rj. ■ ivtü'-.-x-'.-ir; j. , yß 'im Vol. IV March, 1915 No. 13 Biochemical Bulletin Edited, for the Columbia University Biochemical Association, by Her^an M. Adler, John S. Adriance, David Alperin, Carl L. Aisberg, D. B. Armstrong, George Baehr, J. C. Baker, Louise C. Ball, Charles W. Ballard, Louis Baumann, George D. Beal, S. R. Benedict, William N. Berg. Josephine T. Berry, Robert Bersohn, Isabel Bevier, Louis £. Bisch, A. Richard Bliss, Ernst Boas, Helene M. Boas, Charles F. Bolduan, Samuel Bookman, Sidney Born, O. C. Bowes, William B. Boyd. J. Bronfenbrenner, Jean Broadhurst, H. E. Buchbinder, Leo Buerger, Gertr. Burlingham, J. G. M. Bullowa, R. Burton-Opitz, A. M. Buswell, R. P. Calvert, A. T. Cameron, Herbert S. Carter, Russell L. Cecil, Arthur F. Chace, Hardee Chambliss, Ella H. Clark, Ernest D. Clark, Alfred E. Cohn, Kath. R. Coleman, Helen C. Coombs, Burrill B. Crohn, Glen E. Cullen, Louis J. Curtman, William D. Cutter, C. A. Darling, William Darrach, Norman E. Ditman, Ula M. 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Howe, Anton R, Rose, Jacob Rosenbloom, William Salant, W. S. Schley. Oscar M. Schloss. H. von W. Schulte, Fred W. Schwanz. C. A. Schwarze, Emily C. Seaman, Fred J. Seaver, A. D. Selby, A. Franklin Shull. J. Buren Sidbury. C. Hendee Smith. Clayton S. Smith. Edward A. Spitzka. Matthew Steel, Ralph G. Stillman, Chas. R. Stockard, Edward C. Stone, Mary E. Sweeny, Arthur W. Thomas, Helen B. Thompson, M. K. Thornton. Grover Tracy, F. T. Van Buren, Eliz. G. Van Hörne, Philip Van Ingen, Chas. H. Vosburgh, D. M. Ward, Hardolph Wasteneys, Edwin D. Watkins, William Weinberger, F. S. Weingarten. J. W. Weinstein, Charles Weisman, William H. Welker, C. A. Wells, Harry Wessler, Isabel Wheeler, H. L. White, Geo. H. Whiteford, David D. Whitney. Ethel Wickwire, Herbert B. Wilcox, Guy W. Wilson, Louis E. Wise, William H. Woglom. L. L. Woodruff, I. O. Woodruff, H. E. Woodward. Anna B. Yates, Hans Zinsser, William A. Perlzweig NEW YORK £ntered as second-clas* matter in the Post Office Rt Lancaster, P«. ,— IM'»" ' ANNOUNCEMENTS Future issues of the Biochemical Bulletin Pursuant to a further change of plan, which is referred to edi- torially on page 270, this (March) number is the first issue of Volume IV of the Biochemical Bulletin. Hereafter, the volumes of the Biochemical Bulletin will coincide, in periodicity, with the calendar years, instead of the academic years as heretofore. The new plan will enable us to issue the quarterly numbers promptly hereafter. — in March, June, September and December. Professional assistance offered to Biological Chemists The members of the Columbia University Biochemical Association will coöperate confidentially with any one who desires the Services of biological chemists or who seeks a position in biological chemistry. Address inquiries to William J. Gies, 437 West ^pth St., New York. The Biochemical Bulletin is a quarterly biochemical review. 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BiocHEMiCAL Bulletin Volume IV MARCH, 191 5 No. 13 IN MEMORIAM FRANCIS HUMPHREYS STORER Born, March 27, 1832 Died, July 30, 19 14 With the demise of Professor Francis Humphreys Storer, Pro- fessor in the Bussey Institution of Harvard University, on July ßoth last, at the age of 82 years, there ended a long and useful career de- voted principally to chemistry in its relation to agriculture. Frank Storer, as he was known in the early days, was born on Boylston Street, Boston, and was the son of David Humphreys Storer, M.D., LL.D., and Abby Jane (Brewer) Storer. He received his early training in private schools and under private tutors, and from 1850 to 1851 was a Student at the Lawrence Scientific School of Harvard University. From 185 1 to 1853 he served as assistant to Professor Josiah P. Cooke, then Professor of Chemistry at Harvard. In 1853 he was chemist with the U. S. North Pacific Exploring Expedition. After his return to Massachusetts, Storer resumed his studies at the Lawrence Scientific School and graduated with the degree of Bachelor of Science in 1855. Although not reared in an agricultural Community, Storer was a profound lover of nature and in his younger days, it is said, he seized every opportunity to visit the countryside. Endowed with a keen Imagination, he was one of the few to realize the need of a better basis for the practice of farming on the American continent. Rule of thumb methods prevailed at the time to the extreme, and in many cases where crops were successfully grown, or where not. 2 Francis Humphreys Storer [March, the outcome was attributed to this or that cause, but hardly ever to the chemical factors operating in the soil. Impressed with the idea of the need of placing agriculture on a plane with the other sciences, Storer went abroad in 1855 to study the European methods of applying chemistry to the study and prac- tice of agriculture. At Tharand he is found working in the labora- tory of the Royal Academy of Agriculture, studying methods under the famous Julius A. Stöckhardt. At Heidelberg he listened to the lectures of the great Robert Wilhelm Bunsen, and last, but not least, we hear of him making observations in Paris under the master Boussingault. Not finding an appropriate opportunity for applying his newly- gained knowledge — the application of chemistry to the Interpretation of biological processes — Storer, on his return to the United States in 1857, while the " panic of 1857" was at its height, established him- self as a Consulting and analytical chemist in Boston. In 1865, however, he accepted a position as chemist with the Boston Gas Light Company, and also became Professor of General and Indus- trial Chemistry at the newly-created Massachusetts Institute of Technology. This was Professor Storer's first real experience as an independent teacher and here he taught chemistry, as he often re- marked later, " better than ever before in America." Prof. William B. Rogers, the Founder of the Massachusetts In- stitute of Technology, was strongly of opinion that the right way to teach the sciences — chemistry, physics, and biology — was the labora- tory way, without rejecting entirely the lecture method, in which he was himself a master. He insisted wlien he started scientific Instruction in the Institute of Technology, that every Student should have abundant opportunity to make experiments himself in properly equipped laboratories. The first man he selected to be Professor of Chemistry in the new Institute was Storer, with whose thorough laboratory training in chemistry, and experience as a practicing chemist, Professor Rogers was familiär. Professor Rogers had also known about some chemical researches Storer and Charles W. Eliot had made together in the early '60s, especially with one pub- lished as a memoir in the series of the American Academy of Arts and Sciences on "The impurities of commercial zinc." Professor 191 5] Lewis William Fetser 3 Rogers knew that Eliot also belle ved in the laboratory method of teaching chemistry. After the selection of Professor Storer had been made, Eliot re- ceived, at Vienna, a letter from Professor Rogers, asking Eliot to be- come Professor of analytical chemistry in the Institute, and telling him that his f riend Storer was to be the other professor of chemistry. Storer and Eliot began, in September, 1865, to teach chemistry by the laboratory method to the first class enrolled in the Massa- chusetts Institute of Technology in a small and poorly equipped room on the second story of a mercantile building on Summer Street, nearly opposite the störe of C. F. Hovey & Co. But Rogers Hall was nearly finished; and in the course of that year President Rogers assigned the rooms in the new building to the Chemical De- partment, and provided the money with which to furnish and equip the laboratories. Storer and Eliot made all the detailed plans, and supervised the constructions on the principle that in all chemical sub- jects every Student was to have desk-room and apparatus for con- ducting experiments several hours a week with his own eyes and hands. Foreseeing the need of laboratory manuals, first in general chem- istry, and then in qualitative analysis, Storer and Eliot soon began the preparation of these books, — first the " Manual for Inorganic Chemistry," and then the " Manual for Qualitative Analysis." These books were written in a manner then novel, though now familiär — some chapters by Storer and some by Eliot. The manuscript hav- ing been put into type, the authors used the proofs in their classes for one year in the Institute laboratories, and in this process dis- covered and remedied some defects, and made many improvements. It is related, by one who knew of the relations existing between these pioneer teachers of chemistry, that when it came to Publish- ing the book, a title page was demanded of them ; and each author maintained that the other's name ought to stand first. Discussion led to no result ; so they tossed up a cent to decide the question by Chance. Storer picked up the cent, and announced that Eliot's name was to stand first. Eliot accused him of not having looked at the cent; but he would not recognize the correctness of Eliot's Observation. So the book became known as Eliot and Storer's ; but the authors succeeded in putting on the back of the book the names 4 Francis Humphreys Storer [March, Eliot and Storer crossed — one on top of the other — as an indication that the order of the title page had no real meaning. The " Manual of Inorganic Chemistry " sold in considerable num- bers for a long term of years, and is still in the market after several revisions. It was at first the only book of the kind in the English language ; and, indeed, there was no equivalent in any language ; but within a few years many manuals appeared which were intended to promote the same laboratory method of Instruction in chemistry. A few years after its first appearance, one of the authors was one day visiting Rugby School in England, and found that the Master who taught chemistry at that famous School was using it in the laboratory which he had set up for the teaching of chemistry. He accounted for the presence of this American text-book in the School by f rankly saying that he had not been able to find an English book which answered the same purpose, or was conceived in the same spirit. In 1869 Eliot became President of Harvard University, and thereafter Storer made all the revisions of the two manuals he and Eliot had written together.^ Judging from his " Cyclopedia of Quantitative Chemical Anal- ysis," prepared during his stay at the Massachusetts Institute of Technology, it is evident that the mind of Professor Storer was an extremely practical one. This book is a silent witness to the work of a pioneer, and from the preface we note that the object of writing it was "not only to provide the Student and working chemist with a comprehensive dictionary of quantitative processes, but to call the attention of the chemical fraternity to the question of the possibility of presenting this branch of chemical art in a more serviceable and manageable form than has been customary hitherto. The experi- ment is certainly worth the trying whether a definite System of classi- fying substances in alphabetical order, and of referring each and every process to the fundamental fact or principle upon which it depends, will not greatly facilitate both the study and the practice of analysis. . . . The tendency of all the works recently published (1869) on quantitative analysis is towards condensation and ab- 1 Dr. Charles W. Eliot was Professor of Analytical Chemistry and Metal- lurgy at the Massachusetts Institute of Technology from 1865 to 1869, and from then on, President of Harvard University. He was Professor Storer's brother- in-law. 1915] Lewis William Fetzer 5 breviation, while the aim of the present book is to show that per- spicuity can be best gained by amplification, if need be, and method- ical arrangement." Thus, for example, in the case of aluminum acetate we find, first, the principles (underlying the method) ; second, appHcations (of the method) ; third, the various methods; and fourth, the precautions (to be observed) . Truly a noble viewpoint, and one which undoubt- edly called for many sacrifices. As a bibhographer Storer had few equals. One is especially impressed with this f act when examining " The First OutHnes of a Dictionary of Sohibilities," prefaced in 1862 and published in 1864. This work, probably the only one of its kind in the EngHsh lan- guage at the time, and today still a veritable mine of information, surely was a labor of love, and a monument to one who deemed it a pleasure to lessen the bürden of others. When the Bussey Institution, a School of Agriculture and Horti- culture, was finally organized in 1870, Francis Humphreys Storer was chosen on November 25, 1870, to be its Professor of Agricul- tural Chemistry, and in 1871 he became Dean. In this capacity Storer was at his best and it marked the beginning of an era of much fruitful and fundamental agricultural research. The found- ing of the Bussey Institution was, to Storer, " the nearest thing to an agricultural experiment Station in Massachusetts." The Status of Professor S. W. Johnson of the Sheffield Scien- tific School of Yale University, was very similar to that of Profes- sor Storer at Harvard. Many of the ideas of the two savants were alike.^ Thus in 1878, under date of April 26, Storer writes : "I noted (even before you wrote) what you say of Miller's cows vs. meal. 'Tis just what I would have said myself." Again, in a letter dated April 3, 1880, to Samuel W. Johnson, we note the f ollowing : " I am glad you hold your ' luff ' in respect to the con- ventional method of stating analyses of fodder. There is no sense in trying to refine this thing beyond the possibly practical. We are hardly more ripe than Einhof and Sprengel were for the complete analysis of rough fodders, and there is a semblance of — (let us say 2 See " From the Letter Files of S. W. Johnson," by Elizabeth A. Osborne, Yale University Press, 1913. 6 Francis Humphreys Stör er [March, ignorance) in Holding up the names of too many chemicals to the gaze of the great and unsoaked public. It is bad enough to have to report the ' fat' of hay as if it were really oil. What we really need is a critical sifting of all the analyses with the view of dis- covering the best possible means, in the light of existing knowledge. The question is one of chemistry far more than of arithmetic. There are manifold instances of 'maxima' and 'minima' which could be thrown out at once, for cause." The first results of Storer's labors were published in 1874, which was during pre-experiment Station (federal) days, in the Bussey Bulletm. The first bulletin emanating from an experiment Station in the United States was published in August 1877, by Samuel W. Johnson. The first Bussey Bulletin was entitled " A report of the results obtained on examining some commercial fertilizers, by way of analysis," by F. H. Storer (in 1874). The analyses reported were incidental to field experiments un- dertaken by the Bussey Institution in behalf of the trustees of the Massachusetts Society for Promoting Agriculture. The field tests were made for the purpose of testing the efficiency of a variety of substances sold in Boston and supposed to possess fertilizing power. The early bulletins on agricultural subjects are not mere reports of analyses, but worthy discussions of the topics and they are easily comparable with the ones issued by the best agricultural experiment stations of today. Thus in Bussey Bulletin No. 2 we find the f ollowing : " As regards the item * cellulose,' for example, the table shows conclusively that while hay contains 30 percent of Woody fiber, so compact that it can withstand the tolerably long- continued action of dilute acid and alkali ; that while oats contain 10 and one-third per cent, brewers' grains 6.2 per cent (in a total of only 22 and one-quarter per cent of dry organic matter), and dry whiteweed 31 per cent of this resisting substance, bran yields no more than eight and one-third per cent of it when exposed to precisely similar treatment, and maize only about three per cent. The method ordinarily used for determining cellulose is undoubt- edly far from being perfect, as I may have occasion to show in a future communication." Professor Storer had a prof ound respect for the work done by 1915] Lewis William Fefser 7 others. This fact is indelibly recorded in the Bussey Bulletins, which, in almost all cases contain an account of the literature per- taining to the subject under discussion. The work of the Bussey Institution included some of the earliest well-planned and systematic experiments on the field valuation of fertilizers, and in which chemi- cal analysis played an important but subordinate part. This is clearly shown by the results reported in Bulletin No. 7, entitl^d " A record of trials of various fertilizers upon the plain-field of the Bussey Institution." This third report gives the results obtained in 1873 and also reviews the three-year course of experiments. I doubt very much if the Bulletins of the Bussey Institution have been read or consulted to the extent that they should have been by agriculturists — they contain much that affects agricultural con- ditions today. The financial condition of the Bussey Institution was affected considerably by the Boston fire and the financial crisis in 1873. From these inroads into the Bussey fund the Institution never re- covered. Despite these setbacks, Professor Storer kept on with his investigations and teaching, often receiving no salary for his Services, or an amount so small that it was in no way commensurate with his ability and the Services he rendered. To what extent Pro- fessor Storer's financial condition suffered by his interest in agri- culture I am unable to say, but a reply to a letter sent to Storer by Professor Johnson^ from New Haven, Conn., December 15, 1879, may give us some light on the subject. "NewHaven^ Ct., Dec. 15, 1879. My dear Storer: I am most profoundly sorry at the State of Bussey in general and ... in particular. As to the questions — I only know what I got or rather I know nothing beyond that. I can't certainly say whether it was $35 per column that I first worked for, for the Tribüne, or not, but I think it was that. I Struck for $50 per column, had it for 6 months, then declined to go on. ... I shall at once see if I can't suggest to some good parties that they may get you to write for their papers, etc. Yours most faithfully." 2 See f ootnote, page 5. 8 Francis Humphreys Storer [March, The Bussey Institution closed its doors in 1907 to those for whom it was intended, i. e., first, young men who intended to be- come practical f armers, gardeners, Aorists, or landscape gardeners; second, young men who would naturally be called upon to manage large estates or who would make good Stewards or overseers of gentlemen's estates ; and third, persons who wished to study some special brauch of agriculture, horticulture, botany, or applied zoology. Professor Storer's three-volume work on " Agriculture in some of its relations with chemistry " was probably the crowning success of his literary and agricultural career. It has passed through seven editions. The esteem in which this authentic treatise was held after its issue can best be gleaned from the following Statement taken from the Harvard University Report of 1889, "The work combines very happily the Statement of scientific principles with due regard for financial and other practical considerations ; and it is written in an easy, populär style that should render its perusal most pleasurable for any intelligent agriculturist, however slight his acquaintance with chemical terminology. . . . His new work is a splendid contribution to agricultural science, is in fact almost monumental in character, and it must be many years before it can possibly be superseded by anything better." One who knew him intimately has said that all his life Storer was an omnivorous reader ; and as he had a very retentive memory and an unusual alertness and vivacity in conversation, he was a very instructive and inspiring companion for his intimates, of whom, however, there were but few. He gradually ceased to attend the scientific societies of which he was a member, withdrew more and more from society, and lived in his books, and in the circle of his immediate relatives. His habits were always simple and abstemi- ous ; so that he lived to be eighty-two years of age with unimpaired mental interests and powers, though with some bodily infirmities and limitations. His conscience was quick, his intelligence keen and rapid, and his temperament sensitive and impetuous, but not sanguine and serene enough for steady happiness. As a man of science he was spotless — a candid, devout, lover and seeker of truth. 1915] Lewis William Fetzer 9 I can not but think that too little has been made of Professor Storer's scientific Services to agriculture. Of the multitude who know nothing of his work this was not to be expected, but from the American agriculturist, plant physiologist, agricultural chemist, etc., it can command nothing but gratitude and respect. At the April meeting in 1907 of the President and Fellows of Harvard College, the resignation of Francis Humphreys Storer as Professor of Agricultural Chemistry of Harvard University, and Dean of the Bussey Institution, was accepted. The minutes of that meeting on the Services of Professor Storer are as f ollows : "The Services of Professor Storer to the Bussey Institution began with his appointment to the Professorship of Agricultural Chemistry on November 25, 1870, and have continued without any intermission to the present day. They comprehended stated teach- ing in the lecture room and laboratory; the production of a com- prehensive and durable treatise on Agricultural Chemistry ; and the general administration of the Institution, including its library and Bulletin. As a teacher, Professor Storer was highly interesting and helpful, because of his wide ränge of knowledge and his wealth of illustrative material. As an administrator, he was diligent, frugal in expenditure, and especially sympathetic with students whose means and attainments were limited, and whose early oppor- tunities had been few. He devoted himself without reserve to the Bussey Institution in spite of the fact that the Boston fire in 1872 greatly and permanently reduced its resources and changed its prospects." Lewis William Fetzer. Office of Expt. Stations, U. S. Dep't of Agriculture, and Georgetown University, Washington, D. C. PUBLICATIONS OF FRANCIS HUMPHREYS STORER Bulletins of the Bussey Institution Volume I — 1874-1876. {Cambridge: John Wilson and Son) Page Report of results of examination of commercial fertilizers 8 10 Francis Humphreys S torer [March, PAGE Record of results obtained on analyzing American " shorts " and "middlings," with remarks on the composition of bran. ... 25 Agricultural value of the ashes of anthracite 50 Record of trials of fertilizers upon the plain-field of the Bussey Institution : First Report; results obtained in 1871 80 Record of trials of fertilizers upon the plain-field : Second Re- port ; results obtained in 1872 103 Record of trials of fertilizers upon the plain-field: Third Report; results obtained in 1873. With a review of the three years' course of experiments 116 Analyses of several foreign superphosphates of lime, with remarks on the cost of importing superphosphates from Europe. . . 170 On the valuation of the soluble phosphoric acid in superphosphate of lime 185 Average amounts of potash and phosphoric acid in wood-ashes from höuse fires 191 On the importance as plant-food of the nitrogen in vegetable mould 252 Record of trials of fertilizers upon the plain-field of the Bussey Institution : Fourth Report ; results obtained in 1874 300 Report on analyses of salt-marsh hay and bog hay 339 On the fodder value of apples 362 Composition of date-stones, and of the stones of peaches and prunes 373 Analyses of potassic fertilizers 378 On the occurrence of ammonia in anthracite 398 Volume II — 1877-1900. {Boston: John Allyn) Amounts of potash and of phosphoric acid in several kinds of rocks 7 Agricultural value of spent dye-woods and tan 26 Composition of buckwheat straw 51 Fertilizing power of roasted leather 58 Notes of experiments in which buckwheat plants were watered with Solutions of peat in alkalis 72 Composition of certain pumpkins and squashes 81 Record of results obtained on analyzing the seeds of broom corn. . 94 Record of analyses of several weeds that are used occasionally as human f ood 115 iQis] Lewis William Fetser ii PAGE Chemical composition of blue joint-grass (Calamagrostis Cana- densis), as contrasted with that of reed canary-grass {Phalaris arundinacea) 130 Remarks on American fodder rations, with hints for the improve- ment of some of them 137 Results obtained on growing buckwheat in equal vveights of pit- sand and of coal-ashes 159 Chemical composition of the common field horse-tail or scouring rush (Equisetum arvense) 166 Results of a chemical examination of the shells of crabs and lobsters, and of those of oysters, clams, mussels, and other shell-fish 176 On the prominence of carbonate of lime as a constituent of Solu- tions obtained by percolating dry cultivable soils with water 195 Supplementary note to an article on the composition of pumpkins. 221 Results of fodder-analyses of leaves of the yellow- or curled- dock (Rumex crispiis), and of sprouts of the common milk-weed (Asdepias Cornuti) 255 Trials to determine the rates at which some fertilizers may be scattered by band 261 Experiments on feeding mice with painter's putty and other mix- tures of pigments and oil 264 Experiments on feeding plants with the nitrogen of vegetable mould 280 Experiments on the germination of weed seeds 289 An attempt to assay soils by the method of sand culture 292 A special instance of the resistance of clover seeds to water 317 Cherry stones eaten by the domestic pigeon 324 Some items of American experience which suggest that potassic fertilizers may perhaps act in several different ways 343 Observations on some of the chemical substances in the trunks of trees 386 Laboratory notes : (a) Doty birch wood yields little wood-gum 409 (&) Estimations of cellulose, lignic acids, xylan, and wood- gum in peach-stones 410 (c) Cold dilute alkaline Solutions dissolve very little wood- gum from the trunks of coniferous trees 414 (d) Not much wood-gum from the strawberry 417 12 Francis Humphreys Storer [March, PAGB (e) As to the presence of xylan in the membranous covering of the starch grain ? 419 (/) Analysis of ashes of bamboo sugar-baskets 420 On the systematic destruction of woodchucks 422 Results of a search for other sugars than xylose and dextrose in the products of the hydrolysis of wood from the trunks of trees 437 (a) Examination of the products of the acid hydrolysis of wood from the trunk of a sugar maple tree 440 (b) Acid hydrolysis of wood from the root of a sugar maple tf ee 443 (c) Acid hydrolysis of wood from the trunk of a birch tree. 451 (d) Experiments on the acid hydrolysis of cotton 454 (e) Experiments in which pure dextrose was treated with strong sulphuric acid 460 Volume III — 1901-1906. (Cambridge: University Press) Testing for mannose 13 Notes on the occurrence of mannan in the wood of some kinds of trees, and in various roots and fruits 47 A Supplement to the article on the occurrence of mannan in trees, roots, and fruit 69 Remarks on the " popping " of Indian com 74 Observations on a malt-glucose, known as "midzuame," made in Japan from rice and millet (with George W. Rolfe) 80 Experiments made to test the question whether mannite can be re- garded in any large and general way as serving as reserve f ood in flowering plants 98 American Agriculturist Ensilaging night soil, 1881, 40, p.470 Artificial milk, 1881, 40, p. 486 Waste of food from nonassimilation, 1882, 41, p. 756 Memoirs of the American Academy of Arts and Sciences Memoir on the alloys of copper and zinc, 1863, 8, p. 27 (Also in Chemical News), 1860, 2, p. 303 (Also in Chemical News), 1861, 3>PP- 22, 37, 51, 70, 149, 164 4> P-338 I, P-253 80, p.44 38, p.148 1915] Lewis William Fetser 13 (Also in Jour. Prakt. Chem.), 1861, 82, p. 239 (Also in Jour. de Pharmacie), 1860, 38, p. 234 Memoir on the impurities of com- mercial zinc (with C. W. Eliot), 1863, 8, p. 57 Naphtha from destructive distillation of lime soap (with C. M. Warren), 1867, 9, p. 177 (Also in Jour. Prakt. Chem.), 1867, 102, p. 436 Naphtha from Rangoon petroleum (with C. M. Warren), 1867, 9, p.208 Proceedings of the American Academy of Arts and Sciences Detection of Cr in presence of Fe, 1860, (Also in Chemical News), 1860, (Also in Jour. Prakt. Chem.), 1860, (Also in Jour. de Pharmacie) , 1860, Frozen well at Brandon, Vermont (with J. M. Ordway), 1860-62, 5, p. 296 Amounts of Pb in silver coins (with C.W.Eliot), 1860-62, 5, p.52 (Also in Chemical News), 1861, 3» PP- 318, 343 (Also in Repert Chim. Appl.) 1861, 3, p. 152 Difficulty of removing the last traces of CO2 from large quantities of air (with C. W. Eliot), 1860-62, 5, p.62 Chromate of Cr, etc., constituents of black oxid of Mn (with C. W. Eliot), 1860-62, 5, p. 192 (Also in Chemical News), 1S62, 6,pp. 121, 145, 157, 169, 183, 207, 217 {Aisoin Jour. Prakt. Chem.), 1863, 90, p.288 (Also in Repert. Chim. Appl), 1861, 3» P- 39° On the simultaneous occurrence of a soluble lead salt and free sul- phuric acid in sherry wine, etc., 1873, 8, p. 59 (Also in Chemical News), 1870, 21, p. 17 (Also in Philosophical Magazine), iS/O, 39, p. 154 Obituary notice of William Ripley Nichols (with a list of works by Professor Nichols), 1887, 22, pp. 528, 534 14 Francis Humphreys Stör er [March, American Journal o£ Science Second scries Behavior of the carbonates of Ca and Ba, 1858, 25, p.41 Arsenic not injurious to larvs of flies,i859, 28, p. 166 Review of Dr. Antisell's work on photogenic oils, etc., 1860, 30, p. 112 Infiuence of arsenious acid on the waste of the animal tissue, 1860, 30, p. 209 Loss of Hght by glass shades, 1860, 30, p.420 New anesthetic (kerosclene), 1861, 32, p. 276 Arsenic as an impurity of metallic zinc (with C. W. EHot), 1861, 32, p.380 Technical chemistry (notices and ex- tracts), 1861, 32, pp. 114,276,416 American process of working plati- num, 1862, 33, p. 124 Nitric acid and chlorate of K as an oxidizing mixture, 1869, 48, p. 190 Third series Ammonia a constant contaminant of sulphuric acid, 1875, 10, p. 438 Schönbein's test for nitrates, 1876, 12, p. 176 Ferment theory of nitrification, 1878, 15, p. 444 (Also in Chemical Nezvs), 1878, 37, p.268 Mode of action of shell and rock bor- ing mollusks, 1884, 28, p. 58 Obituary notice of Dr. Robert Angus Smith, 1884, 28, p.79 Chemical News Quick wet assay of galena, etc., 1870, 21, p. 137 Examples for practice in quantitative ehem. analyses, 1870, 22, pp. 89, 99, 109, 163, 187 Experiments on the gases occluded by coke (with D. S. Lewis), 1883, 4, p.409 On the oxidation of cork stoppers and rubber joints, 1883, 5, p.68 1915] Lewis William Fetzer 15 Memoranda of methods employed by fishermen for "barking" and in other ways preserving nets and sails, 1883, 5» P-430 (Also, with sliglit additions, in Re- port of U. S. Commissioner of Fish and Fisheries for 1882 ; 10, P- 295). Cultivator and Country Gentleman On the härm done by earth worms, 1882, 47, p. 108 A hint for fruit preservation, 1887, 52, p. 129 Harvard Register Some of the uses of agricultural study, Feb., 1880, i, p.88 The agricultural school as a prepara- tion for the study of medicine, June, 1880, i, p. iii Bussey School of Agriculture and Horticulture, Feb., 1881, 2, p.63 Agricultural education a means of political enlightenment, June, 1881, 3, p. 332 ^ö' Miscellaneous Cherry blossoms destroyed by squir- rels, Nature, Nov., 1875, 12, p.26 Epsom salts versus strawberries, American Journal Pharmacy, July, 1878, 8, p. 321 An item of history as to the idea of making the parts of guns inter- . changeable, Journal Franklin In- stitute, Nov., 1884,118, p.385 Sur le Substitution du verre soluble au savon resineus de fabrication des savon ordinaires, Repert. Chim. Appl, 1863, 5, p. 5 Rural New Yorker Reclamation of bog-land by the Ger- man method of burying with gravel, Feb., 1880, 39, p. loi i6 Francis Humphreys Storer [March, Dr. Angus Smith on the waste of ammonia in coke making, Feb., 1880, 39, p. 120 Maximum yield of wheat, Apr., 1880, 39, p. 246 Money value of leached ashes, Apr., 1880, 39, p. 262 Hurtfulness of chlorids to the tobacco crop, May, 1880, 39, p.277 New evidence in respect to weevil- eaten peas, May, 1880, 39, p. 294 Bone chewing by cattle, May, 1880, 39, p. 311 Indian corn as a starch crop, June, 1880, 39, p. 33^ Deer's horns eaten by cattle, June, 1880, 39, p. 376 The so-called process of ensilage, July, 1880, 39, p.424 A scientific view of composts, Aug., 1880, 39»PP- 503> Si?» 549 The valuation of "reverted" phos- phoric acid, Sept., 1880, 39, p. 586 Significance 01 active nitrogen for grain, Oct., 1880, 39, p.673 Experiments on gravelled bog-land, Oct., 1880, 39, p. 686 Which form of nitrogen is most as- similableby plants? Nov., 1880, 39, p. 766 Preservation of apples, Nov., 1880, 39, p. 784 Some merits of the Ailanthus, Dec, 1880, 39, p. 814 Power of woodland to hold water, Jan., 1881, 40, p. 37 Influence of manures upon the potato disease, Jan., 1881, 40, p. 53 Fodder value of frozen potatoes, Jan., 1881, 40, p. 67 The prevention of infection, Feb., 1881, 40, p. 116 Economy of using appetizing food for fattening cattle, Apr., 1881, 40, p. 222 Disinfection versus Ventilation, Apr., 1881, 40, p. 241 Worthless Compounds of nitrogen in some commercial fertiHzers, May, 1881, 40, p. 350 Preservation of brewers' grains, July, 1881, 40, p.49^ Why blue grass is so called, Aug., 1881, 40, p. 546 Soft wheat in Germany, Sept., 1881, 40, p.622 Tree trunks as water conductors, Sept., 1881, 40, p.662 The Massachusetts law of trespass, Oct, 1881, 40, p.669 Experiments on the use of wilted potatoes as seed, Oct, 1881, 40, p. 729 Temperature of the body after eating,Jan., 1882, 41, p. 10 1915] Lewis William Fetzer 17 Healthfulness of ensilage, Jan., 1882, 41, p. 59 Skim milk and whey, Nov., 1882, 41, p.765 Fermentation of new-made hay, Sept., 1883, 42, p. 555 White bread and brown, Nov., 1883, 42, p. 772 Phosphatic land plaster, Dec, 1883, 42, p.212 Distribution of fat in the bodies of animals, Dec, 1883 ; Jan., Feb., 1884 ; 42, p. 6; 43, pp. 9, 25, 41, 50 and 89 Science First series Rock disintegration in hot, moist climates, Feb., 1883, i, p. 39 Domestic ducks that fly abroad like pigeons, Feb., 1883, i, p.67 "Mother of petre" and "mother of vinegar," Mar., 1883, i, p.98 A caterpillar-eating hen-hawk. Mar., 1883, i, p. 168 Robins, sparrows, and earth-worms, May, 1883, i, p. 457 Symmetrical linear figures produced by reflection along a river bank, July, 1883, 2, p. 37 A populär prejudice: The mad stone, Aug., 1885, 6, p. 163 Books The result of the destructive distillation of bituminous substances. A Report presented to the annual meeting of the American Pharma- ceutical Association at New York, Sept. 10, 1860. With an essay on the history of the manufacture of paraffine oils (with Wm. Henry Whitmore). Boston,. 1860. First outlines of a dictionary of solubilities of chemical substances. Cambridge, Mass. : Sever and Francis, 1864. *A manual of inorganic chemistry (with Chas. W. EUot). New York: Ivison, Phinney, Blakeman & Co., 1866. *A compendious manual of qualitative chemical analysis (with Chas. W. Eliot). New York City (published by the authors), 1868. A cyclopedia of quantitative chemical analysis. Part I. Boston and Cambridge: Sever, Francis & Co., 1870. Part II, Boston: John Allyn, 1873. * Agriculture in some of its relations with chemistry. Volumes I and IL New York: Charles Scribner's Sons, 1887. t w F * These publications have passed through several editions. ON THE BEHAVIOR OF KERATIN SULFUR AND CYSTIN SULFUR, IN THE OXIDATION OF THESE PROTEINS BY POTAS- SIUM PERMANGANATE. I* TH. LISSIZIN (Laboratory of Medical Chemistry, University of Moscow, Russia.) Introduction. Previous researches on the products f ormed f rom proteins in general and from keratin in particular, by the action of potassium permanganate, give almost no indication of the f ate of the sulfur in this oxidation process. Since it is now known, as a result of Maly's^ investigations, that the total sulfur of egg-white remains in oxy-proto sulfonic acid, during permanganate oxidation, it is natural to ask: how does the sulfur of keratin, and of other sul- fur-yielding albuminoids, behave in such oxidation? Following a Suggestion of Prof. Dr. VI. Gulewitsch, I have undertaken the oxidation of human hair and of cystin, and have investigated the oxidation-products in relation to the sulfur content. Experimental. A. First I determined the sulfur-content of dry, fat-free, human hair. I. 0.4106 gm. of human hair was fused with a mixture (1:8) of potassium hydroxid and potassium nitrate, over a small alcohol flame. The fused mass was dissolved in water; the Solution was acidified with hydrochloric acid, after the addition of a few drops of bromin water, and evaporated to dryness on a water-bath; the residue was dissolved in water, filtered, precipitated in the usual way with barium chlorid, and the precipitate ignited and weighed. The amount of barium sulfate obtained was 0.1661 gm. (0.02282 gm. sulfur). The hair contained, therefore, 5.56 percent of sulfur. Then the sulfur-content of the oxy-proto sulfonic acid derived from the hair was determined. For this purpose I took 200 gm. of ♦Translated from the author's manuscript, in German, by Dr. Edgar G. MiHer, Jr. iMaly: Sitzungher. d. k. Acad. d. Wiss., 1885, xci (II Abteil), p. 157. 