentosans and only traces of galacfan. After precipitation with lead acetate, the Juice gave the anihne acetate reaction for pentose, but none for galactose. The presence of fructose and gliicose in considerable amounts was quite definitely estabhshed by several reactions char- acteristic of these sugars. The dried mucilage of the prickly pear, when separated by pre- cipitation with alcohol from a two per cent. Solution, contained 15 per cent. of galactan, 31 per cent. of pentosan and 12 per cent. of ash. The mucilage in the aqueous extracts could not be separated completely from cell fragments, starch, crystals of calcium Oxalate and other solid particles that caused opalescence and turbidity. A dilute Solution containing 1.5 per cent. of solid matter, rendered fairly clear by repeated filtration through silk, had no effect on polarized light. This was true of all the Solutions of mucilage ob- tained in this work, both before and after subjecting them to acid hydrolysis. Harley^^ reports having found a specific rotation of + 38° for Opuntia mucilage, but places little confidence in his own results, since the reading was made on a very dilute opalescent Solu- tion and calculated from an observed rotation of + 6 minutes. Hydrolysis of the mucilage by digestion for several hours with 1,25 per cent. sulfuric acid Solution produced a sugar that had properties similar to arabinose. When its osazone was formed, oily globules rose to the surface. The precipitate was darker than glucosazone, readily soluble in hot water and melted at about 160° C. A 95 per cent. alcoholic extract of the dried stems, previously treated with ether, contained a sugar with specific rotations made on three separate Solutions of — 6.6°, — 8.25°, and — 7.1°. The osazone produced from this sugar had properties similar to those of glucosazone. These results indicate the presence of glucose and fructose^ in this extract. A 60 per cent. alcoholic extract of the dried stems contained a suhstance apparently intermediate in character hetween mucilage and sugars. It did not reduce Fehling Solution before hydrolysis, but was very readily hydrolyzed by dilute acid Solutions. Alcohol stronger than 60 per cent. reprecipitated this material as a flocculent " Harley : Journal de Pharmacie, iii, pp. 6-193. I9I2] 'Alfred P. Lothrop 17 S mass, quite different in appearance and properties from the precipi- tate of the mucilage obtained with alcohol. The precipitate was readily soluble in water, but its Solution was not mucilaginous. When hydrolyzed, it gave a plus rotation to polarized Hght. The coloring matter can be concentrated and made into a mar- ketable product, of value for coloring certain foods, by first remov- ing mucilages and gums with alcohol, and precipitating the pigment from the filtrate with acetone. The pigment is evidently a gluco- side. When separated from the juice with alcohol and acetone, and then precipitated with lead acetate, the coloring matter liberated by sulfuric acid gave a glucose-like sugar on hydrolysis. The lead salt produced by precipitating the purified pigment with lead acetate con- tains 61.42 per cent. of lead. 26. The relation of acapnia to shock.^*^ Henry H. Janeway AND William H. Welker. Henderson has published a number of papers on the relation of acapnia to shock. He maintains that a diminution of the normal amount of carbon dioxide in the blood to a sufficient degree, and maintained for a sufficient length of time, produces an irreparable disturbance of the normal balance of osmotic forces between the blood and the cytoplasm of the body cells, and that this disturbance leads to tissue asphyxia, acidosis, and fatal oligemia, accompanied by Symptoms indistinguishable from shock. He be- lieves that the essential cause of shock is acapnia. He supports this theory, not only by very thorough work on the relation of acapnia to shock from several different Standpoints, but also by furnishing control experiments, as it were, in which shock is prevented by con- servation of the animal's störe of carbon dioxide and also by suc- cessful treatment of animals, already in a condition of shock, with injections of Ringer Solution containing carbon dioxide. Whether this theory fails to stand in whole or in part, its originator deserves the greatest credit for calling attention to the possibility that härm may arise from neglect to conserve the body 's störe of carbon di- oxide, the important functions of which, in the body, have long oxide, the important functions of which, in the organism, have long been appreciated by physiologists. This theory has been of the greatest interest to one of us because of the relation of acapnia to ^ Some of the work was done in the Surgical Research Laboratory of the College of Physicians and Surgeons. 176 Procccdings Columbia Biochemical Association [Sept. artificial respiration, and to the production of shock in connection with intrathoracic surgery. It has prompted us to investigate the degree of acapnia and the associated shock produced by excessive artificial respiration. We soon found that the diminution of carbon dioxide in the blood in ordinary intrathoracic insufflation was neghgible. On the other hand, it has been quite an easy matter for us to reduce the amount of carbon dioxide in the blood to from one-third to one-half the normal amount by forced rapid inflation and deflation of the lungs. The artificial respiration was performed 45 to 90 times a minute and was continued for periods varying from 30 minutes to 3 hours. These experiments have differed from those of Hender- son in that the animals were allowed to recover. The trachea was not divided but respiration was performed by inserting a large, rather tightly fitting, tube through the larynx into the uninjured respiratory tract. The blood pressures in our experiments were not accurately measured, the animals being left as nearly normal as possible after the Operations. The degree of shock was estimated entirely from the condition of the animals after the Operation and the manner in which they recovered from it. Judged in this manner there was nothing about these animals to indicate a serious degree of shock or any greater disturbance than could be accounted for. by three other factors to which we desire to call attention in connection with these experiments and which, unless guarded against, can alone cause considerable depression and even death. ( I ) In all experiments in which excessive artificial respiration is employed there is a great reduction in the animal's body heat. The temperature can easily fall to 85° F. (2) There is a very evident possibility (which we believe to be a fact) that the rapid and complete filling of the lungs exercises a definite interference with the return of the blood to the heart. The fall of the blood pressure, as estimated with the finger, and the rapidity of the heart's actioncoin- cide closely with the pressures used to inflate the lungs; indeed, a scarcely perceptible pulse may be immediately improved by slightly lowering the latter pressures. (3) The duration of the apnea fol- lowing these experiments depends as much upon the amount of mor- phin and ether administered as upon any other factor. We do not I9I2] 'Alfred P. Lothrop i77 believe that it is possible to produce death by apnea, caused in turn by acapnia, without the assistance of the toxic effects of morphin and ether. The toxic effects of these drugs must be included as factors contributing to the shock, This report deals with only one of the phases of the relation of acapnia to shock, namely the relation of acapnia, produced by ex- cessive artificial respiration, to shock ; and as it is only a preliminary report, it is not intended as an answer to Henderson's contention. Its purpose is mainly to record two general f acts : (A) That we have reduced the amount of carbon dioxide in the blood to nearly 40 per Cent, of the normal amount, and have maintained this reduc- tion for a period of 3 hours, without producing Symptoms of shock; and (B) that there are other factors than depletion of the störe of carbon dioxid, which, unless properly guarded against, can in them- selves cause the death of the animal under experimentation. 27. Biochemical studies of sulfocyanate.^^ Max Kahn. A. The ferric chlorid colorimetric test for sulfocyanate in saliva is inexact and unreliable. A negative result by the Bunting suction method is no evidence of the absence of sulfocyanate but a positive result is suggestive of the presence of a comparatively large amount. The pink color spontaneously disappears from the ethereal layer in positive tests by the Bunting suction method. Various medicinal substances, and also certain Compounds that result from biological transformations of proteins and carbohydrates, if excreted in the saliva, give a very marked red coloration in the ferric chlorid test, similar to that produced by sulfocyanate. B. Sulfocyanate occurs in the saliva and salivary glands of man, in the salivary glands of oxen, but apparently not in the salivary glands of dogs. It occurs in the blood, but the spieen, the pancreas, the thymus, the thyroid and the testicles of dogs do not contain it. The liver seems to be the gland in the body that contains most sulfocyanate, which is also present in bile and in the small intestines. The stomach contents of dogs on an ordinary diet were free from sulfocyanate. When, however, sodium sulfid was given, the gastric mixture contained sulfocyanate. '^Kahn: Dissertation, Columbia University, 1912. Conducted under the auspices of the Dental Society of the State of New York. lyS Proceedings Columbia Biochemical Association [Sept. C. Siilfocyanate is excreted in the urine and feces. Its elimina- tion in the urine is not dependent upon the amount in the saliva. Althoiigh dog saliva is apparently always free from sulfocyanate, dog urine invariably contains it. The ingestion of amino acids (alanin) and of nitriles (acetonitrile) increases the amount of sul- focyanate in the body, as well as in the excreta. Sulfocyanate seems to be produced in the body from protein. Results with a fasting dog harmonize with this conclusion. The ingestion of sulfur, so- dium Sulfid, thioacetic acid, thiourea and taurin did not increase the Output of sulfocyanate. D. Potassium sulfocyanate is toxic to both plants and animals. Its toxicity is so marked that indiscriminate dispensation of the sub- stance to people is dangerous. The growth of molds is enhanced by potassium sulfocyanate. Yeast fermentation is not affected or is stimulated by moderate proportions of potassium sulfocyanate. Biological proportions of potassium sulfocyanate have no inhibiting influence on the growth of bacteria. The souring of milk is inhib- ited by large proportions of sulfocyanate. 