18 1915] Th. Lissizin 19 fat-free human hair, and digested it with 6 1. of 2 percent sol. of potassium permanganate. The mixture was allowed to stand for several days, with occasional shaking. The clear fluid was filtered, and the filtrate acidified with hydrochloric acid. The resulting pre- cipitate was washed, first by decantation and then on a filter, redis- solved in dilute soda sol. and precipitated by acidification. The precipitate was thoroughly washed, and dried, first in the air, then in an air-bath at 110° C. The dry substance was weighed and analyzed in the manner described above. IL 1.3399 gm. of the substance gave 0.1407 gm. of barium sul- fate (0.01933 gm. of sulfur). III. 0.1900 gm. yielded 0.1980 gm. of barium sulfate (0.00272 gm. of sulphur). The substance contained, therefore, judging from the results of the two analyses, 1.44 percent and 1.43 percent of sulfur, respec- tively. The oxy-proto sulfonic acid from egg-white contains, ac- cording to Maly,^ 1.77 percent of sulfur. Since only a small amount of oxy-proto sulfonic acid resulted from the oxidation of the hair, it is clear that only a small part of the sulfur of the keratin remained in the oxy-proto sulfonic acid. In Order to determine how much sulfur is held in the Solution as sulfuric acid, and how much is held in the form of some organic combination, after oxidation by permanganate, I have carried out two parallel experiments on the products of the oxidation. In the first experiment, 9.2160 gm. of dry, fat-free hair were digested in 700 c.c. of 2 percent sol. of potassium permanganate. After four days the liquid over the hair was perfectly clear. It was filtered, and from it two portions of 100 c.c. each were taken (IV-V). IV. The first portion was evaporated to dryness, treated with dilute hydrochloric acid, and the resultant precipitate of oxy-proto sulfonic acid was repeatedly extracted with water; the filtrate, together with the wash-water, was evaporated, and precipitated with barium chlorid. It yielded 0.040 gm. of barium sulfate (0.0055 gm. of sulfur). Therefore, in the 700 c.c, 0.0385 gm. of sulfur was held as sulfuric acid. 2 Maly : Monatsch. f. Chemie, 1884, viii, p. 255. 20 Keratin Sulfur and Cystin Sulfur [March, V. The second portion of the same fluid was evaporated to dry- ness, and the sulfur-content determined, after fusion with potas- sium hydroxid and potassium nitrate. The result was : 0.5 172 gm. of barium sulfate (0.07105 gm. of sulfur). Hence, the sulfur-content of 700 c.c. was 0.4974 gm. It is clear that a much greater amount of the sulfur (92.3 percent) is contained in the filtrate from the oxy-proto sulfonic acid in the form of some organic combination, and only a small part {y.y percent) is present in the Solution as sulfuric acid. For the second experiment, 10.2085 gm. of dry fat-free human hair were digested with 800 c.c. of 2 percent sol. of potassium per- manganate and allowed to stand for 3-4 days, until the fluid was perfectly clear. This was then filtered and, of the filtrate, two por- tions of 100 c.c. were taken. VI. The first portion was treated as in determination IV, with the difference, that this time the sulfur-content of the oxy-proto sulfonic acid precipitate was also determined. The filtrate and wash-water were treated with barium chlorid after the removal of the oxy-proto sulfonic acid, and yielded 0.4230 gm. of barium sul- fate (0.00581 1 gm. of sulfur). Hence, in 800 c.c, 0.0465 gm. of sulfur was present as sulfuric acid. VII. After the fusion of the oxy-proto sulfonic acid precipi- tates, with a mixture of potassium hydroxid and potassium nitrate, 0.0117 gm. of barium sulfate (0.00161 gm. of sulfur) was found. The sulfur in the oxy-proto sulfonic acid amounted, therefore, to 0.0129 gm. VIII. The second portion was treated as in determination V. After fusion, 0.4694 gm. of barium sulfate (0.06449 gm. of sulfur) was obtained. Hence, 0.5159 gm. of sulfur was present in 800 c.c. From these experiments it follows, unquestionably, that the greater part of the sulfur occurs, after oxidation with permanga- nate, as water-soluble organic substance. Of the total sulfur-con- tent in the dissolved oxidation products (0.5159 gm. of sulfur) there were found: 9 percent as sulfuric acid, 2.5 percent as oxy- proto sulfonic acid, and 88.5 percent as a water-soluble organic substance. This water-soluble substance gives a precipitate with lead ace- 1915] Th, Lissizin 21 täte, dissolves slightly in dilute alcohol, and is almost insoluble in 95 percent alcohol. I have used the following method, based on these properties, f or the Isolation of this substance : 70 gm. of hair were digested with 5-6 1. of 2 percent potassium permanganate sol. and allowed to stand for several days. The clear fluid was filtered, acidified with hydrochloric acid, and the precipitate separated by filtration. The filtrate was made slightly alkalin with dilute soda sol., and precipitated with lead acetate. The precipitate was washed, first by decantation and then on a filter, suspended in water, and treated with hydrogen sulfid. Precipitated lead sulfid was removed by filtration, the filtrate evaporated to a syrupy consistence, and extracted several times with 95 per cent alcohol. The residue was dissolved in a small amount of water, and the Solution yielded a pre- cipitate with a large excess of alcohol. This precipitate was dis- solved in a little water and re-precipitated with alcohol. The final product contained 12.5 percent of ash and 8.81 percent of sulfur. In Order to purify the product it was re-dissolved several times in water, re-precipitated with alcohol, and washed with alcohol and ether. This purified material was dried at 110° and analyzed (IX-XVI). IX, 0.1437 gm. gave, after ignition, 0.0089 gm. of ash, which consisted of silicic acid, with traces of potassium, sodium, calcium, iron and manganese. X. 0.0743 gm. gave, after ignition, 0.0046 gm. of ash. XL 0.1881 gm. gave, after burning with lead Chromate, 0.2537 gm. of carbon dioxid and 0.0928 gm. of water. XII. 0.1808 gm. burnt with lead Chromate, yielded 0.2385 gm. of carbon dioxid and 0.0908 gm. of water. XIII. 0.2166 gm. gave 26.3 c.c. of nitrogen, at 23.5° and 763 mm. Hg. XIV. 0.1762 gm. gave 21.1 c.c. of nitrogen, at 22.5° and 762 mm. Hg. XV. 0.1578 gm. gave, on fusion by the method described above (determination I), 0.1121 gm. of barium sulfate. XVI. 0.1787 gm. gave 0.1247 gm. barium sulfate. 22 Keratin Sulfur and Cystin Siilfur Summary: Found (percent) [March, IX X XI XII XIII XIV XV| XVI Average percent c 36.78 35-98 36.4 H 5-52 5.62 5-6 N 13-70 13-57 13-6 S 9.76 9-59 9-7 O 28.S Ash 6.19 6.19 6.2 Summary: Calculated. Ash-free subst. ptrcent C10H17N3SO8 percent C30H54N10S3O18 percent C 38.8 39-1 38.3 H 5-9 5-6 5-8 N 14-5 13-7 14.9 S 10.3 10.4 10.2 0 Ash 30.5 31.2 30.8 The substance so obtained is acid in reaction, is very hygro- scopic, and gives, with lead acetate, a precipitate which is soluble in an excess of the reagent, and in lead acetate sol. The substance gives the biuret reaction. Its aqueous Solution gives, on treatment with alcohol, a milky turbidity which, on evaporation, deposits small crystals. The material is very stable and, on heating with mineral acids, is decomposed with the formation of sulfuric acid. To determine its basici'ty the aqueous sol. was titrated with 0.0983/iV soda sol., with lacmoid-naphthol-green as the indicator. XVII. 1.3 c.c. of the soda sol. was required to neutralize 0.1053 gm. of the substance dissolved in water. For a monobasic acid (CioHi7N3S06)3, 1.2 c.c. would be necessary, but it must be con- sidered that the ash of the substance contained a large amount of silicic acid. B. In Order to ascertain the sulfur-distribution among the oxi- dation products of cystin, I have oxidized, with potassium perman- ganate, cystin prepared from human hair. About 2.3 gm. of cys- tin, containing 0.61 gm. of sulfur, were oxidized with i 1. of per- manganate sol. All of the filtrate, together with the washings, was evaporated and the contained sulfuric acid determined (XVIII). XVIII. I obtained 2.0931 gm. of barium sulfate (0.2875 S^- of sulfur). Therefore, 47 percent of the total sulfur was changed to sulfuric acid in the oxidation. iQis] Th. Lissizin 23 To 40 c.c. of the filtrate, which was obtained in the oxidation of the hair with 2 percerit potassium permanganate sol. (p. 19) and which contained 0.0022 gm. of sulfur as sulfuric acid, was added 0.2300 gm. of cystin (with a sulfur-content of 0.0614 gm.), and 45- 50 c.c. of 2 percent permanganate sol. After the reaction was ended, the fluid was filtered, the precipitate washed and the filtrate together with the washings was evaporated to dryness. The residue was acidified with hydrochloric acid, and the precipitate of oxy-proto sulfonic acid separated by filtration. The new filtrate, with the washings, was then precipitated with barium chlorid in the usual way (XIX). XIX. From this precipitate 0.2266 gm. of barium sulfate (0.031 1 gm. of sulfur) was obtained. Thus, from the oxidation of cystin I obtained 0.031 1-0.0022, 0.0289 gm. of sulfur, or 46.9 percent of the total sulfur, as sulfuric acid. From the results of these experiments it follows that a much larger amount of sulfur is split off as sulfuric acid by the oxidation of cystin with permanganate than by the similar oxidation of keratin. It is f urther to be noted that, in the process, some cystin always re- mains unoxidized and that, among the oxidation products of cystin, a small amount of hydrogen sulfid is always present, while in the dis- tillates of the oxidation products of hair, hydrogen sulfid is never found. General conclusions. These data suggest the following con- clusions: In the oxidation of keratin the largest part of the con- tained sulfur remains in organic combination, and only one tenth is converted into sulfuric acid. The oxidations of cystin and of keratin proceed along different lines. I have undertaken further work on this subject. SERUM DIAGNOSIS OF ROUS'S CHICKEN SARCOMA, BASED ON CHEMICAL METHODS CASIMIR FUNK Introduction. Serum diagnosis o£ tumors is, at present, in a preliminary stage. During the last few years there have been de- scribed several methods which are mostly based on biological prop- erties of serum. None of these methods, however, has given satis- factory results. Among the procedures tried by the author were the method of Freund, the meiostagmin-reaction of Ascoli, and the optical method of Abderhalden. In view of the failure of these biological tests to give a reliable method for tumor diagnosis, it seemed advisable to ascertain whether purely chemical methods would serve better for this purpose. This investigation was conducted with serum of chickens inocu- lated with Rous's chicken sarcoma. The large amount of serum obtainable from these animals was a great advantage for our pre- liminary studies. The tumor used being very malignant, the animals died in a few weeks. One would therefore expect the chemical changes to have been very pronounced. In the first part of the work, the serum was precipitated with absolute alcohol ; and the amount of precipitate, and its nitrogen and phosphorus contents, were estimated. The filtrate was analyzed in the same way. Throughout the whole inquiry, normal and tumor sera were treated simultaneously. The results tend to show that the tumor serum was poorer in proteins and phosphorus. Better results were obtained by analyzing serum itself. In this case the proportions of nitrogen, phosphorus, sulfur, chlorin, amino- nitrogen, and sugar, and the molecular depression, were taken into account. Of 22 tumor sera, 20 gave practically identical results. Tumor serum was, as a rule, poorer in nitrogen, phosphorus and sulfur than normal, though richer in amino-nitrogen ; the molecular depression was greater in normal serum. Sugar was determined in serum which had been kept, for some time, in an incubator; 24 1915] Casimir Funk 25 hence the figures are not exact. There was, perhaps, more sugar in tumor serum, but not enough cases were investigated to Warrant, a definite conclusion. Rolly and Oppermann found, in the few cases at their disposal, an increase of sugar in the plasma.^ The two sera which gave contradictory results were, strangely enough, taken from animals in which inoculation occurred much earher than in other cases. Whether these results were due to the resistance of the animal to tumor growth, or to the weakness of the tumor, must be left for further study to determine. For these two sera the tumors were very small and entirely encapsulated. In summarizing the results the author feels justified in conclud- ing that in the case of Rous's sarcoma, chemical analysis gives much more reliable results than other diagnostic methods. Sev- eral objections can, however, be put forward, The most important of these is that Rous's chicken sarcoma differs in many respects from other tumors; it is regarded by Rous himself as being of infective origin. In a few instances rat serum was used — of rats inoculated with Jensen's sarcoma. In each case 5-6 rats were bled and the bloods combined, so that the figures in the Tables represent good averages. Here, too, the differences were very much of the same order as for chicken sera. This matter awaits further study. A second objection, no less important, is the fact that the same chemical differences may be found in other pathological conditions. The animals regarded as normal were brought up in town, and were kept for a long time in the laboratory. Everybody who has worked with fowls knows that, under such conditions, fowls do not develop normally. Incidentally, a few cases of avian tuber- culosis were investigated among the non-tumor animals and nor- mal figures were obtained. We see, from the results in the accompanying Tables, that the size of the tumor does not seem to have an effect on the data, though possibly the length of the "inoculation period" played a part. This point will be studied in the near future. The chlorin content was found to be practically the same in tumor and normal sera, and therefore can be disregarded. Although one is practically able to diagnose Rous's chicken sar- 1 Rolly and Oppermann : Biochem. Zeit., 1913, xlviii, p. 471. 26 Serum Diagnosis of Chicken Sarcoma [March, coma from chemical data for the serum, it does not foUow that analogous results can be obtained for human serum. Our study will be extended in this direction as soon as f urther clinical material is available. By using micro-methods, the amounts of blood to be taken can be very greatly diminished. Very interesting, also, will be the analysis of serum in pregnancy, where differences in other directions may be revealed. The method is slightly inconvenient because normal serum must be taken as a control. From the figures obtained it is evident that, by working in pairs (one normal and one tumor serum), both re- sults were relatively either high or low. This was due very likely to the fact that the birds were bled completely, and practically the whole serum was used for analysis. TABLE I Data pertaining to the alcoholic extraction method on fowls {Blood from the throat: values per loo gm. of serum) ___ Alcoholic precipitate Filtrate Nature of the tumor Number Quantity N P N P 2< 3' 4' 5' R 27 N 4 R 12 ' R 79 . . . . ,N 6 ^R 5 lN 7 [ R 86 [N 8 3-73 5-37 4-S6 3-62 4.00 5.85 4.16 4-63 4.27 6.45 0.518 0.764 0.656 0.5H 0.566 0.859 0.585 0.659 0.577 0.887 0.0116 0.0239 0.0207 0.0150 0.0099 0.0093 0.0245 0.0104 0.0144 0.0124 0.0475 0.0475 0.0408 0.0353 0.0303 0.0197 0.0179 0.0129 0.0383 0.0246 0.0141 0.0239 0.0295 0.0175 0.0244 0.0156 0.0422 0.0154 0.0258 0.0134 Both sera hemolytic. Hemolytic. Slightly hemolytic. Slightly hemolytic. Tumor small and firm. Small, firm tumor. Average R . . . N... 4.14 S.18 0.588 0.736 0.0162 0.0142 0.0349 0.0273 0.0272 0.0171 Experimental. i. The method used was as follows : The blood was taken from pairs (one normal and one tumor animal), and was obtained by cutting the throat of the animal. Both bloods were left for the same length of time in an incubator. The separated serum was weighed and precipitated with 10 times its weight of alcohol. Both precipitates were left in the same desiccator and weighed, and an aliquot part taken for nitrogen and phosphorus estimations. The filtrate was diluted, with the alcoholic washings of the precipitate, to 250 cc. ; 100 cc. were taken for each determi- IQISI Casimir Funk 27 nation. In estimating small amounts of phosphorus, in a total vol- ume of 50 c.c, by the method described in Hoppe-Seyler-Tierfelder, precipitation frequently failed to occur but could be obtained at greater dilutions. These experiments (Table i) show a larger quantity of alco- holic precipitate for normal animals, a corresponding increase in nitrogen, and a slight decrease in phosphorus. In the alcoholic filtrate for tumor animals, there were increases in nitrogen and phosphorus. The experiments were deficient, however, in the fact that, by cutting the throats, the Contents of the crops possibly con- taminated the blood. All the subsequent experiments on fowls were made with blood from the heart, taken by means of a canula, with the animal under anesthesia. TABLE 2 Data pertaining to the alcoholic extraction method on rats {Blood from the throat: values per 100 gm. of serum) Alcoholic precipitate Filtrate Quantity N P N P Sarcoma Normal 6.07 7-54 0.828 1.07 0.0104 0.0240 0.064 0.051 0.019s 0.0201 2. There were eight rats with Ehrlich's sarcoma and six normal ones in this series (Table 2). The method was that described above (i). TABLE 3 Data pertaining to the alcoholic extraction method on fowls {Blood from the heart: values pe'r 100 gm. of serum) Alcoholic precipitate Filtrate Remarks Inoculation period. Quantity N P N P Weight of tumor, grams days 'R22.. 3-39 Lost 0.0089 0.0347 0.0183 40 42 X s N 9.. 3-94 0.547 0.0081 0.0193 0.0162 Trace of hemolysis R59.. 3-12 0.424 0.0104 0.0182 0.0160 145 45 , Nio.. 4.82 0.690 0.0130 O.0211 0.0271 3' [R65.. 4-74 Lost 0.0107 0.0215 0.0145 20 10 1 Nu.. 3-71 0.535 0.0093 0.0184 0.0124 [RiS.. S-3S 0.779 0.0219 0.0318 0.0304 S (pneumonia?) 20 4 ' 1 N12.. 4-47 0.616 0.0130 0.0293 0.0172 S' R s.. 2.32 0.31S 0.0063 0.0154 0.0113 SO 77 LN13.. 2.91 0.407 0.0078 0.0219 0.0134 Aver. R . . 3.78 0.S05 0.0116 0.0243 0.0181 N.. 3.99 0.559 0.0102I 0.0220 0.0172 28 Serum Diagnosis of Chicken Sarcoma [March, TABLE 4 Data pertaining to serum of fowls analysed directly (Blood front the heart: values per loo gm. of serum) 10 II 12 13 14 IS i6 17 i8 19 No. RiS. Nis. RS3. N 17. RS.. N 18. R49. N 16. ■R 134 N19. R 132 N 20. R 118 N21. R 131 N 22. R 133 N 23. R 137 N24. R 136 ,N25. R 122 N26. R I. . N 27. R 29. ,N28. R4.. N 29. R 66. N30. R 8. . N31. R 120 N32. R 109 N33- Serum N 0.602 .784 .588 .875 .616 .889 .728 .871 .546 .812 .770 .819 • 749 .812 .580 .882 .609 •749 .714 .819 .798 .840 .672 I-I55 •693 •833 1.018 .878 .791 1.008 •654 .966 .616 .714 .805 .668 .770 1.036 .644 •763 .819 .868 .500 .840 0.019 .024 .020 .028 .028 .029 .021 .022 .025 .028 .026 .030 .018 .028 .026 .028 .020 .020 .031 •037 •035 •037 .022 .028 .029 •033 .035 .028 •039 .028 .037 .029 •039 •039 .035 .031 •033 .046 .032 .032 •057 .o6i .052 .072 0.070 .066 .061 .077 .071 •093 .077 .042 .062 .076 .070 .090 .058 •054 .077 .050 .068 •059 • 054 .058 .058 .077 .071 .069 .090 .071 .069 .088 •053 .081 .058 .066 .062 .066 •059 .084 .066 .063 .063 .079 •057 .085 Cl 0.255 .199 ■383 .286 •355 •340 •344 •394 •397 .284 •390 •354 •354 .291 •425 •383 •397 •255 .256 •340 •369 •383 •390 •390 •397 .411 .390 .397 •383 •397 •369 .411 •372 •397 •397 NH2-N 0.065 .066 .046 •073 •055 • 031 •032 .032 .038 .019 .010 .014 .013 .020 .016 •059 .016 .020 .014 Sugar •154 .282 .156 .168 • 256 .206 •342 •308 • 165 .098 .029 .156 .205 • 205 .154 0.64 ■65 67 64 68 74 71 74 73 75 74 81 •63 .66 •71 •70 •75 •75 .64 .67 .67 .64 .70 .67 .68 .66 .64 .66 •65 .67 .64 .66 Remarks Weight of tumor, grams Inoculation period, days 120 27 22 Avian tuberculosis 15 50 10 50 Lipemic 100 100 150 60 50 50 50 17 12 14 16 19 21 23 26 28 Avian tuberculosis IS 20 74 Firm, encapsulated, slowly growing 70 0.5 100 26 20 33 65 Slow growth, encap- sulated 10 I 25 Metastases in the muscle SO IS 17 12 Hemolytic 75 I 22 Cysts in the liver Aver. R . N. 0.694 0.858 0.031 0.033 0.066 0.070 0.349 0.350 0.037 0.030 0.203 0.182 0.68 0.69 I9I5] Casimir Funk 29 3. In the third series (Table 3), where the inoculation period, and the size of the tumor, were noted, the average result was simi- lar to that in the previous experiment ( i ) ; but we see that the size of the tumor had no effect, on the numerical values. 4. In the f ourth series of experiments serum was always taken from pairs of animals, to insure rehable results. (Table 4.) We see, from the foregoing data, that nitrogen was very much higher, and phosphorus, sulfur, chlorin and molecular depression slightly higher, in normal serum. In the Rous sera, amino-nitrogen and sugar were slightly higher. Two tumor sera (Nos. 14 and 18) gave contradictory values. This may be due to the resistance of the body to tumor growth. In these two animals the tumor was older than in others but attained only a very small size, with a capsule separat- ing it entirely from the rest of the muscle tissue. TABLE 5 Data pertaining to serum analysed directly. {Serum of rats with Jensen's sarcoma: values per 100 gm. of serum.) Serum N P S Sugar A Remarks Sarcoma Normal 1.050 0.955 0.075 0.078 0.356 0.072 0.070 0.051 0.61 0.63 Tumors 17 days old, hemolytic sera. Hemolytic. 5. In the fifth experiment six rats with small tumors were bled by cutting the throats; the blood volumes were combined. Five normal rats were treated in the same way. (Table 5.) We see, also, chemical changes in the same direction for rats. This result will be checked by f urther study. 324 West Ena Avenue, New York City. CONVENIENT METHODS FOR DEMONSTRATING THE BIOCHEMICAL ACTIVITY OF MICRO- ORGANISMS, WITH SPECIAL REFER- ENCE TO THE PRODUCTION AND ACTIVITY OF ENZYMES C. H. CRABILL and H. S. REEDi (WITH PL ATE l) Introduction. A large amount of work has been done on the biochemistry of microorganisms and many reliable tests f or demon- strating these activities have been evolved. Some of the tests are not well adapted for ocular demonstration to classes, however, because of their transitoriness or for other reasons. We have thought it worth while to describe in the following paragraphs a few methods, for making semi-permanent demonstrations of these activities, which seem adapted to the needs of class room or labo- ratory instruction. Some of the methods given here are adapted from those of other investigators ; other methods are original with the present writers. The methods are designed to show the presence and action of products of cellular activity upon appropriate substances incor- porated in thin layers of agar in Petri dishes. Agar seems to serve very well for this purpose, since it is not difficult to prepare a clear Solution, and most solutes diffuse readily through the gel which it f orms. When solid zymolytes are used they are suspended in the agar. In order to procure uniform distribution of these solids through the medium, the plates are poured directly from the flask in which the medium is cooked and sterilized. By frequent shak- ing between pourings, settling of the solids is prevented. With this procedure, tubing of the medium is entirely unnecessary, if not detrimental to the best results. Stain reduction cannot be well 1 Paper No. 29 from the Laboratories of Plant Pathology and Bacteriology,. Virginia Agricultural Experiment Station, Blacksburg. 30 1915] C. H. Crabill and H. S. Reed 31 shown by this Petri dish method, because the process of chemical reduction is usually counteracted by rapid oxidation of the products in contact with atmospheric oxygen. For a number of the experiments a stock agar was prepared according to the following formula, filtered, and steriHzed in the autoclave: Distilled water, 1000 c.c. ; magnesium sulfate, 0.5 gm.; di-potassium hydrogen phosphate, i.o gm.; potassium chlorid, 0.5 gm.; ferroiis sulfate, o.oi gm.; agar, 20.0 gm. This stock medium is sHghtly acid in reaction. It presents no carbon-containing nutrient and consequently does not support microorganic growth. To this various zymolytes in the form of carbon containing Com- pounds are added and inoculated with the organisms whose activities are to be tested. TABLE I Data pertaining to amylolytic tests on starch-agar. Organism Glomerella rufomaculans . . . Spharostilbe coccophila. . . . Pseudopeziza ribis Helminthosporium turcicum Alternaria sp Phyllosticta pirina Septoria lycopersici Aspergillus niger Oospora Scabies Streptothrix sp Diplococcus sp Micrococcus citricus B. fluorescens liquifaciens . . B. pyocyaneous B. cerogenes B. denitrificans Bad. tumefaciens . B. coli Bact. lactis acidi B. mycoides B. prodigiosus B. vulgaris B. putidum B. hartlebii B. butyricus B. campestris Growth Starch dissolution beneath centre Halo produced Good Poor Good None Good Fair Slight Fair Good Weak Good Good Slight None Weak Excellent None None Amylase. The action of this enz)rme may be conveniently demonstrated by cultivating organisms on starch-agar, made by adding to 500 c.c. of the melted stock agar, 10 gm. of corn starch 32 Biochemical Activity of Microorganisms [March, suspended in a little cold water. The medium is then sterilized in the autoclave and poured with frequent shaking directly from the flask into sterile Petri dishes. This gives a clear white substratum in which the starch is suspended. As soon as the agar has solidi- fied, the centers of the dishes are inoculated with the organisms to be tested. The dishes are inverted and incubated for two to five days under bell jars to prevent loss of moisture. Typical results are shown in Table i and Plate i, Fig. i. Some of the organisms produce extracellular amylase which dif- fuses from the colony, dissolves the starch suspended in the agar, and renders the space about the colony clear. Such an appearance is designated as a " halo." The presence of a halo, then, around the edge of a bacterial or fungous colony on starch-agar indicates the production, by that organism, of a readily diffusible extracellular amylase. Some organisms, especially fungi, do not produce a halo on starch-agar, but grow well and dissolve the starch from the agar immediately in contact with them. In such a case the secretion of extracellular amylase is apparently weak or the amylase is not so diffusible as that produced by other organisms. Among the organisms so far tested, Streptothrix, Oospora Scabies and Aspergillus niger were f ound to be the most active pro- ducers of extracellular amylase. The first of these is able to pro- duce abundant amylase and to dissolve large quantities of starch even in the presence of an abundance of sugar. Inulase. Inulin is hydrolyzed by the enzyme inulase to reduc- ing sugars, and as such may be utilized by microorganisms. TABLE 2 Data pertaining to growth of fungi on inulin agar Organism Growth Spore production Glomerella rufomaculans Fair Fair Phyllosticta pirina Good Good Coniothyrium pirinünt Good Poor Aspergillus niger Good Good Streptothrix sp Poor Almost none Inulift-agar. To 1000 c.c. of the stock agar, 0.5 percent of pure inulin was added, and Petri dish cultures made as in the case of the iQisl C. H. Crabill and H. S. Recd 33 starch-agar. Inulin is soluble in hot water and consequently dis- solves during sterilization. Evidence of the ability of the organ- isms to dissolve inulin in this medium is afforded only by the amount of growth and spore production. Only a few fungi have been tested, but some of them grew well upon this medium. See Table 2. Emulsins : glucoside-splitting enzymes. The glucosides are complex organic Compounds capable of hydrolysis. Various end products, one of which is always a sugar, are the result. Since the glucosides are readily soluble, the agar containing them is trans- parent and the action of enzymes can be determined only indirectly. If the organisms can use the glucosides as sources of carbon, it is assumed that cleavage of the glucosides occurs in such consumption. The glucosides in a pure State are added to the stock agar to the amount of i percent, and plates poured and inoculated. Esciilin-agar. A bluish color pervades the agar made with esculin. In the event of the successful growth of the organism this blue color is reduced, sometimes throughout the plate. The suc- cessful growth of the organism is, however, a better indication of its ability to produce emulsin than is the color reduction. Arbutin-agar. Arbutin, by hydrolytic cleavage, yields sugar and hydroquinone, which gives a brown color. The successful growth of the organism, and production of a brown stain on agar containing this chemical as the only source of carbon, are regarded as evidences of its ability to produce emulsin. Amygdalin-agar. Success or f ailure to grow on this medium is our only indication of the ability or inability of an organism to pro- duce emulsin. Typical data are given in Table 3. LiPASE. Demonstration of the presence of lipase depends upon its power to split fats into glycerol and free fatty acids. The pres- ence of the acids may then be shown by a convenient indicator. Litmus-cream-agar. Fifty c.c. of 48 percent Separator cream are diluted to 600 c.c. with distilled water and fractionally sterilized in an Arnold sterilizer. Twenty gm. of agar-agar are melted in 400 c.c. of water; the liquid is filtered, sterilized and added to the di- luted Cream while hot ; and enough sterile litmus Solution is poured into the fluid to impart a deep blue color. Plates are poured, and 34 Biochemical Activity of Micro organisms [March, TABLE 3 Data pertaining to tests for emulsin-production on amygdalin-agar Organism Growth Fusarium culmorum Excellent Alternaria sp Fair Oospora Scabies None Streptothrix sp Good B. fluorescens liquifaciens Good B. coli Good Bact. lactis acidi Good B. denitrificans Good B. prodigiosus Fair Bact. tumefaciens Fair B. mycoides Slight B. catnpestris Slight M. citricus Slight TABLE 4 Data pertaining to tests for lipase-production on litmus-cream-agar Organism Growth Reddening of litmus* Sphcerostilbe coccophila Good None H elminthosp orium turcicum Good Good, dififusing Macrosporitim sp Good Sharp Fusarium culmorum Good Deep, diffusing Stysanus capitata Good Slight Penicillium sp Good Sharp Oidium lactis Good Good, diffusing Streptothrix sp Good Good, diffusing Asotobacter chroococcum None B. megatherium Good Good, diffusing B. butyricus Good Good, diffusing B. fluorescens liquifaciens Good Deep B. mycoides Good Good, diffusing B. Proteus vulgaris Good Sharp, brilliant B. pyocyaneus Good Good, diffusing B. coli Good Good, diffusing B. prodigiosus Good Deep Bacteria of ropy milk Good None, blue stain reduced M. citricus Good Slight, diffusing Sarcina alba Good Deep Diplococcus sp Good None inoculated with various organisms. A medium containing approxi- mately 2.5 percent of butter-fat and a very small amount of other *" Sharp," in the above table, indicates that the red area produced by the organism is sharply defined and in marked contrast to the surrounding blue. " Diffusing " indicates a gradual gradation f rom red through purple to blue, the acid apparently diffusing more rapidly than in the case of sharp contrast. 1915] C. H. Crabill and H. S. Reed 35 cream-constituents is thus prepared. Lipase excreted by the or- ganism splits the butter-fat and the resulting free fatty acids turn the htmus red. In the case of several organisms the Htmus is subsequently reduced to a colorless substance. Ropy-milk bacteria reduce the blue stain without first turning it red, and Diplococcus sp., although it grows fairly well, produces no change in the litmus. See Table 4. All but one of the fungi tested cause production of acid from the Cream- fat. Macrosporium, Penicillium and Fusarium show very marked lipolytic activity. Nearly all the bacteria tested are active in breaking up fat. Among the best are B. proteus vulgaris, B. prodigiosus, and B. fliiorescens liqiiifaciens. The fat in the cream-agar, prepared as above but without litmus, may be stained with alkannin. The lipolytic action of the bacteria growing on this medium brings about a reduction of the stain. Those organisms which show the strongest acid forming ability on the litmus-cream-agar give also the strongest reactions on alkannin- cream-agar. A third series of plates of unstained cream-agar may be em- ployed. The organisms most active on litmus and alkannin in the above tests are also most active in this series. No halo is produced however. Most of the fungi tested digest all of the fat in the agar immediately under the culture and leave it colorless. Ethyl butyrate-litmus-agar, prepared as follows, may also be used : To 500 c.c. of the stock agar, 20 c.c. of saturated litmus Solu- tion are added. Enough sodium hydroxid sol. is introduced to give a slight alkaline reaction. Five c.c. of ethyl butyrate are then added, and the medium sterilized and poured into plates. B. mycoides, M. citriciis and Oidiuni lactis give a decided red- dening of the agar as a result of acid production by hydrolysis of the ester. Nearly all of the organisms tested grow to some extent on this medium. It is however much less satisfactory than the litmus Cream agar. Proteases. The production of proteases is a familiär process, numerous examples of which may be seen daily in the laboratory. The peptonizing action of many organisms, when grown upon gelatin-media, is familiär to all. 36 Biochemical Activity of Microorganisms [March, The solvent action of enzymes is commonly apparent when microorganisms are grown upon Heyden Nälirstoff-agar. This medium almost invariably contains particles of protein material which have passed through the cotton filters and give Petri dishes a slight turbidity. Colonies of organisms usually dissolve these par- ticles through the action of proteases, the diffusing enzymes acting for some distance beyond the margin of the colony. More con- spicuous examples of this power may be afforded by using some of the following methods. TABLE 5 Data pertaining to tcsts for proiease-production on fibrin-agar Organism Growth Dissolution of fibrin Phyllosticta pirina Good Rapid Helminthosporium turcicum Excellent Very rapid Aspergillus sp Good Rapid Aspergillus niger Good Slow B. pyocyaneus Very slight Very slight Sarcina lutea Excellent Good Diplococcus sp None B. hartlebii Slight if any Slight if any B. lactis ccrogcncs Slight if any Slight if any B. fiuorescens liquifacicns Slight if any Slight if any M. citricus Excellent Good B. subtilis Excellent Slight B. megatherium Excellent Good Bact. lactis acidi Fair Slight B. prodigiosus Good Good B. campestris Good Good B. mycoides Good Good B. putidum Slight if any Slight if any B. cyanogcnus Slight if any Slight if any Bacteria of ropy milk Fair Fair B. butyricus Excellent Excellent B. amylovorus None Microspira tyrogena Slight if any Slight if any B. vulgaris Slight Slight Bact. denitrificans Slight Slight B. coli Slight Slight Bact. tumcfacicns Slight Slight B. radiatum Fair Slight Streptothrix sp Excellent Fair Oidium lactis Poor Slight Fibrin-agar. Some blood fibrin is pulverized in a mortar and added to the stock agar to the amount of i percent. Agglutination of the fibrin takes place and although it is almost impossible to get it evenly distributed through the medium, many fungi thrive on it satisfactorily and dissolve fibrin. Some bacteria show marked action on the fibrin but produce no distinct halo; others grow poorly I9I5] C. H. Crabill and H. S. Rced 37 or not at all. Perhaps the failure of many of them to grow is not due to their inability to produce proteases but to the fact that the uneven distribution of the fibrin clumps makes it difficult for the organisms to get started. See Table 5 for typical data. Protein-agar. A sample of commercial protein prepared from wheat is ground very fine in a mortar and added to the stock agar to the amount of i gm. per 100 c.c. Table 6 presents typical results. TABLE 6 Data pertaining to tests for protease-proditction on protein-agar Organism Growth Fniiting Dissolution of protein particies Phyllosticla pirina Good Good Excellent Poor Poor Fair Fair None None None None None None None Slight Slight Good Good Excellent Poor Poor None Good Rapid Rapid Very rapid Slight Slight Rapid Slight Coniothyrium pirinum Helminthosporium turcicum Aspergillus sp. (green) Aspergillus sp. (white) Rndothia parasitica Aspergillus niger Glomerella rufomaculans Rhizoctonia solani Ascochyta colorata B. prodigiosus B. suhtilis B. pyocyaneus B. amylovorus B. campestris Bad. tumefaciens Erepsin, Erepsin hydrolyses the simpler proteins into amino acids, such as leucin and tyrosin. It is best demonstrated by its solvent action upon the simpler proteins. Casein-agar. To the stock agar technical casein, in a finely divided condition, is added to the amount of i percent. This me- dium shows in an excellent manner the action of ereptic enzymes. Many of the organisms tested secrete erepsin in such quantity as to dissolve entirely all the casein in a wide band around the colonies, producing thereby a notable halo. A series of plates of casein agar was made in which the agar was rendered neutral to rosolic acid with ammonium hydroxid. All the organisms tested made a much weaker growth and produced much poorer halos on this series than on the slightly acid series. Erepsin is elaborated with greater rapidity and works best in the presence of a slight amount of acid. The bacteria were grown only on the alkaline agar because they 38 Biochemical Activity of Microorganisms [March, could not survive on the acid medium. Typical data are given in Table 7. TABLE 7 Data pertaining to tests for erepsin-production on casein-agar Organism Growth and fruiting Dissolution of casein particles beneath culture Halo produced Phyllosticta pirina Good Good Good Good Good Good Good Good Poor Good Good None Fair Good None Asi?ereillus nieer None Good but narrow Crlomerella rufomaculans Good but narrow B. aroeenes B. -bvocvaneus B. C(11fli>€StYis B CLtyivlovoYus Bact. tutnefaciens M. citricus TABLE 8 Data pertaining to tests for erepsin-production on skim-milk-agar Organism Sphcerostübe coccophila Stysanus capitata Pseudopeziza ribis Colletotrichum gloeosporoides . Glomerella gossypii Helminthosporium turcicum . Macrosporium solani Alternaria sp Fusarium culmorum Septoria lycopersici Phyllosticta pirina Penicillium pinophylli Oidium lactis Actinomyces bovis Streptothrix sp Azotobacter chroococcum . . . . B. campestris B. amylovorus B. hartlebii B. fiuorescens liquifaciens . . B. butyricus B. prodigiosus B. mycoides B. pyocyaneus B. megatherium B. putidum Bact. lactis acidi B. cerogenes M. citricus M. candicans Sarcina alba Microspira tyrogena Diplococcus Urea coccus Growth Good Good Good Good Fair Good Good Good Good Poor Good Good Good Good Good Good Good None Good Excellent Good Excellent Good Good Excellent Poor Good Good Good Poor Excellent Poor Excellent Good Dissolution of coag^lum Slight Good Good Good Fair Fair Good Good Good None Good Good Halo produced None Good None None Poor None None None None None None Fair Poor Poor Poor Poor None None None Good Good Good Good Good Excellent None None None Excellent None None None None None 1915] C. H. Crabill and H. S. Reed 39 Skitn-milk-agar. Some Separator skim milk is diluted with an equal volume of water and 20 gm. o£ agar added per liter. This mixture is heated for half an hour in an autoclave to coagulate the casein. The medium is then boiled in an Arnold sterilizer for 4 hrs., when the casein is in a very finely divided condition. Plates are prepared in the usual way. See Table 8. This medium is most excellent for the demonstration of erepsin secretion as shown in Plate i, Figs. 2, 3, and 4. The most satis- factory organisms to use for the demonstration of ereptic activities on skim-milk-agar are: B. megatherium, M. citriciis, B. hutyricus, B. fluorescefis liquifac'iens, B. prodigiosus and B. pyocyaneits in the Order named. Among those tested which showed least activities are : B. campestris, M. candicaiis, B. hartlehii, Bad. lactis acidi, and B. lactis aerogcnes. Peptone-agar is also very useful in the demonstration of erepsin, It is prepared by adding Witte peptone (i percent) to the stock agar. Good halos are produced by many organisms. Trypsin. One of the most convenient methods of demonstrat- ing the action of trypsin, produced by microorganisms, depends upon the use of egg-albumen-agar. Many microorganisms grow readily upon egg-albumen-agar and digest the coagulum. TABLE 9 Data pertaining to the action of certain organisms on egg-albumen-agar Organism Growth Digestion B. campestris Good Good Sarcina lutea Good Very good B. pyocyaneus Great Very good Oidium lactis Great Slight B. subtilis Good Slight B. mycoides Good None B. prodigiosus Good Good B. m.egatherium Good Good Streptothrix sp Good Slight Bact. lactis acidi Fair Slight Bact. tumefaciens Small None B. amylovorus Small None B. vulgaris Slight None B. lactis (crogenes Slight None B. hutyricus Small Slight B. putidum Small Slight Egg-alhumen-agar is prepared by taking the " whites " of three eggs and beating them in 75 c.c. of water with a Dover egg-beater. This material is then added to 500 c.c. of melted stock agar, shaken, 40 Biochemical Activity of Micro organisms [March, and cooked for a half hour in an autoclave. The cooking process breaks the coagulated albumen into small particles and gives a cloudy medium. This agar is then poured into Petri dishes and, when soHd, inoculated with the desired organisms. As the colonies develop they digest the coaguhim and a halo of clear agar surrounds the colony (Plate i, Fig. 5). The results of tests of the tryptic activity of certain organisms on egg-albumen-agar are given in Table 9. Amidase. Amidase is an enzyme capable of attacking amino Compounds and forming ammonia along with other cleavage prod- ucts. Upon this liberation of ammonia is based the following test. Asparagin-rosolic-acid-agar. To the stock agar is added 0.5 percent of asparagin and 5 c.c. of 2 percent rosolic acid per liter. The rosolic acid, which is yellow with acid, gives this medium a slight yellow tint. The liberation of ammonia from the asparagin renders the medium alkaline, and the rosolic acid takes on a brilliant red color. This is a beautiful reaction and a highly satisfactory test for the production of amidase by lower organisms. See Table 10. TABLE 10 Data pertaining to tcsts for amidase-production on asparagin-rosolic-acid-agar Organism Growth Reaction B. fluoresccns liquifaciens . . . Good Deep brilliant red, diffusing B. putidum Very good Deep red B. ccrogenes Good Red, little diffused B. prodigiosus Fair Slight red, little diffused Bact. lactis acidi Very good Deep red, widely diffused B. denitrificans Very good Deep red, widely diffused Bact. tumefaciens Very good Deep red, widely diffused B. pyocyaneus Very good Deep red, widely diffused B. coli Very good Deep red, widely diffused Actinomyces bovis Very good Deep red, widely diffused B. hartlebii None B. butyricus Slight or none No change B. megatherium Slight No change B. radiatum Slight No change B. campestris Slight No change B. amylovorus Slight No change B. vulgaris Slight No change B. subtilis Slight No change B. mvcoidcs Slight No change M. citricus Slight No change Sarcina alba Slight No change Sarcina lutea Slight No change Oospora Scabies None No change Streptothrix sp None No change Pseudopeziza ribis Good Slight red Glonicrella rufoniaculans Very poor No change Hehninthosporium turcicum . . Poor Very deep red, not diffused I9IS] C. H. Cr ahm and H. S. Reed 41 Cultures showing active production of ammonia from asparagin and reddening of the agar were kept for 8 to 10 days. Some of them, at the expiration- of this time, showed a yellowing of the medinm aboiit the centre of the cidture. This is notably true of B. putiduui and B. pyocyancus. The indications are that subse- quent to the production of ammonia an acid is produced, due, no doubt, to bacterial action on the cleavage products of the asparagin. Cytase. The presence and action of celkdose-dissoh'ing en- zyme has been for a long time demonstrable microscopicahy. Re- cently, however, Kellerman and McBeth- have described a conven- ient Petri-dish rnethod for demonstrating the celhilose-dissoh'ing action of bacteria and fnngi. The medium used in our exeriments was prepared according to Kellerman's method, as follows : Exactly 400 c.c. of ammonium hydroxide ( sp. g. 0.90) were dikited to 600 c.c. and an excess of copper carbonate added. After standing all night, the excess of copper carbonate settled to the bottom and the supernatant Solution was siphoned off. Seven and one half gm. of dry filter paper were added and the product shaken up at intervals. In a few minutes Solution of the cellulose was complete. The Solution was diluted to 5 liters and a i : 5 Solution of hydrochloric acid added, a few c.c. at a time, until the cellulose settled to the bottom. Water was added to make 10 liters, the cellulose allowed to settle and the su- pernatant liquid decanted. The cellulose was then washed with repeated changes of water containing a little hydrochloric acid, until the blue color of the Solution disappeared. It was then washed with distilled water until a silver-nitrate test showed the washins^s to be free from chlorid. The cellulose was suspended in 500 c.c. of water containing magnesium sulphate, 0.25 gm.; potassium Ijiphosphate, 0.5 gm.; potassium chlorid, 0.25 gm.; ferrous sulfate, trace; sodium nitrate, i.o gm.; agar agar, lo.o gm. The mixture was autoclaved and poured into plates. 2 Kellerman and AlcBeth : The fermentation of cellulose. Ccntralbl. f. Bükt., Abt. 2, 34: 485 (1912). Kellerman: The excretion of c}^tase hy Pcnicillhim t>iiiophiluiii, Circ. 118. Biir. PI. Ind., U. S. Dcpt. Agr., 1913. McBeth and Scales : The destruction of cellulose by bacteria and filamentous fungi, Bull. 226, Biir. PI. Ind., U. S. Dcpt. Agr., 1913. 42 Bioclicniical Activity of Microorganisiiis [March, It is not always easy to obtain organisms which produce cytase abundantly. Kellerman has described, in the papers cited, some species of ^erobic bacteria and fungi which he foiind to be active. Mixed cultures of these organisms may be obtained by selective ciiltivation on the fohowing medium : fiher paper, in strips 2.0 gm. ; ammonium chloride, o.i gm.; potassium biphosphate, 0.05 gm.; calcium carbonate, 2.0 gm. ; water, 100. o c.c. This medium is placed in large Erlenmeyer flasks to form a layer about one centimeter in depth. The flasks are inoculated with fresh horse-manure, slimy mud from a pond, or with sewage from a septic tank in active Operation. After a month's incubation trans- fers are made to new flasks containing the same medium. In this way the cellulose-destroying organisms are selected. The strips of filter paper first become brown and then perforated with holes, and a brown color is usually imparted to the Solution. Transfers may then be made to cellulose-agar in tubes or Petri dishes. Solingen-"' has recently described a method for demonstrating the cytolytic powers of bacterial colonies by the use of manganese Compounds. A sheet of filter paper is dipped in a manganese sulfate sol. and then in sol. of potassium permanganate. The resulting manganic oxid is held in the paper and gives it a brown color. This sheet is dried and sterilized in a Petri dish, then moistened with a nutrient Solution and inoculated with cellulose-destroying bacteria. The acids formed in course of the destruction of cellulose combine with the manganic oxid to form light colored salts, which present a conspicuous contrast on the dark background. Formation of lactic acid and other acids. It has been known for some time that the addition of calcium carbonate to agar gives a method for demonstrating the action of acids produced by bacteria. Frequently failures have resulted from the difliculty of adding just the proper amount of calcium carbonate. The method which we have employed obviates this difliculty. Beef-peptone agar containing i percentof lactose is made and put into test tubes as usual. Before plugging the tubes a small quan- tity of calcium carbonate (0.5 to 2.0 gm.) is added to each tube. The 3 Söhngen : Umwandlungen von Manganverbindungen unter dem EinHuss mikrobiologischer Prozesse, Ccniralbl. f. Bakt., 2tc Abt., 40: 545 (1914)- 1915] C. H. Crahill and H. S. Reed 43 agar is then given triple sterilization in an Arnold sterilizer. Most of tlie carbonate settles to the bottom of the tubes, but a small amount of finely divided material remains in Suspension in the agar and renders it distinctly turbid. After the final sterilization, the snpernatant, turbid, agar is poured into sterile Petri dishes, care being taken to prevent carbonate in the bottom of the test tube from passing into the dishes. The turbid agar was inoculated with Bactcrinin lactis acidi and placed in the incubator for three days. At the end of that time the colonies showed distinct halos due to the solvent action of acids upon the calcium carbonate. (Plate i, Fig. 6.) Another convenient method of demonstrating the presence of acids is the familiär one of adding litmus Solution to medium con- taining lactose. The method is so well known to all bacteriologists that it is unnecessary to repeat it here. Another indicator which is useful in this connection is rosolic acid. A few drops of 0.5 percent Solution are added to tubes of sterile lactose-agar before melting and pouring it into Petri dishes. The results are shown in Table 1 1 . TABLE II Data pcrtaining to cffccts of organisms upon the color of rosolic acid Organism After 13 days After 28 days Glomcrclla riifomaculans Deep pink Beginning to fade Fusarium lycopcrsici Unchanged Colorless Stcrigmatocystis niger Slight change Deep pink Bacillus subtilis No growth B. pyocyaneus No growth The alkaline conditions observed were probably due to the for- mation of ammonia from protein constituents. Numerous other applications of these various methods will sug- gest themselves to students of these problems. When only quali- tative results are required, it is believed these methods will be found highly satisfactory. An added advantage lies in the ease with which permanent records may be made for the student's note book. The Petri dishes may Ije set on Photographie or blue-print paper and exposed to light. The paper, after development, may be inserted in the note book as a part of the record. 44 Biochcmical AcHvity of Microorganisms [March, EXPLANATION OF PLATE i Fig. I. Solvent action of Strcptothrix on starch-agar. Fig. 2. Colony of Macrosporium solani on milk-agar. Fig. 3. Large colony of B. nicgathcrium on milk-agar. Fig. 4. Action of M. citricus on milk-agar. Fig. 5. Solvent action of Sarcina lutea on egg-all)umen-agar. This print was made by placing the Petri dish on the Photographie paper and illuminating it for the proper time. Fig. 6. Solvent action of Bact. lactis acidi on calcium carbonate suspended in the agar. BiocHEMicAL Bulletin (Vol. IV) Plate 1 CRABILL AND REED: CONVENIENT METHODS FOR DEMOXSTRATIXG THE BIOCHEMICAL ACTIVITY OF MICROORGAXISMS REACTION OF RABBITS TO INTRAVENOUS INJEC- TIONS OF MOULD SPORES^ A. F. BLAKESLEE and ROSS AIKEN GORTNER (Coiui. Agric. College) {Univ. of Minucsota) (WITH PLATE 2) The work outlined in this paper was imdertaken in connection with experiments having as their object a determination of the chemical differences which may exist between the two sexual races in the fungous group commonly known as the mucors. In these forms the majority of the species are dioecious, having separate male and female races, which may be propagated independently by means of vegetative spores. As is weh known the repeated injection of red blood corpuscles and of certain other cells is capable of producing in the blood of rabbits cytolytic antibodies that will dissolve these cells when they are subsequently mixed with the sermii of the treated animal. It was thought that a similar cytolysin might be developed for mould spores, and that the action might be foiind to be sexually specific. Althoiigh agglutinins apparcntly are fonncd, 110 cytolysins for fungus spores coiild be induced by intravenotis injections. Despite the largely negative character of the results obtained, they seem to be sufiiciently controlled to show positively that rabbits are incap- able of producing cytolysins for the spores of the mucor tested. The fact that the cell wall is highly resistant throughout the fungi renders it extremely improbaljle that cytolysins can be developed for spores of any other fungus form. Preliminary tests showed that spores of Cunninghamella echinu- lata, when injected intravenously, kill a rabbit within a week's time (four instances). Postmortem examination demonstrated thepres- iThe major part of the work embodied in this paper was carried out at the Station for Experimental Evolution of the Carnegie Institution of Washington, Cold Spring Harbor, New York. See Proceedings of the Cokmil)ia University Biochemical Association, Dec. 4, 1914; BiocHEM. Bull., 1915, iv, p. 212. 45 46 Intravenous Injectiom of Mould Spores [March, ence of g-erminated spores in the liuigs. This mould is a tropical form growing readily at temperatures above 31° C, and its growth in the rabbits may have cansed death by mechanically interfering with the functions of the organs infected. Most species of the miicors will not grow at temperatures over 30° C. Among the forms that grow only at relatively low temper- atures, " Mucor V " — a form similar to M. hicmalis, if not identical with this species — gives an especially strong sexual reaction. Its spores are relatively small ( about 8X3-5/^) and can offer little interference to the circulation. No strong toxins, moreover, are developed by this mould such as have been found in the allied form Rhizopus nigricans (i, 2). Altogether, the species seemed espe- cially favorable and has been used in the present investigation. The mold was grown, generally on agar, in shallow pie-tins pro- tected with paper covers. Water was poured over the mature cul- ture and filtered through linen which allowed the spores to pass while keeping back fragments of the aerial mycelium which it was feared might block the capillaries. The spore-water was centri- fuged and the resulting compacted mass of spores was mixed with 0.9 percent salt Solution and used at once for the injections. Centrifuged spores were dried in a vacuum desiccator and in a few instances were used later when f resh spores were not available. The injections were all made in an ear vein, the usual aseptic precautions being observed. The dose varied from 3 to 4 c.c. The spore- water was always very dark with spores and, although counts were not made each time, the individual injections can be considered to average about 500,000,000 spores. Rabbit No. 5, beginning April 2, 191 3. received at intervals of about 4 days, 28 preliminary injections of the spores of the ^ race (3) of "Mucor V." Rabbit No. 55, beginning April 17, similarly received 27 preliminary injections of the $ spores of the same spe- cies. On August 13, five days after the last injection, rabbits Nos. 5 and 55 received approximately 800,000,000 spores, respectively, of the (^ and ? races ; and at the same time two control rabbits, Nos. 6 and 66, previously untreated, were similarly injected with like doses of the -1-» +J cd t; CS 3 O r, M QO 1-. '•" i ■" s 5 o '^' •;: o cd D 00 rc ro C> t^ O On 00 O \0 t^ ro O M N ro O 0\ c<) 00 ro i-i ■"öö lö O •* ro O ■* t- ro O 0\ r~ -t o 00 0\ 00 ^ I the calculation of which is facilitated by tables published else- where in this Journal: 3, p. 259-263, 1914. 4. Discussion of data. The individual constants are given in the accompanying series of tables (2-21). Numbers in the left hand columns are the laboratory numbers of the samples. A. Specific gravity and concentration. (Data, Tables 2-6 and 17-21.) These are the simplest possible measures of the properties of the sap of the two kinds of tissues. It would be surprising if there were not distinct differences be- tween the means of the specific gravities of samples taken from separate cultures and at different times. The data for mean spe- cific gravities in the appended summary are for the three larger series : Experiment Wall Prolificatioa C 1.020168 + 0.000154 1.017831 + 0.000156 D 1.019225 + o.oooiis 1.018125 + 0.000119 E 1.020636 + 0.000231 1.019500 ± 0.000247 6o Physico-Chemical Properties of Vegetable Saps [March, TABLES 2-6 Data pertaining to mean weight of parts of fruit and specific gravity of sap Table 2, Serie s A Sample Mean weight Specific gravity of sap number Wall Prolification Difference Wall Prolification DiflFerence 76-86 90 207 292 1.424 1-445 1.666 1.526 0.640 0.724 0.858 1-073 -0.784 — 0.721 -0.808 -0.453 I.02IS 1.0202 I.OI86 I.OI9I I.O195 I.OI93 I.O168 I.OI80 — 0.0020 — 0.0009 — 0.0018 — O.OOII Table 3, Serie s B I a+5 1.0194 1.0202 1.0187 1.0213 — 0.0007 +0.0011 34 1-233 1-073 — 0.160 1.0230 1.0223 — 0.0007 35 I-I33 0.950 -0.183 1-0234 1.0229 — 0.0005 64 1-300 0.882 —0.418 1.0207 1.0198 — 0.0009 189 1.582 0.88s -0.727 1.0204 1.0179 — 0.0025 286 1.463 I-I4S -0.318 1.0199 I-0I93 — 0.0006 Table 4, Serie s C 210 1.676 1.212 —0.464 1.0221 1.0204 — 0.0017 211 1.970 1.280 —0.690 1.0201 1.0182 — 0.0019 213 1.710 1-235 -0.475 1.0195 I.0178 —0.0017 214 1.780 1-330 -0.450 1.0200 1.0172 —0.0028 221 228 1.654 1.122 -0.532 1.0220 1.0215 1.0193 1.0183 —0.0027 —0.0032 237 1-507 1-030 -0.477 1.0193 1.0170 —0.0023 238 2.160 1-233 -0.927 1.0199 1.0173 —0.0026 245 I-915 1.165 -0.750 I.0191 1.0173 —0.0018 246 2.077 1.209 -0.868 1.0198 1.0170 — 0.0028 247 2. 181 1.272 — 0.909 1.0197 1.0171 — 0.0026 248 1.956 1.217 -0.739 1.0190 1.0169 — 0.0021 260 1.827 1.438 -0.389 1.0201 1.0177 — 0.0024 261 1-855 1-305 -0-550 1.0198 1.0179 —0.0019 262 1.406 1-031 -0.375 1.0204 1.0173 —0.0031 269 2.041 1-352 -0.689 1.0204 1.0186 —0.0018 These values furnish the data for the accompanying compari- sons for the wall and the abnormal mass. Comparison Difference 'il^d Comparison Difference ^l£d C and D C and E D and E 0.000943 ±0.000192 0.000468 ±0.000277 O.OOI411 ±0.000258 4.91 1.68 5-46 C and D C and E D and E 0.000294 ±0.000196 0.001669 ±0.000292 0.001375 ±0-000274 1.50 5-71 5.02 There can be no reasonable question of the significance of four of these differences. The factors involved in producing them are far too complicated to justify any attempt to explain them at present. Possibly they are in part due to differences in the strains IQISI /. Arthur Harris and Ross Aiken Gortner 6i TABLES 2-6 (continued) Data pertaining to mean weight of pdrts of fruit and specific gravity of sap Table 5, Series D Mean weight Sp :cific gravity of sap Sample number Wall Prolification Difference Wall Prolification Difference 304 1.768 1.468 -0.300 I.OI97 I.OI91 — 0.0006 305 1.990 1-585 -0.405 I.OI96 I.OI82 — 0.0014 310 1-405 I.IOO -0.305 1.0187 I.OI75 — 0.0012 3" 1-455 1.090 -0.365 I.OI83 I.O171 — 0.0012 3IS 1.917 1-352 -0.565 I.OI96 I.OI82 — 0.0014 316 1.710 1-035 -0.675 I.OI95 I.OI90 — o.ooos 321 1.760 1.005 -0.755 I.O188 I.O176 — 0.0012 324 2.010 1-255 -0.755 I.O185 I.0177 — 0.0008 328 1.861 1.076 -0.785 I.018S I.OI76 — 0.0009 329 1-352 0.910 -0.442 I.020I I.OI88 — 0.0013 330 1.620 1-285 -0.335 I.OI99 I.OI82 — 0.0017 347 2.031 I.318 -0.713 I.OI95 I.O185 — O.OOIO Table 6, Series E 21 1.411 1.283 -0.128 1.0200 1.0195 -0.0005 24 1-205 0.92s —0.280 1.0214 1.0202 — 0.0012 28 1.200 0.805 -0.395 — 0.480 1.0230 1.0187 1.0221 — 0.0009 40 1-253 0.773 41 1-500 1.070 -0.430 1.0183 1.0177 — 0.0006 102 1.550 0.683 -0.867 1.0208 1.0188 — 0.0020 103 1-550 0.683 -0.867 1.0210 1.0191 —0.0019 118 1-576 0.881 -0.695 1.0194 1.0186 —0.0008 119 1-576 0.881 -0.695 1.0203 1.0188 — 0.0015 131 1.512 0.752 — 0.760 1.0226 1.0210 — 0.0016 141 1.650 0.696 -0.954 1.0219 1.0205 —0.0014 146 1.772 0.813 -0.959 1.0186 1.0174 — 0.0012 147 1-656 1.036 — 0.620 1.0191 1.0170 — 0.0021 153 1-756 0.712 -1.044 1.0203 1.0173 — 0.0030 364 1-835 1-195 — 0.640 1.0190 1.0180 — O.OOIO 365 1.760 1.450 -0.310 1.0211 1.0206 —o.ooos 377 1-795 1.282 -0.513 1.0202 1.0202 dbo. 392 1-773 1.226 -0.547 1.0218 1.0211 —0.0007 407 1.462 1-537 +0.075 1.0194 1.0198 +0.0004 408 1.866 1.411 -0.455 1.0239 1.0228 — O.OOII of plants employed ; probably they are in part due to diff erences in cultural and meteorological conditions. The mean concentrations in the three large series are shown in the accompanying summary. Prolification 0.03591 + 0.00049 0.03705 + 0.00028 0.04009 + 0.00069 These data give differences for the constants for the juice from the wall and from the mass : Experiment Wall c 0.04065 + 0.00043 D 0.03943 + 0.00029 E 0.04224 + 0.00061 62 Physico-Chemical Properties of Vegetdble Saps [March, Wall J uice from Prolification Comparison Difference dlE^ Comparison Difference dlE^ C and D C and E D and E 0. 00122 ±0.00052 o.ooiS9±o.ooo75 0.00281 ±0.00068 2.35 2.12 4-13 C and D C and E D and E O.OOII4±O.OO056 0.00418 ±0.00085 0.00304 ±0.00074 2.04 4.92 4.12 The results are in substantial agreement with the findings for spe- cific gravity. We have been considerably surprised at the narrow limits of Variation in specific gravity. The observed ranges are : Experiment A B C D E Wall I.O186 — I.O215 =r 0.0029 I.Ol 94 — 1.0234 = 0.0040 I.OI9O — I.022I = 0.0031 I.OI83 — I.020I = 0.0018 I.O183 — 1.0239 = 0.0056 Prolification 1.0168 — I.OI95 = 0.0027 I.Ol 79 — 1.0229 = 0.0050 1.0169 — 1.0204 = 0.0035 I.OI71 — I.OI91 = 0.0020 1.0170 — 1.0228 = 0.0058 If one expresses the Variation, in specific gravity, in the more sci- entific terms of the Standard deviation,® the results are, for the three large series: Experiment Wall Prolification Difference c 0.000910 0.000926 4-0.000016 ±0.000108 ±0.000110 ±0.000141 D 0.000594 0.000612 +0.000018 ±0.000081 ±0.000084 ±0.000100 E 0.001497 0.001600 +0.000103 ±0.000163 ±0.000175 ±0.000244 The entries in this summary show, as do the ranges given above, that the Variation in specific gravity within a particular series is very low indeed. For both wall and prolification ,the variability in specific gravity, as measured in terms of the Standard deviation, is higher in exp. E than in either of the others. Thus, numerically the differences between exp. C and E are : For wall, 0.000587 + 0.000195, d/Ei = 2.99 ; For prolification, 0.000674 + 0.000207, d/Eä = 3.26 ; while that between the same constants, for D and E, are : 8 For the method of calculating the Standard deviation, and the probable errors of means of series of observations as given in this paper, the reader must consult texts on higher statistics. IQISI /. Arthur Harris and Ross Aiken Gortner 63 For wall, 0.000903 + 0.000182, d/Ei = 4.96 ; For prolification, 0.000988 + 0.000194, d/Ea = 5.09 ; There can be no reasonable question of the trustworthiness of the diff erence. Compare the same constants for exp. C and D : For wall, 0.000316 + 0.000135, c?/.Ed= 2.34; For prolification, 0.000314 + 0.000138, (//£d = 2.27; Note that the absolute differences are much lower and that the prob- able errors are relatively higher. The ranges of Variation observed in the concentration of solutes are: Wall Prolification 0.0377 — 0.0429 = 0.0053 0.0342 — 0.0379 = 0.0037 0.0400 — 0.0482 = 0.0083 0.0357 — 0.0462 = 0.0105 0.0376 — 0.0463 =: 0.0087 0.0329 — 0.0437 = 0.0107 0.0374 — 0.0412 = 0.0038 0.0347 — 0.0391 = 0.0044 0.0358 — 0.0490 = 0.0132 0.0333 — 0.0482 = 0.0149 Experiment A B C D E Or, expressed in terms of the Square root of mean square deviation from the mean (Standard deviation), the fluctuation is Experiment Wall Prolification c 0.00240 + 0.00031 0.00270 + 0.00034 D 0.00150 + 0.00021 0.00142 + 0.00020 E 0.00383 + 0.00043 0.00432 + 0.00049 Series E shows a greater variability in concentration, just as was found to be true for specific gravity. The explanation of these results probably lies in the wider ränge of external environmental conditions and internal physiological States prevailing during the collection of certain of the series as explained in a preceding section of this paper. When one turns to a comparison of wall and prolification he finds that the entries in the individual tables show that there are 55 cases in which the specific gravity of the sap of the prolification is lower than that of the wall, to 2 cases in which it is higher. In one instance they are identical. The means for the three series in which the number of observations is large enough to justify the calculation of a probable error are : Experiment Wall Prolification Difference c D E 1. 02017 ±0.00015 1. 01923 ±0.00012 i.o2o64±o. 00023 1. 01783 ±0.00016 i.oi8i3±o. 00012 i.oi950±o.ooo2S — 0.00234 ±0.00022 — o.ooiio±o.oooi7 —o.ooi 14 ±0.00033 64 Physico-Chemical Propertics of Vegetdble Saps [March, TABLES 7-1 I Data pertaining to specific electrkal conductwity, and to ratio of electrical con- ductivity to depression of the freesing point Table 7, Series A Sample Number Specific Conductivity ' or diagnosis SO 122 27 4 2 2 II 3 10 113 No tuberculosis nor typhoid in history or diagnosis As this summary shows, of 122 cases in which the history of tuberculosis and typhoid were ruled out, 113 gave negative tests by both methods. The 4 cases in which both diazo and Weiss tests were positive were clinically cases of diabetes, Cholecystitis, gangrene and pleural effusion. Whether in these conditions, the findings for neutral sulfur are constant we cannot teil, for we had only one case of each in this series. C omparisons of urinary findings with the results of the serum test. Having thus ascertained the degree of efficiency of the tests as compared with each other, and having found that the tests are usually negative for cases where both typhoid and tuberculosis are excluded, we proceeded to compare the findings of the urinary examination with those of the serum test. TABLE 2 Data pertaining to comparative urinary findings and results of the serum test d + tn tu + 0 N tt P 1 '5 + N P + «1 n '5 1 0 N a P 1 'S 1 0 N P Percentage of positive findings m tfi 0 1 0 N a P M V tfi 2 'S Clinically tuberculous. . . i Clinically non- j tuberculous \ Serum test : Positive .... Serum test: Negative. . . Serum test: Positive. . . . Serum test: Negative. . . 89 40 6 I 12 3 0 0 2 7 2 I 6 42 0 9 69 43-5 74-4 9.1 13-5 46.7 74-4 9.1 15-7 Sil 100 1.20 2.2s Total 200 59 5 16 120 28. 30.5 37.8 84 Diagnosis of Tuberculosis [March, The serum test was that described in detail before.^ The anti- gen was that of Besredka.^ The total number of cases compared was 200. The results are given in Table 2. It was previously shown by one of us^^ that, in cases of certain tuberculosis, only 90-95 percent of the cases give positive serum reactions. It was also shown that, in the advanced cases, the reac- tion is usually absent, so that it was even suggested that a failure of the serum test in an advanced case of tuberculosis may be taken as a bad prognostic sign. Comparing the earlier findings with those of the present series, we notice the same phenomenon, namely, in 8 out of 100 cases the serum reaction is negative in spite of the fact that the cases are undoubtedly tuberculous. The value attributed previously to these negative findings seems to be justified by the present results, in spite of the fact that the urinary findings appear to contradict the serological, inasmuch as out of 92 cases of positive serum results the urinary findings in 42 cases, at least, were negative by both methods, and in only 40 cases, or less than 50 percent, the urinary findings confirmed, by both methods, the serum findings. On the other hand, out of 8 cases of tuberculosis in which serum findings were negative, every case gave a positive Weiss test and 6 cases also gave positive diazo tests. Remembering that the presence of neutral sulfur, according to Weiss, is due to the destruction of tissue, and that the intensity and frequency of the occurrence of the reaction run parallel with the progress of the disease, the findings above may be of great value in confirming the opinion, stated earlier, that cases of tuberculosis in which there is no circulating antibody are cases in which there is considerable destruction of tissues, as indicated by the excess of sulfur in the urine. The finding of 11 positive serum reactions among the cases which do not present any Symptoms of tuberculosis clinically, should not be attributed, as was explained before,^^ to non-specificity of serum diagnosis, but rather to the fact that in its earlier stages, 8 Bronfenbrenner : Zeitschr. f. Immunitätsforsch., 1914, xxiii, p. 221. ö Besredka and Manouschine: Compt. rend. soc. hiol., 1914, Ixxvi, p. 180. 10 Bronfenbrenner : Arch. of Intern. Med., 1914, xiv, p. 786. I9I5] /. Bronfenbrenner, J. Rockman and W. J. Mitchell, Jr. 85 the tuberculous process does not induce Symptoms enough for clin- ical diagnosis. A positive serum test in such cases may indicate its extreme diagnostic value. Although urinary findings in these II cases were all negative, with the exception of one, which was a case of typhoid, they indirectly explain the failure of clinicians promptly to discover the tuberculous process, since in incipient cases the destruction is so insignificant, that no increase of sulfur can be detected.^^ SuMMARY. Comparisons of the diagnostic values of the uri- nary findings for neutral sulfur with those for the serum reaction in tuberculosis reveal the f ollowing f acts : 1. The diazo or Weiss test in tuberculosis is less constant, in gen- eral, than the serum reaction. 2. Positive results with the diazo or the Weiss test are of value only if typhoid is excluded. There are also a few other patho- logical conditions in which these tests are positive, but the data at hand are inadequate for conclusions regarding the constancy of these findings. 3. The urinary findings are not sufficiently frequent in tuber- culosis to be of special diagnostic value, even when other possible complications giving rise to positive tests can be excluded. 4. The occurrence of increased amounts of urinary neutral sulfur, in advanced stages of disease, is quite constant and may be of prognostic value, especially in connection with corresponding negative findings in the serum, 11 These examinations were made during the spring and summer of 1914. At that time we had the opportunity of niaking the tuberculin test on only 6 cases of the ii reported above. Since then, however, in two more cases the tuberculin test was made and in all 8 cases it was positive. Moreover, very recently we received a report that one of the patients of this series had a hemor- rhage and also has a very distinct consolidation at present— about 8 months after the first serum test. We take this opportunity to thank Dr. Marks, who was kind enough to give us this information. THE ROLE OF SERUM ANTI-TRYPSIN IN THE ABDERHALDEN TEST* J. BRONFENBRENNER, W. J. MITCHELL, Jr., and PAUL TITUS (Pathological and Research Laboratories of the Western Pennsylvania Hospital, Pittsburgh, Pa.) In previous Communications (i, 2) we outlined the mechanism of the Abderhalden test as an aütodigestion of serum protein of the patient due to the removal of antitryptic inhibition. This removal of anti-trypsin, although quite apparent in our experiments, has not thus far been demonstrated directly. In this preliminary report we wish to record the fact that actual measurement of the anti-trypsin in the serum, before and during the progress of the Abderhalden reac- tion, reveals the fact that the anti-tryptic titer of the serum is actu- ally involved. The diminution of the anti-tryptic activity of the serum, as tested against trypsin Solution, takes place in a specific manner, inasmuch as it occurs only in cases where the serum used is that of pregnant individuals and is parallel with the intensity of the Abderhalden test; so that the estimation of anti-trypsin in serum undergoing digestion, after its removal from contact with placenta, may be used as a method of diagnosis of pregnancy parallel with, and complementary to, that of the Abderhalden test. Moreover, it is evident that this inactivation of anti-trypsin takes place at ice-box temperature as well as at the temperature of the incubator. If the Abderhalden test is divided into two periods, as was shown before (3), over 30 percent of the anti-trypsin is removed during the first part of the reaction, The comparison of the anti-tryptic index of the serum, before and during the Abderhalden test, with the index obtained by measur- ing the effect of kaolin and other substances capable of adsorbing anti-trypsin in a non-specific manner, confirms our contention (i) that the appearance of dialyzable cleavage products in serum may be determined by specific as well as by non-specific mechanisms, and that the esseritial part of this phenomenon is the removal of serum anti-trypsin, which in turn liberates the normal proteases of the serum, thus setting the serum into aütodigestion. * Proceedings of the Columbia University Biochemical Association, Dec. 4, 1914; BiocHEM. Bull., 1915, iv, p. 211. 1 Bronfenbrenner : Proc. Soc. Exp. Biol. and Med., 1914, xü, pp. 4 and 7. 2 Bronfenbrenner : Jour. Exp. Med., 1915, xxi, p. 221. 3 Bronfenbrenner, Mitchell and Schlesinger : Biochem. Bull., 1914, iii, p. 386. 86 ON THE NATURE OF THE ABDERHALDEN REACTIONi J. BRONFENBRENNER According to Ehrlich's theory the parenteral introduction of foreign protein causes the cells of the body to produce an excess of specific receptors, which, at a certain period of the process, circu- late freely in the blood, and are known to the Student of immunity under the name of amboceptors, antibodies, or substances sensibili- satrices. These specific antibodies are complex in character; and, although they are directly responsible for the specificity of the pro- tective processes in the body, they are not of themselves active prin- ciples. It is to complement that Ehrlich and his school attribute the power of action on antigen. The properties of the antibodies resemble those of enzymes in very few respects, while they differ from them at many points. According to Abderhalden, however, the parenteral introduction of foreign protein results in the production of specific enzymes capable of directly digesting antigen in vitro. I disagree with those who think that Abderhalden has proved that these substances are enzymic in character. On the one hand it is difficult to believe that the organ- ism is able to supply so many specific enzymes; on the other, it is improbable that the enzymes circulating in the blood are strong enough to digest coagulated protein, as is the case in the Abder- halden test. In our own experiments we tried to produce a specific enzyme by repeated inoculation of rabbits with egg-white; and, although the serum of these animals contained a very large amount of antibodies, a Mett-tube filled with coagulated egg-white failed to show even the slightest traces of digestion of the egg-white after suitable Im- mersion in such serum. This and the results of other experiments led US to conclude that either the enzymes on which the supposed di- iDiscussion at a meeting of the Pennsylvania State Medical Society, at Pittsburgh, September 22, 1914- See also Proceedings of the Columbia Univer- sity Biochemical Association, Dec. 4, IQM; Biochem. Bull., 1915, iv, p. 211. 