28. The chemical Constitution of renal calculi. Max Kahn. Sixteen stones of nephric origin were analysed according to the method of Mackarell, Moore and Thomas. ^^ Most of the stones were composed mainly of salts of calcium. All of the stones -con- tained uric acid or urates in varying amounts, but no stone was wholly composed of urates. The shape, color and consistency of a stone are not criteria of its chemical composition. Three gouty tophi were examined by the murexid test for urates. A negative response was obtained in each case, showing that not all gouty deposits are composed of uric acid salts. 29. The colloidal nitrogen in urine from a dog with a tumor of the breast. Max Kahn and Jacob Rosenbloom. (Published in füll in this issue of the Biochemical Bulletin ).2^ 30. A non-protein, colloidal, nitrogenous substance in milk. Max Kahn and Frederic G. Goodridge. Since the figure ob- tained for " total " nitrogen in milk exceeds the sum of the values for the known nitrogenous constituents, unknown nitrogenous sub- ^ Mackarell, Moore and Thomas : Bio-Chemical Journal, 1910, iv, p. 179. ^ Kahn and Rosenbloom : Biochemical Bulletin, 1912, ii, p. 87. 1912] "Alfred P. Lothrop 179 stance must be present. The urines of man and dog contain col- loidal nitrogenous material.^* It was thought probable that such material is present in all the secretions. After a careful process, including the removal of protein without hydrolysis, substance was obtained f rom milk which is white, amor- phoiis, odorless and tasteless; insoluble in the lipin solvents, but forms in water an opalescent Solution which falls to flocculate on boiling. This material does not respond to any of the protein "color tests." It contained about 5.3 per cent. nitrogen; also car- bon, hydrogen, oxygen, and sulfur, but no loosely combined am- monia radicals. 31. A biochemical test for free acid, with a review of the methods for estimating the various factors in gastric acidity.^^ John L. Kantor. The author presented details along the lines of our original publication on this subject.^^ The test is a microscopic one and depends upon the immediate expansion of moist collagen fibrils when they are immersed in aqueous Solutions containing free organic or mineral acids of the kinds that ordinarily appear in gas- tric Contents. " Combined " acid^^ and acid salts fail to induce such effects. The test may be satisfactorily conducted with a drop of liquid and a single collagen fibril. Comparative observations indicate that for free mineral acid (HCl) the collagen-fibril test is equal in delicacy to the Töpfer and Günzberg tests, but that for free organic acid (lactic), or for mix- tures of free mineral and organic acids, it is more delicate than the latter tests. Comparative studies of common factors of interfer- ence with the several tests indicate that the collagen-fibril test ex- hibits the greater delicacy. The color of the Solution under exam- ination had no effect on the test. Further details from the clinical Standpoint, and an abstract of the historical discussion, will be pub- lished at an early date. 32. A study of modifications of the biuret reagent. Mar- GUERiTE T. Lee. This investigation was made in the endeavor to ^ Kahn and Rosenbloom : Biochemical Bulletin, 1912, ii, p. 87. ^ Kantor : Dissertation, Columbia University, 1912. ^ Kantor and Gies : Proceedings of the American Society of Biological Chemists, 1911, ii, p. 20; Journal of Biological Chemistry, 1911, ix, p. xxvi. " Goodridge and Gies : Proceedings of the Society for Experimental Biology and Mediane, 1911, viii, p. 107. i8o Proceedings Columbia Biochemical Association [Sept. discover, if possible, a more effective alkali for the biuret reagent than the Standard sodium hydroxid — or a combination of alkalies that might be better. Fairly strong Solutions o£ the following alkalies, when substi- tuted for sodium hydroxid in the biuret reagent,^^ yield Solutions that give the biuret test when they are added to dilute Solutions of Witte peptone : potassium hydroxid, ammonium hydroxid, calcium hydroxid, sodium carbonate, conin, piperidin, ethylene di-amin, tri- methyl amin, piperazin, and tetra-ethyl ammonium hydroxid. Sodium hydroxid, potassium hydroxid, ammonium hydroxid, tri- methyl amin, and tetra-ethyl ammonium hydroxid are excellent as alkalies in the biuret reagent. Tri-methyl amin appears to be more effective than sodium hydroxid. Tetra-ethyl ammonium hydroxid is seemingly as effective as sodium hydroxid when the reagent is fresh, but the efficiency of the Solution decreases on standing. Piperazin and tetra-ethyl ammonium hydroxid give most satisfac- tory tests when an excess of copper is present. There is apparently an Optimum amount of copper (sulfate) for each alkali. The study is in progress. 33. A chemical study of salivary mucin. Alfred P. Loth- ROP, Salivary mucin from the submaxillary glands of oxen.was prepared by the Hammarsten-Levene method. It is a white powder, insoluble in water, acid in reaction and readily soluble in dilute alkalin Solutions. The sodium salt can be prepared by dissolving mucin in nine parts of 0.5 per cent. sodium bicarbonate Solution plus one part of 0.5 per cent. sodium carbonate Solution. The thick Solution is then dialysed until it no longer reacts alkalin to phenolthalein but is still alkalin to litmus. (Prolonged dialysis completely hydrolyses the salt and precipitates the mucin. ) The dialysed Solution may be pre- cipitated by the addition of about six volumes of alcohol, although electrolyte (NaCl) must be present for complete flocculation. The product, washed with alcohol and ether, dries to a fine powder. ^ Gies : Proceedings of the American Society of Biological Chemists, 1910, i. P- ^7Z'> Journal of Biological Chemistry, 1910, vii, p. Ix. Also, Kantor and Gies : Biochemical Bulletin, 1912, i, p. 264. I9I2] ^Alfred P. Lothrop i8i The Salt, having an ash content of 2.7-3.3 per cent., is completely soluble in water. A 0.2 per cent. Solution is very much like a rela- tively thick natural saliva. The Solution is faintly alkalin to litmus, gives all the usual protein tests, including the Molisch test for the carbohydrate group, and is precipitated in stringy masses by acetic acid. Quantitative determinations of nitrogen and ash in mucin prep- aration III and its sodium salt gave the f ollowing typical results : Ask Nitrogen Per Cent. Found Per Cent. Calculated (Ash Free) Per Cent. Preparation III Sodium Salt III 0.28 3.27 12.49 12.20 12.53 12.61 The potassium salt was prepared in the same manner, Fre- quent reprecipitations by alcohol render the salts decreasingly solu- ble in water. These products have been made preparatory to experiments on the possible relation of salivary mucin to dental caries, in contin- uance of our studies under the auspices of the Section on Stoma- tology and Research, of the First District Dental Society, State of New York. 34. A study of some of the more important biochemical tests.^^ C. A. Mathewson. Representative substances from the following groups were studied in their influence on the tests named below: neutral inorganic salts, neutral organic Compounds, acids, acid salts, bases, basic salts, biological mixtures and miscellaneous materials. Over seventy-five substances or products were used in each case. It was found that the ten tests under examination could be arranged in the following sequence according to the percentage of factors causing interference with them: Sudan III, o; xantho- proteic, 4; Hopkins-Cole, 4; Seliwanoff, 5; MoHsch, 6.5; iodine, (for starch) 6.5 ; Fehling-Benedict, 10; biuret, 13; Millon, 22; Bar- foed, 60. The acid salts were the most potent interfering substances, the neutral organic Compounds the least potent. Of the salts, ferric chlorid was the most active agent of interference. An extension of the study is in progress. ''Mathewson: Dissertation, Columbia University, 1912. i82 Procccdings Columbia Biochemical Association [Sept. 35. A quantitative study of the lipins o£ bile obtained from a patient with a biliary fistula. Jacob Rosenbloom. Through the kindness of Dr. William Weinberger, of the Lebanon Hospital, there was placed at my disposal 3180 c.c. of human bile obtained from a patient with a biliary fistula. The fluid had the appearance of typical human bile. Its specific gravity was 1.020. The follow- ing data were obtained in a quantitative lipin analysis, the results being expressed in parts per thousand: Water, 970.2; total solids, 29.8; cholesterol, 2.61; lecithans, 6.42; fat, 6.85; fatty acids, 1.2; soaps, 2.6. Total lipins, 19.68 (1.97 per cent.). 36. Effects of intraperitoneal injections of epinephrin on the partition of nitrogen in urine from a dog. Jacob Rosenbloom AND William Weinberger. (Published in füll in this issue of the Biochemical Bulletin. )^^ 37. A case of allergy to common foods.^^ Oscar M. ScHLOSs. In a boy now 8 years old marked urticarial lesions were caused by the Ingestion of eggs, almonds and oatmeal. The idiosyn- crasy to egg was not congenital but was acquired at some time be- tween the ages of 10 days and 14 months. Symptoms due to the ingestion of oats appeared some time after the child had first eaten oatmeal when he was 22 months old. As far as can be ascertained, the idiosyncrasy to almonds was manifested the first time this food was eaten. It was found that cutaneous inoculation of these and certaln related food substances produced an urticarial wheal at the site of inoculation. The cutaneous reaction was produced only by the protein constituents of eggs, almonds and oats. Different proteins from the same source varied in activity, some being incapable of causing a reaction. Some of the active proteins caused Urticaria by mere contact with the unbroken skin. It was possible passively to sensitize guinea-pigs to ovo-mucoid (one of the active proteins from eggs) by intraperitoneal injections of the patient's blood-serum. By feeding ovo-mucoid, in gradually increasing doses, the patient became immune to egg. At the same time immunity to oatmeal and an apparently decreased susceptibility to almonds occurred. ^"Rosenbloom and Weinberger: Biochemical Bulletin, 1912, ii, p. 123. ^ Schloss : American Journal of Diseases of Children, 1912, iii, p. 341. I9I2] 'Alfred P. Lothrop 183 38. The comparative enzyme content of green and varie- gated leaves of Tradescantia.^^ Carl A. Schwarze. The re- sults of the experiments made to determine the relative enzyme con- tent of green and variegated leaves of Tradescantia show that there is a marked difference between Juices expressed from them. Etio- lated leaves are yellow in their rudimentary stage ; that is, an entirely yellow leaf presents this condition when first formed. The etio- lated leaves are free from chloroplasts and therefore possess no starch. The juice extracted from yellow leaves gives a negative Fehling test; that from green portions, a positive test. When yel- low leaves are ground in a mortar, and the juice is expressed through cheese cloth, a dark brown liquid results. Green leaves similarly treated yield a dark green liquid. Alcoholic extracts of crushed green and yellow leaves, when filtered, assume a brown color. The filtrate from yellow leaves is at first pink but the liquid gradually assumes a brown color. The filtrate from the green leaves comes through brown immediately. The juice of yellow and green leaves, when filtered, gives in both cases a brown filtrate, that from the yellow leaves being a reddish brown, When unfiltered green juice desiccates, a glossy dark green residue is deposited, at the periphery of which a few needle-shaped crystals are seen. The juice from the yellow leaves, upon desiccation, deposits a brown crystallin mass, the long crystals of which make a figure which resembles a polyaster seen in plant cells. Extracts in alcohol (80 per Cent.) deposit the greatest amount of crystals. The crys- tals from yellow leaves are darker than those from green leaves. Such reagents as guaiac and trikresol show the presence of oxidase and peroxidase in yellow and green Juices. The yellow juice seems to be richer in oxidase and peroxidase. When green juice was heated to 72° C, and tested the following day, oxidase proved to be present, that temperature having failed to destroy it. (Subjecting green juice to high temperatures results in the produc- tion of a flocculent precipitate, which Sediments promptly under a clear supernatant liquid.) Juice from yellow leaves was injected into the nodes and inter- ** Conducted in the Botanical Laboratory under Dr. Gies' guidance. 184 Proceedings Columbia Biochemical Association [Sept. nodes of healthy green Tradescantia stems. No discoloration or yellowing of the injected stem coiild be detected. 39. Biochemical studies of beryllium sulfate.^^ Emily C. Seaman. The experiments vvith beryllium sulfate have shown very conclusively that the substance has a marked effect on biochemical processes. When administered with the food it produced in dogs decided nutritive disturbances, which manifested themselves in loss of body weight, total inorganic matter, nitrogen, sulfur and phos- phorus. When large doses were administered per os the substance caused vomiting before a sufficient amount was absorbed to pro- duce any other obvious toxic Symptoms. When the calculated lethal dose was administered by a single siihcutaneous injection, the substance produced edema and necrosis of the tissue extending over a large area. No other decided Symp- toms were produced by this method. Very gradual intravenous injections of the salt produced decided toxic effect. The action of the heart became irregulär — unusually rapid and very weak: the respiration also became irregulär and shallow. During the course of the injection, there was decided tremor but this disappeared soon after the Operation. As a direct effect of the injection the temperature increased, sometimes to 105° F., but about 24 hours before the death of the animal the tem- perature began to decrease and steadily feil. After intravenous injections there was increased elimination of urine followed by re- tention. The feces became diarrheal and bloody. Vomiting began about the time the dog refused food or water. Beryllium sulfate had a decided inhibitory effect on the action of ptyalin, pepsin, and trypsin. It also retarded the action of sucrase but not to so great an extent. Solutions of the salt (i per cent. or less) did not precipitate proteins from neutral or acid Solutions. Below the concentration of M/512 Solution, beryllium sulfate did not inhibit the growth of lupin or timothy seedlings, but more con- centrated Solutions prevented growth. When present in propor- tions less than 0.5 per cent., beryllium sulfate had very little, if any, bactericidal action. 40. Chemical changes in fish during long periods of cold storage. Clayton S. Smith. Fresh fish were delivered directly ^Seaman: Dissertation, Columbia University, 1912. 1912] 'Alfred P. Lothrop 185 frort! the boat. Specimens of the same catch were immediately placed in storage and delivered to us, at intervals, in a frozen State, when they were thawed under uniform conditions and promptly subjected to analysis. Comparative data were obtained regarding moisture, organic matter, inorganic matter, and total solids; ammonia nitrogen, solu- ble nitrogen, insoluble nitrogen, coagulable nitrogen, non-coagulable nitrogen and total nitrogen; "proteose" nitrogen, both before and af ter autolysis ; f at content and f atty-acid number ; and the reducing power of the aqueous protein- free extract, as determined by the Benedict method. The flesh of fish which had been refrigerated not less than four months and not more than six months was unaltered in composition. After a period of nine months in cold storage there was a sHght, almost imperceptihle, increase in the content of ammonia nitrogen, but no other change was noted. The work is in progress. 41. An attempt to sharpen the end point in Benedict's method for the quantitative determination of sugar in urine. William Weinberger. In Benedict's modification of FehHng's sugar titration method "instead of the reduced copper being pre- cipitated as the red sub-oxid, which of its own color obscures the end point of the reaction, the copper is precipitated as cuprous sulfo- cyanate, a snow white Compound, which is rather an aid than a hin- drance to accurate Observation of the disappearance of the last trace of blue color," However, in applying Benedict's method to urine of low sugar content (below 0.5 per cent., as it frequently occurs in cases of glycosuria), one is Struck by the fact that the blue color of the mixture does not persist until the reaction is ended, for the Contents of the porcelain dish assume a dirty brownish-green hue that gradually merges into brown. This renders the correct estima- tion of the end point very difficult if not impossible. Clarifying the urine by the addition of lead acetate previous to the titration might overcome the difficulty, but this procedure would require additional manipulations and calculations ; and there is also the danger of a chemical change in the copper Solution. None of these objections apply to the simple method proposed by the author. It consists in the addition, just before heating, of approximately 10 i86 Proceedings Columbia Biochemical Association [Sept. grams (two heaping teaspoonsful) of powdered calcium carbonate to the Contents of the porcelain dish (25 c.c. of Benedict's Solution, 5-10 grams of anhydrous sodium carbonate, and a small amount of powdered pumice). The titration is then made in the usual manner. The snow white calcium carbonate, insoluble and suspended in the alkalin Solution, appears to act like the copper sulfocyanate in that it efTectively obliterates all colors except the blue color of the copper Solution. The end point obtained is sharp, the blue color being visible up to the addition of the last two drops of urine that are necessary for complete reduction. A sufficient amount of cal- cium carbonate (10 grams) must be added, otherwise the precipitate will be gray and the end point less distinct. In order to prevent sudden ebullition of the concentrated Solution, it is advisable to dilute the latter with a little distilled water, Experiments have shown that the addition of the calcium carbonate does not introduce any noticeable error. The author demonstrated these facts. 42. Diffusibility of protein through rubber membranes, with a note on the disintegration of collodion membranes by common ethyl ether and other solvents. William H. Welker. {Puh- lished in füll in this issue of the Biochemical Bulletin, )2* 43. A further study of the Bardach test for protein. Charles Weisman. {Piihlished in fiill in the June issue of the Biochemical Bulletin ).2^ 44. A study of the surface tension of dog blood-serum by the drop-weight method.^^ Harold E, Woodward. These ex- periments, about twenty in number, were planned to answer the question whether ordinary variations in the blood supply and nutri- tive condition of an individual affect the surface tension of the blood.^'^ Serum could be handled better than blood and serum from ** Welker: Biochemical Bulletin, 1912, ii, p. 70. ** Weisman : Ibid., 1912, i, p. 538. ^The animals were fed and controlled, and the blood was withdrawn and the serum coUected, by Dr. Gies and Mr. Chris Seifert. The drop-weights were made by the author in the laboratory of physical chemistry under the direction of Prof. J. L. R. Morgan. ^ These experiments were a logical preliminary to the work described else- where: Woodward, Dissertation, Columbia University, 1912. I9I2] 'Alfred P. Lothrop 1S7 clotted blood was more satisfactory than serum obtained by centri- fuging defibrinated blood. The normal surface tension of dog serum (five dogs), from the blood of animals on the usual diet in metabolism experiments in this laboratory, is about 45.5 dynes per centimeter. A daily hemorrhage of 3 per Cent, or more of the body weight, on two successive days, was without material effect on the surface tension. ^^ Small addi- tions of salt to the food raised, whereas additions of sugar lowered, somewhat the surface tension. The Ingestion of extra quantities of meat, several hours before blood was withdrawn, caused a decrease of about 1.5 per cent. in the surface tension. Fasting (1-2 days) raised the surface tension about i per cent. Copious water drink- ing (2 hours before withdrawal of blood) and the administration of magnesium sulfate, with resultant marked diarrhea (a short time prior to removal of blood from another dog), were without appre- ciable effect on the surface tension of the serum. These results suggest that the nutritive State of a given individual must be defi- nitely established before accurate conclusions can be drawn regard- ing the significance of data for surface tension of the subject's blood (or serum). [The December issue of the Biochemical Bulletin will pre- sent abstracts of the scientific Communications at the meeting of the Biochemical Association to be held on December 6, at the Columbia Medical School.] Biochemical Laboratory of Columbia University, College of Physicians and Surgeons, New York. '^When the second bleeding occurred in much less than 24 hours after the first, the surface tension was above normal. BIOCHEMICAL NEWS, NOTES AND COMMENT Contents. I. General: Necrology, i88; in memoriam, i88; anniversary celebrations, 189; honors, 190; retirements, resignations and appointments, 190; prizes, grants, endowments and funds, 193; meetings of congresses and societies, 194; buildings and general equipment, 195 ; acts of congress, 196; miscellaneous, 197. IL Columbia University Biochemical Association: General notes, 200; pro- ceedings, 201 ; biochemical department, 201. I. General Necrology. Dr. W. W. Daniels, emeritus professor of chemis- try at the University of Wisconsin. — Thomas Doliber, president of Mellin's Food Co., and one of the best known manufacturing drug- gists in America. — Dr. Morris Loeb, professor of chemistry at New York University and president of the Chemists' Club. — Dr. Her- mann Munk, formerly professor of physiology at the veterinary Col- lege in Berlin. — Dr. E. A. Holmström, Sweden's foremost pharma- cist. — Dr. Edmund von Neusser, professor of internal medicine at Vienna. — Prof. Melville Amasa Scovell, director of the Kentucky Agricultural Experiment Station and dean of the College of Agri- culture of the Kentucky State University. — Dr. Henry Adam Weber, professor of agricultural chemistry, Ohio State University. — Dr. Thomas Winter, professor of agriculture in University Col- lege of North Wales, Bangor. In memoriam. Lord Lister. A memorial to Lord Lister will be established at University College Hospital. It was in 1843 that Joseph Lister entered the College as an arts Student and graduated bachelor of arts in 1847. He then became a Student of medicine and entered the hospital to complete his studies. A special commit- tee has been formed under the presidency of the Duke of Bedford, President of the hospital. The exact nature of the tribute will be largely decided by the amount of the subscriptions received, but it has been suggested that either a bust or a tablet should be placed in both the hospital and the College. It is understood that the memo- rial will be entirely local in character, and only those who have been 188 I9I2] General 189 in some way connected with University College or the hospital are being asked to subscribe. The presidents of the Royal Society and the Royal College of Surgeons some weeks ago took the necessary steps for the forma- tion of a large and representative committee for the purpose of es- tablishing a memorial to the late Lord Lister. A meeting of the committee, which was largely attended, was held on July 22 at the rooms of the Royal Society, under the chairmanship of Sir Archi- bald Geikie. The following were appointed an executive commit- tee to recommend to a future meeting of the general committee a scheme for the memorial to Lord Lister and to organize an appeal for subscriptions : The Archbishop of Canterbury, the Lord Chan- cellor, Lords Iveagh, Rayleigh, Rothschild and Alverstone, the dean of Westminster, the Lord Mayor, the Lord Provosts of Edinburgh and Glasgow, the Master of the Rolls, Mr. Lewis Harcourt, M.P., Sir T. Barlow, Sir W. W. Cheyne, Sir R. J. Godlee, Sir H. Morris, Sir A. Geikie, Sir D. MacAlister, the Hon. Sir C. Parsons, Sir W. Turner, Sir J. Wolfe-Barry, Sir J. R. Bradford, Sir A. P. Gould, Sir A. Kempe, the Hon. W. F. D. Smith, Mr. F. M. Fry and Mr. Edmund Owen. Lord Rothschild and Sir W. W. Cheyne were appointed treasurers and Sir J. R. Bradford was appointed secretary of the Lister Memorial Committee. Dr. Paul C. Freer. The Bureau of Science of the Philippine Government has adopted resolutions in memory of Dr. Paul C. Freer, director of scientific work in the bureau, who died last April. The resolutions express the sense of his associates that " the Bureau of Science has suffered a very great loss and that the cause of sci- ence in the Philippine Islands has been deprived of one of its most zealous and conscientious advocates." Anniversary celebrations. June 30: Professor Gad, formerly director of the Physiological Institute at Graz, a pupil of Du Bois- Reymond, celebrated his seventieth birthday. — July i: Prof. Carl Binz, formerly director of the Pharmacological Institute at Bonn, celebrated his eightieth birthday. — August ß: Professor Bernstein, formerly director of the Institute of Physiology at Halle, celebrated the fiftieth anniversary of his doctorate. — September 14: Prof. W. 190 Biocltemical News, Notes and Comment [Sept. O. von Leube, the distinguished clinician, celebrated his seventieth birthday. Professor Leubc has been living at Stuttgart since last year, when he resigned his directorship of the Würzburg medical clinic. Honors. 'Awards of prizes. Dr. Alexis Carrel has been awarded the Nobel prine in medicine, in recognition of his achieve- ments in the suture of blood-vessels and the transplantation of Organs. — The Vienna Academy of Sciences has conferred its Liehen prise for 191 2 on Dr. Oswald Richter for his work on the food of algse. Honorary degree. The University of St. Andrews, Dundee, Scotland, has conferred the degree of LL.D. on Dr. S. J. Meltzer. Foreign associates. Sir William Ramsay and J. Reverdin have recently been elected foreign associates of the Paris Academie de Medecine. Retirements, resignations and appointments. Retirements. Col. Martin V. Calvin, for the past six years director of the Georgia Agricultural Experiment Station. — Prof. H. J. Wheeler, former act- ing-president of the Rhode Island State College, at Kingston, R. I., and, during the past eleven years, director of the government agri- cultural experiment Station at that Institution. Leave of ahsence. Dr. W. P. Bradley, professor of chemistry at Wesleyan University, has been granted leave of absence for the year 1912-13, to organize a department of research for the United States Rubber Goods Company. — Dr. A. F. Blakeslee has a year's leave of absence from the Connecticut Agricultural College. He has a tem- porary appointment on the staff of the Carnegie Station for Experi- mental Evolution at Cold Spring Harbor, L. L, where he will study lower fungi. Appointments have lately been announced, as follows '} Bryn Mawr College: Dr. Don R. Joseph (associate in physiology and pharmacology at the Rockef eller Institute), associate professor of physiology. Carnegie Institution, Boston Nutrition Laboratory: Mr. Joseph C. Bock (instructor in chemistry at Michigan Agricultural College), chemist. ^In the appended summary, institutions from which resignations occurred are named in parenthesis. I9I2] General 191 College of Agriculture and Mechanic Arts (Mayaguez, P. R.) : Dr. B. E. Ray (N. C. Experiment Station and College of Agriculture), Professor of chemistry. Columbia University: Mr. Ernest L. Scott (University of Kansas), instructor in physiology; Dr. Otto von Huffman (Cincinnati Hospital and Ohio Miami Medical College), instructor in clinical pathology. Commission for the study and prevention of malaria in the South : Dr. William S. Thayer, member. English Government Laboratory, London : Mr. E. Grant Hooper, deputy-government chemist (promoted), vice Mr. H. W. Davis, retired. Hamburg Botanical Institute: Dr. Hans Winkler (associate Pro- fessor of botany at Tübingen), director. Harvard University: Dr. Geo. R. Lyman (assistant professor of botany in Dartmouth College) will take the work of Professor Roland Thaxter during a sabbatical leave of absence. Institute for experimental research on Cancer, established by the Kaiser Wilhelm Society for the promotion of science: Prof. A. von Wassermann, director. Margaret Morrison School for Women of the Carnegie Institute (Pittsburgh) : Miss Mary D. MacKenzie (professor of biology at Western College, Oxford, Ohio), head of the department of biology. McGill University (Montreal) : Prof. Francis E. Lloyd (professor of botany in the Alabama Polytechnic Institute and plant physiologist to the Alabama Experiment Station), MacDonald professor of botany; Dr. F. R. Miller, lecturer in physiology. Medico-Chirurgical College (Philadelphia) : Dr. H. Lowenherg, assistant professor of infantile dietetics and also pediatrist to Mount Sinai Hospital, succeeding the late Dr. Edwin Rosenthal. Municli medical clinics : Prof. Friedrich Müller, instead of retain- ing the second clinic, has taken the first, left vacant by the death of Professor Bauer; Prof. E. v. Romberg (Tübingen) succeeds Professor Müller. N. Y. State Food Laboratory (Ithaca) : Mr. /. T. Ciisick (assistant in nutrition investigations, N. Y. Agricultural Experiment Station), analyst. N. Y. State School of Agriculture (Alfred University at Alfred) : Prof. W. J. Wright (Pennsylvania State College), director. N. C. Agricultural Experiment Station (West Raleigh) : Dr. Joseph F. Brewster, chemist. Ohio State University: Dr. W. G. Stover (Oklahoma Agricultural Experiment Station), assistant professor of botany. 192 Biochemical Nezvs, Notes and Comment [Sept. Ontario Agricultural College : Mr. R. E. Stone, lecturer in the botan- ical department. Pennsylvania Chestnut-Tree Blight Commission: Dr. F. D. Heald (professor of botany in the University of Texas), pathologist; Miss Caroline Rumbold (Missouri Botanical Garden), physiologist in charge of tree medication ; Mr. Joseph Shrawder, chemist. Reed College (Portland, Oregon) : Dr. Harry Beal Torrey (asso- ciate professor of zoology in the University of California), professor of biology. Skin and Cancer Hospital of Maryland : Mr. /. M. Codd, chemist. State University of Oregon Medical College (Portland) : John M. Co;mo//y, Ph.D., M.D. (Harvard Medical School), professor of physio- logical chemistry. U. S. Bureau of Animal Industry : Dr. Frederick J. Birchard (as- sistant in chemistry at the Rockefeiler Institute), research chemist in the Dairy Division. U. S. Bureau of Mines (Pittsburgh) : Dr. /. K. Phelps (U. S. Bu- reau of Chemistry, Washington, D. C), chemist. U. S. Bureau of Plant Industry : Dr. R. Kent Beattie (professor of botany in the State College of Washington), expert in the office of forest pathology; Dr. Neil E. Stevens (assistant pathologist in Kansas Experiment Station), forest pathologist. University College, Reading: Dr. vS". M. T. Auld (lecturer in the chemical department of the Southeastern Agricultural College atWye), professor of agricultural chemistry; Mr. John Goding (Midland Agri- cultural College), research chemist in dairy ing. University of Bonn: Professor Johannes Fitting (director of the State Botanical Institute at Hamburg), successor of Professor Stras- burger. University of Illinois: Dr. /. Howard Beard, instructor in physi- ology (promotion). University of Maryland : Dr. Jsaac M. Macks, pathologist. University of Minnesota : Dr. Robert B. Gihson, assistant professor of physiological chemistry (promotion) ; Dr. Rodney M. West, assist- ant professor of agricultural chemistry (promotion). University