87 88 Natur e of Abderhalden ReacHon [March, gestion of placenta depends in the Abderhalden test are essentially different from the substances obtained by immunisation of rabbits, or that they are both ahke, but not enzymic in character. Further experiments along this hne convinced us that the latter alternative is the correct one. We corroborated the earlier findings of Stephan and Hauptmann, that the complement plays an important part in the Abderhalden test, but also found that the specific enzymes (so- called) of Abderhalden behave, in every way, like antibody, as under- stood in the terminology of immunity. We also succeeded in ex- hausting the serum of pregnant individuals of its specific elements, and in actually sensitizing placenta-protein so as to obtain a positive ninhydrin test after the addition of fresh human or animal (male or female) serum. Having thus convinced ourselves that the Abderhalden test did not depend on any enzyme specifically able to digest placenta-pro- tein (since the addition of any serum favored a positive ninhydrin test, provided the serum was added to previously sensitized placenta) we concluded that the ninhydrin test is nothing but a new expression of the phenomenon which previously had been brought to light by the indicator of Bordet-Gengou, viz., hemolysis. Viewed in this light the Abderhalden test, without offering anything new on the theory or mechanism of immunity, introduces a very effective indi- cator of the occurrence of the reaction. As to the mechanism of the test proper, I wish to State without going into the details of our experiments, that I have proof of the fact that in the Abderhalden test placenta is not digested, but that the amino-acids and Polypeptids, which dialyse through the wall of the thimble, come from the serum. I have noted their appearance in a serum after it had been incubated with placenta-protein for some time, and under certain conditions. These dialysable products result from the digestion of the globulin in the serum by the accompanying serum protease; in other words, as a result of the autodigestion of the patient's serum. The proteolytic ferment responsible for this auto-digestion is not specific, but is present in all fresh sera, in vivo as well as in vitro. The action of this ferment, while in the body, is arrested by the antitryptic action of serum constituents, among which are non-sat- urated fatty acids. The combination of any specific antibody (not 1915] /. Bronfenbrenner 89 of a ferment natura) with its antigen, in vitro, is also capable of re- moving the antitryptic inhibiting principle from the serum, setting free the protease which, in turn, digests the globuHn fraction of the serum and produces dialysable substances, Incidentally I wish to call attention to the fact that this auto- digestion of serum may explain the mechanism of the phenomenon of the complement-deviation or complement-fixation, for, in each case where complement is fixed, there appear dialysable products that give a positive ninhydrin test and, vice versa, wherever the Abder- halden test is positive, the complement (as can be proved) is inac- tivated. The auto-digestion of serum, induced by the removal of anti- trypsin in Jobling's experiments, can be stopped by returning non- saturated fatty acid to the serum. The auto-digestion of the serum in the Abderhalden test (which is due to the removal of the anti- tryptic Inhibition from the serum of the patient, by the combination of serum antibody with placenta-antigen) can also be stopped by the addition of non-saturated fatty acids. According to my experi- ments, moreover, self-digestion of the serum results in the produc- tion of a toxic substance which appears to be identical with Fried- berger's anaphylatoxin and, when occurring in vivo, is probably the cause of eclampsia. I am inclined to think from the results of some of our experiments, that here we have the clue to possible prevention of this much dreaded occasional accompaniment of child-birth. In Short, the Abderhalden reaction is specific, but depends not as Abderhalden believes, on the presence of specific enzymes, but on the presence in the blood of pregnant women of the specific antibody that Combines with placenta antigen, and thus sets free the only proteolytic enzyme which is always present in the serum of every animal. When considered from this point of view, the Aberhalden test should be positive wherever the complement-deviation test is positive. I have obtained, in many instances, a positive reaction with the sera of syphilitics, using pure lipoid antigen, in which the only source of protein cleavage products was the serum of the patient. This again proves that not the Substrate, but the serum itself, is digested in the Abderhalden test. Western Pennsylvania Hospital, Pittsburgh, Pa. A NOTE ON THE ABSENGE OF MORPHINE FROM THE LIVER IN A GASE OF GHRONIG LAUD- ANUM ADDIGTION JACOB ROSENBLOOM (Biochemical Laboratory of the Western Pennsylvania Hospital, Pittsburgh, Pa.) There is considerable doubt regarding the nature of the transfor- mations through which morphine may pass af ter its introduction into the animal body. It is possible that such morphine may be changed into oxidimorphine or some other derivative, or that a Compound of morphine with cell material may be formed. However, in many cases of undoubted poisoning by opium or morphine, it has been im- possible to detect this drug or alkaloid in the tissues or organs. Witthaus^ States that Lesser, in a case of post-mortem analysis, f ound morphine in the urine but not elsewhere in the cadaver. Las- saigne^ could not lind morphine in the liver of a dog poisoned with 8 oz. of Sydenham's laudanum. Christison^ mentions four cases of death due to poisoning from laudanum where no trace of the poison could be detected. Woodman and Tidy* could not detect any alkaloid in a case of laudanum poisoning. Haines^ could find no trace of morphine in the stomach in a case where lo to 15 grains were taken. Haines also quotes the report of Surg.-'Maj. Ross, who writes that in Bengal, in 1869, there were 45 fatal cases of poisoning by opium, and an analysis was made in each instance, yet in only two was opium detected in the stomach.^ The failures to detect morphine in the urine^ in cases of un- 1 Witthaus and Becker: Med. Juris., Forens. Med. and Toxicol., 191 1, iv, p. 977. 2 Laissaigne : Jr. de chim. Med., 1841, p. 448. 3 Christison : On poisons, 1845, pp. 57, 58, and 537. ^ Woodman and Tidy : Forensic Med. and Toxicol., 1877, p. 27^' 5 Haines : Hamilton's Legal Med., 1894, i, p. 446. ^ It is possible that the methods of detection in these cases were faulty. '^Kreyssig: Dissertation, Leipzig, 1856; Vogt: Arch. d. Pharm., 1875, vii, p. 23; Landsberg: Pflüger's Arch., 1880, xxiii, p. 413; Burkart: Weit. Mitth. u. ehr. Morph. Vergift, Bonn, 1882; Donath: Pflüger's Arch., 1886, xxviii, p. 528; Von Jaksch : Prag. med. Woch., 1897, xxii, p. 477. 90 1915] Jacob Rosenbio om 9^ doubted opium poisoning, as well as in the urine of morphinists, has also strengthened the idea that the alkaloid is modified or rendered undetectable in the System.^ However, Marquis^ after the injection of morphine into the circulation of cats, recovered from the liver 30 percent of the injected amount; and Antheaume and Mouneyrat/" in a case of morphine poisoning in an individual who had previously used 62 grains daily (recently 30 grains daily), but who had taken no morphine f or the preceding two weeks, f ound morphine in large quantity in the liver. As morphine, through its phenolic hydroxid, combines with Sul- fate to form a Compound similar in structure to the ethereal sulfate normally contained in urine, the possibility of such a formation in the body suggests itself. Eliasso w^^ and Stolnikow^^ have shown that the proportion of ethereal sulfates is increased under treatment with morphine.^^ The results of Rubsamen's^* experiments tend to show that a certain percentage of injected morphine disappears in the bodies of rats, and that this proportion is increased by habitua- tion. The changes said to occur are effected by oxidation or by "pairing." There has been considerable controversy^^ about these experiments, however, and the matter is still unsettled. 8 Cloetta : Virchow's Arch., 1866, xxxv, p. 369 ; Taylor : On Poisons, 3d ed., pp. 556 and 559; Buchner: N. Rept. f. Pharm., 1867, xvi, p. 43; Landsberg: Pflüger's Arch., 1880, xxiii, p. 413; Puschmann : Dissert, Göttingen, 1895; Wel- mans: Pharm Ztg., 1898, xliii, p. 908; Stursberg: Arch. Int. de pharmacodyn., 1898, iv, p. 333; Bougault: Compt. rend. Acad. Sei., 1902, cxxxiv, p. 1361 ; Gerard, Delearde et Ricquet: Jr. de pharm, et de chim., 1905, 6S, xxii, p. 49; Stolnikow : Dissert., Lausanne, 1899 ; Marquis : Arh. a. d. pharm. Inst. 2. Dorpat, 1896, xiv, p. 118; Strzyzowski: Dissert, Lausanne, 1899. 9 Marquis : Chem. Centralbl., 1897, i, p. 249. 10 Antheaume and Mouneyrat: Compt. rend., 1897, cxxiv, p. I47S- " Eliassow : Dissert, Königsberg, 1882. 12 Stolnikow : Zeit. f. physiol. Chem., 1884, viii, p. 235. 13 This might be due, however, to the constipating action of morphine. 14 Rubsamen : Arch. f. e.vp. Path. u. Pharm., 1908, lix, p. 227 ; see also Faust, ibid., 1900, xliv, p. 217. 15 Marme : Deut. med. Woch., 1883, ix, p. 197 ; Polstorff : Berichte, 1880, xiii, p. 86; 1886, xix, p. 176; Brookmann and Polstorff: ibid., 1880, xiii, p. 88, Pelletier: Ann. de chim. et de phys., 1835, xvi, p. 50; Hesse: Liebig' s Ann., 1867, cxli, p. 87 ; 1875, clxxvi, p. I95 ; 1883, ccxxii, p. 234 ; 1886, ccxxxiv, p. 253, ccxxxv, p. 229; Vongerichten: ibid., 1896, ccxciv, p. 206; Lamal: Bull. Ac. r. de Med. de Belg., 1888, 4S, ii, p. 639 ; Jr. de pharm, et de chim., 1904, xix, p. 61 ; Diedrich : Diss., Göttingen, 1883 ; Donath : /. /. prakt. Chem., 1886, xxxiii, p. 559 ; Pflüger's Arch., 1886, xxxviii, p. 528. 92 Transformation of Morphine in the Body [March, BabeP^ claims that morphine is oxidized by brain pulp in vitro, Cloetta^'^ previously supposed that nerve tissue is vitally active in this direction. Rübsamen^^ could not verify in rats or rabbits the results of Babel's experiments. Tauber/^ by perfusion experiments on the Hver and kidney of pigs, found that these organs could not oxidize morphine, but Gerard and Ricquet^^ showed that, by macera- tion with horse kidney pulp, morphine is oxidized to oxidimorphine and the latter is also reduced to the former. It may be readily noted that there is considerable difference of opinion on the question of the transformation of morphine in the body. I recently obtained the liver, three hours after death, of a woman who had used large amounts of laudanum for about five years. It seemed of interest to determine whether morphine was present in this organ. A careful search for morphine by Dragen- dorfif's process, as described by Witthaus,^*^ showed that it was absent. As a control, 150 mg. of morphine sulfate were added to a liver; the same amount of morphine sulfate was isolated, proving that the technic was good. This result indicates the possibility that morphine is so changed in the body, that, under conditions as yet unknown, it cannot be recovered. However, I have shown with Dr. S. R. Mills^^ that, under certain conditions, morphine withstands decomposition in the presence of putrefying material. Ogier^^ states that he has frequently failed to detect morphine in viscera, which had contained it, after putrefac- tion for f rom two weeks to one month. Tardieu^^ found morphine in putrefying viscera after 45 days; Nagelvoort'^^ after 50 days; Marme^^ after 8 weeks; Marquis^® after 2 months; Proelss^'^ after 16 Babel : Arch. f. exp. Path. u. Pharm., 1905, lii, p. 262. ^"^ Cloetta : Virchow's Arch., 1866, xxxv, p. 369. 18 Tauber : Arch. f. exp. Path. u. Pharm., 1890, xxvii, p. 336. 19 Gerard and Ricquet : Compt. rend. soc. bioL, 1904, Ivi, p. 904. 20 Witthaus : Loc. cit. 21 Rosenbloom and Mills : Jour. Biol, Chem., 1913, xvi, p. 327. 22 Ogier : Chim. Tox., 1899, p. 567. 23 Tardieu : Empoisonnement, 2d ed., p. 1043. 24 Nagelvoort : Amer. Jr. Pharm., 1896, Ixviii, p. 374. 25 Marme : Zeit. f. anal. Chem., 1883, xxii, p. 635. 26 Marquis : Dissert., Dorpat, 1896, p. 159. 27 Proelss : Apoth. Zeit., 1901, xvi, p. 492. I9IS] Jacob Rosenhloom 93 260 days; Taylor^^ after 14 months; Kauzmann^^ after 40 days; Stevenson^" after 60 days ; Tidy^^ after 90 days ; Pauzer/^ in two cases, after 6 months; Witthaus,^^ in two cases, after 43 and 53 days, respectively; Autenreith^^ after 15 months and Strzyzowski^' after 5 months. Faust's^® experiments are also of great interest in this connection. He found that, after the hypodermic injection of moderate amounts of morphine into dogs, about 66 percent could be extracted from the feces. By gradually increasing the dose, this amount dimin- ished until, after a time, no morphine was excreted in the urine or feces; and after the death of the animals, none could be extracted from the organs. He thinks that habituation to morphine is due to increased capacity of the tissues to destroy it. From the results of Autenreith's and Strzyzowski's experiments it appears, however, that morphine undergoes decomposition, which is more extensive with aerobic than with anaerobic putrefaction. Strzyzowski estimates that under certain conditions of putrefac- tion, 0,5 gm, of morphine mixed with putrefying material would be detectable after 800 days. However it is possible that the effect of the dead cells on morphine is not comparable to the effects of living cells in regard to its oxidation or change into a form or forms that would not be detectable. The absence of morphine from the liver in the case studied by myself indicates (i) that the morphine was so changed in the or- ganism, under conditions as yet unknown, that it was impossible to detect morphine, and (2) that such a change is marked in cases of habituation to the alkaloid. 28 Taylor : On Poisons, 3d ed., p. 556. 29 Kauzmann : Dragendorflf's Beiträge, p. 131. 30 Stevenson : Lancet, 1903, ii, p. 1443. 3iTidy: Med. Times and Gazette, 1868, i, p. 497. 32 Pauzer : Zeit. f. Unt. d. Nähr. u. Genuss., 1902, v, p. 8. 33 Witthaus: Toxicology, 191 1, p. 982. 3* Autenreith : Ber. d. deut. pharm. Gesell., 1901, xi, p. 494. 35 Strzyzowski : Dissert, Lausanne, 1899. 36 Faust : Arch. f. exp. Path. u. Pharm., 1900, xliv, p. 217. STUDIES OF SOME COMPOUNDS OF CINCHONA ALKALOIDS, CERTAIN METALS AND PHOSPHORIC ACID* EDWIN D. WATKINS {University of Tennessee, Memphis) During the year 1900, under the direction of Prof. J. W. Mallet of the University of Virginia, I undertook some studies of Com- pounds of alkaloids and metals. The alkaloids were principally those of cinchona, and the metals were of several groups. This work was abandoned before anything definite was accompHshed, although I had reason to beheve that some Compounds had been made. In an attempt to obtain better means of treating gonorrheal Urethritis than was available, I again turned my attention, in 1910, to a study of alkaloidal and metalhc Compounds. Cinchona alkaloids, especially quinin, were studied because of their protoplasmic poisonous qualities, and the fact that there had been some success with quinin in the treatment of infections. The success of Helmholtz and others with quinin as an antiseptic war- ranted a close study of it. The known gonococcidal effect of silver commended that metal. Many efforts to combine different acids and various radicals with quinin and silver resulted in failure until orthophosphoric acid was tried. An aqueous sol. of silver nitrate was treated with a conc. sol. of sodium phosphate to complete precipitation of the silver as phosphate. The yellow silver phosphate was washed by decan- tation and then on a filter. It was then treated with syrupy ortho- phosphoric acid to complete Solution. The resulting sol. was treated with pure quinin until no more of the alkaloid was taken up. As the point of Saturation was reached, the sol. changed to a darker color. This Solution was used clinically, diluted as found best by trial. There is no Intention of going into a clinical discussion in this com- * Proceedings of the Columbia University Biochemical Association, Feb. 5, 1915; BiocHEM. Bull., 1915, iv, p. 227. 94 igi5] Edwin D. IVafkins 95 munication. Suffice it to say that the results from the use of the sol. in the treatment of gonorrhea have been most gratifying to those who have used it. My colleagues here and in other places have reported to me splendid success with it. Extending its use I tried it on chancroids, tonsilHtis, ulcers of various kinds, and in one case of amebic infection of the colon. This last case was reported in the Journal of the American Medical Association, vol. Ix, pp. 1357 and 1358(1913). It has been found especially beneficial in chronic gonorrheal Urethritis and in gonorrhea in women. Until recently I had no positive evidence that I had made a Com- pound of silver, quinin and the acid. All attempts to obtain crystals met with failure. Almost by accident a crystal was found in a conc. sol. which had stood unmolested in a dark cabinet from October, 191 3, to December, 1914. On closer investigation two complex crystals were found in the bottom of the flask containing the conc. sol. which had stood 15 months. These were removed, carefully washed and dried. They were very dense; their color was dark yellow. One of them was used in demonstrating the presence of silver, quinin and phosphoric acid; the other is now in safe keeping for further investigation. The crystal was decomposed in heated strong nitric acid, and the presence of silver demonstrated by precipitation with sodium Chlorid and the character of the resulting precipitate. An ammo- nium phosphomolybdate precipitate was then obtained. On addi- tion of strong ammonia to the nitric acid sol. of the crystal, a heavy yellow precipitate was thrown down, which was dissolved with excess of ammonia when, in the top of the sol., there appeared a flocculent white precipitate that proved to be quinin. One of my associates went over the work with me, so that there could be no mistake in it. A quantitative analysis of the crystal has not been made. That will be done at an early opportunity. In place of silver I have made sol. of copper phosphate and zinc phosphate with quinin, quinidin, cinchonin and cinchonidin. The relative merits clinically of these sol. remains for future determina- tion. No crystals of these Compounds, if they be such, have been obtained. ON THE ACCELERATION OF THE OXIDATION OF ALUMINIUM BY MEANS OF MERCURY J. F. McCLENDON (Laboratory of Physiology, University of Minnesota) Our knowledge of oxidations in the body is so meagre that any observations on rapid oxidations at room temperatures outside the body may be of interest. Although many accelerators (enzymes) have been extracted from living cells, such extracts, after being cen- trifuged, are incapable of oxidizing any of the ordinary food stuffs to carbon dioxid and water. With the aid of adsorption surfaces, carbon dioxid may be produced by some tissue extracts, but the complicated relations involved are very difficult to investigate. Un- saturated fatty acids and their Compounds (such as lecithin) oxidize spontaneously in the air but no carbon dioxid is produced. OxaHc acid is completely oxidized by blood charcoal and oxygen in water ; but in this case one active oxygen atom is sufficient to oxidize a whole molecule of the acid, or the molecule of formic acid, if it is split into carbon dioxid and formic acid. A less complete oxida- tion would hardly be expected. A number of inorganic accelerators have been found and I wish to add one to the list. If a trace of mercury is driven into a piece of aluminium by means of an electric spark, the aluminium will burn in dry air (humidity lo percent at 20° C.) at a rapid rate. A volu- minous oxid is formed so fast that its increase may be easily de- tected by continuous Observation for a few seconds with the naked eye or a low-power lens. The masses of white oxid grow out of the metal as plants grow out of the ground, attaining the height of a millimeter in a few minutes. In this process, the energy liber- ated by oxidation is partly expended in lifting the weight of the oxid against gravi ty, in the same way that part of the energy of oxidations in the body is ultimately expended in lifting the body during growth. 96 THE DETOXICATING EFFECT OF THE LIVER OF CATHARTES AURA UPON SOLUTIONS OF ^-IMIDAZOLYLETHYLAMIN* ALLAN C. EUSTIS (Department of Dietetics and Nutrition, College of Mediane, Tulane University, New Orleans) This research was undertaken with an idea of explaining some of the clinical phenomena observed in cases of intestinal toxemia. Many cases are observed by clinicians in which there is very marked indicanuria, but in which there are no subjective Symptoms; while other cases may present decided subjective Symptoms with only mod- erate indicanuria. Most physiologists overlook a very important function of the liver which, to the writer, appears to be its chief function so far as Prolongation of life is concerned. We know how soon an animal may die after the Institution of an Eck fistula, and yet we meet cases in which the glycogenic (diabetes) and biliary functions (biliary cir- rhosis) of this organ are greatly disturbed, or altogether lacking, with little impairment of health for a long time. The presence of indican in the urine is an example of the results of the detoxicating action of the liver cells upon an intestinal toxin. That such action obtains in the case of other intestinal poisons is shown by the experiments of Ewins and Laidlaw (i) who have shown that />-oxyphenylethylamin, when perfused through the liver of a cat, is broken up into /)-oxyphenylacetic acid and urea, which are non-toxic. The liver of the common turkey buzzard, Ca^hartes aura, was chosen for the following experiments on account of its well-known fondness for Carrion, upon which it apparently thrives. An adult bird, after having been trapped and kept in a cage for 3 days on a diet of fresh raw meat, was killed by a rifle bullet through its head. It was then immediately skinned, care having been taken to avoid * Proceedings of the Columbia University Biochemical Association, Feb. S, 1915; BiocHEM. Bull., 1915, iv, p. 224. 97 98 Detoxicating Effect of the Liver [March, opening of the peritoneal cavity. With the carcass lying on its back, the muscles and fascia to the right of the median Hne of the abdomen were thoroughly cooked with a soldering iron. An in- cision was made, with a sterile scalpel, through the cooked tissues into the peritoneal cavity. The liver was removed piecemeal, with sterile scissors and forceps, and placed in a sterile mortar with ster- ile broken glass, and then ground to a pulp. No effort was made to weigh the liver-substance used, all endeavors aiming at transference to sterile flasks as soon as possible, to avoid contamination. Ap- proximately 10 gm. of the liver pulp and glass were placed in one Erlenmeyer flask (a) and about 20 gm. in another (b), while the third flask was a control (c) : Liver pulp A, lo gm. B, 20 gm. C, none /3-ImidazolyIethylamin in saline sol., i-iooo (c.c.) ... lo c.c. 15 c.c 5 c.c. Toluene (c.c.) 4 4 4 All flasks were incubated at 2)7° C., for 24 hr. Inoculations from all flasks were then made on agar and in bouillon to test the sterility, which showed no growth in 48 hr. The Contents of the flasks were filtered and the filtrates used for injections into guinea-pigs. Dale (2), as well as the writer (3), has shown that 0.5 mg. of yö-imidazo- lylethylamin, injected into the blood stream, kills a 300 gm. guinea- pig in 6 min., from spasm of the bronchioles and suffocation. In- jected into guinea-pigs on a basis of 0.5 mg. per 300 gm. of weight, there was no effect for the Solutions that were incubated with liver, büt for the control Solution there were the usual fatal Symptoms. That this action is due to some enzyme seems probable, for heating to boiling inhibits the detoxicating action. Further study will no doubt elucidate this problem but the scarcity of material has, for the present, required postponement of the experiments. It is also of interest to note that the power of causing urticarial lesions, possessed by this amin, to which the writer called attention last year (4), is also destroyed by heat. Füll protocols of these experiments will be published as soon as a sufiicient quantity of the amin can be obtained for final tests, but justification for this preliminary note is found in the hope that some one more fortunate than the writer may have sufiicient of the amin to be able to complete the study ; or that these results may lead 1915] Allan C. Eustis 99 to further experimental work on the several amins of intestinal origin, with a view to extending our knowledge on the detoxicating function of the Hver. BIBLIOGRAPHY 1. EwiNS and Laidlaw: Jour. of Physiol., 1910, xli, p. 78. 2. Dale and Laidlaw: Ibid., p. 318. 3. Eustis : Amer. Jour. Med. Sciences, 1912, cxliii, p. 862. 4. Eustis : New Orleans Med. Surg. Jour., 1914, Ixvi, p. 730. THE ORGANIC PHOSPHORUS COMPOUNDS OF WHEAT-BRAN CHARLES J. ROBINSON and J. HOWARD MUELLER {Lahor atory of Physiological Chetnistry, University of Louisville, Ky.) Introduction. The organic-phosphorus materials, or phytins, obtained by alcoholic precipitation of aqueous or dilute acid ex- tracts from various sources, are not identical, but ultimate analyses show a fair degree of similarity. Thus, the phosphorus content varies between 14 and 17 percent, and there are varying propor- tions of magnesium, potassium and calcium. There has been pre- pared, also, from the phytins from many sources, the free phytic acid, corresponding to the formula CsHgPoOg (anhydro-oxy- methylene phosphoric acid, Posternak),^ or C6H24P6O27 (Neu- berg,^ Starkenstein^). In the case of the material extracted from wheat bran, how- ever, there has been difference of opinion regarding its identity with phytin and its ability to yield phytic acid. Patten and Hart* claimed to have obtained an acid containing 10.63 percent of carbon, 3.38 percent of hydrogen, and 25.98 percent of phosphorus, figures agreeing very well with the formula C6H24O27P6. They therefore called their product phytic acid. Anderson,^ on the other hand, was unable to obtain such a Compound, and ascribed Patten and Hart's supposed error to contamination with inorganic phosphates and phosphoric acid. Anderson obtained his material by a method of procedure different from that used by Patten and Hart, a fact that may explain the divergent results. It was with a view to Clearing up this matter that the work described in this paper was undertaken. Wheat-bran contains a 1 Posternak : Rev. gen. de bot., 1900, xii, pp. 5 and 65. 2 Neuberg : Biochem. Zeitschr., 1908, ix, pp. 551 and 557. 3 Starkenstein : Ibid., 191 1, xxx, p. 56. * Patten and Hart: Compt. rend. de l'acad. des sei., 1903, cxxxvii, Nos. 3, 5 and 8. 5 Anderson : Jour. Biol. Chem., 1912, xii, p. 447. 100 1915] Charles J. Robinson and J. Howard Mueller 10 1 much larger percentage of organic-phosphorus extractives than most other materials so far examined, and probably is the best source of phytin and phytic acid for further investigations. We have repeated Patten and Hart's work. Their so-called tri-barium phytate has been prepared from wheat bran, with care to insure the absence of inorganic phosphates by means of the method recommended by Anderson, viz., repeated Solution of the Salt in dilute hydrochloric acid sol. and reprecipitation with alcohol. With Anderson's new method,^ we have been able to prepare this barium salt in crystalline form and identical in properties with that obtained by him from cotton-seed meal, oats and corn, but corre- sponding more closely in composition with the formula, CeHigOj*- PgBaa, than with Anderson's formula C6Hi2024P6Ba3. Our data leave no question as to the presence of substances in wheat-bran which yield, by the usual treatment to be described in the experi- mental part, a substance very similar to phytic acid, but apparently having the composition represented by the formula C6H24024P6- From his crystalline tri-barium salt from cotton-seed meal, oats and corn, Anderson obtained an acid to which he ascribed the for- mula, C6H18O24P6. Hence, both in the case of the barium salt and the free acid, our Compounds appear to contain six more hydro- gen atoms to the molecule ; while in carbon, barium and phosphorus Contents, they agree very well with Anderson's Compounds. In comparing the results of the analyses, the method used in combustion must be taken into consideration. It is a well known fact that, in the combustion of organic Compounds containing phosphorus, the phosphorus is converted into metaphosphoric acid, HPO3, which remains as a glossy coating in the boat, and may occlude more or less carbon. Anderson states that in decomposing his crystalline barium salts, it was necessary to burn a second time with chromic acid, in order to insure combustion of all the carbon, but that this was unnecessary with the amorphous barium salts. Since he does not say that he burned the free acid with chromic acid, we presume he did not do so. It is inevitable, if t'his is true, that his hydrogen analyses gave low results for phytic acid. It is a noteworthy fact that his formula shows six atoms less in the 6 Anderson : Jour. Biol. Chem., 1914, xvii, p. 160. 102 Organic Phosphorus Compounds of Wheat-Bran [March, molecule than ours; and, since the molecule contains six atoms of phosphorus, the formation of the metaphosphoric acid residue would account for the discrepancy, if our substance is identical with his. In the case of the barium salt, the explanation is less evident, for, of course, barium phosphate or metaphosphate would be formed, together with some barium carbonate and metaphos- phoric acid, ahhough a reaction between the latter two substances might take place, liberating both the hydrogen and carbon. In each of our combustions, we burned the material a second time: in the case of the free acid and the brucine salt to be de- scribed, with well dried, powdered lead Chromate; in the case of the barium salts, with a mixture of lead Chromate and potassium dichromate. There was always an increase in weight in both the potash bulbs and the calcium chloride tube after the second burn- ing. It is probable, therefore, that our Compounds from wheat- bran are identical with those obtained by Anderson from various other sources. We believe, however, that in addition to the phytic acid deriv- ative in our extracts of wheat-bran, there were at least two other organic-phosphorus Compounds, which we have been prevented from investigating completely by lack of time. It was one of these substances which Anderson'^ investigated, and found to yield an acid, to which he ascribed the formula, C20H65O49P9, combined with the elements of a pentose. In regard to this substance, we wish to point out that his analytical results show rather wide de- partures from the calculated formula, and that none of the barium salts were obtained crystalline ; hence may not have been pure. It is also noteworthy that the analytic data for the crystalline brucine salt [to which he ascribed the formula CooH55049P9- (C23H26O4- ^2)10]» accorded better (except in the case of carbon which is low) with brucine phosphate, (C23H2604N2)3- (H3P04)2 than with his calculated formula. Anderson^ himself has shown that ph5rtic acid is broken down into phosphoric acid and other substances by drying at 100° C, and even to some extent by drying at ordinary temperatures. The new acid prepared by him from wheat-bran ' Anderson : Jour. Biol. Chem., 1912, xii, p. 450. 8 Anderson : Ibid., 1914, xvii, p. 171. iQisl Charles J. Robinson and J. Howard Mueller 103 was found to yield inosite and phosphoric acid on hydrolysis with acid, and hence possibly also on drying. At any rate, we have been unable to obtain a crystallina salt of brucine by using a prep- aration which had not first been heated. After drying about i gm. of the acid at 100° C. for several hours, hovvever, we obtained a good yield of crystals, corresponding in physical properties with, and approximating in composition, Anderson's brucine salt; and also with pure brucine phosphate, prepared and analyzed by us. The description of the experimental work is divided into three parts. The first part deals with an investigation of a precipitate obtained by adding copper acetate sol. to an extract of wheat-bran. The second part relates to the material resulting from alcoholic precipitation of bran extract. The third part describes a combina- tion of the two methods. Experimental part. i. Precipitate obtained with copper ACETATE. Preparation of the impure harium salt. Five kilos of wheat-bran were extracted over night in 30 1. of 0.2 percent hydro- chloric acid sol., the liquid then strained and pressed out of the residue, and 16 1. more of the 0.2 percent acid sol. added. After stirring at intervals for 2 hr., this was strained out, and the two cxtracts united. After standing for some time, to allow suspended matter to settle out, the supernatant liquid was strained through cotton. To the filtrate was added an excess of conc. sol. of copper acetate, containing some acetic acid. A heavy precipitate was pro- duced. This was allowed to settle over night, the precipitate filtered on a Buchner funnel, and washed two or three times with water. It was then suspended in water, and hydrogen sulfid gas run in for several hours, the mixture being stirred constantly by means of a water motor. The liquid was then filtered from the precipitated copper sulfid. To the filtrate was added a sol. of 100 gm. of barium chlorid, and then barium hydroxid sol. to strong alkaline reaction. A heavy precipitate was obtained. This was filtered out, dissolved in dil. hydrochloric acid sol. and filtered from a slight insoluble residue. To the filtrate was again added some barium chlorid and barium hydroxid sol. to alkaline reaction. After filtering and dissolving the precipitate in dilute hydrochloric acid sol, it was precipitated with 3 vol. of alcohol. The precipitate, I04 Organic Phosphorus Compounds of Wheat-Bran [March, after resolution in dil. hydrochloric acid sol., was again preclpitated with alcohol. This process was repeated three times more. The material was now free from inorganic phosphates, i. e., it gave no yellow precipitate when warmed with molybdic sol. After wash- ing in alcohol and ether, and drying, the product weighed 57 gm. Dried at 130° C. for analysis, this material turned slightly gray. Analytic data : 0.2080 gm. gave 0.1235 gm. MgjPoO,. 0.4736 gm. gave 0.2853 gm. BaSO«. 1.0058 gm. gave 0.00098 gm. N, by Kjeldahl method. Found: P, 16.55%; Ba, 35-54%; N, 0.098%. Calculated: for tri-barium inosite-hexaphosphate, CeHizOjiPeBaa — P, 17.44% ; Ba, 38-65%. In elementary composition this material approaches the Constitution of a phytin derivative more closely than does Anderson's product, but it is low in both phosphorus and barium. Believing it to be a mixture of tri-barium phytate and some other substance, a means of effecting a Separation was sought. Separation by dialysis. One gm. of the material, dried at 100° C, was dissolved in dil. hydrochloric acid sol., and placed in a S. & S. No. 579, dialyzing capsule, the latter being put into a beaker of distilled water. After 48 hr., the dialysate and the material remaining in the capsule were precipitated with 3 vol. of alcohol. After filtering out both precipitates, and washing with alcohol and ether, and drying, it was found that the undialyzable material weighed 0.2083 S^- Although precipitation was evidently incomplete, it was piain that some, at least, of the substance was capable of dialysis. Barium was determined in both f ractions : 0.2083 gm. gave 0.1213 gm. BaSO« (undialyzed fraction). 0.3820 gm. gave 0.2408 gm. BaSO« (dialysate). Found: in the dialysate, 37.09 per cent. Ba; in the non-dialyzable fraction, 34.27 per cent. Ba. From these data it is evident that there are at least two sub- stances in the crude barium-product obtained from the wheat bran, and that the one having the higher percentage of barium is dialyz- able. Since no attempt was made to effect complete Separation, by changing the water in the outer Container, the undialyzed material 1915] Charles J. Robinson and J. Howard Mueller 105 was, of course, contaminated by some of the dialyzable portion, so that the true barium content must be lower. Separation hy extraction with water. This method was sug- gested and used by Anderson** in purifying the phytin derivative from oats. Ten gm. of crude barium preparation were rubbed up in a mortar with about 50 c.c. of water and, after standing for a time, filtered. The residue was extracted twice more in this way with small quantities of water, and finally washed with water, alcohol and ether, and dried. The aqueous extract was shghtly yellow. Addition of alcohol produced, in the first two portions of extract, a rather abundant precipitate; but in the third portion, only a faint turbidity, showing that the extraction was fairly com- plete. The precipitate, after being filtered out and dried, weighed i.i gm. The water-insoluble material was pure white, while the water-soluble f raction was slightly yellow. Analytic data : Water-insoluble Fraction. Dried for analysis at 100° C. 0.4972 gm. gave 0.3115 gm. BaSOi. 0.3210 gm. gave 0.0650 gm. CO2 and 0.0516 gm. H2O. 0.2946 gm. gave 0.1676 gm. MgjPjOj. Found: Ba, 36.87% ; P, 15.86% ; C, 5-52% ; H, 1.80%. Calculated: for tri-barium inosite-hexaphosphate, CgHiaOziPcBas — Ba, 38.65%; P, 17.44%; C, 6.75%; H, 1.12%. Water-soluble Fraction. Dried for analysis at 100° C. 0.4448 gm. gave 0.2560 gm. BaSO«. 0.2300 gm. gave 0.0630 gm. CO2 and 0.0410 gm. HjO. Found: Ba, 33-87%; C, 7A7%; H, 2.00%. This substance has not been investigated further, Preparation of crystalline barium sali. Five gm. of the water- insoluble fraction were dissolved in the smallest possible quantity of dil. hydrochloric acid sol., a sol. of pure barium hydroxid was added to nearly neutralize the free acid, together with 10 gm. of barium chlorid in conc. sol. The mixture was filtered, and alcohol added until a slight permanent precipitate resulted. This precipi- tate was amorphous. After standing over night, it was crystalline er pseudo-crystalline, the material having aggregated in the form 8 Anderson : Jour. Biol. Chetn., 1914, xvii, p. 160. io6 Organic Phosphorus Compounds of Wheat-Bran [Mardi, of microscopic globules, similar to those described by Anderson from cotton-seed meal, oats and corn. After filtration, second, third and fourth crops of these crystals, as we shall call them, were obtained by adding more alcohol and allowing to stand. These were united, and recrystallized by the same procedure. About 2 gm. of pure white substance were thus obtained. The crystals were dried for analysis at 105° C, in vacuum over phosphorus pentoxid. Analytic data: 0.2124 gm- gave 0.1274 gm. MgoPjOr. 0.2053 gm. gave 0.1347 gm. BaSO*. 0.3099 gm. gave 0.0708 gm. CO, and 0.0392 gm. HoO. Found: F, 16.72%; Ba, 38.61%; C, 6.23% ; H, 1.42%. Calculated: for tri-barium inosite-hexaphosphate, CeHijOjiPsBaj — P, 17.44%; Ba, 38.65%; C, 6.75%; H, 1.12%. Calculated; for CeHisO.iPgBas — P, 17.35%; Ba, 38.43%; C, 6.72%; H, 1.69%. There is little choice between these two calculated formulas. While the results for this material correspond to those for a tri- barium salt, the crystalline substance obtained by Anderson (by the same method) gave analytic data corresponding to the hepta- barium salt, (RgBay). It is possible that our Solution contained more free acid than his. When portions of this salt that had been dried in vacuum over sulfuric acid at room temperature, or in a water oven at 100° C, were further dried at 105° C, in vacuum over phosphorus pentoxid, a slight loss in weight resulted. This was, however, variable; the crystal form of the salt was not in- jured by it. It hardly seems probable, therefore, that water of crystallization was present, hence the percentages of moisture lost by this drying are not quoted. Crystallisation of the barium sali from dilute acid Solution. All of the remaining impure barium salt was extracted with water as described above, and the insoluble portion, weighing about 20 gm., purified as follows. It was dissolved in 0.2 percent hydrochloric acid sol. and, after filtering from a slight insoluble residue, alcohol was added until a fairly heavy precipitate resulted. This required considerably less than i vol. of alcohol. The precipitate was amorphous but, after standing over night, it became crystalline, similar in appearance to that already described. It was filtered 1915] Charles J. Robinson and J. Howard Mueller 107 out, washed with alcohol and ether, and dried. After securing second and third crops of crystals, all were united and recrystallized in the same way. After drying in a water-oven for some time at 100° C, the product, weighing about 7 gm., was a light, powdery material. Part of this was dried at 105° C, in vacuum over phos- phorus pentoxid, and analyzed. Analytic data : 0.1400 gm. gave 0.0860 gm. MgaPsOi. 0.1596 gm. gave o.iooi gm. BaSO«. 0.2275 gm. gave 0.0575 gm. COo and 0.0399 gm. HjO. Found: F, 17.12%; Ba, 37-77% ; C, 6.89% ; H, 1.96%. Calculated: for CeHisOiiPeBaj — P, 17-35% ; Ba, 38.43% ; C, 6.72% ; H, 1.69%. Preparation of the free acid from the crystallized material. The entire amount of the crystalline material remaining, a little less than 7 gm., was decomposed as follows. Somewhat more than the calculated amount of normal sulfuric acid sol. was added to pre- cipitate the barium and, af ter warming for some time, the liquid was filtered. To this was added an excess of copper acetate sol, and the precipitate filtered out and washed thoroughly with water. It was finally suspended in water, and decomposed with hydrogen Sulfid gas. After filtering from the copper sulfid, the liquid con- taining phytic acid was concentrated to a small bulk by boiling in vacuum, the temperature not rising above 65° C. The residue was finally dried for ten days in vacuum over sulfuric acid at room temperature. The residue, weighing about 3 gm., was a very thick, amber colored syrup. For analysis a portion of it was dried at 105° C. in vacuum over phosphorus pentoxid. Analytic data: 0.1208 gm. gave 0.1199 gm. MgjPoOr. 0.2893 gm. gave 0.1156 gm. CO2 and 0.0917 gm. HjO. Found: P, 27.67% ; Q 10.90% ; H, 3-54%. Calculated: for QH^^OsiP« — P, 27.92%; C, 10.81%; H, 3.63%. Calculated: for QHisOjiPs — P, 28.18% ; Q 10.90% ; H, 2.73%- Drying at 105° C. caused blackening, and, presumably, partial decomposition of the material. Anderson^ ° shows, in the case of 10 Anderson : Jour. Biol. Chem., 1914, xvü, p. 171. io8 Organic Phosphorus Compounds of Wheat-Bran [March, the similar acid f rom cotton-seed meal, corn and oats, that such dry- ing causes the formation of phosphoric acid. We have found that this is true for our product from wheat-bran. Analytic data: 0.1919 gm. unheated acid gave 0.0070 gm. MgoPjO,. 0.2360 gm. acid heated to 105° C. gave 0.0381 gm. MgoPjOj. 0.2570 gm. unheated acid gave, after heating at 105° C, 0.0210 gm. HjO. The unheated acid therefore contains 8.17 percent of water. Stating the results on the dry basis: before heating, 4.00 percent of the total phosphorus was present as phosphoric acid, a part of which may have been produced by the nitric acid of the molybdate Solution. After heating for 2 hr. at 105° C, 16.26 percent of the total phosphorus was present as phosphoric acid. Attempt to prepare brucine phytate. Hoping that it might be possible to prepare a crystalline brucine salt of phytic acid, that could be compared with the brucine salt prepared by Anderson from his more complex acid, we undertook to make it by the method used by him, except that to begin with, we did not use the dried acid. This work was done before we effected a Separation of the crude barium salt by water extraction, and the mixture of the water soluble and insoluble materials was therefore used. To 10 gm. of this material, the calculated amount of normal sulfuric acid sol. was added to precipitate the barium. After filtering, the filtrate was concentrated, by boiling in vacuum, to a small bulk. An ex- cess of crystallized brucine was added and, in turn, 150 c.c. of alcohol, 15 c.c. of Chloroform, and ether until a permanent turbid- ity resulted. After standing for two weeks with occasional addi- tion of ether, at a temperature most of the time below freezing, there was not a trace of crystalline deposit, although there was a small amount of gummy material on the bottom of the flask. To 3 gm. of the impure barium salt was added the calculated amount of normal sulfuric acid sol., and the liquid, after filtering, evaporated on a water bath, and dried in a water oven for 24 hr. The black residue was dissolved in a small amount of water, alcohol added and the Solution filtered from a small amount of insoluble, carbonaceous matter. Brucine, Chloroform and ether were then added as before and, after standing for i hr. in the laboratory, there iQisl Charles J. Robinson and J. Howard Mueller 109 began to form a deposit of fine needles. After standing in the cold for several days, these were filtered out, and recrystallized by the same procedure. About 0.8 gm. of crystals were obtained. These were soluble in water and alcohol, but insoluble in ether and Chloro- form. No sharp melting point could be obtained, the substance gradually melting with decomposition between 187° C. and 200° C. The remaining material was dried for analysis at 100° C. Analytic data : 0.3207 gm. gave, by Kjeldahl method, NH3 to neutralize 12.84 c.c. n/io H.SO«. 0.2916 gm. gave 0.0684 gni. MgiPoOr. 0.1087 gm. gave 0.2224 gm. CO2 and 0.0624 gm. H.O. Found: €,55.80%; H, 6.42% ; P, 5-55% ; N, 5.61%. Found: by Anderson from the other acid, (C20H55O48P») — C, 56.24% ; H, 6.26% ; P, 4-69% ; N, S-88%. Calculated: for brucine phosphate, (C23H26N204)3(H3P04)2 — C, 59-53% ; H, 6.08% ; P, 446% ; N, 6.04%. Obtaining thus a crystalline Compound agreeing closely, both in properties and composition, with that obtained by Anderson from a different acid, we can account for the result only by supposing that the acids were decomposed by heat into some other material, com- mon to both — which could be only phosphoric acid. Prcparation of brucine phosphate. An unweighed amount of syrupy phosphoric acid was diluted somewhat with water, brucine added, then alcohol, Chloroform and ether. Almost immediately a deposit of fine needles appeared, which increased in amount on Standing over night. These were filtered out, recrystallized as be- fore, and dried for analysis at 100° C. Their physical properties were identical with the material previously prepared. Analytic data: 0.5658 gm. gave 0.0872 gm. MgoPiOj. 0.3400 gm. gave, by Kjeldahl method, NH3 to neutralize 14.97 c.c. n/io H2S0<. 0.4198 gm. gave, by Kjeldahl method, NH3 to neutralize 18.25 c.c. n/io HzSO«. 0-I550 gm- gave 0.3301 gm. CO2 and 0.0891 gm. H2O. Found: C, 59-66%; H, 6.43%; P, 4-53% ; N, 6.18% and 5-8s%. Calculated: for brucine phosphate, (C23H28N204)3(H3P04)2 — C, 59-53% ; H, 6.08% ; P, 4.46% ; N, 6.04%. It seems probable, therefore, that the substances obtained by Anderson and by us, from the organic-phosphorus acids, is nothing HO Organic Phosphorus Compounds of Wheat-Bran [March, but impure brucine phosphate, the phosphoric acid being produced by hydrolysis. Attempt to prepare the ethyl est er of phytic acid. An attempt was made to prepare the ethyl ester of this organo-phosphoric acid, by treating the silver salt, prepared from silver oxid, with ethyl iodid; but only an impure product, containing free iodin, was ob- tained. This impure product was soluble in nitrobenzene. 2. Precipitate obtained by means of alcohol. The work represented in this part of the paper is of interest chiefly by comparison with the results of part III. Wheat-bran extract was treated by the method used by Anderson for the prep- aration of the Compound CssHggOggPgBag, and we obtained a sub- stance giving an analysis fairly close to that obtained by him. Preparation of the crude phosphorus Compound by "Anderson's method. Five kilos of wheat-bran were extracted over night in 0.2 percent hydrochloric acid sol. To the extract, after straining through cloth, was added dry tannic acid to precipitate the protein material. A very heavy purplish precipitate was produced. This was filtered out, and to the filtrate was added one vol. of alcohol. A heavy white precipitate appeared at once. This was allowed to settle, the liquid siphoned off, and the precipitate collected on a large Buchner funnel, without suction (the method which was found to be most satisfactory in working with all these Compounds). The pre- cipitate was redissolved in dil. hydrochloric acid sol., the Solution ob- tained being milky and filtering only with difficulty. In attempting to overcome this trouble, more tannic acid was added, producing a precipitate which soon became gummy, and from which a perfectly clear filtrate could be obtained. One vol. of alcohol was added, and the resulting precipitate was purified by dissolving in dil. acid sol. and precipitating with alcohol, repeating five times. The precip- itate, instead of being light and flocculent, was rather heavy, and soon settled into a gummy mass, which could be removed from the Solution with a glass rod. In the last precipitation, it was found that 3 vol. of alcohol were necessary to throw down the sub- stance completely, so that considerable of the material was lost. This Statement may have some bearing on the results in the third part of this paper. The gummy substance was dried in a vacuum I9I5] Charles J. Robinson and J. Howard Mueller iii desiccator, a white, opaque solid being produced, weighing lo gm. This could be readily powdered in a mortar. It gave a streng acid reaction to litmus paper. The substance was dried for analysis at 100° C Analytic data: 0.3052 gm. gave 0.1563 gm. MgoP^Or. 0.2163 gm. gave 0.1385 gm, CO2 and 0.0828 gm, H^O, Found: P, 14.27%; C, 17.46%; H, 4.32%, Quantitative determinations 9f nitrogen were not made, but qualitative tests showed its presence in traces only. Qualitative tests for bases sliowed mag- nesium and potassium in fairly large quantities, calcium and sodium in traces. This material, after treatment with boiHng hydrochloric acid sol,, reduced Fehling sol, but not before. The vapors from the boiling mixture colored a strip of anilin-acetate paper pink, indicating the production of furfurol from pentose. Polariscopic examination of a 10 percent sol, showed optical inactivity. This Solution was boiled for some time with an equal vol. of conc. hydrochloric acid so!., the water lost by evaporation being replaced, and the Solution, which had darkened considerably, was decolorized with animal charcoal. Polariscopic examination now showed what was judged to be a very slight dextro-rotation, but so slight as to be uncertain. Preparation of the hariiim sali. All the remaining material, weighing 5,5 gm., was dissolved in 200 c.c. of dil. hydrochloric acid sol. and, after heating nearly to boiling, barium hydroxid sol. was added to alkalin reaction and the precipitate filtered out. The filtrate was reserved for further examination. The precipitate was dissolved in dil. hydrochloric acid sol. and barium hydroxid again added to alkalin reaction. The precipitate was again dis- solved in dil. acid sol. and reprecipitated with 3 vol. of alcohol. After undergoing three more purifications by precipitation with alcohol, the substance was washed with alcohol and ether, and dried. The product, weighing 2.5 gm., was a white amorphous powder, having an acid reaction. Dried for analysis at 130° C, the material turned slightly gray. Analytic data: 0.3017 gm. gave 0.1568 gm. BaSO« and 0.1430 gm. Mg^PjO,. Found: Ba, 30.59%; P, 13.16%. Found: by Anderson — Ba, 31.29%; P, 12.71%. 112 Organic Phosphorus Compounds of Wheat-Bran [March, We did not make carbon and hydrogen analyses of this material. So far as examined, however, this substance appeared very similar to that prepared by Anderson. Examination of filtrate front barium precipitation of phytin Solution. This filtrate was freed from barium with carbon dioxid, filtered and concentrated on a water bath to a small volume, and again filtered from traces of barium carbonate. The residue, after further concentration, was a yellowish, somewhat viscous liquid, with a very peculiar odor, somewhat like old, but not putrid, egg- yolk. The taste was not marked. It reduced Fehling sol. on boil- ing, but did not give the anilin-acetate test for furfurol on boiling with hydrochloric acid. It gave heavy precipitates with phospho- tungstic acid, picric acid and tannic acid. Phosphorus was present, as was also nitrogen. Dried in vacuum, the material seemed some- what crystalline, but was sticky and hygroscopic. 3. Precipitates obtained by a combination of treatments WITH copper acetate (i) AND ALCOHOL (2). From the fact that the two different methods used in the first and second parts of this paper yielded different products, it appeared possible that neither method alone was sufficient to secure a complete removal of all the organic-phosphorus Compounds in the wheat-bran extract. Supposing this to be true, it should be possible by combining the two methods, to obtain two fractions of precipitate, thus not only securing a more nearly complete precipitation, but also establishing the presence of two different substances in the extract. By pre- cipitating the acid extract first with 3 vol. of alcohol, removing this precipitate, and treating the filtrate with copper-acetate sol., we hoped to effect this Separation. The results obtained are somewhat isurprising in the light of the data in the first two parts of this paper, and we are unable at this time to offer an adequate explana- tion for them. The precipitate produced by alcohol, upon purifi- cation and formation of the barium salt, yielded, instead of the 31 percent barium salt of part 2, prepared by the same method, the 36 percent barium salt of part i, prepared by the copper acetate method, and like it, separable into water-soluble and water-insoluble materials, the latter obtainable only in an impure form, but semi- crystallizable. The copper acetate product from the alcoholic iQisl Charles J. Robinson and J. Howard Mueller 113 filtrate gave a heavy precipitate, consisting almost entirely of in- organic phosphate, since, after being converted to the barium salt, it failed to precipitate with alcohol. Preparation of the alcoholic precipitate (2). Two and one-half k. of wheat-bran were extracted as before with 0.2 percent hydro- chloric acid sol. over night, and the extract, after filtering, precip- itated directly with 3 vol. of alcohol, without previous purification with tannic acid, which would have interfered with the copper pre- cipitation of the filtrate. The precipitate, after settling, was filtered out, dissolved in 0.2 percent hydrochloric acid sol., and tannic acid added in excess. The resulting precipitate was filtered out, and the filtrate precipitated with alcohol. The precipitate was then purified by four more alcoholic precipitations, and was finally washed with alcohol and ether, and dried. Yield : 40 gm. ; free f rom inorganic phosphates and slowly but perfectly soluble in water. Copper acetate precipitation of filtrate (i). To the alcoholic filtrate was added a conc. sol. of 100 gm. of copper acetate. A very heavy precipitate was produced. This was filtered and washed, suspended in water, and decomposed with hydrogen sulfid. The filtrate from the copper sulfid was made alkalin with barium hy- droxid sol., after the addition of a sol. of 100 gm. of barium chlo- rid, a heavy white precipitate resulting. This was filtered out, dis- solved in 0.2 percent hydrochloric acid sol., and 3 vol. of alcohol added. Only a faint turbidity was produced, and after long Stand- ing a slight precipitate formed which, after filtering and drying without further purification, weighed only about 0.2 gm. It was discarded. It is possible that the copper precipitate at first con- tained more organic material, but it stood in the laboratory at 20° C. — 25° C. for 2 or 3 days and may have undergone decomposition, although this does not seem probable. Preparation of the barium salt by direct precipitation with barium hydroxid. Twelve gm. of the crude material were dissolved in water to which a small amount of hydrochloric acid was added, and the Solution boiled. Barium hydroxid sol. was now added to strong alkalin reaction. The precipitate was filtered out, dissolved in dil. hydrochloric acid sol., and reprecipitated with barium hydroxid sol. It was then purified by three alcoholic precipitations in the usual 1 14 Organic Phosphorus Compounds of Wheat-Bran [March, manner. The resulting precipitate weighed, after drying, 12.8 gm. It was dried for analysis at 105° C, in vacuum, over phosphorus pentoxid. Analytic data: 0.2050 gm. gave 0.1261 gm. BaSOi. 0.2019 gm- gave 0.1186 gm. MgoPjOj. 0.3348 gm. gave 0.0834 gm. CO, and 0.0555 gm. HoO. Found: Ba, 36.20%; P, 16.37%; Q 6.79%; H, 1.85%. This method of preparation does not seem to replace entirely the bases originally present for, on fusing the salt with potassium hy- droxid and potassium nitrate for the determination of phosphorus, a faint trace of green was produced, showing the presence of a trace of manganese, which was present in somewhat greater quantity in the original alcohoHc precipitate. Preparation of the barium salt by the copper acetate method. Tv/elve gm. of the alcohoHc precipitate were dissolved in water, a few drops of hydrochloric acid sol. added, and then an excess of a conc. sol. of copper acetate. The conversion of the resulting copper salt to the barium salt was accomplished by the procedure described previously for this method. The product free from phosphates, weighed 12.2 gm. It was dried for analysis at 105° C, in vacuum, over phosphorus pentoxid. Analytic data : 0.2333 gm. gave 0.1418 gm. BaSO«. 0.2042 gm. gave 0.1160 gm. MgoPjO;. 0.3342 gm. gave 0.0862 gm. CO, and 0.0497 gm. H^O. Found: Ba, 36.90%; P, 16.32%; C, 7.03%; H, 1.66%. The material was free from manganese, and probably this method insures a more thorough Separation of the bases than the former method. Purification of the barium salt by water extraction. Ten gm. of this material were extracted with five successive 25 cc. vol. of water, being rubbed up thoroughly in a mortar with each portion. The final extract gave only a cloudiness with alcohol. The united extracts were treated with 3 vol. of alcohol and the precipitate col- lected, washed with alcohol and ether, and dried. Weight : 4 gm. It was dried for analysis at 105° C, in vacuum, over phosphorus pentoxid. Analytic data : ipis] Charles J. Robinson and J. Howard Mueller 115 0.1974 gm. gave 0.1211 gm. BaSOi. 0.2068 gm. gave 0.1116 gm. MgoPsOj. 0.3655 gm. gave 0.0794 gm. CO; and 0.0629 gm. H2O. Fotind: Ba, 36.10%; P, i5-53%; C, 5-93%; H. 1.93%- 'Attempt to crystallize the water-insoluble fraction. All the water-insoluble material was dissolved in 0.2 percent hydrochloric acid sol., and alcohol added until a fairly heavy amorphous precip- itate was obtained. After Standing several days, this precipitate had in part crystallized, but there was considerable amorphous matter mixed with the crystals. The form of the crystals was identical with that of the crystals obtained before in pure form. Upon Standing for several days, complete crystallization failed to take place, and the mixture of crystals and amorphous matter was filtered out and dried for analysis at 105° C, in vacuum, over phosphorus pentoxid. Analytic data : 0.1569 gm. gave 0.0942 gm. BaSO,. 0.1525 gm. gave 0.0889 gm. MgoPoOi. 0.2455 gm. gave 0.0570 gm. CO, and 0.0391 gm. H^O. Found: Ba, 35-35%; P, 16.87% ; Q 6.33%; H, 1.78%. None of these substances bears any resemblance to the material obtained by us as described in the second part of this paper. No satisfactory reason suggests itself for this fact, although there were three differences in the methods of preparation: first, a larger amount of alcohol was used, securing a more complete precipitation ; second, tannic acid was not added to the original extract ; and third, the extraction and precipitation of the Compound were completed in three days, whereas two weeks were consumed for the first prepara- tion. The latter fact could make a difference only if the material tends to decompose. Believing that these differences of procedure are not adequate to explain the disparities in composition, we leave the question open for f urther investigation, with the Suggestion that there may possibly be differences in wheat-brans, one of the Com- pounds being formed first and converted gradually, by the metabo- lism of the plant, into the other. Conclusions, A large part of the organic phosphorus of wheat- bran exists as phytin, similar to that from many other sources. A crystalline tri-barium salt may readily be prepared from it. This material is most readily obtained by the copper acetate method. ii6 Organic Phosphorus Compounds of Wheat-Bran [March, There is, in addition, a considerable amount of another sub- stance, very similar in composition, the barium salt of which con- tains only 34 percent of barium, instead of the 38 percent in barium phytate. The f act that this substance does not dialyze indicates that its molecule is larger than that of barium phytate. There is, finally, a Compound differing widely from phytin in having more carbon and less phosphorus in the molecule, which by hydrolysis splits off a reducing sugar (pentose), and whose barium salt contains only about 31 percent of barium. We do not believe the composition of this substance has been definitely fixed. It has not been obtained in crystalline form, the analogous crystalline bru- cine salt prepared by Anderson probably being simply brucine phosphate. The formulas, C6Hi8024P6Ba3, for the tri-barium salt, and C6H24O24P6, for the acid, accord more closely with our analytical results than any other formulas, although the agreement is not en- tirely satisfactory. Two tables of analytic results are appended. TABLE I P c H From wheat bran, found: Percent 27.67 10.90 3-54 For inosite hexa-phosphate, C.Hi8024Pe, calcu- lated: Percent 28.18 10.90 2.72 For CeHaiÜMPt, calculated: Percent 27.92 10.81 3-63 Found by Patten and Hart: Percent 25.98 10.63 3.38 For CeHsiOwPs, calculated: Percent 26.07 10.08 3-39 TABLE 2 For tri-barium inosite From wheat bran, found hexa-phosphate. For CeHisOjiPsBaj. For CsHisOrPuBaj. (crystallized sample): C(sHi202iPeBa3, calcu- calculated: Percent calculated: Percent Percent lated: Percent p 17.12 17.44 17-35 16.62 Ba 37-77 38.65 38.43 36.78 C 6.89 6.75 6.72 6.43 H 1.96 I.I2 1.69 1.62 ADDENDUM After the proof of the foregoing paper had been corrected and returned to the editor, Anderson* published a new series of papers, ♦ Anderson : Jour. Biol. Chetn., 1915, xx, pp. 463, 475. 483. 493- 1915] Charles J. Robinson and J. Howard Mueller 117 in which the disagreement between his findings for wheat-bran, and those of Patten and Hart and ourselves, is explained. The existence in wheat-bran of a phytin-spHtting enzyme, a "phytase," active in dilute hydrochloric acid sol., accounts fully for the failure to isolate in all cases from wheat-bran the inosite hexaphosphate Compound. We refer above (p. 115), to the possible occurrence of such an agent. As to the reason why this enzyme has been active in some instances and not in others, there appear to be two possibilities. Either the hy- drochloric acid used for extraction, having been made up roughly to 0.2 percent, was somewhat stronger and therefore (as has been shown) inhibitory to the phytase; or, in the case of our work, which was conducted during the winter months, the original extraction hav- ing been made in a cold room at a temperature not above 10° C, the enzyme was inactivated by the low temperature. THE NEUTRAI^SULFUR AND COLLOIDAL-NITRO- GEN TESTS IN THE DIAGNOSIS OF CANCER* FREDERIC G. GOODRIDGE and MAX KAHN (Biochetnical Laboratories of Columbia University and the Beth Israel Hospital, New York City) Introduction. During the past few years a number of uri- nary tests have been suggested for the early diagnosis of Cancer. These tests have originated f rom German and Austrian laboratories ; and, immediately after their publication, scientific workers in all parts of the world have endeavored to confirm or disprove the value of these tests, which, if specific, would aid greatly in the conquest of Carcinoma. The reports of various observers have been either very favorable or totally discouraging. Accordingly, it is impos- sible to draw definite conclusions, at present, regarding the efficiency of these laboratory methods. We have attempted to determine the relative values of the uri- nary colloidal-nitrogen and neutral-sulfur tests; to study the per- centage of positive results obtained with these methods in known cases of malignancy; and to discover, if possible, whether the re- sults of these tests run parallel in Cancer and non-cancerous diseases. Colloidal-nitrogen test. In 1892, Töpfer (i) 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 quantity of total nitrogen and then subtracting, from this amount, the sum of the nitrogen values for Urea, uric acid, and ammonia, of the same urine. Bondzynski and Gottlieb (2), five years later, reported that the nitrogen in oxy- proteic acid, in the urine, was 2 to 3 percent of the total urinary nitrogen. Salkowski (3), and Hess and Saxl (4), using different procedures, concluded that the oxyproteic acid portion of the alco- hol-precipitable substances is increased in the urine of human beings suffering from Carcinoma. * Proceedings of the Columbia University Biochemical Association, Dec. 4, 1914; BiocHEM. Bull., 1915, iv, p. 217. 118 1915] Frederic G. Goodridge and Max Kahn 119 Salkowski and Kojo (5), in a preliminary communication, re- cently suggested several methods for the determination of colloidal nitrogen in the urine. A year later, Kojo (6) published the results of a comparative study of the various procedures suggested in this connection. Kahn and Rosenbloom (7) studied the zinc-sulfate- precipitable, colloidal, nitrogenous material from the urine of nor- mal subjects, as well as of carcinomatous patients, and concluded that the amount of colloidal nitrogen was invariably increased in Carcinoma. They also found that diseases like myocarditis, diabetes, leukemia, and anemia, likewise gave a high coUoidal-nitrogen index. They concluded that this quantitative test was not specific for Cancer. Kahn and Rosenbloom (8) studied the amount of col- loidal nitrogen in the urine of a dog suffering from a malignant neoplasm. In this case they used dialysis as a part of the method, and found that the quantity of colloidal nitrogen was much greater in the urine of the diseased dog than the amount present in the urine of normal dogs. Volpe (9) found that the colloidal-nitrogen index is of special value in Cancer diagnosis. Mancini (10), using the Salkowski method, found that there were increased ehminations of colloidal nitrogen in the urines of patients afflicted with Cancer, but this in- crease also occurred in pneumonia and pleurisy. Semionov (11) reported that the colloidal nitrogen Output is low in normal in- dividuals and is increased in Cancer patients. He concluded that although the normal index excliides the possibility of a malignant growth, the increased amount of colloidal nitrogen in the urine is not specific for Carcinoma. Konikov (12) found that the average amount of colloidal nitrogen in the urine, as determined by the Sal- kowski-Kojo method, was 1.68 percent of the total nitrogen in normal cases, and 2.47 percent in carcinomatous individuals. Of 73 cases of Cancer investigated by him, only 9 showed a higher coeffi- cient than 2.5 percent. According to Marcel, Labbe, Dauphin (13) and others, on the other hand, increase in the urinary colloidal nitrogen is an index of a derangement of nitrogenous metabolism; and while it may serve to detect functional insufficiency in the liver, it is not at all specific for cancerous states. Carforio (14), also, concluded that the col- loidal nitrogen index is not pathognomonic of Cancer. 120 Tests in the Diagnosis of Cancer [March^ Neutral-sulfur TEST. Salomon and Saxl (15) have de- scribed a neutral-sulfur reaction in the urine. Like all the other tests in this connection, it has given excellent results in some hands but, in others, has proved valueless. The abnormal constituent in the urine of carcinomatous patients is a neutral-sulfur fraction, the sulfur of which can be split off by means of hydrogen peroxide, and can be determined as barium sulfate. Positive urines yield o.oio to 0.018 gm. of barium sulfate from this fraction, for 100 cc. of urine. Of 41 Carcinoma cases examined by Salomon and Saxl, 30 Werte positive, 4 faintly positive, i questionable, and 6 negative. Of 182 normal urines, 6 were positive, 3 faintly positive, i questionable and 172 negative. Petersen (16) divided his cases into three classes. {A) Clin- ically non-cancerous suspects: of 26 patients examined, 25 gave a negative Salomon and Saxl neutral-sulfur reaction. {B) CHnically Cancer suspects : of 20 cases examined, 5 were negative, 2 alternately positive and negative reactions, and 13 cases positive. (C) Manifest Cancer: of 19 cases, 17 always gave a good positive reaction; the two negatives were icteric and cachectic. Dozzi (17) found that the test was invariably negative in all his patients free from Cancer or tuberculosis, but the frequency of the positive responses in tuber- culous patients detracted from its value as a sign of Cancer, although Cancer is rarely mistaken for tuberculosis. The only Cancer cases that gave negative results were those in which the Cancer had been excised. Murachi (18), also, found an increase in the neutral sulfur from Cancer patients. The coefficient, according to him, may be 3.8 percent of the total sulfur. In contrast to the foregoing, Pribram (19) found that only 60 percent of Cancer patients gave a positive Salomon-Saxl test and that the test is, therefore, far from specific. Alekseev (20) came to a similar conclusion. Mazzitelli (21) has studied this test in 50 cases of Cancer, with and without cachexia. Of 18 cases of the latter variety, the test was positive in 14; but also in 8 of 10 cases of tuberculous cachexia, and 16 of 23 cases of cachexia of various orig- ins, including 11 with Cancer and 4 with tuberculosis, Greenwald (22) concluded that this test has no value in the diagnosis of Cancer. Stadtmüller and Rosenbloom (23) studied sulfur metabolism, in general, in Carcinoma. They found that the lowest average total- 1915] Frederic G. Goodridge and Max Kahn 121 sulfur excretion (0.88 gm. per day) occurred in a series of 13 cases of Carcinoma. The same series also showed the lowest average neu- tral-sulfur excretion (not by the Salomon and Saxl method) — 0.20 gm. per day. The proportion of neutral sulfur to total sulfur in the series was considerably higher than the normal proportion. They conclude, however, that " it is a precarious undertaking to diagnose a malignant tumor on the basis of the absolute or relative amount of neutral sulfur in the urine." Experimental. The following methods were used by us for determinations of the colloidal nitrogen and the neutral sulfur in the urine. CoLLOiDAL-NiTROGEN. The urine was first tested for coag- ulable protein, which, if found, was removed by means of heat coagulation, with addition to the boiling liquid of a few drops of dilute acetic acid sol. To 100 cc. of mixed, filtered, 24-hr. specimen of urine, zinc sulfate was added in sufficient quantity to effect Satu- ration. The saturated liquid was allowed to stand for 24 hours, then was filtered through ashless paper, and the precipitate washed several times on the paper with saturated zinc sulfate Solution, to remove nitrogenous substances adherent to the precipitate. The paper and precipitate were then placed in a Kjeldahl flask and the nitrogen content determined by the Kjeldahl method. The total nitrogen in 5 cc. of urine was also determined by the Kjeldahl method. The ratio of the nitrogen in the zinc sulfate precipitate to the total urinary nitrogen was computed. Neutral-sulfur. The technic of the Salomon and Saxl neu- tral-sulfur test is the following: 150 cc. of urine, freed from coag- ulable protein by heat and acid, are diltited with 100 cc. of water. A mixture of 100 cc. of sat. aqueous sol. of barium hydroxid and 50 cc. of sat. aqueous sol. of barium chlorid is added, the liquid filtered and the filtrate tested with barium to see if precipitation is complete. In Order to remove the ethereal sulfates, 300 cc. of the filtrate are treated with 30 cc. of conc. hydrochloric acid sol., and boiled for 15 min. in an Erlenmeyer flask, using a funnel condenser. The flask is then placed on a water-bath for 24 hr. Of the clear filtrate, 200 cc. are mixed with 3 cc. of hydrogen peroxide (perhydrol- Merck), and boiled for 15 min. with a funnel condenser. After boiling, the liquid is transferred to a conical graduate, whcre, at the 122 Tests in the Diagnosis of Cancer [March, end of 6 hr., the amount of precipitate is observed. Antipyrin and creosote medications interfere, according to certain authors, with this test. TABLE I Data pertaining to normal cases No. Name Diagnosis Total N in IOC cc. urine gm. Colloid- N in 100 cc. urine gm. Per- Cent: col- loid-N of total N Total S in 100 CC. urine gm. Salomon- Saxlneutral- S in 100 CC. urine gm. Percent: neutral-S in total S I A. I. Normal 0.7459 0.01006 1.35 0.II2 0.0019 1.72 2 A. I. 0.7875,0.0098 1.25 0.109 0.0018 1.65 3 J. s. 0.8132 0.0109 1.84 0.097 not w'g'd less than i 4 M. K. 0.7986 0.0167 2.10 0.124 0.0027 2.07 5 D. F. 0.9178 0.0164 1.74 O.I71 0.0033 1.94 6 J. s. 0.93560.017s 1.87 0.195 0.0026 1.34 7 S. H. 0.9471 0.0136 1.44 0.172 not w'g'd less than i 8 R. L. 0.7344 O.OII8 1.62 0.155 not w'g'd less than i 9 B. C. 0.5467 0.0103 1.90 0.137 0.0017 1.22 10 J. H. 0.8264 0.0158 1.92 0.208 0.0044 2.14 II D. F. 0.8326 0.0146 1-75 0.170 not w'g'd less than i 12 W. S. 0.8521 O.OII3 1-33 o.iis not w'g'd less than i 13 M. K. 0.7287 0.0153 2.IO O.IIO 0.0025 1-75 14 M. K. 0.9812 0.0210 2.IS 0.13s 0.0023 1.90 IS J. G. 0.924s 0.0138 1.50 0.152 0.0024 1.62 i6 A. P. 0.8352 0.0129 1.55 0.162 not w'g'd less than l 17 V. L. 0.6992 O.OII8 1.70 0.178 not w'g'd less than i i8 B. H. 0.9272 0.0124 1-34 0.096 not w'g'd less than x 19 D. R. 0.9817 0.0124 1-35 0.087 not w'g'd less than i 20 S. H. 0.8228 O.OI16 1.42 0.135 0.0025 1.90 21 R. L. 0.8298 0.0142 1.72 0.109 0.0020 1.85 22 M. B. 0.8218 0.0156 1.90 0.17s not w'g'd less than i The accompanying tables present our comparative results. Table i shows the results obtained in normal subjects. The nitrogen values for the zinc-sulfate precipitate, as compared with those for total nitrogen, varied from 1.25 percent as a minimum, to 2.15 percent as a maximum, with an average of 1.67 percent. This agrees with the results obtained by Salkowski and Kojo, and Ein- horn, Kahn and Rosenbloom, who obtained respectively averages of 1.75 percent and 1.9 percent. Of 22 urines examined, 10 gave a precipitate by the Salomon and Saxl method that was so light as not to be weighable. The other 12 cases gave sulfate precipitates which varied between 1.22 percent of the total sulfur as a minimum, and 2.14 percent of total sulfur as a maximum. In general the Salomon and Saxl test was negative in all cases in which the neutral sulfur TABLE 2 Data pertaining to Cancer cases Total N Per Cent: Salomon- in Coiioid- col- Total S Saxlneutral- Percent : No. Name Diagnosis 100 cc. N in 100 loid-N in 100 S in IOC neutral-S urine cc. urine ot CC. urine cc. urine in total S gm. gm. total N gm. gm. 23 T. A. Cancer of Uterus 0.9756 0.0419 4-3 0.085 0.0031 3-7 24 M.W. Gastric Cancer I.IO71 0.0636 5-75 0.087 0.0035 4-1 25 F. C. Gastric cancer 1. 