of South Dakota : Mr. Herbert Otto Lussky (assistant in physiology at the University of Chicago), director of the department of physiology in the College of arts and sciences and the College of medicine. University of Vienna: Prof. Wilhelm Türk, temporary successor to Prof. E. von Neusser in the medical school (page 188). I9I2] General 193 Washburn College : Dr. Edith M. Twtss, head of the department of botany ; Mr. James P. Poole, instructor in botany. Washington State College (Pullman) : Dr.IraD. Cardiff (professor of botany in Washburn College), professor of plant physiology. Prizes, grants, endowments and funds. Prizes. The Col- lege of Physicians, Philadelphia, announces that the next award of the Alvarenga prise, amounting to about $180, will be made July 14, 191 3. Essays may be devoted to any subject in medicine but must not have been published, and shonld be received by May i, 1913, by the secretary of the College, Dr. Thomas R. Neilson, 1937 Chestnut Street, who will furnish particulars, on request. — Madame Dieula- foy, widow of the late clinician, has given to the Academy of Medi- cine, of Paris, in memory of her husband, the sum necessary to found the Dieulafoy prise of $400, which will be awarded every two years to the author of the best work on the subject of internal pathology. — The Riberi prise, amounting to $4,000, will be awarded by the University of Turin, after the close of the year 1916, for the work which is adjudged to have most advanced the science of medicine. Grants. Grants for research, at the recent meeting of the Brit- ish Association: Mr. A. D. Hall, plant enzymes, £30; Prof. E. A. Schäfer, the ductless glands, £40; Prof. E. H. Starling, oxy-hemo- globin, £15 ; Prof. F. Gotch, mammalian heart, £20; Sir W. Ramsay, for the International Commission on Physical and Chemical Con- stants, £40. Endowments and funds. The London School of Tropical Med- icine is making an appeal for $500,000 to provide for the equipment and more efficient conduct of its work. — The late Dr. J. E. Robinson, first governor of Kansas, bequeathed $100,000 to the University of Kansas. The gift will be used for the medical school. — Mr. James B. Brady, of New York, has given the sum of $220,000 to the Johns Hopkins Hospital, for the establishment of a ward for the treatment of diseases of the kidney. — The late Mr. Allan Octavian Hume, well known as an ornithologist and botanist, lately bequeathed about £14,000 to the South London Botanical Institute, to which in 1907 he gave £10,000. — Under the will of the late Augustus W. Open- hym, Columbia University will receive a third of a trust fund of 194 Biochemical News, Notes and Comment [Sept. $275,000 for the endowment of research into the cause, prevention and eure of Cancer. Mr. Openhym's will stipulates that if at any time further investigation of Cancer is not required, the income of the fund may be used for research in any brauch of medicine or sur- gery. The endowment under Mr. Openhym's will is to be known as the Openhym Research Fund, and the terms of the gift are sub- stantially the same as those of the Crocker Research Fund, which amounts to $1,440,777.13. Meetings of congresses and societies. The Fifteenth Inter- national Congress on Hygiene and Demography was officially opened in the Continental Memorial Hall on September 23 and continued until September 27. President Taft delivered an address at the opening exercises. The delegates numbered about 3,000, represent- ing 33 foreign governments, every American State and territory, over 300 American cities, and leading Colleges and universities and many scientific, medical and social institutions throughout the world. The congress was divided into eleven sections and four general ses- sions were held. President Taft was honorary president, Dr. Henry P. Walcott, of Massachusetts, was president, and Dr. John S. Fulton, of Maryland, was secretary-general, of the congress. A füll account of the proceedings is given in the Journal of the Amer- ican Medical Association, beginning at page 1207 (September 28). The proceedings of the biochemical section — " dietetic hygiene ; hy- gienic physiology" — are reported at page 129 of this issue of the Biochemical Bulletin. The Eighth International Congress of Applied Chemis'try was officially opened at Continental Memorial Hall, in Washington, on September 4, and continued in New York from September 6-13, in- clusive, where the work was centralized at Columbia University and the College of the City of New York. About 2,500 members were in attendance. Dr. Edward W. Morely was honorary president, Prof. William H. Nichols was president, and Dr. Bernhard G. Hesse was secretary, of the congress. The scientific work of the congress was organized in twenty-four sections. Among the gen- eral addresses was one by Prof. Gabriel Bertrand on " The part played by infinitely small quantities of chemicals in biological chemistry." igi2] General i95 Professor W. H. Perkin delivered a lecture on " The polymeriza- tion of butadiene and isoprene," before the Sections on Organic Chemistry and India Rubber. Prof. Perkin outlined bis original method of making synthetic rubber,^ and then described the follow- ing new method : Take ethyl alcohol, which may be easily oxidized to acetaldehyde. This is Condensed by means of potassium carbon- ate to aldol and the aldol can be quantitatively converted into butyl- idine gycol. All the yields of these reactions are practically quan- titative. The butylidine glycol is then converted into a chlorid and passed over soda-lime, when practically the same product is pro- duced as the isoprene from isoamyl chlorid and, when treated with sodium, gives even better rubber than isoprene. Professor Perkin exhibited samples of what he called the first synthetic rubber ever made (the product of Tilden). A general review of the proceedings of the Congress will appear in the October issue of the Journal of Industrial and Engineering Chemistry (pages 706-719). The proceedings of the biochemical section are reported at page 150 of this issue of the Biochemical Bulletin. The eighty-second annual meeting of the British Association for the Advancement of Science, which opened at Dundee on Septem- ber 4, had a registration of 2,504 members, which is considerably larger than the average. At the opening session the President, Prof. E. A. Schäfer, delivered a notable address on the "Nature, origin and maintenance of life," which has been published in Nature (90: 7-19) and Science (36: 289-312). It was announced that Dr. J. K. Caird, of Dundee, had given £10,000 to the funds of the association. A general account of each sectional meeting will ap- pear in Science (36 : 446-452). The Royal Society recently celebrated its 25oth anniversary. The I4th meeting of the Australasian Association for the Ad- vancement of Science will be held in Melbourne in January, 19 13. Buildings and general equipment. The work of the Herriman Dispensary of the Brooklyn Hospital was inaugurated on July 17. The dispensary will be open daily. It is a two and one-half story * Biochemical Bulletin, 1912, i, p. 566. 196 Biochemical News, Notes and Comment [Sept. brick and marble structure and was given by Mr. William H. Herri- man in memory of his wife. Mr. Herriman donated $100,000 for this purpose, $25,000 of which will be used as an endowment fund. — Messrs. Jacob H. Schiff, Sei. R. Guggenheim, Ferdinand Sulz- berger and Samuel Sach have each given $50,000 to a fund for the construction of a private hospital for persons suffering f rom chronic diseases, to be built by the Montefiore Home, in the Bronx, New York City. — The Medical Faculty of the University of Utah is re- questing the Regents of the University to ask the Legislature for a special appropriation of $25,000 for the medical school. It is not generally known that the State of Utah is doing better by its Uni- versity, proportionately, than any other State, in that this Institution receives 28 per cent. of the state's income in taxes. The State of Utah contains about 400,000 inhabitants. Acts of Congress. Public Health Service. The following is the text of the act of congress concerning the Public Health Ser- vice : Be it enacted by the Senate and House of Representatives of the United States of America in Congress assembled. That the Public Health and Marine-Hospital Service of the United States shall hereafter be known and designated as the Public Health Serv- ice, and all laws pertaining to the Public Health and Marine-Hos- pital Service of the United States shall hereafter apply to the Public Health Service, and all regulations now in force, made in accord- ance with law for the Public Health and Marine-Hospital Service of the United States, shall apply to and remain in force as regula- tions of and for the Public Health Service until changed or rescinded. The Public Health Service may study and investigate the diseases of men and conditions influencing the propagation and spread thereof, including sanitation and sewage and the pollution either directly or indirectly of the navigable streams and lakes of the United States, and it may from time to time issue Information in the form of publications for the use of the public. 'Amendment to the food and drug act. Congress, before ad- journment, passed an amendment to the food and drug act which the President has signed, making it illegal " if its package or label shall bear or contain any Statement, design, or device regarding the curative or therapeutic effects of such article, or any of the ingredi- I9I2] General 197 ents or substances contained therein, which is false and fraudulent." It will be remembered that the act of 1906 declared that a drug is misbranded "the package or label of which shall bear any Statement . . . which shall be false or misleading in any particular . . . " ; but the supreme court, by a majority of five to three, decided that this did not refer to false Statements regarding the curative effect of a drug. Miscellaneous items. Proposed State medical service in Eng- land. During the recent meeting of the British Medical Associa- tion at Liverpool, a State Medical Service Association was formed under the inspiration of Dr. B. Moore, professor of biochemistry at the University of Liverpool. Prof. Moore lately produced a book entitled " The dawn of the health age," in order to demonstrate the necessity for entirely remodeling the present System of medical prac- tice in the interests of the whole Community. The object of the new association is to advocate a State medical service on the follow- ing basis : (i) the whole profession to be organized on the lines of the other State Services now in existence; (2) entry to the profes- sion to be by one state examination; (3) each member of the serv- ice to be paid an adequate salary, increasing gradually according to the length of service and position in the service, and to be entitled to a Pension after a specified number of years or in case of perma- nent disablement; (4) members of the public to have, as far as possible, free choice of physicians, but no physician to be called on to have charge of more than a specified number of patients; (5) one of the primary objects of the State service to be to unite preventive and curative medicine ; all hospitals to be nationalized and used for the purpose of consultative, operative and therapeutic work at the request of and in conjunction with the patient's own physician; (6) the Services of the state physicians to be open to €very one, rieh or poor; (7) the state medical service to be administered by a board of health under a minister of public health with cabinet rank, assisted by expert medical advisers. This movement was started before the insurance act was passed and is quite independent of the present impasse. It is intended that the work of the association shall form a brauch of sociologic science, and membership will be open to all prominent sociologists, whether lay or medical. 