1950 0.0652 4.62 0.108 0.0041 3-8 26 A. R. Cancer of breast 1. 2104 0.0568 4-7 0.152 0.0045 2-9 27 S. G. Gastric cancer 0.9260 0.0361 3.9 0.095 not w'g'd less than 1 28 T. S. Cancer of liver 0.5762 0.0247 4-3 0.104 0.0035 3-4 29 T. A. Cancer of Uterus 1-3550 0.0469 4.2 0.087 0.0032 3-7 30 M.W. Gastric cancer 0.8722 0.0305 3-5 0.1055 0.0025 2.5 31 S. G. Gastric cancer 0.9128 0.0447 4-9 0.1243 0.0045 4-4 32 A. R. Cancer of breast 1.0424 0.0458 4.4 O.II75 0.0037 3-2 33 A. G. Cancer of rectum 0.4728 0.0212 4-5 0.1480 0.0050 3-4 34 C.J. Cancer of cervix 1.1307 0.0431 4-7 0.0875 0.0030 3-5 35 W.J. Gastric cancer 0.9246 0.0388 4.2 O.O98Ö 0.0036 3-7 36 B. M. Cancer of liver 1.1108 0.0377 3-4 0.097 0.0031 3-2 37 K. B. Cancer of liver 0.8229 0.0427 5-2 0.1452 0.0062 4-3 38 E. F. Cancer of stomach I-I055 0.0608 5-5 0.1445 0.0059 4-1 39 B. J. Cancer of pancreas 1.1782 0.0494 4.2 0.1378 0.0060 4-4 40 E. M. Cancer of stomach 1-1363 0.0441 3-8 0.1025 0.0043 4.2 41 B. M. Cancer of liver 0.8912 0.0427 4.8 0.1644 0.0070 4.0 42 C.J. Cancer of cervix 0.77550.0334 4-3 0.1552 0.0067 4-5 43 P. B. Cancer of uterus 0.57370.0252 4.4 0.1275 0.0049 4-1 44 M. G. Cancer of stomach 0.93450.0345 3-7 0.09865 0.0035 3-4 45 E. F. Cancer of stomach 0.85480.0435 5-1 O.II43 0.0038 3-5 46 M.W. Gastric cancer 0.98450.0502 5-1 0.0975 0.0026 2.7 47 F. 0. Cancer of pelvis 0.9642 0.0424 4-4 0.0956 0.0039 4.2 48 M. F. Cancer of cervix 0.875O1O.0411 4-7 O.II40 0.0031 2.9 49 C.J. Cancer of cervix 0.6437 0.0270 4.2 O.1231 0.0037 3-1 50 R.W. Cancer of rectum 0.8821 0.0379 4-3 0.1242 0.0041 3-4 Si CS. Cancer of appendix 1.0632 0.0404 3-8 0.0983 0.0031 3-2 52 R.W. Cancer of rectum 1.0452 0.0387 3-7 0.0875 0.0031 3-5 53 C. S. Cancer of appendix 1. 2371 0.0507 4-1 0.0844 0.0031 3-7 54 Z, H. Cancer of stomach 1.1685^0.0561 4.8 0.0847 0.0026 3-1 55 A. T. Cancer of stomach 0.7325J0.0370 5-05 0.0934 0.0031 3-4 56 LS. Cancer of liver o.45iO|0.0237 5-2 0.0895 0.0029 3-3 57 A. I. Cancer of liver 1.47840.0784 5-3 0.0880 0.0034 4.2 58 R. A. Cancer of breast 0.69050.0359 5-2 0.0975 0.0041 4-3 59 G. T. Cancer of esophagus 0.90750.0472 5-2 0.1302 0.0043 4-1 60 G. F. Cancer of esophagus 0.6470I0.0349 S-4 0.0888 0.0031 3-6 61 M. B. Cancer of intestine 0.69840.0356 5-1 0.1207 0.0055 4.6 62 B.J. Cancer of pancreas 0.97500.0536 5-5 0.1405 0.0053 3-8 63 E. 0. Cancer of esophagus 1. 17500.0564 4.8 0.0995 0.0030 3-2 64 D.S. Cancer of breast 0.8705 0.0409 4-7 0.0894 0.0050 4-5 65 T. G. Cancer of breast 0.6095:0.0289 4-75 O.III4 0.0051 4-7 66 G. H. Cancer of uterus 0.52760.0243 4-6 0.1237 0.0039 3-3 67 I. S. Cancer of liver 0.76540.0260 3-4 0.1047 0.0040 3-9 68 Z. H. Cancer of stomach 0.85050.0366 4.3 0.0997 0.0038 3-8 69 A. T. Cancer of stomach 0.9472 0.0360 3-8 0.0896 0.0029 3-3 70 B.J. Cancer of pancreas 1.15600.0541 4.7 0.0945 0.0035 4.8 71 D. S. Cancer of stomach 1.423 0.0448 3-2 O.II44 0.0047 4.2 72 T. A. Cancer of uterus 1.2025 0.0408 3-4 0.0795 0.0024 3-1 73 M.W. Gastric cancer 0.87010. 0296 3.4 0.0994 0.0034 3-5 74 F. C. Gastric cancer 0-75750.0271 3-6 0.0774 0.0028 3.6 75 A. G. Cancer of rectum 1.3645 0.0525 4.6 0.0985 0.0033 3-4 76 C.J. Cancer of cervix 0.6443 0.0248 3-4 0.0863 0.0031 3-6 77 W.J. Gastric cancer 1.2380 0.0516 4.2 0.0784 0.0035 4-5 78 B. M. Cancer of liver 0.9522 0.0432 5-6 0.0952 0.0027 2.8 79 E. F. Cancer of stomach 1.202 0.0528 4-4 0.1205 0.0040 3-7 80 M. G. Cancer of stomach 0.75400.0337 4-5 O.IIO4 0.0041 3.8 81 K. B. Cancer of larynx 0.6884 0.0353 5-2 0.0948 0.0039 4.4 124 Tests in the Diagnosis of Cancer [March, TABLE 3 Data pertaining to non-cancerous cases No. Name 82 L. S. 83 A. H. 84 L. L. 85 P. B. 86 C. H. 87 G. J. 88 S. B. 89 S. E. 90 B. I. 91 H. R. 92 B. R. 9i J. B. 94 A. I. 95 H.W. 96 A. K. 97 M. R. 98 J. R. 99 C. S. 100 child lOI C. F. 102 R. K. 103 F. G. 104 A. E. 105 R. E. 106 B. B. 107 H. S. 108 J. M. 109 J.A. HO B. S. III I. B. 112 I. B. 113 A. K. 114 H. H. 115 S. H. 116 M. F. 117 M.M. 118 B.W. 119 C. Z. 120 J.J. 121 R. K. 122 child 123 «( 124 P. B. 125 E. L. 126 N. R. 127 H. F. 128 N. K. Diagnosis Total N in 100 cc. urine gm. Colloid- N in IOC cc. urine gm. Per- cent: col- loid-N of total N Total S in 100 cc. urine gm. Salomon- Saxlneutral- S in 100 cc. urine gm. Percent: neutral-S in total S Lung Tbc. Nephritis Nephritis Nephritis Myocarditis Myocarditis Typhoid Typhoid Typhoid Empyema Empyema Empyema Endarteritis obliter. Endarteritis obliter. Endarteritis obliter. Endarteritis obliter. Sarcoma of leg Leukemia Hemophilia Pernicious anemia Atrophie cirrhosis Atrophie cirrhosis Pneumonia Pneumonia Pneumonia Pneumonia Diabetes Diabetes Diabetes Diabetes Diabetes Syphilis Syphilis Syphilis Gastric ulcer Gastric ulcer Gastric ulcer Gastric ulcer Gastric ulcer Gastric ulcer Chorea Chorea Endocarditis Tbc. of glands Lung Tbc. Lung Tbc. Lung Tbc. 0.872 0.695 0.723 0.646 1.078 1-055 1.072 0.9465 0.6435 0.7281 0.8253 0.7642 0.6895 0.7321 o.82i8|o 1.0878 o 1.046 0 1.0975 o 0.784 o 1.095 0.5965 0.7234JO 0.65461O 1.0725 0 1. 13470 1.1485 o i-0953|o 1.20750 0.964210 1.2007 0.6435 0.7114 0.7227 0.5835 0.6444 0.9007(0 0.8767 o o.6ii4jO 0.6275 0 0.8649 o 0.965310 0.75560 I-0755 0.6443 0.7627 1.2005 0.9234;o. 0117 0118 0144 0115 0363 0221 0139 0118 0081 0122 0139 0083 0108 0102 0147 0097 0468 0239 0109 0130 0106 OIOI Olli OI6I 0x25 OI6I 0466 0465 0501 0552 0290 0263 0296 0222 0077 0126 0075 0109 0100 0130 0II6 0128 0258 0I4I 0137 0281 0139 1-35 1-75 2.0 1.8 3-4 2.1 1-3 I.2S 1-35 1-7 1-75 i.i 1.6 1.4 1.8 0.9 4.5 2.2 1.4 1.2 1.8 1.4 1-7 i-S I.I 1.4 4-25 3-75 5-2 4.6 4-S 3-7 4.1 3-8 1.2 1.4 0.85 1-7 1.6 1-5 1.2 1-7 2.4 2.2 1.8 1.4 1-5 0.1409 0.1875 0.1482 0.1843 0.1077 0.1255 0.1586 0.1755 0.1641 0.1974 0.1722 0.1645 0.0047 not w'g'd 0.0031 not w'g'd 0.0033 0.0045 0.0029 0.0025 not w'g'd not w'g'd 0.0036 0.0042 not w'g'd 0.0068 0.0072 0.0063 0.0046 3-4 less than i 1-7 less than 2.4 2.5 2.8 2.1 less than i less than i 2.4 2.5 less than i 4-3 3-8 3-7 2.9 ipis] Frederic G. Goodridge and Max Kahn 125 was less than 2 percent of the total sulfur. Considering it from this point of view, 90.9 percent of normal cases gave a negative Salomon and SaxI reaction. In the results for 59 urinary examinations of cases of Cancer (Table 2), the colloidal-nitrogen percent was generally increased to as high as 5.75 percent of the total nitrogen, the minimum being 3.4 percent. Fifty-eight of the 59 cases of Cancer gave a positive Salomon and Saxl reaction. We are doubtful whether the case in which it was negative (case 27) was one of true malignancy, the diagnosis having been made clinically. Table 3 is the most interesting. Forty-seven cases of diseases other than Cancer were studied. We obtained positive results with the colloidal-nitrogen estimations in cases of myocarditis, diabetes and Syphilis. The colloidal nitrogen is constantly increased in amount in diabetics. Wallace,^* basing his conclusion on the find- ings in only two cases, states that this increase is not constant and that there is no relationship between the colloidal-nitrogen outptit and the severity of the diabetes. Tuberculosis and the other diseases gave negative results. On the other hand tuberculosis, hemophilia, pernicious anemia, and atrophic cirrhosis of the liver, gave positive Salomon and Saxl neutral-sulfur reactions, whereas the other dis- eases reacted negatively. General conclusion. We conclude that positive results with either the colloidal-nitrogen test or the neutral-sulfur test, alone, are not indicative of Carcinoma. When performed conjointly on urine of the same case, however, positive results with both methods are strongly indicative of malignancy. Further work along these lines is desirable. BIBLIOGRAPHY 1. Töpfer: Wiener klin. Woch., 1892, v, p. 49. 2. BoNDZYNSKi and Gottlieb: Zentralb. f. d. med. Wiss., 1897, XXXV, p. 577. 3. Salkowski: Berliner klin. Woch., 1910, xlvii, p. 1746. 4. Hess and Saxl : Beitrag, z. Carcinomforsch., 1910, part IL 5. Salkowski and Kojo: Berliner klin. Woch., 1910, xlvii, p. 2297. 6. Kojo: Zeit. f. physiol. Chem., 191 1, Ixxiii, p. 416. 7. Einhorn, Kahn and Rosenbloom : Arch. f. Verdauungsh nk., 1911, xvii, p. 557. 126 Tests in the Diagnosis of Cancer [March, 8. Kahn and Rosenbloom : Biochemical Bulletin, 1912, ii, p. 87. 9. Volpe: Practiceski Vratch, 1913, xii, 84, 105. 10. Mancini: Deut. Arch. f. klin. Med., 191 1, ein, p. 288. 11. Semionov: Russki Vratch, 1913, xii, p. 576. 12. KoNiKOv: Ibid., 1913, xii, p. 927. 13. Marcel, Labbe and Dauphin : Compt. rend. soc. bioL, 1913, Ixxv, P- 391. 14. Carforio: Berl. klin. Woch., October, 191 1. 15. Salomon and Saxl: Deut. med. Woch., 1912, xxxviii, p. 58. 16. Petersen: Ibid., 1912, xxxviii, p. 1536. 17. Dozzi: Gas. degli Ospedali e delle Cliniche, 1912, xxxiv, p. 1007. 18. MuRACHi: Biochem. Zeit., 1913, xii, p. 138. 19. Pribram : Wien. klin. Woch., igi2, xxiv, p. 1235. 20. Alekseev: Russki Vratch, 1913, xii, p. 319. 21. Mazzitelli: Jour. Amer. Med. Assoc. (abstract), 1913, lix, p. 978. 22. Greenwald: Archiv. Int. Med., 1913, xii, p. 283. 23. Stadtmüller and Rosenbloom : Ibid., 1913, xii, p. 276. 24. Wallace: Proc. Soc. Exp. Biol. and Med., 1914, xi, p. 113. ACTIVE IMMUNIZATION TO HAY FEVER* MARK J. GOTTLIEB and SEYMOUR OPPENHEIMER (Laboratory of Biological Chemistry of Columbia University] at the College of Physicians and Surgeons, and the Laboratory of Clinical Research, 45 East öoth'St., New York, N. Y.) Introduction. Hay fever, or pollinosis, is a disease which manifests itself in the spring, from the latter part of May or the early part of June to the early part or middle of July ; and in the autumn, from the middle of August to the end of September or early October. It is characterized by itching of the eyes and lach- rymation, sneezing, serous discharge from the nose, obstructed breathing, and itching of the palate and face. If the attack is very severe, sooner or later there is coughing, and difficult breathing accompanied by wheezing. It is caused by the action of poUen grains from flowering plants, the pollen being carried by air cur- rents and thus inhaled. If the recipient is susceptible to a partic- ular pollen, an attack of hay fever promptly ensues. In 1906 Wolff-Eisner (i) suggested that this disease was a con- ditionof anaphylaxis. Dunbar (2) has studied the subject exhaust- ively and Claims that, besides hypersusceptibility to the pollen "toxin," there must be, in patients subject to this condition, an abnormal permeability of the skin and mucous membranes for the pollen substances. This last fact we have demonstrated to my own satisf action by dropping a small quantity of pollen on the skin of the face, when redness and itching were soon manifest; also by dropping a minute quantity of pollen on the conjunctiva, in a very short time redness and swelling of the lids occurring. Riebet and Hericourt (3) in 1898 applied the name of anaphy- laxis to a Symptom complex of vomiting, diarrhea, respiratory dis- tress, and sometimes death, w^hich was produced in animals by a sub- lethal dose of toxic protein, or by a dose of non-toxic protein, fol- * Proceedings of the Columbia University Biochemical Association, June i, 1914; BiocHEM. Bull., 1915, iv, p. 205. 127 128 Active Immunization to Hay Fever [March, lowed in twelve days by a second dose of the samesubstance, which did not cause any Symptoms in control animals not previously so treated. Since then much research has been conducted, and many theories suggested, regarding the mechanism of the phenomenon. Our present conception of the modus operandi of anaphylactic shock has been evolved from the work of Vaughan and Wheeler (4) on " spHt proteid," of Sleeswijk (5) and others on the role of complement during anaphylactic shock, and that of Friedberger and Hartoch (6), and Ulrich Friedman (7), on the production of anaphylatoxin in vitro. These investigators have given us the f ollowing hypothesis : When foreign protein is injected into an animal, there is a produc- tion of antibody or amboceptor specific for that particular protein. This amboceptor unites with the antigen. By the action of com- plement in the blood, the antigen then undergoes proteolysis, the proteolytic products inducing the Symptoms known as "anaphyl- actic shock." The antibody is formed after the first injection. If the second injection is given at the proper time, the proteolysis goes on very rapidly, with the production of fractions, or anaphylatoxin, in large proportion, and consequent pronounced Symptoms. Hay fever is due, as previously stated, to a sensitization of an individual by the conveyance of pollen contents through the res- piratory tract. There must be, at the time of sensitization, an abrasion of the mucous membrane so as to make parenteral absorp- tion possible. In all likelihood, there exists in the patient an indi- vidual susceptibility to this particular disease, which seems to have some relation to heredity, for this and other allied ailments are fre- quent in given families. Among our patients there are two brothers with hay fever; a brother and sister with hay fever; a woman, with hay fever, whose son suff ers from asthma ; two cases in which a f ather and one or more of his children suffer from hay fever ; a young woman with hay fever who had intense eczema as a child and whose mother suffers with eczema that is rebellious to treatment. An attack of hay fever is comparable, in effect, with the Wolff- Eisner (8) tuberculin reaction in the skin or with the Calmette (9) reaction in the eye. During the flowering season of plants, pollen is transported by air currents and is inhaled by all of us. The sus- 1915] Mark J. Gottlieh and Seymour Oppenheimer 129 ceptible person becomes ill from the action of the pollen contents on his respiratory mucous membrane and the skin of the face. If a quantity of air laden with pollen is directed into the stomach or rectum, the Symptoms are localized in the stomach or rectum and do not appear in the nose, eyes, mouth or face, If a large dose of pollen extract is injected subcutaneously into a susceptible individ- ual, typical Symptoms of anaphylaxis result, as has been observed in a patient to whom we administered an excessive dose of the ex- tract. Within ten minutes thereafter, this patient feit a sense of oppression in the ehest, a suffusion of the face, her breathing be- came labored, there was marked palpitation of the heart, and within forty-five minutes, a giant urticarial rash covered her entire body. All of the Symptoms subsided within two hours and the patient feit well enough to get up. Pollen grains of many varieties are capable of producing this condition, and not all individuals are sensitive to the same pollen. Among the most common plants in this country whose pollen induces hay fever are timothy, red-top and blue grass, and ragweed and golden-rod. The grasses cause the early or spring variety, whereas ragweed and golden-rod produce the late or autumnal variety. Our experience has been mainly with the autumnal variety of hay fever. The majority of our patients were susceptible to rag- weed alone; a few were markedly sensitive to ragweed and also slightly to golden-rod. There are three methods by which it is possible to determine which kind of pollen is operative in a given case. A drop of each of a given series of weak pollen extracts may be instilled into the lower conjunctival sac of the eye. The one which produces con- gestion and swelling of the caruncle and mucous membrane of the lid is the one to which the patient is sensitive. Very minute quan- tities of the available extracts may be injected intracutaneously and the extract of the pollen to which that patient is anaphylactic will cause swelling and redness around the point of introduction. When a very minute quantity of pure pollen is gently rubbed into a small scarification wound of the skin, a wheal will develop at and around this point of scarification, if the patient is susceptible to that pollen. Some patients are sensitive to more than one pollen; and it seems 130 Active Immunization to Hay Fever [March, that there may be, in some cases, a general susceptibility to all pollen, so that only when a given reaction is marked is it possible to conclude which pollen is specifically causative of hay fever in a particular case. To be sure that no other factor than the pollen causes the reaction in a given instance, it is advisable to establish a negative control by simultaneous vaccination of another patient. No swelling should occur in the control. Theoretical considerations. According to Rosenau and An- derson ( 10) , Otto, (11), and others, if on the seventh, eighth or ninth day after the first injection, a massive dose of antigen is injected into the experimental animal, Symptoms of anaphylaxis do not occur with a dose of antigen on the twelfth day. This refractory condition, so produced, is called awfi-anaphylaxis. This same ani- mal will, twenty to thirty days later, become slightly sensitive to antigen, the Symptoms being mild, fatal reactions rarely occurring. The reason for this refractory condition, so produced, was revealed by the researches of Neufeld and Dold (12), Kraus (13), Ritz and Sachs (i4),Izar (15), Friedberger andMita (16), Zinsser (17) and Bordet (18), who, working on the quantities of antigen, ambocep- tor, and alexin, which are most favorable for the production of anaphylatoxin in vitro, found that large proportions of anitgen as compared with the other factors inhibited the production of ana- phylatoxin. They also showed that an excess of amboceptor pro- duced the same result. In view of these facts, they concluded that the great concentration of antigen in the blood of the re- fractory animal inhibited the production of sufficient anaphylatoxin to cause Symptoms. Zinsser and Dwyer (19), working with typhoid anaphylatoxin, showed that guinea pigs treated with sub-lethal doses of anaphyla- toxin, developed a tolerance which enabled them to resist one and one-half to two units of the poison, the tolerance developing within three days and lasting to a slight degree for as long as two months. From the foregoing facts, it should be possible to treat patients suffering with pollinosis by one of f our methods : I. By injecting a dose of pollen extract just before the hay fever time and repeating the procedure in twenty to thirty days. 2. By injecting a large quantity of immune serum during the attack. 1915] Mark J. Gottlieb and Seymour Oppenheimer 131 This we have accomplished in one of our cases. From G. G., a patient who received forty-five injections of ragweed extract, we took about two ounces of blood from a vein. After the proper precaution of a Wassermann reaction, we injected subcutaneously 8 c.c. of the serum into a patient thirteen years of age, during a violent attack of hay fever. Before the expiration of thirty-six hours all Symptoms of hay fever disappeared from this little patient and no signs of the disease returned during the entire season. 3. By injecting very small amounts of pollen extract at inter- vals of ten days or less, so that only minute quantities of anaphyla- toxin are formed and the patient's tolerance is raised. 4. By injecting very small doses of anaphylatoxin made in vitro to produce the same results as in method 3. Practical considerations. It has been our object to im- munize our patients by injecting gradually increased doses of pollen extract to produce tolerance to the anaphylatoxin formed in the body, Beginning with 1-5 units of pollen extract, the dose was gradually increased until a local reaction appeared at the site of the injection. This dose was then continued until the patient showed no more reaction. Then the dose was gradually increased as before. One Unit of pollen toxin was the amount of antigen dissolved in I c.c. of extract at a dilution of i : 20,000,000. Method of preparing Vaccine. Flowers were dried, stripped from their stems, and crushed by hand. This material was then enclosed in muslin bags of suitable size and thoroughly shaken in a large bottle. This bottle contained a cheese-cloth-covered, in- verted, funnel connected by rubber tubing to a suction flask with the outlet in the latter protected by a silk filter. As the fine powder was shaken from the bag into the bottle, the air current carried it into the funnel and thence into the flask, where the silk filter helped to prevent loss of pollen grains. This method was partly success- ful with ragweed but of no use with golden-rod. It was thought likely that golden-rod flowers needed a greater pulverization to free the pollen from the anthers. Flowers were accordingly put into a ball-mill and a fine dust was obtained. Sedi- mentation experiments were then undertaken with this powder to determine what concentration of alcohol in water, and alcohol 132 Active Immunizaüon to Hay Fever [March, with ether in water, would give the greatest concentration of pollen grains in the sediment. It was found that a 20 percent Solution of alcohol in water sedimented most pollen, and that, from powdered flowers containing about 8 percent of pollen, a sediment containing 15 percent of pollen could be obtained in this way. This method is not satisfactory, however, for the reason that immersion in such Solutions of alcohol, for the time required for Sedimentation, rup- tured the pollen grains, with consequent loss of their contents. Many pollen grains were found, by microscopic examination, to be in this condition. The greatest concentration of pollen derived, by the suction method, was about 80 percent for the ragweed powder, in only one sample of 1.75 gm. All other samples contained 50 percent or less. Extracts of the pollen were made as f ollows : It was thoroughly triturated with sand in a mortar and treated with a moderate ex- cess of 5 percent sodium chlorid Solution containing 0.5 percent of phenol to prevent putrefaction. This mixture was kept in an incu- bator for 72 hr. at 37° C. and then filtered. None of the extracts by this method gave the biuret reaction and few gave a positive ninhydrin reaction. The filtered extract was then precipitated with 8 parts of alcohol and filtered quickly in a Buchner funnel to avoid denaturation, if possible, of the active principle by the strong al- cohol. The precipitate was promptly dried and weighed. This precipitate failed to give a biuret or ninhydrin reaction. It was partly soluble in 0.85 percent sodium chloride Solution and physi- ologically active in very weak Solutions. A total content of nitrogen in one of the extracts of ragweed was 0.066 percent. This same Solution, on December 20th, 191 3, gave a positive ninhydrin reaction, whereas on March 24th, 1914, three months later, the test was doubtful. The dry precipitate was dissolved in 0.85 percent sodium chlorid Solution with 0.25 percent of phenol, and serial dilutions were made. With these Solutions patients were treated by hypodermic injections. The method described above for the Separation of pollen grains from the flowers was cumbersome and the positive results hardly justified the time expended. But the negative outcome in this re- 1915] Mark J. Gottlieh and Seymour Oppenheimer I33 spect has suggested the Substitution of a method that is less labori- ous and time consuming, and on which we are now working. The f act that the product is not completely soluble shows that denatur- ation occurred. For this reason we are now endeavoring to perf ect a method of extraction which will prevent such a result. Results of treatment with the Vaccine. Eleven cases were treated in 1 914, before and during the season for autumnal catarrh. Six cases were treated in advance of the attack. One of these was cured for the season, four had very mild Symptoms, and one was not improved. Five cases were treated during the attack. The Symptoms of four subsided after one to four injections, whereas one patient received no benefit. Altogether, there were five eures for the season. In four cases there was marked improvement. In two cases there was no improvement. Of the two cases that were not improved, one had a polypoidal degeneration of the middle turbinate with underlying bone necrosis. The patient had distinct asthmatic attacks every night and it was impossible to say whether the attacks were due to his hay fever or his local nasal condition. The other was a physician who reacted to both ragweed and golden-rod pollen. He received in all thirty-three injections, alter- nating the ragweed extract and the golden-rod extract. He came very irregularly. It is possible that at times the treatment was too intensive. His physical condition was so poor that possibly he could not develop a tolerance. DiscussiON. Nine of our cases reacted to ragweed pollen and two reacted to that of both ragweed and golden-rod. Both of these latter cases received both golden-rod and ragweed antigen hypodermically. One was cured but "the other was not improved. When a patient is sensitive to more than one pollen, individual doses of each extract should be administered, in order to determine when the tolerance is sufficiently raised for each. Mixing the antigen is too empirical, There are two ways of determining when a patient has become sufficiently immune to Warrant discontinuance of the treatment. 1. With the complement-fixation test. 2. From the size, intensity and duration of the wheal produced by skin scarification, at different times, namely, before and during the treatment. 134 Active Immunkation to Hay Fever [March, Clowes (20) was one of the first investigators In this country to immunize hay fever patients with pollen extracts. He performed complement-fixation tests, before and during the treatment, and showed that an increase in antibodies is produced in a few weeks. The scarification method is the one we have generally used to diagnose and determine the degree of immunity induced. The wheal produced by the initial vaccination is measured, its time of appearance and its duration noted. After five or six treatments the patient is revaccinated and the wheal is observed again as before, and compared with the former results. When the wheal is very small or does not appear, the patient is sufficiently immune and probably will go through the season with very mild Symptoms or none at all. Naturally the question arises whether such immunization is permanent. We believe it is safe to say that, while immunity may not be successfully carried over to the succeeding year, recurrences are much milder at least and require less re-immunization. An attack the following year can probably be overcome by a few injections. The best time to begln treatment is probably about ten weeks before the attack may be expected to occur. Regulär ity of at- tendance at about weekly intervals is important. We feel that eures were not accomplished in two cases because treatment was begun too early; and in two other cases, because the patients were treated too irregularly. Furthermore, it is probable that some of these cases were susceptible to pollen other than that from ragweed and golden-rod. At the time of our initial work, we were not pr epared with as large a variety of pollens as we now possess for the continuance of this work. Our heartiest thanks are due to Dr. Wm. J. Gies, for assistance in the conduct of the work done at the College of Physicians and Surgeons; also to Dr. E. P. Bernstein, for many valuable sug- gestions. 1915] Mark J. Gottlieh and Seymour Oppenheimer 135 BIBLIOGRAPHY 1. WoLFF-EiSNER : Das Heufieber, sein Wesen und seine Behand- lung, 1906. 2. Dunbar: Berl. klin. Woch., 1905, Nos. 26, 28, 30; Zeitschr. f. Immunitätsforsch., 1907, 7; Deutsche med. Woch., 191 1, 37, P- 578. 3. RicHET and Hericourt: Compt. rend. de la Soc. bioL, 1898. 4. Vaughan and Wheeler: Jour. Inf. Dis., 1907, 4. 5. Sleeswijk: Zeitschr. f. Immunitätsforsch., 1909, 2. 6. Friedberger and Hartoch : Ibid., 1909, 3. 7. Friedman : Ibid., 1909, 2. 8. WoLFF-EisNER : Berl. klin. Wach., 1904, Nos. 42 and 44. 9. Calmette: Compt. rend., de l'acad. des sciences, 1907, June. 10. RosENAU and Anderson : U. S. Pub. Health and Marine Hospital Service, Hyg. Lab. Bull., 36, 1907 ; Btdl. 64, 1910. 11. Otto: Miinch. med. Woch., 1907, No. 34. 12. Neufeld and Dold: Berl. klin. Woch., 191 1, Nos. 2, 24; Arb. aus d. Kais. Gesundheitsamt, 191 1, 38. 13. Kraus: Zeitschr. f. Immunitätsforsch., 191 1, 8. 14. Ritz and Sachs: Berl. klin. Woch., 191 1, No. 22. 15. Izar: Zeitschr. f. Immunitätsforsch., 191 1, 10. 16. Friedberger and Mita: Ibid. 17. Zinsser: lour. Exp. Med., 1913, 17. 18. Bordet: Ann. de VInst. Fast., 1903, 17. 19. Zinsser and Dwyer: Proc. Soc. Exp. Biol. and Med., 1914, 11, p. 74; Jour. Exp. Med., 1914, 20, pp. 387, 582. 20. Clowes: Soc. for Exp. Biol. and Med., 1913, 10, p. 48. THE INFLUENCE OF LOW TEMPERATURES UPON ENZYMES A review JOSEPH SAMUEL HEPBURN (University Fellow in Biological Chemistry, Columbia University, 1912-1913) Introduction. The influence of low temperatures on enzymes is a subject of growing importance to the chemist, the biologist, and the bromatologist. Problems in this field may be studied from either the potential or the kinetic side; for either the resistance of an enzyme to, or its activity at, low temp. may be investigated. Various researches, conducted during the last half Century, have demonstrated that enzymes survive exposure to low temp. and also act as catalysts at such temp. The reports of these researches are widely scattered in the literature ; and f requently the original papers may be obtained for consultation only with difficulty. It is the pur- pose of this paper, which is based on primary sources, to give a re- sume of our present knowledge of this subject. One section is de- voted to the resistance of enzymes to low temp., and one to their activity at such temp. In the first section, the following data for each enzyme 'are given, so far as they have been recorded in the original literature: source of enzyme; temp., time and mode of ex- posure. In the second section, the data given for each enzyme, so far as recorded by the various observers, are: source of enzyme, temp. and time of incubation, substratum, degree of progress of re- action, and results of comparative experiments carried out at higher temp. 2. Resistance of enzymes to low temperatures. The re- searches reviewed below demonstrate that the following enzymes survive exposure to low temp. and again exert their usual catalytic power when brought into a suitable environment : — lipase, protease of plants, pepsin, trypsin, rennin, thrombin, zymase, invertase, mal- tase, diastase, inulinase, oxidase, peroxidase, catalase, and simple 136 iQisl Joseph Samuel Hepburn i37 and aldehyde reductase. This order will be followed in presenting the data. LiPASE. According to Kastle and Loevenhart ( i ) the lipase of a pig pancreas, which had been held in cold storage at 4° C. f or 7 days, retained 40 percent of its power to produce hydrolysis of ethyl buty- rate. A 10 percent aqueous extract of pig pancreas was held at 1° C. for 72 hr., and a 10 percent aqueous extract of pig liver was kept on ice for 48 hr. During holding at these temp., both extracts gained in power to hydrolyze butyric ester, a zymogen having be- come activated. Pennington and Hepburn (2) demonstrated the presence of active lipase in the crude abdominal fat of chickens of known history, held hard frozen for periods of 12}^, 13, 16, 28, 29 and 4:* months at a temp. of — 9.4° to — 12.2° C, and of chickens, whose history prior to freezing was unknown, kept at that temp. for periods of 54 and 89 months. The chickens held for 28 or more months were not marketable and are of scientific interest only. As the period of holding hard frozen grew longer, the activity of the hpase toward esters usually became greater, and the acidity of the crude fat in- creased. Apparently a zymogen became converted into its active form, thus giving rise to increased activity of the lipase. The increase in activity of the lipase, and in the acidity of the crude fat, occurred less rapidly in hard frozen chickens than in birds held at higher temp., e. g., room temp. Active lipase was also found in the crude abdominal fat of a chicken kept at 0° C. for 24 hr. af ter death. Pennington and Robertson (3) detected lipase in eggs which had been held at a temp. of 0° C. for 66 days. Proteases of plants. Kovchoff (4) demonstrated the power ot these enzymes to survive freezing. Wheat seedlings which had germinated for 17 days, excoriated peas, peas excoriated after ger- mination for 5 days, and certain tissues of the bean, Vicia faha — etiolated caulis tops, etiolated leaves and green leaves — were studied separately. Each sample was frozen for 24 hr., then permitted to undergo autolysis at room temp., in the presence of toluene as a bac- tericide, for a period varying f rom 2 days to 5 weeks. The amounts of protein and non-protein nitrogen were then determined. Almost invariably the former decreased and the latter increased during the autolysis. Therefore, these proteases had survived freezing and had 138 Influence of Low Temperatur es upon Enzymes [March, converted protein nitrogen into non-protein nitrogen during the autolysis. The proteolysis was most marked in the frozen wheat seedHngs kept at room temp. for 5 weeks, 48.6 percent of the protein nitrogen becoming non-protein. Microscopic examination showed the absence of bacteria during the autolysis. Pepsin. Pozerski (5) exposed a glycerol sol. of pepsin to the temp. of Hquid air (approximately — 191° C.) for 45 min. The enzyme retained ahnost unchanged its power to digest albumin (con- tained in a Mett tube) in the presence of hydrochloric acid. Trypsin. Pozerski (5) also exposed an aqueous sol. of trypsin (pancreatin) to the temp. of liquid air (app. — 191° C.) for 45 min. The trypsin retained unaltered its power to digest albumin contained in a Mett tube. Rennin. Chanoz and Doyon (6) exposed samples of commer- cial rennet to a temp. of — 180° C, obtained by means of liquid air, for periods of i, 5, 10 and 30 min. These samples coagulated milk with the same speed as did the unfrozen rennet. The curds appeared to be entirely similar. Pozerski (5) found that a Solution of rennin, kept for more than I hr. at the temp. of ebullition of liquid air (app. — 191° C), re- tained completely its power to clot milk. Thrombin. From the following experiment of Chanoz and Doyon (6), the conclusion may be drawn that thrombin survives exposure to a temp. of — 180° C. Fresh blood of a dog, containing 1.5 part of Oxalate per 1000, was kept in liquid air at a temp. of — 180° C. for 13 min. After thawing at ordinary laboratory temp., the blood coagulated upon addition of calcium chlorid, in the same manner as did an unfrozen control sample. Zymase. Zymase survives exposure to extremely low temp. Buchner (7) obtained the enzyme by grinding yeast beneath a layer of liquid air. He also prepared zymase by grinding 500 gm. of Berlin bottom yeast S with its own weight of solid carbon dioxid (carbon dioxid snow) for y^ hr. ; the stone-like mass gradually be- coming soft and slightly liquid. The plasma was then obtained from the triturated mass by filtration on a hardened filter with the aid of suction; it fermented sucrose. Zymase also survived com- plete freezing of yeast press-juice, in fact a process for the con- centration of the enzyme was based on that fact. Press-juice, con- 1915] Joseph Samuel Hepburn i39 tained in a tall glass cylinder, was completely frozen with a mixture of ice and rock salt, and the frozen mass permitted to thaw slowly but completely. During liquefaction, the juice separated into two layers ; a colorless Upper layer, consisting of almost pure water, and a deeply reddish-brown lower layer, the conc. press-juice. The Upper layer possessed but slight fermentive power and the lower layer a fermentive power considerably greater than that of the orig- inal press-juice, the conc. juice at the very bottom of the lower layer being the most active. Sucrose was used as Substrate in these tests. Ahrens (8) concentrated yeast press-juice, in order to increase its zymase content, by cooling to a temp. not lower than — 2° C, while stirring. Ice crystals, which contained but slight quantities of the constituents of the juice, separated and were removed by rapid filtration with the aid of pressure. The zymase was in the filtrate. In Order to attain a still greater concentration of the enzyme, in some of the experiments, the filtrate was cooled and the entire procedure just outlined was repeated several times. Macfadyen (9) subjected yeast-cell plasma to the temp. of liquid air, —182° to —190° C, for a period of 20 hr. After this ex- posure, the zymase remained unchanged in its power to produce alcohol and carbon dioxid. Invertase (sucrase) retains its activity after exposure to the temp. of solid carbon dioxid; for the yeast-cell plasma, obtained by Buchner (7) by means of carbon dioxid snow, produced alcoholic fermentation of sucrose during incubation at 22° C. Pozerski (5) held Solutions of invertase, prepared from beer yeast and from Aspergillus niger, at the temp. of liquid air (app. — 191° C.) for 45 min. The enzyme completely retained its power to invert sucrose. Maltase (glucase) retains its activity after repeated exposure to a temp. as low as — 2° C. The yeast press-juice, concentrated by the process of Ahrens (8) and incubated at a temp. of 5° to 18° C, with a wort prepared from starch paste and kiln-dried malt, induced alcoholic fermentation of the latter. DiASTASE. Pozerski (5) studied the action of liquid air (temp. app. — 191° C.) on Solutions of diastase. Two varieties of the enzyme were used, salivary diastase contained in filtered mixed 140 Influence of Low Temperatures lipon Enzymes [March, human saliva, and amylase from Aspergillus niger. After the Solu- tion of each variety had been subjected to that temp. for 4-5 min., it retained unaltered its power to hydrolyze starch. Inulinase. Pozerski (5) subjected a Solution of inulinase, obtained from Aspergillus niger, to the temp. of liquid air (app. — 191° C.) for 45 min. The inulinase completely retained its power to hydrolyze inulin. OxiDASE. Pennington, Hepburn, St. John, Witmer, Stafford and Burrell (10) held, at 0° C, both milk and cream containing formaldehyde (o.i percent) and which were bacteriologically sterile; the milk for 35 days, the cream for 28 days. Analyses were made at weekly intervals. During the entire period of holding, the trikresol oxidase of both the milk and the cream retained its activity. Hepburn (11) records the occurrence of oxidases in the crude fat of a chicken held at 0° C. for 15 days after death, and in the crude fat of chickens of known history held hard frozen at — 9.4° to — 12.2° C. for 9 months; and also of birds, whose history prior to freezing was unknown, kept at — 9.4° to — 12.2° C. for periods of 23 and 63 months. Peroxidase, The presence of peroxidase in the crude fat of all the chickens mentioned in the preceding paragraph was demon- strated by Hepburn (11). Catalase. This enzyme was detected by Hepburn (11) in the crude fat of chickens of known history kept hard frozen for 9 months at — 9.4° to — 12.2° C. Pennington and Robertson (3) found that, after eggs had been held at 0° C. for 65 days, the catalase of both the white and the yolk was still active. The work of Pennington, Hepburn, St. John, Witmer, Stafford and Burrell (10) shows that the catalase in both milk and cream, rendered bacteriologically sterile by formaldehyde (o.i percent), re- tained its activity during holding at o°C. for as long as 21 days. Van Driest (12) reports the presence of catalase in frozen sole, held at — 2° to — 9-5° C, for periods of 19 to 21 days, and in frozen cod kept at — 2° to — 6.5° C. for 30 days. Reductase. Hepburn (11) found reductases in the crude fat of chickens subjected to prolonged hard freezing at — 9.4° to — 12.2° C. Simple reductase, which decolorized methylene blue, was found in chickens of known history kept for 9 months, and 1915] Joseph Samuel Hepburn 141 in birds, whose history prior to freezing was unknown, held for 23 months. Aldehyde reductase, which decolorized methylene-blue- formaldehyde sol., occurred in chickens whose history prior to freezing was unknown and which had been in a f reezer for periods of 23 and 63 months. According to Pennington, Hepburn, St. John, Witmer, Stafford and Burrell (10), the simple reductase retained its activity as long as 28 days in both milk and cream rendered bacteriologically sterile by formaldehyde (o.i percent) and held at 0° C. The aldehyde reduc- tase of the Cream likewise remained active during that period of holding. 3. Activity of enzymes at low temperatures. Studies have been made of the activity of the following enzymes at low temp. : lipase, diastase, invertase, maltase, zymase, pepsin, trypsin, galactase, urease, rennin. This order will be followed in presenting the data. At times the enzyme studied was permitted to produce autolysis, at times to act in Solution on an artificial medium, at a given low temp. Lipase, Kastle and Loevenhart (i) studied the influence of temp. as low as — 10° C. on the lipolysis of ethyl butyrate. For lipase, I cc. each of 10 percent aqueous extracts of liver and pan- creas from a pig were used. In each experiment, this quantity of lipase was permitted to act on 0.23 gm. of the ester, in the presence of toluene as a bactericide, the total volume being 5 cc. After a reaction period of 30 min., the percentage of the ester hydrolyzed by the enzyme was : Temp. of the Percentage of ethyl butyrate hydrolyzed by Reaction Pancreatic lipase Hepatic lipase 40° C. 2.82 11.29 30° C. 3- 16 5.96 20° C. 2.51 5.27 10° C. 1.88 3.89 0° C. 1.25 2.26 — 10° C. — 0.70 Richardson (13) ground 150 gm. of perfectly fresh hogpancreas with 500 cc. of water, emulsified with 3 k. of neutral lard, and stored the emulsion at a temp. of — 9° to — 12° C. The pan- creatic lipase caused the initial acidity of 0.25 percent free acid to rise to 2.42 percent after 2 months, and to 4.30 percent after 3 months. 142 Influence of Low Temperatur es upon Enzymes [March, In the course of a study of the influence of temp. on the hydrol- ysis of esters, Hepburn and Pennington (14) demonstrated the activity, in vitro, of the lipase of the crude abdominal fat of the chicken at 0° C. and at — 6.7° to —9.4° C. The increase in acid- ity due to the action of the Hpase for 3 days, in the incubator at 40° C, was chosen as a Standard for comparison. With this were compared the increases in acidity, due to the action of the Hpase, for 3 days in a house refrigerator (average temp. 17.2° C.) ; for 18 days in a mechanically refrigerated chill room at 0° C. ; and for 45 days in a mechanically refrigerated freezer at — 6.7° to — 9.4° C The crude fat was extracted with ten-fold its weight of water, and 50 cc. of the extract were permitted to act on i cc. of an ester. The ratios of increase in the acidity of the Substrates were expressed throughout on a basis of the action of the enzyme for a period of 3 days, and the following data were obtained. The lipolysis of ethyl acetate in the incubator was twice as rapid as in the refrigerator, 15 times as rapid as in the chill room, and 37>4 times as rapid as in the freezer. Ethyl butyrate was hydro- lyzed by lipase in the incubator 2}^ times as fast as in the refriger- ator, 12 times as fast as in the chill room, and 40 times as fast as in the freezer. Ethyl benzoate was split by the enzyme in the incu- bator 8>^ times as rapidly as in the refrigerator, 25^^ times as rapidly as in the chill room, and 255 times as rapidly as in the freezer. The hydrolysis of amyl salicylate by lipase in the incubator was 63/2 times as rapid as in the refrigerator, 13 times as rapid as in the chill room, and 97^^ times as rapid as in the freezer. Although the rate of lipolysis was decreased by a lowering of the temp., lipolysis took place even at the temp. of the freezer, while the reaction mixture was frozen solid. DiASTASE. Müller (15) prepared a glycerol Solution of dias- tase from the liver of the carp, and used i percent starch paste as Substrate. The volumes of enzyme extract and starch paste were kept constant in the entire series of experiments. The opalescence of the mixture vanished after digestion for i}i min. at 25° C, for 5 min. at 8° C, or for 20 min. at 0° C. The Solution then reacted violet to iodin. The Solution first gave a red color with iodin after 3M hr. at 25° C, 18 hr. at 8° C, or 32 hr. at 0° C. The Solution lost its power to react with iodin after digestion for 8>^ hr. at I9IS] Joseph Samuel Hepburn I43 25° C, 44 hr. at 8° C, or 72 hr. at o*^ C. Therefore, even at the latter temp., diastase transformed starch through the stages of soluble starch and erythrodextrin to maitose. Invertase, maltase^ zymase. These enzymes are active at the temp. of an ice box, for, according to Buchner (16), yeast press- juice produced alcoholic fermentation of s'ucrose, maitose, glucose, and fructose at that temp. Pepsin (gastric Protease). Murisler and Fick (17) ex- tracted finely divided gastric muscosae from various animals with 40-fold their weights of water, and permitted these extracts to act at various temp. on small cubes of coagulated albumen in the presence of hydrochloric acid. The extracts of the gastric mucosae from pigs and dogs rarely acted upon coagulated albumen at temp. below 10° C. and never acted, even in the slightest degree, at 0° C. On the other hand, pepsin extracts prepared with gastric mucous mem- branes from frogs, pike and trout, regularly digested coagulated albumen at 0° C, and were fully as active at 40° C. as were the ex- tracts obtained with mucosae from pigs and dogs. From these ex- periments, which were qualitative, Murisier and Fick concluded that the gastric protease of cold blooded animals is not completely iden- tical with that of warm blooded animals. This opinion was shared by Hoppe-Seyler (18), who studied the action, upon fibrin shreds, of artificial gastric juice prepared with mucous membrane from pike stomachs. The Optimum temp. for digestion was approximately 20° C. Proteolysis was more rapid at 15° C. than at 40° C, and became somewhat less rapid when the temp. was reduced from 15° C. to several degrees above 0° C, Di- gestion was very energetic at temp. between 5° and 20° C. Flaum (19), however, demonstrated that at 0° C. the pepsin of warm blooded animals gives rise to complete proteolysis of oval- bumin, and that the same products are formed as at higher temp., although digestion is greatly retarded. He studied the action of artificial gastric juice, prepared from gastric mucosae of swine, on coagulated egg white at various temp. between 40° C. and 0° C. In all the experiments of a given series, the volume of gastric juice and the quantity of Substrate were kept constant. In the prelim- inary series, the artificial gastric juice had been rendered free from acid metaprotein. The time required for the appearance of acid 144 Influence of Low Temperatures upon Ensymes [March, metaprotein in the reacting mixture was: at 40° C, i^ to 2 hr. ; at 16.5° C, 2^ hr.; at 10° C, 3 to zYa hr.; at 5 to 6° C, 8 hr.; at 0° C, 2 to 3 days. In Flaum's final series of experiments, the artificial gastric juice was prepared with mucous membranes from the fundus of pig stomachs, and purified until free from proteoses and peptones. In this series, the study of proteolysis was continued until the acid meta- protein disappeared. At 40° C, decomposition of the coagulated protein began in 30 min. After 50 min., soluble protein was pres- ent; after 2 hr., acid metaprotein. Traces of proteoses were noted after 2 hr. and peptone was present after 2^ hr. Acid metaprotein disappeared after 48 to 50 hr. At 16° to 17° C, acid metaprotein formed after 2% hr., and proteoses and peptone after 2^- hr., while 4 days (about 94 hr.) were required to carry digestion to the stage at which acid metaprotein was no longer present. At 10° to 10.5° C, after the lapse of 4 to 5 hr., acid metaprotein made its ap- pearance. After 5^ to 6 hr., proteoses and traces of peptone were detected; and acid metaprotein disappeared at the end of the 5th day. At 5° to 6° C, 8 to 10 hr. were required for the formation of acid metaprotein, and about 20 hr. for the definite appearance of Proteose and peptone, while 7 to 8 days elapsed before acid metapro- tein completely vanished. At 0° C. in 3 to 4 days the coagulated protein was decomposed with the formation of acid metaprotein, Proteose and peptone; after 14 to 15 days, acid metaprotein was no longer present. Flaum also prepared an artificial gastric juice from frog stom- achs by extraction with 2 percent hydrochloric acid sol. at 0° C. This juice digested both fibrin and ovalbumin at 0° C, and at 16° to 17.5° C. (room temp.). However, when the stomachs of living frogs were flushed, and coagulated tgg white was then introduced, no digestion of the protein took place in the living ani- mals held at 0° C. for as long as 14 days, or at 4° to 5° C. for 6 days. Digestion occurred in i day in frogs held at 10° C. The failure of digestion to occur in vivo at the lower temp. is ascribed by Flaum to the fact that no gastric juice was secreted, the lower temp. limit for its secretion being 8° C. "Müller (15) permitted the gastric protease of pike to act on 100 mg. of heavy fibrin particles. The same volume of enzyme extract igisl Joseph Samuel Hepburn 145 was used throughout each series of experiments, and the time of digestion was noted. A glycerol extract of the gastric mucosae re- quired 40 min. at 24° C, 100 min. at 8° C, and 230 min. at 0° C An extract of the gastric mucous membrane in 0.25 percent hydro- chloric acid sol. was more active, and required 20 min. at 24° C, 65 min. at 8° C, and 140 min. at 0° C, for digestion of the fibrin. Oguro (20) studied the influence of temp. as low as 0° C. on the peptic digestion of ricin. From 0.05 to i.o cc. of o.i percent sol. of pepsin was permitted to act on 2 cc. of 0.2 percent Suspension of ricin in the presence of 0.5 cc. n/io hydrochloric acid sol.; the total volume of the reacting mixture always being made 4.5 cc. by dilution with water. The temp. of incubation were 38°, 20° to 21°, 8°, 5° and 0° C. The progress of digestion was followed by noting the degree of cloudiness of the Suspension from time to time, the results being recorded as " clouded," " a little clouded," " traces of cloud," " almost clear," " nearly clear," " clear." The pepsin di- gested the ricin at all the temp. of incubation, gradually producing a clear Solution, although the digestion proceeded more slowly at the lo wer temp. Thus, when o.i cc. of the pepsin sol. was used, the ricin Suspension became clear after 50 min. at 38° C, 50 min. at 20° to 21° C, or 24 hr. at 0° C, while merely a trace of a cloud re- mained after incubation for 2 hr. at 8° C, or for 2 hr. at 5° C. Trypsin. Müller (15) prepared a glycerol extract of trypsin from the intestinal tracts of carp, a fish which is without a stomach and secretes no peptic enzyme. Pieces of nutrient gelatin of equal size, and fibrin particles, were used as Substrates. A given volume of the trypsin sol. dissolved the piece of gelatin after incubation for 2}i hr. at 20° C, 16 hr. at 8° C, or 34 hr. at 0° C. The fibrin particles (100 mg.) were dissolved by a given volume of enzyme ex- tract after digestion for 5^ hr. at 20° C, 31 hr. at 8° C, or 72 hr. at 0° C. The trypsin, therefore, showed a distinct activity at 0° C. Galactase. Babcock (21) and his collaborators (22) have demonstrated the activity of galactase, the native trypsin-like, pro- teolytic enzyme of milk, during the ripening of Chedder cheese at low temp. Fresh Cheddar cheese were carried at a temp. of 25 to 30° F. for periods of 14 and 17 months. Progressive increase occurred in the total soluble nitrogen of the cheese during holding. In another series of experiments, Cheddar cheese were manufactured 146 Influence of Low Temperatur es upon Enzymes [March, with 3, 6 and 9 ounces of rennet per 1000 pounds of milk, and were permitted to ripen at temp. of 15°, 33°, 40°, 50° and 60° F. for periods of 6, 10, 12, and 14^ months. Total soluble nitrogen in- creased progressively in the cheese held at each of these temp., the increase being more marked at any given time in a cheese kept at a higher temp. than in one held at a lower temp. A marked increase in soluble protein was noted even in cheese stored at a temp. below freezing (15° F.). At a given temp. and in given time, a greater rise in the total soluble nitrogen occurred v^hen larger quantities of rennet had been used in the process of manufacture. This influ- ence of the rennet was due to the pepsin content of the latter, and was observed in the samples held at 15° and 33° F. as well as in those stored at higher temp, The action of the galactase at all the temps., including 15° and 33° F. was absolutely demonstrated by progressive increase in amino-acid nitrogen, This increase was independent of the quan- tity of rennet used in the process of manufacture, and became more marked as the cheese were carried at a higher temp. Ravenel, Hastings and Hammer (23) held a sample of "barn milk," the best milk obtainable, and one of a fair grade of dairy milk at — 9° C. for 203 days. At the end of that period the water soluble nitrogen, expressed as percent of the total nitrogen of each sample, was: barn milk, 17.97 percent; dairy milk, 22.38 percent; while the average for fresh milk is 10 percent, The higher per- centage in the milks subjected to prolonged holding at this low temp. is Said to be due, probably, to the action of galactase, Pennington, Hepburn, St. John, Witmer, Stafford and Burrell (10) held a milk, rendered bacteriologically sterile by formalde- hyde (o.i percent), at 0° C. for 35 days, and studied the partition of the nitrogen at intervals of 7 days, The nitrogen present as lactalbumin and syntonin, and as peptone, tended to decrease but the caseose nitrogen and the amino-acid nitrogen increased, the casein nitrogen remaining practically constant, Proteolysis was due mainly, if not entirely, to the action of galactase, Urease, Van Slyke (27) demonstrated that the urease of the soy bean hydrolyzes urea at temperatures as low as 0° C, The temperature coefficient of the reaction was found to be 2.80 for 1915] Joseph Samuel Hephurn 147 the interval 0° C. to 10° C, while it remained nearly constant, with an average value of 1.9 1, for each interval of 10° between 10° C. and 50° C. Rennin. Selmi (24) noted that small quantities of rennin coagulated milk, stored at 1° to 2° C, within 4 to 5 days. Camus and Gley (25) demonstrated that rennin exerts some action on the casein of milk at 0° C. When rennin and milk were mixed and held at that temp. for periods of ^ hr. or longer, no precipitation of protein took place. Either lactic, acetic or hydrochloric acid was then added in quantity insufficient to produce a precipitate of protein, yet such a precipitate immediately formed, showing that the rennin had given rise to the conversion of casein into paracasein, the first stage of the enzymic curdling. Morgenroth (26) states that, even after the prolonged action of very great quantities of rennet on milk at 0° C, the milk is not curdled; however, it clots immediately if this mixture be brought to a higher temp. The rennin, therefore, produces certain chem- ical changes in milk during holding at 0° C, but the definite trans- formation of the casein into its insoluble modification occurs only at higher temp. Müller (15) prepared a sol. of rennin by extraction of the gas- tric mucosae of pike with 0.25 percent hydrochloric acid sol. Five drops of this extract were able to curdle 5 c.c. of unboiled milk in 2 min., at 40° C. ; in 53^ min., at 25° C. ; in 25 min., at 15° C, and in 18 hr., at 7° C. At 0° C. no distinct curdling occurred, but a finely flocculent condition of the milk was noted after incubation for 5 days at that temp. This flocculation was apparent when the tube was slowly moved to and fro. The time of curdling of milk is the sum of two factors, the time required for the conversion of casein into paracasein and that re- quired for the deposition of a visible coagulum; and the latter phenomenon may require several days at lower temp., while occur- ring in a few min., at most, at higher temp. Experiments were carried out in such a manner that the first stage, or formation of paracasein, occurred at 0° C, while the second stage, or Separation of a visible coagulum, followed at a higher temp. (40° C). Both the diluted rennin sol. and the milk were cooled at 0° C. Several 148 Influence of Low Temperatures upon Ensymes [March, tubes were then prepared, each containing i c.c. of the enzyme sol. and 5 c.c. of the Substrate. The tubes were incubated at 0° C. for periods differing between o min. and 96 hr., then were held at 40° C, and the time required at the latter temp. for the production of a coagulum was noted, with the f ollowing results : Time of inca- Time required for subsequent bation, at o° C. coagulation, at 40° C. Minutes Minutes 0 7 10 6, 30 sec. 20 5, 30 sec. 30 4, 45 sec. 45 4 60 2 75 I, 50 sec. 90 1, 45 sec. 105 I, IG sec. 120 55 sec. 150 45 sec. (Hours) 24 45 sec. 48 ,45 sec. 96 45 to so sec, In the last experiment of this serles — held at 0° C. for 96 hr. — the flocculent precipitate was so finely divided that the period of time required for its Separation at 40° C. was determined with difficulty. Another series of experiments was carried out as described above, with the single exception that the temp. of mixing the rennin and the milk, and of the preliminary holding, was 15° C. In these experiments the transformation of the casein into paracasein pro- ceeded more rapidly than at 0° C, and consequently less time was required for the Separation of a visible precipitate. For instance^ after holding at 15° C. for periods of o, 10 and 30 min., the co- agula were formed at 40° C. after 6 min., 2^ min., and 55 sec, respectively ; while after 45 min. at 15° C. coagulation had already occurred. Müller concludes that rennin exerts its characteristic action, to a certain degree, at 0° C. 4. Summary. The power to survive prolonged exposure to jQis] Joseph Samuel Hepburn 149 low temp. is possessed by various enzymes, including those produc- ing hydrolysis of fats, carbohydrates, and proteins; those con- cerned in biochemical oxidations and reductions; the clotting en- zymes; and that of alcoholic fermentation. The enzymes retain their catalytic power after exposure, either in situ or in Solution in vitro, to temp. varying from a few degrees above 0° C. to the temp. of liquid air ( — 180 to — 191° C). The shortest periods of holding — invariably less than i day and usually less than i hr. — were at the temp. of liquid air. The longest period of holding was 89 mo. at a temp of —9.4° to — 12.2° C. The activity of certain of these enzymes, including rennin, zy- mase, and those hydrolyzing fats, carbohydrates and proteins, has been studied at low temp., varying from that of an ice box to one of — 9° to — 12° C. While the enzymes induce autolytic diges- tion, or act on artificial media, at these temp., the velocities of their reactions are always diminished to a considerable degree. BIBLIOGRAPHY 1. Kastle and Loevenhart: Amer. Chem. Journ., 1900, xxiv, p. 491. 2. Pennington and Hepburn : Jottrn. Amer. Chem. See., 1912, xxxiv, p. 210; U. S. Dep't of Agri., Bur. of Chem., Circular 75, 1911. 3. Pennington and Robertson: U. S. Dep't of Agri., Bureau of Chem., Circular 104, 1912. 4. KovcHOFF : Ber. d. deutsch, botan. Gesellschaft, 1907, xxv, p. 473. 5. PozERSKi : Campt, rend. de la soc. de hiol., 1900, lii, p. 714. 6. Chanoz and Doyon : Campt, rend: de la sac. de hiol., 1900, lii, P- 453. 7. Buchner: Die Zymasegährung. München und Berlin. Druck und Verlag von R. Oldenbourg, 1903, pp. 67, 226. 8. Ahrens: Zeitschr^ f. angewandte Chem., 1900, 483. 9. Macfadyen: Proc. Royal Soc. af London, 1900, Ixvi, p. 180; Lancet, 1900, Ixxviii (i), p. 849. IG. Pennington, Hepburn, St. John, Witmer, Stafford and Burrell: Journ. Biol. Chem., 1913, xvi, p. 331. II. Hepburn: U. S. Dep't of Agri., Bureau of Chem., Circular 103, 1912, p. 6. ISO Influence of Low Tempcratures upon Enzymes [March, 12. Van Driest: Third Int. Congr. of Refrig., jd See. (Nether- lands). Notes on the investigation of preserving fish by arti- ficial cold; preHm. report, 1913, p. 30. 13. RiCHARDSON : Premier Congrcs Internat, du Froid, 1908, ii, p. 261. 14. Pennington and Hepburn : U. S. Dep't of Agri., Bureau of ehem., Circular 103, 191 2, p. i. 15. Müller: Arch. f. Hygiene, 1903, xlvii, p. 127. 16. Buchner: Ber. d. deutsch, ehem. Gesellschaft, 1897, xxx, p, 117. 17. Murisier and Fick: Verhandlungen der physikal.-med. Gesell- schaft, Würzburg, 1873, iv, p. 120. 18. Hoppe-Seyler : Arch. f. d. gesammte PhysioL, 1877, xiv, p. 395, 19. Flaum : Zeitschr. f. BioL, 1891, xxviii, p. 433. 20. Oguro: Biochem. Zeitschr., 1909, xxii, p. 278. 21. Babcock: Second Intern. Congr. of Refrig., English Edition of the Rep. and Proc, 1910, p. 430, 22. Babcock, Russell, Vivian and Baer: Eighteenth Ann. Rep., Agr. Exp. Sta., Univ. of Wis., 1901, p. 136. 23. Ravenel, Hastings and Hammer: Journ. Inf. Diseases, 1910, vii, p. 38. 24. Selmi : Ber. d. deutsch, ehem. Gesellschaft, 1874, vii, p. 1463. 25. Camus and Gley: Arch. de physiol. norm, et path., 1897, (V) ix, p. 810. 26. Morgenroth : Arch. intern, de pharmacodynamie et de therapie, 1900, vii, p. 272. 27. Van Slyke: Journ. Biol. Chem., 1914, xix, p. 174. PLANT PIGMENTS The chemistry of plant pigments other than chlorophylP CLARENCE J. WEST Accompanying Chlorophyll in the chloroplasts of green plants and leaves there are two yellow pigments, Carotin and xanthophyll. Isomers of each of these have been found in lycopin, the color- ing matter of the tomato; and lutein, the pigment found in the yolk of eggs. Fucoxanthin, a xanthophyll-like substance, found in brown algae, has also been described. The principal result of the studies thus far made upon these pigments is that a satisfactory method for their Isolation and purifi- cation has been worked out. Very little if anything is known con- cerning their Constitution. Owing to the difficulty of obtaining them in large quantities, to their ease of oxidation during the process of purification, and to the fact that upon decomposition they yield only amorphous products, it may be a long time before their Constitution is established. Carotin. Carotin is widely distributed, being generally asso- ciated with Chlorophyll in the chloroplasts. It is also found in various parts of many plants. The color of yellow or orange petals is frequently due to it, e. g., the Corona of the common narcissus. It is largely responsible for the color of the carrot root, being pres- ent as innumerable small intracellulaf crystals. The tint of many fruits is due to amorphous granules of Carotin. The most recent chemical study of Carotin has been made by Willstätter,^ who isolated it from the leaves of stinging nettle and f rom carrots, and showed the complete identity of the two prepara- ^ A review of recent work on the chemistry of Chlorophyll will be found in the BiocHEMiCAL Bulletin, iii, pp. 229-258, 1914. These two reviews include all the work on plant pigments published by Willstätter and his pupils. A later review will discuss the work on flower pigments, the anthocyanins and related Compounds. 2 Willstätter and Mieg : Ann. d. Chan., 355, i, 1907. Willstätter and Escher : Ztschr. f. physiol. Chem., 64, 47, 1910; 76, 214, 1912. Escher: Ibid., 83, 198, 1913. 151 152 Plant Pigments [March, tions. The following methods were used : loo k. of dry leaves were extracted with 120 1. of petroleum ether (b. p. 40°-70°) in the cold for two days, the extract filtered off and the residue washed with 60 1. of petroleum ether. The extract was shaken with a Httle conc. alcohohc potash sol. to remove Chlorophyll, then with water, con- centrated to about 3 1. and allowed to crystallize. After shaking with I 1. of low-boiling petroleum ether, the product was purified by repeated precipitation from carbon disulfid sol. with absolute alcohol. Finally it was recrystallized from petroleum ether (b. p. 30°-50°). The yield was 3.1 gm.-0.03 gm. per k. of dry leaves. Data pertaining to some plant pigments Carotin, Lycopin, Xanthophyll, Lutein, Fucoxanthin, C40H56 C40H56 C40H58O2 C40H56O2 C40H54O6 Appearance Copper col- Carmine-red, Pleochroic, Brownish- Needles. ored, rhom- long pointed dark red- yellow bic leaflets. prisms or dish-brown plates or needles. plates or prisms. leaflets. Color by trans- mitted light Red. Brownish to carmine-red. Yellow to orange. Melting point . . . 167.5-168° j68-9° 172° 195-6" I59-5-I60.5» Solubility in petroleum ether . Appreciably Slightly solu- Insoluble. Insoluble in soluble (i g. ble (i g. in cold. in 1.5 1., 10-12 1., boiling). hot solvent). Alcohol Practically insoluble in Slightly soluble. Sparingly sol- uble in cold; I g. in I 1. of 100 gm. of boiling methyl alco- cold; very fairly read- methyl alco- hol dissolves sparingly in ily in hot. hol. 0.41 gm. at hot. I g. in 700 c.c. of hot methyl alco- hol. 0°; 1.66 gm. at boiling temperature. Acetone Very spar- ingly sol- uble. Readily sol- uble. Carbon disulfid . . Very readily I g. in so Sparingly I g. in 400. Fairly sol- soluble. c.c, at room temperature. soluble. c.c, warm uble. Ether I g. in 900 c.c, hot. I g. in 3 1.. hot. I g. in 300 c.c. Easily sol- uble. Slightly sol- uble. Modifications of the above method, in which mother liquors from the preparation of Chlorophyll were used, are given by Will- stätter and Stoll.^ The Carotin was found in the petroleum ether 3 Willstätter and StoU : Untersuchungen über Chlorophyll, p. 239. 1915] Clarence J. West i53 mother liquor, from which it was precipitated by alcohol. This gave a much larger yield, 0.15-0.20 gm. per k. of dry leaves. The preparation from cärrots was carried out by extracting the dry material with petroleum ether in a percolator and purifying as above; 5000 k. of fresh carrots (473 k. of dry material) gave 125 gm. of pure Carotin. Carotin forms quadratic or four-sided reddish-yellow plates, which exhibit the phenomenon of dichroism, being orange-red by transmitted Hght and greenish-blue in refracted Hght. Dilute Solu- tions are yellow, conc. Solutions orange-red but Solutions in carbon disulfid, or in other solvents upon the addition of carbon disulfid, are red. Analyses of carefully purified products indicate the formula (C5H7)x, which, from molecular weight determinations, becomes C40H56. Earlier workers gave C^Hg," Ci8H240,^ CzßHgs« and other formulae.'^ It should be mentioned that in the precipitation with absolute alcohol, a product is obtained which contains from Yz io Yz oi 2. molecule of alcohol ; this may be removed by recrys- tallization from petroleum ether. With conc. sulfuric acid it gives an indigo-blue color; upon diluting this Solution, green flakes pre- cipitate. Solutions of Carotin readily absorb oxygen. Willstätter found that Carotin took up 34.3 percent of its weight (11 atoms), forming a colorless Compound. Various other values, from 21 percent to 37.8 percent, have been given. Shaken with ^ of its weight of iodin in ether, a di-iodid is formed, C40H56I2; rosettes of dark violet prisms. However, if benzene, carbon disulfid or carbon disulfid-ether is used, and a larger amount of iodin, a tri-iodid^ re- sults; dark violet leaflets, melting at 136-7°. Carotin, shaken with bromin at 0°, and then allowed to stand at room temperature, forms a bromid, C4oH36Br22, decomposing about 171-174°. During the process, about 20 molecules of hydrobromic acid are evolved, so that probably 2 atoms of bromin are added and 20 atoms of hydrogen substituted by bromin. *Zeise: Ann. d. Chem., 62, 380, 1847. ßHusemann: Ihid., 117, 200, 1861. «Arnaud: Compt. rend. acad. sc, 100, 75, 1885; Bull. soc. chint., 48, 641, 1887. 7 Immendorf : Landwirtschaftliche Jahrb., 18, 507, 1889. »Arnaud: Compt. rend. acad. sc, 102, 11 19, 1886. Willstätter and Escher: Ztschr. f. physiol. Chem., 64, 59, 1910. 154 Plant Pigments [March, Carotin is of interest because of its probable physiological sig- nificance. The work of Tammes^ and Kohl^° shows that Carotin absorbs certain rays of radiant energy, which can be made use of in photosynthesis. It may also be of importance in respiration, acting in a manner comparable to the hemoglobin of the blood. Palladin^^ supposes that, by the action of an oxidase, Carotin is changed into xanthophyll (C40H56O2), which in turn is acted upon by a reduc- tase, yielding Carotin. In cases where large amounts of Carotin occur in organs of storage, such as the roots of the carrot, it may be of value as a reserve food material. Finally, where the colors of flowers are due to its presence, it is of importance in floral biology. Experiments by Iwanowski/ ^^ in which Chlorophyll Solutions containing various amounts of yellow pigments ( Carotin and xan- thophyll) were subjected to the action of sunlight, show that with the increase of the relative content of yellow pigments the stability of the Chlorophyll towards light also increased. While this protec- tive action is exercised by both Carotin and xanthophyll individually, a more favorable effect is obtained by a mixture of the two. This action is probably due to the absorption of the blue and, especially, the violet rays, whose chlorophyll-destroying power is very high. It is not yet established whether the oxygen absorption of these pig- ments plays a röle in this process. Mention may be made here of the recent studies of Palmer and Eckles^^ on Carotin and xanthophyll. They have shown that the fat of cow milk owes its natural yellow color to the presence of Caro- tin and xanthophyll (principally Carotin), which are taken up from the food and subsequently secreted in the milk fat. The same pig- ments are found in the body fat, blood serum, corpus luteum, and human milk. Carotin is assimilated from the food of the cow in pref- erence to xanthophyll, partly because of its greater stability toward the digestive Juices.^ ^ It probably forms by far the greater part 9 Tammes : Flora, 87, 205, 1900. 10 Kohl : Ber. d. deutsch, bot. Gesellsch., 24, 222, 1906. iiPalladin: Ibid., 26a, 125, 378, 389, 1908; 27, iio, 1909. "a Iwanowski : Ibid., 31, 600, 613, 1913-1914. 12 Palmer and Eckles : Jour. Biol. Chem., 17, 191. 211, 223, 237, 245, 1914; Research Bulletin, No. 10, Missouri Exper. Station. 13 Cf. Willstätter and Mieg (2), who State that xanthophyll is very sensitive to acids. 1915] Clarence J. West ^55 of the lipochrome of the cow body, chiefly on account of its ability to form a Compound with one of the proteins of the blood. Xan- thophyll apparently is not capable of forming such a complex. It is much more soluble in bile than carotin^^ which accounts for its appearance in the fat of the blood. While Palmer did not isolate Carotin, it has been separated by Escher/ ^ who obtained 0.45 gm. of pure pigment from 10,000 cow ovaries (corpus luteum). Carotin has also been isolated from brown algae.^^ Lycopin. Lycopin is the coloring matter of the tomato. Ear- lier investigators considered this pigment identical with carotin.^^ Schunck,^^ by a careful spectro-analysis of the two Compounds, showed that they were quite different and gave the tomato pigment the name lycopin. The next year Montanari^^ confirmed the ob- servations of Schunck; he recognized it as a hydrocarbon and ascribed to it the formula, C52H74. Willstätter and Escher^^ found that it was isomeric with Carotin, having the formula, C40H56. They used tomato conserve instead of the fresh fruit for the preparation of the pigment. The conserve was treated with 96 percent alcohol (to coagulate it), pressed, dried and extracted with carbon disulfid. The concentrated extract was precipitated with absolute alcohol and then recrystallized several times from petroleum ether and carbon disulfid. The yield was about 0.2 percent of the dry substance, i. e., 74 k. of conserve (5.6 k. of dry powder), yielded 11 gm. of pigment. Lycopin forms light, or dark carmine-red, long, microscopic prisms or hair-like needles, which cannot be mistaken for Carotin. Dilute sol. in carbon disulfid have a hluish-red color, while those of Carotin have a yellowish tinge. The two pigments show the same color reactions with sulfuric and nitric acids. Lycopin differs from Carotin in the following points: Lycopin absorbs oxygen more rapidly and to a greater extent than does Carotin. Und'er 14 Cf. Fischer and Rose : Ztschr. f. physiol. Chem., 88, 331, 1913. 15 Escher : lUd., 83, 198, 1913. 16 Willstätter and Page: Ann. d. Chem., 404, 237, 1914. 17 A. Arnaud: Cotnpt. rend. acad. sc, 102, 1119, 1886. Kohl: Carotin und seine physiologische Bedeutung in der Pflanze, p. 41. 18 Schunck : Proc. Royal Soc, 72, 165, 1903. löMontanari: Le stationi spcrm. agr. ital, 37, 909, 1904. 20 Willstätter and Escher : Ztschr. f. physiol. Chem., 64, 47, IQIO- 156 Plant Pigments [March, the same experimental conditions (in 10 days), lycopin absorbed 30 percent of the oxygen from the air; Carotin 0.25 percent. Ly- copin does not give a crystalHne iodin addition product, but a dark green amorphous product with indefinite iodin content. It reacts with bromin with the evolution of hydrobromic acid, but differs from Carotin in that it takes up far more bromin than corresponds to the hydrobromic acid evolved ; the Compound f ormed is probably C4oH44Br26. It is very evident from these differences that the two isomers must vary considerably in structure. Xanthophyll. The existence of a second class of yellow pig- ments in leaves was first mentioned by Stokes,^^ who supposed the existence of two xanthophylls. Sorby^^ beheved that there were three such Compounds. Borodin^^ divided the yellow pigments into two classes : the Carotins, soluble in benzine and slightly soluble in alcohol ; and the xanthophylls, slightly soluble in benzine but soluble in alcohol. His observations were confirmed by Monteviede,^* Tschirch,^^ Tswett,^^ and Schunck.