198 Biochemical News, Notes and Comnient [Sept. (London correspondent, Journal of the American Medical Associa- tion, 19 12, lix, p. 663 : August 10). Detection of formaldehyde in foods. In view of the introduc- tion of a mixture of nitrite and formaldehyde with the object of masking the reactions of the latter when used as a food preserva- tive, the following experiments may be of interest. A sample of f resh mixture was divided into four portions and treated as follovvs : (i) A small amount of commercial formaldehyde Solution was added; (2) small amounts of formaldehyde and sodium nitrite were added; (3) a small amount of sodium nitrite was added; (4) no addition was made. Portions of each of these were tested with Rimini's test (Phenylhydrazin hydrochlorid, sodium nitroprussid and sodium hydroxid). Prompt reactions for formaldehyde were obtained in i and 2; negative results in 3 and 4. Other portions of the samples were tested with the well-known test for nitrite (sul- fanilic acid and alphanaphthylamin) . The responses of 2 and 3 were prompt and distinct. No color was produced in i and 4. The original mixtures were allowed to stand 24 hours at room tempera- ture and the tests repeated with the same results as obtained at first. It seems easy, therefore, to unmask nitrite and formaldehyde in the presence of each other. Henry Leffmann. {Journal of Industriell and Engineering Chemistry, 1912, iv, p. 626: August.) Joiirnalistic. With the September number Prof. A. R. Cushny, of University College, London, becomes Joint editor with Prof. John J. Abel, of Johns Hopkins University, Baltimore, of the Jour- nal of Pharmacology and Experimental Therapeutics. At the same time, Sir T. Lauder Brunton, of London, Professors J. T. Cash, of Aberdeen, W. E. Dixon, of Cambridge, J. A. Gunn, of Oxford, Sir Thomas R. Fräser, of Edinburgh, J. N. Langley, of Cambridge, C. R. Marshall, of the University of St. Andrews, R. Stockman, of Glasgow, F. Ransom, of London and Dr. H. H. Dale, of London, join the board of associate editors. By this arrangement the ablest representatives of phannacology in Great Britain unite with the American and Canadian colleagues in the conduct of the Journal and the publishers feel confident that it will henceforth serve as the medium of publication for the best pharmacological researches of the I9I2] General 1 99 english-speaking countries. (Publisher's announcement, Septem- ber number, vol. iv, no. i.) Visiting agriadturalists. Mr. Paul Korchoof, agricultural ex- pert, department of the Russian ministry of agriculture, and Mr. Vaseelie Yurieff, assistant director, Kharkow Central Agricultural Experiment Station, have been visiting the agricultural Colleges and stations in this country. — Dr. E. B. Copeland, dean of the College of Agriculture, Los Bafios, P. I., who has been visiting the United States, recently returned to the Philippines. Parsons in Washington. Dr. Charles L. Parsons, secretary of the American Chemical Society, moved from Durham, N. H., to Wash- ington on September i. The headquarters of the American Chem- ical Society may now be addressed, Box 505, Washington, D. C, Remsen to remain at Hopkins. Owing to the difficulty of find- ing a suitable occupant for the post, Dr. Ira Remsen will remain at the head of Johns Hopkins University for the ensuing session, or part of it at least. Petroleum production in the United States, in 191 1, surpassed its own record (made in 1910) by an increase of nearly 11,000,000 barreis. In 1910 the Output was 209,557,248 barreis. The total production of the world also surpassed all previous records, amount- ing to over 345,000,000 barreis. Johns Hopkins limits enrolment. The dean of Johns Hopkins Medical School announces that it has become necessary to limit the number of students owing to the restricted space and facilities in the various laboratories. The present enrolment is 355, the largest in the history of the school, and fifty other students were refused ad- mission prior to the beginning of the session. Standard rations for nutrition experiments. A Conference was held at the Graduate School of Agriculture, Lansing, Mich., on July 24, to discuss the formulation of Standard rations for experi- mental work in determining the comparative value of feed stuffs. Mr. B. H. Rawl, chief of the dairy division, U. S. Department of Agriculture, President H. J. Waters, of Kansas Agricultural Col- lege, Prof. C. H. Eckles, of Missouri Experiment Station, and other leading workers in this field were present and led the discussion. 200 Biochemical News, Notes and Comment [Sept. COLUMBIA UNIVERSITY BIOCHEMICAL ASSOCIATION I. General notes Miscellaneous items. Dr. Carl L. Alsherg was one of the dis- tinguished non-resident scientists to participate, by invitation, in a series of lectures, during the late summer at Fordham University Medical College, in New York, on nervous and mental diseases. — The following members of the Association conducted investigations at Woods Hole, Mass., during the summer: Cora J. Beckwith, H. B. Goodrich, Louise H. Gregory, Charles Packard, Alwin W. Pap- penheimer, Henry J. Spencer, Charles R. Stockard, Isabel Wheeler, and L. L. Woodruff. — Dr. 'A. Richard Bliss is editor-in-chief of TheMask, the official national organ of the Kappa Psi Fraternity. — Prof. R. Burton-Opitz is now in Europe, where he is spending a half-year leave of absence. Officers of societies. Section (V) on Control of Infectious Diseases of the I5th International Congress on Hygiene and Demog- raphy (page 194) : Dr. Charles F. Boldiian, secretary. — New York Post-Graduate Medical School and Hospital : Dr. Arthur F. Chace, secretary (reelected). — Section (IV) on Organic Chemistry of the 8th International Congress of Applied Chemistry (page 194) Dr. Harry L. Bisher, secretary. — N. Y. Entomological Society: Prof. Raymond C. Oshiirn, president. — American Association for the Study and Prevention of Infant Mortality: Dr. Philip Van Ingen, secretary. Appointments. Jefferson Medical College (Philadelphia) : Dr. Philip B. Hawk (professor of physiological chemistry, University of Illinois), professor of physiological chemistry and toxicology. — Rockefeiler Institute for Medical Research: Dr. Michael Heidel- berger (recently returned from Zürich), fellow in chemistry. — Johns Hopkins University: Dr. John Howland (professor of pediatrics in Washington University, St. Louis), director of the Harriet Lane Home for Invalid Children, professor of pediatrics, and physician in charge of the pediatric department of Johns Hopkins Hospital. — ^Cornell University Medical College, Loomis Laboratory : Miss Jessie A. Moore (assistant at the Rockefeiler Insti- tute for Medical Research), chemical assistant. — N. J. Agricultural Experiment Station: Mr. Carl A. Schwarze, assistant plant pathol- ^, Ol*jJJ_ "X-^Wv^n^, 1912] Columbia University Biochemical Association 201 ogist. — Long Island Medical College: Dr. Matthew Steel (assist- ant Professor of physiological chemistry, University of Missouri), as- sistant professor of physiological chemistry and pharmacology. — At a recent annual meeting of the Imperial Cancer Research Fund, in London, Dr. William H. Woglom was appointed first assistant in New York, a position maintained under the auspices of the Crocker Fund for the investigation of Cancer. Dr. Woglom has returned from London, where he had been pursuing a course of study under Dr. Bashford, director of the Imperial Cancer Research Fund. 2. Proceedings of the Association. Abstracts of the scientific proceedings of the third annual meet- ing (June) are pubHshed on pages 156-187 of this issue. 3. Columbia Biochemical Department. The new Assistant Professor, Dr. Paul E. Howe, B.S., A.M., PhD.^ Memorandum which was presented to the Faculty OF Medicine with Dr. Howe's nomination to the Assistant PROFESSORSHIP IN BIOLOGICAL CHEMISTRY. Paul Edward Howe was born in Chicago, Illinois, on July 29, 1885. His early education was received in the public schools of Chicago, Champaign and Urbana, Illinois (1890-1901). He at- tended the Urbana High School (1899-1901) and spent a year (i90i-'02) in the Preparatory School of the University of Illinois. At the end of a four-year course at the University of Illinois he received the degree of B.S. in Chemistry in 1906. Since 1906 he has been a graduate Student and officer at the Uni- versity of Illinois, passing by promotion through the grades of Scholar in chemistry in the graduate school (i9o6-'o7), assistant chemist in the laboratory of physiological chemistry (1907-08), assistant in physiological chemistry (1908-10), and instructor in physiological chemistry (i9io-'i2). In 1907 he received the degree of M.A. ; in 1910, the degree of Ph.D. His major subject for the Ph.D. degree was physiological chemistry, with Professor P. B. Hawk; his minor subjects were physical chemistry, physiology and histology. * Biochemical Bulletin: 1911-12, i, pp. 136, 570, 573 and 574. 202 Biochcmical N'czvs, Notes and Comment [Sept. Dr. Howe is a member of the American Society of Biological Chemists, American Chemical Society, American Society of Animal Nutrition, American Association for the Advancement of Science, Illinois Academy of Science, Sigma Xi, Phi Lambda Upsilon, and the Gamma Alpha Graduate Scientific Fraternity. Dr. Howe's publications. 1907. The electrolytic corrosion of brasses (with A. T. Lincoln and David Klein) ; Journal of Physical Chemistry, 11, 501. 1908. Comparative tests of Spiro's and Folin's methods for the determination of ammonia and urea (with P. B. Hawk) ; Proceedings of the American Society of Biological Chemists, i, 104; Journal of Biological Chemistry, 4, p. x. 1909. Comparative tests of Spiro's and Folin's methods for the determination of ammonia and urea (with P. B. Hawk) ; Journal of Biological Chemistry, 5, 477. — On the preservation of feces (with T. A. Rutherford and P. B. Hawk) ; Proceedings of the American Society of Biological Chemists, i, 196; Journal of Biological Chemistry, 6, p. xlix, 1910. On the preservation of feces (with T.A. Rutherford and P. B. Hawk) ; Journal of the American Chemical Society, 32, 1683. — A study in repeated fasting (with P. B. Hawk) ; Proceedings of the American Society of Biological Chemists, i, 259; Journal of Biological Chemistry, 7, p. xlvi. — Fasting studies on men and dogs (with H. A. Mattill and P. B. Hawk) ; Proceedings of the American Society of Biological Chemists, i, 260; Journal of Biological Chemistry, 7, p. xlvii. — Nitrogen partition in repeated fasting; Dissertation (pp. 42), pre- sented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (University of Illinois). 191 1. On the differential leucocyte count during prolonged fasting (with P. B. Hawk) ; Proceedings of the American Society of Biological Chemists, 2, 15; Journal of Biological Chemistry, 9, p. xxi. — Fasting studies : I. Nitrogen partition and physiological resistance as influenced by repeated fasting (with P. B. Hawk) ; Journal of the American Chemical Society, 33, 215. — Fasting studies: III. Nitrogen partition of two men through two seven-day fasts following the prolonged Ingestion of a low-protein diet : Supplemented by comparative data from the sub- sequent feeding period (with H. A. Mattill and P. B. Hawk) ; Journal of the American Chemical Society, 33, 568. — Fasting studies: V (Studies on water drinking: XI). Influenae of an excessive water Ingestion of a dog after a prolonged fast (with H. A. Mattill and P. B. Hawk) ; Journal of Biological Chemistry, 10, 417. I9I2] Columbia University Biochemical Association 203 1912. A metaboHsm study on a fasting man (with P. B. Hawk) ; Proceedings of the American Society of Biological Cheniists, 2, 65 ; Journal of Biological Chemistry, 11, p. xxxi. — Hydrogen-ion concen- tration of fecal extracts (with P. B. Hawk) ; Proceedings of the American Society of Biological Chemists, 2, 66; Journal of Biolog- ical Chemistry, 11, p. xxxii. — Studies on water drinking: XIII (Fast- ing studies: VIII). Hydrogen-ion concentration in feces ; Journal of Biological Chemistry, 11, 129. — A comparison of the data from two fasts each exceeding one hundred days in length and upon the same subject (with P. B. Hawk) ; Proceedings of the American Physiological Society, American Journal of Physiology, 29, p. xiv. — On the differ- ential leucocyte count during prolonged fasting (with P, B. Hawk) ; American Journal of Physiology, 30, 174. — Fasting studies: VI. Dis- tribution of nitrogen during a fast of 117 days (with H. A. Mattill and P. B. Hawk) ; Journal of Biological Chemistry, 11, 103. — The gen- eral aspects of fasting: Address before the Columbia University Bio- chemical Association, May i, 1912; Biochemical Bulletin, 2, 90. — The distribution of urinary nitrogen as influenced by the Ingestion of moderate and copious quantities of distilled water at meal time (with D. W. Wilson and P. B. Hawk) ; (in press) Journal of the American Chemical Society, 34, Proceedings, p. ^^i- — Addendum. The utiliza- tion of individual proteins by man as influenced by repeated fasting (with P. B. Hawk) ; Proceedings of the Eighth International Congress of Applied Chemistry (preliminary edition), 19, 145. (William J. Gies, Secretary of the Faculty of Medicine. ) Resignations and appointments. The following changes in the staff for the year 1912-13 were officially authorized prior to October i, IQ12: Dr. Paul E. Howe, assistant professor, vice Prof. Wm. H. Welker, resigned; Dr. Clayton S. Smith, instructor (pro- moted), vice Dr. Ernest D. Clark, appointed instructor in chemistry at the Cornell University Medical College; Dr. Frederic G. Good- ridge, assistant, vice Reuben Ottenberg, resigned ; Messrs. E. G. Mil- ler, Jr., and Arthur Knudson, assistants, vice Dr. C. S. Smith (pro- moted) and Mr. A. R. Rose, resigned; and Misses Ethel Wickwire and Tula L. Harkey, assistants (at Teachers College), vice Mr. E. G. Miller, Jr. (promoted) and Miss Blanche Harris, resigned. Summer session. Courses. Professor Gies kept the labora- tory open daily throughout the summer and conducted courses (July 5-August 15) in nutrition at the College of Physicians and 204 Biochemical News, Notes and Comment [Sept. Surgeons with Dr. C. S. Smith's assistance, and at Teachers' Col- lege with the aid of Dr. Emily C. Seaman and Miss B. E. Shaffer. Investigators. The workers named below conducted research, in the biochemical laboratory at the College of Physicians and Sur- geons, during all or part of the summer vacation : Louis Berman, R. J. Cook, Edward Cussler, F. R. Eider, N. B. Foster, Wm. J. Gies, Samuel Gitlow, Isidor Greenwald, W. M. Kraus, Alfred P. Lothrop, H. A. Mattill, H. O. Mosenthal, Jacob Rosenbloom, Emily C. Seaman, C. S. Smith, William Weinberger, Charles Weisman, Wm. H. Welker, Harry Wessler. Miscellaneous notes. Professor Gies was vice president of the Section on Biochemistry including Pharmacology of the 8th Inter- national Congress of Applied Chemistry (page 150). — Dr. H. 0. Mosenthal recently returned from Tübingen, where he had been working in the medical clinic under the direction of Prof. E. von Romberg. — Dr. Jacob Rosenbloom has resigned the affiliated Posi- tion of assistant pathologist at the German Hospital. — Mr. A. R. Rose has lately completed the requirements for the Ph.D. degree and will be publicly examined in October. He has begun a special study of amylase with Prof. H. C. Sherman. — Mr. Joseph Hepburn- has begun work as " university fellow " in biological chemistry. EDITORIALS Ernst Schulze was one of the great pioneers in biological chem- istry. He worked in an inspired way along the zone between the old zoöchemistry and the ancient phytochemistry, and achieved the distinction of removing the barriers between rnst c uze these two fields and uniting them in one great open biochemical territory. He brought Hght and understanding into large domains where darkness and doubt prevailed. His ex- ample in industry, patience, perseverance, devotion, enthusiasm, abil- ity and productiveness has been an inspiration to biochemical work- ers the world over. Schulze's classical achievements and Service will be forever linked with the history of fundamental developments in a great formative scientific era. His name and Service will be justly remembered, as his memory will be venerated, for very many generations. As the methods of chemical analysis become more delicate and refined there appears ever increasing evidence that the maintenance of health and nutrition depends not alone on the caloric values of T . ^ ^1. u food-stuffs and the relativ^e proportions of nitro- Important though ^ . unknown factors in gen and carbon in the diet, but quite as much on nutrition other factors which we are beginning fully to appreciate. Scurvy has long been one of the indications that there are certain unappreciated factors in a normal diet, and the antiscor- butic action of vegetables and vegetable Juices is strong emphasis on this point. The researches of Hart, McCollum, Steenbock and Humphrey/ on cattle, and of Osborne and Mendel,^ on rats, are among the many recent studies that reveal the importance of such unknown though influential factors in their broad bearing. The disease beriberi is a concrete example of the disturbance of such subtle influences. For a considerable time physicians in the ^ Hart, McCollum, Steenbock and Humphrey : University of Wisconsin Agricultural Experiment Station Research Bulletin, No. 17 (June, 1911). * Osborne and Mendel: Carnegie Institution, Puhlication 156. 205 2o6 Important Factors in Nutrition [Sept. Orient have believed that certain foods were responsible for this form of Polyneuritis. Miura believed the noxious agent to be contained in a certain fish, which is much eaten raw ; but more re- cently the blame has fallen on rice. It has been asserted that in the prisons of Java, beriberi occurs in one out of every forty prisoners when shelled rice is eaten; in one out of ten thousand, if the un- shelled grain is used. The classical studies of Schaumann were sug- gested by observations of this kind. Schaumann believed that since polished rice is poor in phosphorus, beriberi is due to a deficiency of certain organic phosphorus Compounds. This hypothesis had some Support in the fact that materials which relieve the pain of neuritis, such as bran, are rieh in phosphorus, but the later investigations of Wieland^ cast doubt on the accuracy of these deductions, since it could not be shown that the total body-phosphorus was much in- fluenced by feeding mice on polished rice. In this connection the researches of Fingerling,^ and of McCollum and Halpin,^ are sug- gestive, for they have shown a synthesis of organic phosphorus Com- pounds from inorganic phosphates. The latest contributions to the study of beriberi were made by Chamberlain and Vedder,^ by whom it has been shown that extracts of rice-bran are effective as therapeutic agents and that these ex- tracts contain mere traces of phosphorus. The active substance in the bran has not yet been identified, but the interesting feature dis- closed by the present evidence as to the etiology of kakki is that a food stuff may contain an ingredient which is essential in order to prevent injury to the tissues by other components of such food ma- terial. Rice grain is harmless when eaten with the pericarp but, if the latter is removed by " polishing," a malady ensues which may be cured by extracts made from the pericarp. These facts present a new face to the idea of " balanced rations " and also remind us of the broad biological significance of Loeb's "balanced Solutions." N. B. F. ' Wieland : Archiv für experimentelle Pathologie und Pharmakologie, 1912, Ixix, p. 293. * Fingerling: Biochemische Zeitschrift, 1912, xxxviii, p. 448; xxxix, p. 239. "McCollum and Halpin : Journal of Biological Chemistry, 1912, xi (Pro- ceedings of the American Society of Biological Chemists, p. xiii). * Chamberlain and Vedder: Philippine Journal of Science, 1912, vi, p. 251. 1912] Editoriais 207 In a circular with this title, Director Russell of the Wisconsin A'gricultural Experiment Station^ has recently given an interesting summary of the perfection of the Babcock quantitative test for milk- rru ~- t ^ fat and the influence which it has exerted on The Coming 01 age of the dairy science and practice throughout the world. Babcock test Milk and its numerous products play so impor- tant a röle in the economy of the home and in the dietary of the sick that the significance of Professor Babcock's contribution cannot re- main unnoticed in the annals of the medical world. The simple, yet highly accurate Babcock method of estimating the fat content of milk and cream finds daily application not only in dozens of analytic laboratories, but likewise in hundreds of creameries, in milk establishments, and even in the office of the busy practitioner of medicine, where a few inexpensive devices enable him to gauge the richness of a breast-milk or a modified milk mixture with facility. Every pediatrist appreciates what the Babcock test means for the exigencies of practice and successful feeding. Today, twenty-two years after the introduction of this procedure which, as Ex-Gover- nor Hoard remarked, has made dairymen more honest than the Bible because it has removed all opportunity for them to profit by any deceit, it is interesting to note that no change has been made in the essential features of the test during all this period. The tech- nic of the Operation remains the same as when the details were pub- lished by Dr. Babcock in 1890. The Stimulus which it has given to scientific dairying, to the standardization and improvement of our milk-supplies, to the possibilities of rational infant-feeding, and to what these in turn involve in the direction of the public health, is scarcely appreciated by the medical profession. Director Russell has written that the Babcock test frees the dairy farmer from the fetters of past traditions, and removes him from the category of "mossbacks." The influences here referred to have in fact been even more far-reaching. An additional feature deserves mention: No patent was taken out on either the method or the apparatus required to carry out the Babcock test. There zuas no copyrighting of a name — no commer- ' University of Wisconsin Agriculitiral Experiment Station, Circular of Information, No. 2^, 1912. 2o8 Babcock Test [Sept. cialism. In accord with a code of ethics now more generally recog- nized than at any time, the discoverer, becaiise of his connection with the State experiment Station, gave his invention freely to the world. We may gladly join in acknowledging our Obligation to the man whom the grate ful State of Wisconsin has presented a medal in recognition of " his iinselfish dedication of these inventions to the public Service." (Editorial : Journal of the American Medical As- sociation, 191 2, lix, p, 544.) The discovery and investigation of the specific secretions of the so-called ductless glands and of other organs make one of the most interesting chapters in physiology. Much has been learned con- cerning these secretions and their röle in the rgano- er py j^^Q^jy These extracts, theoretically, should be of great value in the treatment of diseases in which a certain gland or glands are deficient or entirely lacking in function. But actual ex- perience has been disappointing as a rule, for two reasons : ( i ) The diagnosis of insufficiency of secretion on the part of a certain gland or Organ is usually most difficult; (2) and even when a correct diagnosis is made, it is rarely possible to administer the gland sub- stances in such a way as to develop their specific activity. A notable exception to this experience is the successful use of thyroid extract in thyroid insufficiency or myxedema. Suprarenal substance has also proved highly use ful as a circulatory stimulant and hemostatic, but not for the treatment of Addison's disease. It can safely be said that the administration of gland substance from the thymus, hypophysis, ovaries, pancreas, testicles, etc., for dis- eases of these organs, has hitherto met with failure. Only härm can come from their promiscuous use before careful experimentation fully determines their value. A wholesome skepticism concerning the efficiency of preparations of the digestive enzymes is likewise commended. After years of usage many of our best clinical observers believe that pepsin, " pan- creatin " and the amylases are of little or no value. The use of se- cretin more recently has been similarly disappointing. The con- tinued routine use of these preparations is due chiefly to the claims of manufacturers. I9I2] Editorids 209 We congratulate our English confreres on the successful con- summation of their plans for the formation of a biochemical society and the publication of a biochemical Journal^ under their associated Biochemical So- control. In this coimtry we have long derived ciety, England great benefit from the meetings and activity of the American Society of Biological Chemists and are confident our English colleagues will have a similar experience. We felicitate the biochemical profession at large on this further evidence of the rapid growth in usefulness, and the prominent place of Service, of biochem- ical art and science. The Bio-Chemical Journal has been highly esteemed in America, and we wish it long life and distinguished Service under its new management. " Science is essentially mutual- istic and the success of one Organization is the gratification of all — the triumphs and discoveries of one are shared with the many, and the feeling of pride in the progress of the one may he shared. zvithout loss by sister organizations. As the discovery made in one brauch of science may be the necessary foundation for the Solution of some problem in another, so the contribution from one society may be the stepping stone to advancement in another. It is all hail then, greetings and felicitation — and Godspeed in the accomplish- ments of your future destiny." The name of the writer of this note might suggest a strong par- tiality on his part for the incorporation into biochemical discussions, in English, of such words as " Baustein." He believes, neverthe- " Baustein " or ^^^^' ^^^^ English phrases of equal f orce thoug'h " construction Unit** of more abstract significance, such as "construc- tion Unit" for "Baustein," are more acceptable, especially to stu- dents receiving their introduction, in English, to the subject of pro- tein synthesis and similar processes. The foregoing remarks recall the common use, in English, of " Splitting product " or " split product " as equivalents for " Spal- tungsprodukt," when the substance referred to is neither " Splitting Splitting productsor "^^ "split," but has resulted from cleavage. cleavage products Why not term such substances "cleavage prod- ucts" in conformity with good usage in analogous relationships ? "Halliburton: Biochemical Bulletin, 1912; ii, p. 128. 2IO X-Rays fSept. We received recently, with very great pleasure, a foreign money Order for twelve dollars instead of tzvelve Shillings in payment of Volume I of the Biochemical Bulletin. In view of the fact that this overpayment did not excite a desire to dis- A rare complimen ^^ontinue the subscription, we have proceeded with more enthusiasm than ever with our editorial work, in the hope that future volumes of the Bulletin may be much more deserving of such a compliment. The doing that makes commerce is born of the thinking that makes scholars. — Ruskin. Perhaps the most valuable result of all education is the ability _. _ to make yourself do the thing you have to do, when it ought to be done, whether you like it or not. — Huxley. The fabric of medical progress — indeed, of all progress — is woven from legitimate dreams to a greater extent than the " practi- cal" man is wont to realize or willing to admit. Editorial: Journal of the American Medical Association, 1912, lix, p. 1195. Who is it that, when years are gone by, we remember with the purest gratitude and pleasure? Not the learned or clever. But those who have had the force of character to prefer the future to the present, the good of others to their own pleasure. — Stanley. A fig for yesterday's convictions ! They were the cocksure beliefs of children lost in the dark. This is another day, and we've grown overnight. Do you plead the dignity of fixed opinion? It is enough for us to say : " We believed it when we affirmed it ; we have learned and changed our minds." — Ana Phylactic. The successful man, whether in business, in the professions and trades, or in politics, enjoys the game for its own sake. He is not a conscript in life's battles, but a volunteer, The way interests him as much as the goal. Not only the result, but the exercise of powers necessary to achieve it, gives him satisfaction. — AI I. Phatic. Speaking mentalwise, overfed conceit equals the blind staggers. The easiest kind of intoxication is that which feeds upon the poisons distilled by a self-caressing imagination. Open the floodgates of self-approval and soon you won't know whether you are making good or not, for you won't be able to present an intelligent compari- son of your own achievements with those of others. — Jaun Dice. BOOKS RECEIVED The BiocHEMicAL Bulletin will promptly acknowledge, under this heading, the receipt of all publications that may be presented to it. From time to time, selections will be made for review on pages of the volume to be appropriately indicated here. Reviews will be matter-of-fact Statements of the nature and Contents of the publications under consideration, and will be intended solely to guide possihle purchasers. The wishes or expectations of publishers or donors of volumes will be disregarded, when they are incompatible with our convictions regarding the interests of our colleagues. The size of the printed pages, in inches, is indicated in the appended notices. Practical physiological chemistry. A book designed for use in courses in practica! physiological chemistry in schools of medicine and of science. By Philip B. Hawk, professor of physiological chemistry and toxicology in the Jefferson Medical College of Philadelphia. Fourth edition, revised and en- larged. Pp. 475 — 4J^X8; $2.50 net. P. Blakiston's Sons & Co.. Philadelphia, 1912. The protein element in nutrition. (One of the International Medical Mono- graphs.) By Major D. McCay, professor of physiology, Medical College, Cal- cutta. Pp. 216 — 4X7, with 8 füll page portraits of human subjects; $2.00 net. Longmans, Green and Co., New York; Edward Arnold, London, 1912. Oxidations and reductions in the animal body. (One of the Monographs on Bio chemistry.) By H, D. Dakin, The Herter Laboratory, New York. Pp. 135 — 41^X8; $1.40 net. Longmans, Green and Co., 1912. Researches on cellulose. III (1905-1910). By C. F. Gross and E. J. Bevan. Pp. 173 — 3>