^'^ Other writers thought that Carotin was the only yellow pigment accompanying Chlorophyll.^^ The question was partially settled by the Isolation and analysis of a crystalline representative of the second class of Borodin, by Will- stätter and Mieg.^^ The high yield of the two pigments, Carotin and xanthophyll, makes it very improbable that there are any other Caro- tinoids (this term includes both classes of pigments) accompany- ing Chlorophyll in the land plants. Tswett,^" on the basis of a Chro- matographie adsorption analysis (the pigments in organic solvents, filtered through a column of calcium carbonate, inulin or sugar, are adsorbed in different zones; each zone is considered a chemical 21 Stokes : Proc. Royal Soc, 13, 144, 1864. 22Sorby: Quart. Jour. Science, 8, 64, 1871 ; Proc. Royal Soc, 21, 442, 1873. 23 Borodin : Melanges biologiques tires Bull, de l'Acad. Imper. de St. Peters- burg, II, 512, 1883. 2* Monteviede : Acta Horti Petropolitani, 13, 148, 1893. 25Tschirch: Ber. d. deutsch, bot. Gesellsch., 14, 176, 1896; 22, 414, 1904. 26Tswett: Ibid., 24, 316, 384, 1906; 29, 630, 1911. 27Schunck: Proc. Royal Soc, 63, 389, 1898; 65, 177, 1899; 72, 165, 1904. 28 Immendorf , loc cit., p. 18. Molisch: Ber. d. deutsch, bot. Gesellsch., 14, 18, 1896. Tammes, Flora, 89, 205, 1900. 29 Willstätter and Mieg : Ann. d. Chem., 355, i, 1907. soTswett: Die Chromophylle in der Pflanzen- und Tierwelt, 1910, p. 233 (Warsaw). ipisl Clarence J. West i57 substance, the test being a different adsorption spectrum) distin- guishes four xanthophylls, et, a', a", and ^3. He believes the xan- thophyll of Willstätter and Mieg is an isomorphous mixture of two or three xanthophylls, with the a-form predominating. Unfortu- nately the method does not seem to permit of the isolation of the individual pigments in quantity large enough for chemical investi- gation. It is entirely possible that Tswett is right and that there is present in the chloroplast a mixture of very similar isomorphic and isomeric xanthophylls, for the Separation of which we have as yet no preparative method. This is all the more plausible when we consider the slight differences between Carotin and lycopin; and the similarity of xanthophyll and lutein (described below). On the other hand, these Compounds are rather easily oxidized and the slight differences in the absorption spectra may be due to changes in the xanthophyll by oxidation. Xanthophyll is found in the alcoholic extract of the leaves. Attempts to obtain it pure, in which the Chlorophyll was isolated as the magnesium-free derivative, pheophytin, by treatment of the extract with oxalic acid, always gave negative results. This is prob- ably because the xanthophyll is changed by the acid into a more easily soluble and non-crystalline substance. Better results were obtained when the mother liquor of potassium chlorophyllin was used. For example, the extract from loo k. of nettle leaves, after removal of the potassium salt by filtration and further precipitation with a large quantity of ether, was washed free from alcohol with water, the deep yellow ether sol. evaporated to about 6 1., washed repeatedly with alcoholic potash sol. and water, dried with sodium Sulfate and mixed with 2 vol. of petroleum ether. The xantho- phyll was purified by extraction with 1200 cc. of boiling acetone, and precipitated with 2 vol. of methyl alcohol. Recrystallized from methyl alcohol, about 12 gm. of xanthophyll were obtained. Willstätter and Stoll have also described methods for the prepa- ration of xanthophyll from the mother liquors of Chlorophyll and of crystalline Chlorophyll. These depend upon the removal of xanthophyll, from the petroleum ether sol., with dilute methyl alco- hol (80-90 percent), the Carotin and Chlorophyll remaining in the petroleum ether. This is also the basis of the quantitative estima- tion of the various plant pigments. The two yellow pigments make up from o.i to 0.2 percent of the dry weight of the leaf, of which 15^ Plant Pigments [March, xanthophyll is 0.07 to 0.12 percent and Carotin 0.03 to 0.08 percent, or about i molecule of Carotin to 1.5 to 2 molecules of xanthophyll. Xanthophyll has the formula, C40H56O2, and thus may be con- sidered an oxid of Carotin. Nothing is known of the function of the oxygen atoms ; they are considered ether-like, since xanthophyll does not give a reaction f or ^ COH, = CO or — COOH. It appears to give a very easily dissociable addition product when an ether Solution is treated with methyl alcoholic potash. It shows a tendency to crystallize with alcohol of crystallization and is best obtained solvent-free by precipitation from Chloroform with petrol- eum ether. The typical crystal forms are long tables and prisms. which are pleochroic and often show a steel blue luster. In trans- mitted light they are yellow, and are red only where several crystals Gross. This distinguishes them from Carotin, for the colors of the Solutions are very similar. It melts at 172°. Xanthophyll is rela- tively Stahle towards oxygen in dilute sol., but the pulverized sub- stance takes up 36.5 percent of its weight of oxygen, giving a Com- pound, which, precipitated from methyl alcohol by ether, has the formula, C4oH560i8- Like Carotin, it gives a di-iodid, tufts of thin, dark violet, prisms with metallic luster. It is easily decomposed. The bromid, C4oH4oBr22, is also similar to that of Carotin, It gives the same color reactions with conc. sulfuric acid and alcoholic hydro- chloric acid sol. Lutein.^^ As mentioned above, a Compound isomeric with xanthophyll has been found in lutein, the coloring matter of egg- yolk. This was first isolated in a pure State by Willstätter and Escher,^^ who obtained 4 gm. of very crude pigment from 6000 eggs (iio k.). The yolks were coagulated with alcohol (7 1. to 6 k. of eggs) and the coagulum extracted with acetone (5.4 k. were shaken with 3 1. of acetone and filtered; 2.8 k. of the residue were shaken with 2 1. of acetone for one hour and then washed on a filter with 2 1. of acetone). Phosphatids were removed by shaking the acetone with petroleum ether, washing with water, and mixing the petroleum- ether sirup with two vol. of acetone; the acetone was then removed by washing with water, the petroleum ether concentrated to about 2 1., filtered from cholesterol, diluted to about 6 1. and cooled. 31 Although lutein is an animal pigment, its dose relationship to xanthophyll Warrants its inclusion here. 32 Willstätter and Escher: Ztschr. f. physiol Chem., 76, 214, 1911-1912. I9I5] Clarence J. West i59 The lutein that separated was purified by repeated crystallization from methyl alcohol (i gni. required looo cc. for Solution) or from carbon disulfid. It formed dark, brownish-yellow, compact prisms with blue surface-luster, melting at 195-6°. It differed from xan- thophyll only in its higher melting point, and was called xanthophyll b by Willstätter. Fucoxanthin. Fucoxanthin is the Carotinoid characteristic of the Phaeophyceae, or brown algae. It differs from the other yellow pigment, in its high oxygen content, having the formula, C40H54O6. Many investigators^^ have had more or less pure Solutions of this pigment, but Willstätter and Page^* were the first to obtain a crystal- line product. Fucoxanthin was isolated from the mother liquor of Chlorophyll a (extracted with 85 percent acetone). Four liters of the extract were treated with i 1. of a mixture of petroleum ether (30-5<>°» 3 vol.) and ether (i vol.) and then with 1.5 1. of water. The ether mixture was then carefully washed free from acetone, concentrated to about y2 1. and shaken with i 1. of methyl alcohol (saturated with petroleum ether) four times, then twice with >4 1. of alcohol. The xanthophyll was removed by shaking with an equal vol. of a mixture of 5 vol. of petroleum ether and i vol. of ether. The fucoxanthin was then transferred to a large vol. of ether, and the ether concen- trated to a thick sirup. Fucoxanthin crystallized out upon the addi- tion of low-boiling petroleum ether. The yield from 20 k. of fresh algae was about 2 gm. of a product 85 percent pure. The use of all reagents containing mineral matter must be avoided, if ash-free preparations are desired. It is also essen- tial that all extracts and solutions be kept from the light and from moisture as much as possible. If the algae are dried previous to the extraction, the yield is very much smaller; and if this dry material is kept for some time before being used, little if any fucoxanthin can be isolated. The crude product may be recrystallized from methyl alcohol, forming bluish, glistening, brownish-red, long, monoclinic prisms, containing 3 molecules of alcohol. From methyl alcohol or acetone, in the absence of air, it forms dark red six-sided tables containing 33 Cf. Gaidukow: Ber. d. deutsch, bot. Gcsellsch., 21, 538, 1903. Tswett: Ibid., 24, 234, 1906. Kylin : Ztschr. f. physiol. Chcm., 82, 221, 1912. 3* Willstätter and Page: Ann. d. Chem., 404, 237, I9i4- i6o Plant Pigments [March, 2 molecules of water. These forms are interchangeable. It is ob- tained free from solvent by precipitation from absolute ether with low-boiling petroleum ether, forming compact needles, melting at 159.5-160.5°, depending upon the rate of heating. The ether Solu- tion is orange-yellow ; the alcoholic sol. more red, with a brownish- yellow tinge ; while the carbon di-sulfid sol. is quite red. The pure pigment is stable in an atmosphere of oxygen, but is oxidized in benzene or dilute alcoholic sol., giving a product of approximately the composition, C40H54O16. Fucoxanthin does not show acid properties; it is not extracted from ether by aqueous potassium hydroxid sol. and is not changed by 50 percent hydroxid sol., solid barium hydroxid or metallic sodium. It reacts with alcoholic potash, forming an addition product. This is decomposed by water but gives, instead of fucoxanthin, a product with increased basic properties and a different absorption spectrum.^'' The ether sol. gives a deep blue salt with dilute hydrochloric acid sol. This behavior may indicate the existence of a pyrone ring in the fucoxanthin. The reaction with alcoholic potash appears to consist in the decomposition of a part of the pyrone nucleus; the hydroxyl group thus f ormed would account for the increase in basic properties.^^ A characteristic of fucoxanthin is its marked basic properties. The other Carotinoids give a deep blue color only with conc. sulfuric acid. Fucoxanthin reacts as a weak base with dilute mineral acids. Thirty percent hydrochloric acid sol. decolorizes the ether sol., itself becoming violet blue in color. With a Solution of the acid in dry ether the hydrochlorid, C4oH5406-4HCl, is ob- tained as blue flakes with a copper luster. When shaken with ether and sodium bicarbonate, a Compound is formed with one atom of chlorin, which gives a greenish yellow Solution. The iodid, C40H54O6I4, forms violet-black, short pointed prisms with a copper luster. One k. of fresh algae {Fuchs) contains 0.169 gm. of fucoxan- thin, 0.089 gm. of Carotin, 0.087 g^i. of xanthophyll and 0.503 gm. of Chlorophyll a. Rockef eller Institute for Medical Research, New York City. 35 Xanthophyll is stable towards alcoholic potassium hydroxid. 36 Willstätter and Pummerer: Ber. d. deutsch, ehem. Gesellsch., 37, 3740, 1904; 38, 1461, 1905. PLANT PIGMENTS Their color and interrelationships B. HOROWITZ Introduction. In attempts to explain the action of ammonia on thymol,^ Prof. Gies and the author were led to review the work of Liebermann on the influence of ammonia upon orcinol.^ Lieber- mann's Suggestion that ammonia combines with oxygen of the air to form nitrous acid, and that the latter is the effective agent in the production of pigment, strengthened our view, as already held, that many plant pigments are synthesized in a similar way. Miss Wake- man's study of the pigments in the Monardas,^ whereby she came to the conclusion that these are probably oxidation products of thymol, and its isomer, carvacrol, and Wurster's suggestive paper on the role of hydrogen peroxid in color formation,^ afforded fur- ther evidence in support of this idea. During the past year the thymol problem has been studied side by side with an investigation into the chemistry of some plant pigments (the botanical side of which is engaging the attention of Dr. A. B. Stout, of the N. Y..' Botanical Garden). As an introduction to a description of these studies, we present herewith a brief review of the theories on color and chemical Constitution, as well as an outline of the possible chemical interrelationships of some of the more important plant pigments. Color and chemical Constitution. Perhaps one of the most fascinating chapters in the development of organic chemistry has been the attempt to correlate the chemical Constitution of substances with their physical properties. With the rise of synthetic chem- istry, and especially as a result of the pioneer work of Graebe, Liebermann and Baeyer in the production of synthetic dyes, color ^Gies: Biochem. Bull., 1912, ii, p. 171. Horowitz and Gies: Ibid., 1913, ii, p. 293. Horowitz : Dissertation, Columbia Univ., 1913, pp. 68. 2 Liebermann : Ber. d. d. ehem. Gesell., 1874, vii, p. 247. 3 Wakeman : Bulletin of the Univ. of Wisconsin, No. 448; Science series, 191 1, iv, p. 25. * Wurster : Ber. d. d. ehem. Gesell., 1887, xx, p. 2934. 161 102 Plant Pigments [March, and chemical Constitution began to attract attention. Witt's chro- mogen-chromophore theory, as well as the quinone theory of Arm- strong, held absolute sway f or many years ; and though their use- fulness is far from exhausted, the tendency at the present time seems to be to rely less on the influence of the radical in the altera- tion of color, and more on the relationship of color to the absorp- tion spectra produced. As is well known, colored substances exhibit the phenomenon of selective absorption; that is, whenever a body vibrates so as to emit waves of certain definite periods, any waves of these periods falling upon the body will be absorbed. This gives rise to the ab- sorption spectra that have so often been of use in the Identification of complex colored Compounds. Introduction of radicals into a Compound, transforming it from a colorless to a colored substance, with consequent exhibition of absorption in the visible part of the Spectrum, may be explained by assuming that the oscillation-fre- quency has been altered; for example, benzene, though colorless shows absorption bands in the ultra-violet portion. Introduction of the azo group, — N = N — , gives red azo-benzene, with absorp- tion bands in the visible part of the spectrum. What apparently occurs in this case is a change of the short wave-length with its high oscillation-frequency (as found in the ultra-violet region) into a longer wave-length and a consequent slower oscillation-frequency. Application of the inductive method in attempts to draw general conclusions has been but partially successful. Even early in the course of these studies it was recognized that unsaturation in a Com- pound is essential to the development of color. Attempts were also made to trace relationships between the molecular weights of Com- pounds and the probable colors produced. This culminated in Nietzski's rule : " The simplest colored substances are in the green- ish yellow and yellow, and with increasing molecular weight the color passes to orange, red, violet, blue and green." Like most of the theories in this field, this is at best highly imperfect. Undoubtedly the most fruit ful theory which has so far been advanced connecting color with chemical Constitution is that due to Witt.^ He considered color to be due to the presence of a " chro- mophore" group in the molecule. The resulting "chromogen" 5 Witt : Ber. d. d. ehem. Gesell., 1876, ix, p. 522 ; 1888, xxi, p. 325. 1915] B. Horowits 163 (as the substance containing the " chromophore " group is called) may itself be colored, or yield color by the addition of an " auxo chrome" group. For example, benzene itself is colorless; the ad- dition of a " chromophore " group such as — N ^ N — , gives the " chromogen," azo-benzene, which is red. On the other hand, the " chromophore," =C=0, is so weak that benzophenone, CßHg — CO — CgHg, is a colorless " chromogen." In neither case, however, is the true coloring matter, or dye, f ormed, tili the " auxochrome " is added. Thus, amino-benzophenone (yellow) and amino-azoben- zene, each with the " auxochrome " — NH2, are true dyestuffs. The more important " chromophore " groups are N^ , N = 0, C = 0, C = C, C = N, N = N The first four are decidedly weak in their action. An increase of nitro-groups, instead of increasing color — which is usually the case with increase of chromophoric groups — decreases it; thus, nitro-ben- zene is yellow, but dinitro- and trinitro-benzene are colorless. On the other hand, diphenylethylene, CgHg — CH = CH — CßHg, with a Single C = C group, is colorless but diphenyl-hexatriene, CßHj — CH = CH — CH = CH — CH = CH — CßHg, is yellow. The same is true of the carbonyl group. One C = 0 (as in the alde- hydes, for example) is of no effect; and here even the presence of more than one of these groups will not produce color, if they are separated in the molecule. Acetyl-acetone, CH3 — CO — CHg — CO — CH3, is colorless, but di-acetyl, CH3 — CO — CO — CH3, is yellow. The ring structure seems to have a marked influence on the de- velopment of color. The nitro-paraffins are colorless, whereas a large number of the aromatic nitro-compounds are colored. Tetra- phenyl ethylene, CeHsx _ /CeHs CeHs/^'^NCeHs is colorless, but bis-diphenylene ethylene. C6H4. /C6H4 C6H4 C6H4 is red. On the other hand, in the azo group, which is among the strongest of the chromophoric groups, ring structure seems to inter- 1^4 Plant Pigments [March, f ere with color f ormation. Thus, diazomethane, CH2\ II is yel- low, but tolazone, ^ CH3 CH3 <::>-<":> \ / N N is but slightly colored. 0£ special interest is the influence that the position of the double bonds may have. Benzene, as has been pointed out, though color- less, shows absorption bands in the ultra-violet part of the spec- trum; fulvene, CH = CH. I >C = CH2 CH = CH' an isomer of benzene, with the same nuniber of double bonds, is yellow. The most important " auxochromes " are the amino and hy- droxyl groups, of which the former is the stronger. The color is usually intensified by increasing the number of auxochromes, or, in the case of the amino group, by substituting aklyl and aryl rad- icles for the hydrogen atoms. On the other band, acetylating a hydroxyl group inhibits auxochromic action: O O Fluoran (chromo- Fluorescein, Diacetyl fluorescein, gen), colorless brown colorless That the position of the " auxochrome " group may be of impor- tance is shown by contrasting quinolphthalein, O HO\A/\/OH which is colorless, with its isomer, fluorescein.^ c Hewitt : Chromophores and chromogens, Thorpe's Dict, of Applied Chem., 1912, ii, p. 59- 191 5] B. Horowits 165 No relationship seems to have been worked out with reference to the influence of the position, of the " auxochrome " relative to that of the " chromophore " group. In some cases when the two are in the Ortho position with respect to one another, the intensity of color seems most marked ; in others, exactly the reverse is noticed. Even this brief outline of Witt's theory suffices to show its many shortcomings, though its suggestiveness cannot be questioned. In 1888 Armstrong put forward his quinone theory of color.''' In some respects this theory has shown a distinct advance over Witt's conception. Bearing in mind the fact that dyestuffs in general can be reduced to their colorless leuco bases by the addition of hydro- gen, Armstrong traced all these Compounds to colored quinone, and its reduction product, hydroquinone : O li — > I I II TT I I HC /CH O \/ Y O« II O and he came to regard ortho- and para-quinone as the parent substances : II - There have been many objections to various phases of this theory. Hartley,^ as a result of a careful study of absorption spectra, concludes that color and change of structure do not neces- sarily go hand in hand, nor is a quinoid nucleus essential in a colored substance. Baeyer's studies of tri-phenyl methyl have led him to similar conclusions, in contradistinction to Gomberg.® Silberrad^" 7 Armstrong : Chem. Soc. Proc, 1888, iv, p. 27 ; 1892, viii, pp. loi, 143, 189, 194. ^Hartley: Kayser's Handbuch der Spectroscopie, 1900, iii, p. 170; quoted by Cohen, Organic Chem., 1913, ii, p. 364. ^Gomberg: Jour. Amer. Chem. Soc, 1914, xxxvi, p. 1161. An excellent critical review of this intricate question is given here. 1° Silberrad : Jour. Chem. Soc, 1906, Ixxxix, p. 1787. i66 Plant Pigments [March, has prepared products from melletic and pyromelletic acids, which he cannot under any circumstances regard as quinonic in structure. It is worthy of note, in this connection, that such simple Com- pounds as quinoneimine, O = CqR^ — NH, and quinonediimine, HN = C6H4 = NH, are colorless. Lately Willstätter^^ has suc- ceeded in isolating colorless ortho-quinones. To differentiate these from the isomeric orange variety, the Superoxid f ormula, rv? '\/-o has been assigned to them. This also serves the purpose of em- phasizing the absence of the chromophoric group.^^ Chemical interrelationships.^^ General observations. Un- til recently the chemistry of plant pigments had not become a subject for systematic research. Thus far only a "few Standard types of such pigments have been fairly well identified, the great mass of 11 Willstätter : Ber. d. d. ehem. Gesell, 1904, xxxvii, p. 4744. 12 Many interesting problems of a more specific nature were taken up, among others, by Hantzsch (Ber. d. d. ehem. Gesell, 1906, xxxix, p. 1073) in his study of the colored nitro-phenol ethers, and his subsequent development of " chromo-isomerism " (isomerism exhibited in change of color) ; Willstätter {Ber. d. d. ehem. Gesell, 1908, xli, p. 1465) and Hewitt {Trans. Chem. Soc., 1907, xci, p. 1251; Zeit. Physik. Chem., 1900, xxxiv, p. l), who has attempted to har- monize his theory of fluorescence with that of color. " Symmetrical Compounds," he says, " capable of equal tautomeric displacements in either of two directions, should be those to exhibit the phenomena of fluorescence, for the molecule would Swing between the two extreme positions like a pendulum, the energy absorbed of one wave-length being degraded and given out with slower fre- quency." (Thorpe, Die, Applied Chem., 1912, ii, p. 59.) Thus, in fluorescein, we have : o o o 0= /\/\/\0H _ HO|/\/\/^OH __ HO/\/\f^ \A/\/ c I C6H4— COOK c o I I C6H4— CO =0 c C6H4— COOK This brings fluorescence in relationship to color, the quinonoid structures being the fluorescent substances. 13 For a detailed description of individual plant pigments see West : Biochem. Bull., 1915, iv, p. 151 (preceding paper). 1915] B. Horowitz 167 material still awaiting further study. It follows f rom this that no clearly-defined chemical relationships between many of the pignients can be traced. The many theories regarding the origin of plant pigments — Chlorophyll in particular — have lost much of their force as a result of a more detailed study of the chemistry of the pig- ments. Nothing analogous to Baeyer's beautiful conception of sugar synthesis f rom f ormaldehyde has been traced f or Chlorophyll. Perhaps ere long the master mind of Willstätter will have added this achievement to his many others. From the purely genetic Standpoint colors in flowers, especially those due to anthocyanin, may be traced to the following factors :^* C, a chromogen, colored or not colored — possibly a glucosidal flavone. E, an enzyme (oxidase) which acts on C and produces color, giving product X. e, another enzyme, which acts on X, giving product Y, which dif- fers in color from X. 'A, an anti-oxidase, which inhibits the action of E. R, reductase, which does the exact opposite to that of the enzyme E. " If a flower only possesses C or E, then the color will be white or pale yellow, according to the color of the chromogen, if present. If the flower with the factor C be crossed with a flower with the factor E, then the color of the flowers of the offspring will be red, or a deeper color, if e also be present. If either ^ or i? be present, then there will be no difference in the flowers of the offspring as compared with the parents." Palladin, in developing his conception of the röle coloring mat- ters play in respiratory activity of plants, has this to say:^^ "In plants are to be found pro-chromogens which may be regarded as glucosides, or decomposition products of proteins. Enzymes con- vert the pro-chromogens to chromogens. Oxidases act on the chro- mogen (in the presence of oxygen) yielding pigments which re- ductases are capable of reducing again. For example, an oxidase can convert Carotin, C40H56, into xanthophyll, C40H56O2, a reduc- tase being able to reconvert the latter into the former." ^* Haas and Hill : Chemistry of plant products, 1913, p. 243. 15 Haas and Hill: Ihid., 1913, p. 251. i68 Plant Pigments [March, Keeble, Armstrong and Jones^^ suggest that the higher mem- bers of a flower-color series owe their origin to the presence, with the lower members, of specific substances which, acting as receivers of oxygen, reduce the pigments characteristic of the lower members of the color series, except oxygen, and then become oxidized to other pigments. Wheldale^"'^ classifies pigments other than Chlorophyll as follows : A. Pigments in Solution in cell-sap. (i) Soluble red, purple, blue pigments ( " anthocyanin " ) . Several subclasses. (2) Soluble yellow pigments ( " xanthein " ) . Several sub- classes. B. Pigments associated with specialised protoplasmic bodies — chro- moplastids, the color in this case being usually yellow, orange-yellow, orange or orange-red. Insolubility in water appears to be a constant characteristic of this group. (i) Carotin. (2) Xanthin. A more detailed Classification is that given by Keeble, Arm- strong and Jones, as follows :^^ I. Plastid pigments. (a) Chlorophyll pigments, containing C, H, O, N. (b) Carotin, containing C, H. (c) Xanthophyll (oxidized Carotin), containing C, H, O. IL Sap pigments. (a) Yellow, hydroxy-flavone glucosides or their derivatives, containing C, H, O. (b) Red products of the action of oxidase on hydroxy-flavone (glucoside derivatives containing C, H, O). (c) Red and brown substances {e. g., the plum) produced by oxidation of phenols in the presence of amino acids, con- taining C, H, O, N. 16 Keeble, Armstrong and Jones: Proc. Roy. Soc. London (B), 1914, Ixxxvii, p. 113. 17 Wheldale : Ibid., 1909, Ixxxi, p. 44. 18 Keeble, Armstrong and Jones: Ibid., 1914, Ixxxvii, p. 113. 1915] B. Horowits 169 (d) So-called anthocyan pigments (red and magenta). These may arise in the oxidation of phenols by organic oxygen carriers : contaln C, H, O.^^ Chlorophyll. Willstätter^*^ has shown thät the amorphous and the crystalline varieties of Chlorophyll are esters of the tri-car- boxylic acid, chlorophyllin, C3iH29N4Mg(COOH)3, the amor- phous being the methyl-phytyl ester, COOH C3iH29N4Mg^COOCH3 ^COOC20H39 and the crystalline, the methyl-ethyl ester, COOH C3iH29N4MgfCOOCH3 COOC2H5 The fact that Carotin and xanthophyll are always associated with Chlorophyll has led to attempts to trace the origin of the latter to them. Thus far this has met with no success. However, Mon- teverde and Lyubimenko,^^ in studying the transformation of the green leaves of many plants that turn reddish-brown or red in the autumn, have isolated from the red leaves a red pigment (rhoda- xanthin) which they suppose is isomeric with xanthophyll. Most authors seem to be agreed that light is a most impor- tant factor in the formation of Chlorophyll. Palladin^^ ^nd D'Ar- bamont^^ consider sugar a necessary intermediary, the latter adding starch also. But, even if this were so, the difficulties for the chem- ist in tracing the synthesis of Chlorophyll, with carbohydrate as the starting point, would but begin, now that we know what Chlorophyll is chemically. Carotin, C40H56, and xanthophyll, C40H56O2. It has al- lö It is interesting to note here that Tswett {Ber. d. deut. bot. Gesell, 1906, xxiv, p. 326; 1907, XXV, p. 137), from studies in absorption spectrum analysis, concluded that there are at least seven diflferent coloring-matters in leaf -pigment. 20 See West's comprehensive review of Willstätter's book on Chlorophyll : BiocHEM. Bull., 1914, iii, p. 229. 2iMonteverde and Lyubimenko: Bull. Acad. Sei. St. Petersburg, 1913, P- 1007; Chem. Abstracts, 1914 viii, p. 728. 22Palladin: Ber. d.d. bot. Gesell., 1902, xx, p. 224. 23 D'Arbamont : Ann. Sei. Nat. Bot., 1909, ix, p. I97- I/o Plant Pigments [March, ready been stated that Carotin can be transformed into xanthophyll by oxidation. It is therefore highly probable that the origin of the latter may be attributed to this action. But how and under what conditions, does Carotin arise ? Here again no answer can as yet be given. Flavones. The mother substance of the yellow, water-soluble pigments, flavone, has the Constitution indicated by the formula, H H H I I I I II II \c=c/ H— C\, /Cv /C— H I M ^C^ \C/ H H I II H O Most of the flavones are readily synthesized from phenols and car- boxylic acids. On fusion with alkaH they usually yield phloro- glucinol and protocatechuic acid, and sometimes resorcinol and hydroxybenzoic acid. These Compounds may, therefore, be looked upon as giving rise to the flavone coloring matters. Anthocyanins. The red and blue coloring matters that can be extracted from leaves, flowers, and fruit by means of water are commonly spoken of as anthocyanins or anthocyans. No good Classification of these substances has as yet been suggested. Will- stätter, who has recently begun to investigate them, and whose work promises to shed much light on the subject, in a recent study of the anthocyan of corn flower (cyanin), has found that it hydrolyzes into two molecules of glucose and one molecule of cyanidin (QsHi.OeCl).^^ With regard to the mode of formation of these anthocyanins much of interest has been suggested. Miss Wheldale,^^ by cross- breeding yellow and white forms, obtained anthocyanin products. As the yellow forms were flavone in nature, and the white contained oxidases, Miss Wheldale drew the conclusion that anthocyanins are oxidation products of flavones. Since the flavones are known to 24 Willstätter : Sitsb. preuss. Akad. Wissenschaften, 1914, xii, p. 402. See also Willstätter and Everest, Ann., 1914, cccci, p. 189. 25 Wheldale: Proc. Cambridge Phil. Soc, 1909, xv, p. 137; Proc. Roy. Soc, 1909, B, Ixxxi, p. 44; Biochem. Jour., 1913, vii, p. 87. 1915] B. Horowitz 171 occur as glucosides in many plants, the following scheine of reaction was suggested :^® ( 1 ) Glucoside + water ^ flavone + sugar. (2) X (flavone) + oxygen—>anthocyanin. In addition to oxidation there might be condensation of the flavone molecules. Reaction (i) may be controlled by a glucose- spHtting enzyme, and (2) may be due to an oxidase. Many authors have shown that oxygen or oxidase plays an important part in an- thocyanin formation.^'^ Acid turns anthocyanins red; alkali, blue. This characteristic feature of these pigments naturally suggested that the red modifi- cation behaves Hke a weak acid, and the blue Hke a weak alkali. The fact that an excess of alkali gives a green instead of blue color has been explained by the assumption that anthocyanin is a bi-basic acid. Attempts to trace some relationship between anthocyanin and Chlorophyll have not been wanting. Thus, it had been stated that leaves containing anthocyanin have relatively less Chlorophyll than those which do not contain anthocyanin.^^ Macaire's hypothesis that Chlorophyll is transformed into anthocyanin held sway for many years, tili Mohl disproved it. Mulder was of the opinion that the decomposition of Chlorophyll gave rise both to blue and yellow pigments. ^^ None of these Statements has been substantiated. A close chemical relationship between anthocyans and flavones has been shown by Willstätter.^*' As has been stated Willstätter has f ound that the anthocyan of corn flower can be hydrolyzed into glucose and cyanidin. Now, if quercetin, a hydroxy-derivative of flavone, is dissolved in alcohol, made strongly acid with hydro- chloric acid, and reduced at 35° with Mg-Hg, a small quantity of cyanidin is formed. The Solution can be concentrated and the separated cyanidin and quercetin filtered off, dissolved in alcohol, and the cyanidin precipitated with ether : 2« Wheldale: Proc. Roy. Soc. (B), 1914, Ixxxvii, p. 301. 27 Malvezin : Compt. rend., 1908, cxlvii, p. 348. MoUiard : Ibid., 1909, cxlviii, P- 573- Combes: Ibid., 1910, cl, pp. 1186, 1532. Keeble and Armstrong: Jour. of Genet., 1912, ii, p. 277. See also Czapek, Biochemie der Pflanzen, 1913, i, p. 591. 28 Haas and Hill : Chem. of plant products, 1913, p. 244. 29 See Czapek : Biochemie der Pflanzen, 1913, i, p. 586. 30 Willstätter and Mallison : Sitz, preuss. Akad. Wiss., 1914, xii, p. 769. 1/2 Plant Pigments [March, Cl I OH O OH O OH > HOr^/^-0H + H2 HO/^/\c-<' >0H + HCl HO^,'^\c-<( >0H l^/\/COH ^/\/COH \A/COH HO CO HO C HO C /\ I H OH H This is in direct contradiction to Miss Wheldale's findings. Here the change f rom a flavone to an anthocyanin product involves reduction, whereas Miss Wheldale regarded the process as one of oxidation. The exact influence of Hght in the formation of anthocyanin pigments has yet to be settled.^^ Ewart^^ has shown that, in aquatic plants at least (such as Elodea canadensis) , the red color does not appear if the plant is grown in diffuse sunlight. Overton^^ names temperature as an additional factor : a low temperature, but one above freezing-point, favors the formation of the pigment. This explains the prevalence of red color in alpine plants. That anthocyanin formation is dependent upon the presence of sugar is suggested by Overton's interesting experiments.^* He found that the pigment formation could be artificially induced by immersing the cut leaves of many plants in a 2-3 percent sugar Solution. This finding was later confirmed and extended by Combes^^ and Rose.^^ The fact that anthocyanins are commonly found with tannin-like substances has led Wigand to regard the two as closely allied. Anthocyanins give the iron reaction and, like the tannins, are precipitated by caffein and antipyrin.^^ Biochemical Laboratory of Columbia University, College of Physicians and Surgeons, New York. 31 Fischer: Flora, 1908, xcviii, p. 380. Chartier and Colin: Rev. gen. Botan., 1911, xxiii, p. 264. Landel: Compt. rend., 1893, cxvii, p. 314. 32Ewart: Ann. Bot., 1897, xi, p. 461. 33 O verton : Nature, 1899, lix, p. 296. 3* Overton : Jahr. wiss. Botan., 1899, xxxiii, p. 171. 35 Combes : Compt. rend., 1909, clxviii, p. 790. 3« Rose : Ibid., 1913, clviii, p. 955. 37 See Czapek : Biochemie der Pflanzen, 1913, i, p, 587. For the chemistry of tannins, especially with reference to an attempted synthetic production, see Fischer: Jour. Amer. Chetn. Soc, 1914, xxxvi, p. 1187. FURTHER COMMENT ON MUSCULAR WORK AND RESPIRATORY QUOTIENT In my note on " Muscular Work and Respiratory Quotient," in the last number of the Biochemical Bulletin/ it was erroneously stated that Benedict and Cathcart, in their monograph on this sub- ject, did not mention the rate at which the air was circulated in the apparatus used by them for measuring the gaseous metaboHsm of the bicycle rider. I find now, however, an allusion to this matter in the text, according to which a tremendous Ventilation of 85 liters per minute was maintained during a work experiment, and I am glad to correct the error committed in my note. In discussing the shortcomings of Benedict and Cathcart's tech- nic, I assumed that probably 600 liters of air passed through the sulfuric acid in the course of a work experiment. Since these ex- periments usually lasted ten minutes, a Ventilation of 60 liters per minute, or one liter per second, was postulated in my argument. Considering the peculiar arrangement of the sulfuric acid absorp- tion System in their apparatus, it must have been impossible to free such a rapid current of saturated air of all its moisture. Now, according to Benedict and Cathcart's own Statement, 85 liters of air instead of 60, as I had assumed, were actually passed through the sulfuric acid wash bottle every minute, or practically one and a half liters per second. It is obvious that while I erred in "saying that no information is given in the monograph regarding the rate of Ventilation, my argument is strengthened by the fact alluded to above and which was overlooked in the preparation of my first note on this subject Sergius Morgulis College of Physicians and Surgeons, Columbia University, New York. 1 Morgulis : Biochemical Bulletin, 1914, iii, p. 435. 173 THE BIOCHEMICAL SOCIETY, ENGLAND Scientific programs R. H. A. Flimmer, Secretary October i6.^ Physiol. Lab., Univ. of London, South Kensington, S. W. (5.30 P. M.). /. C. Drummond and C. Funk: Chemical investigation of some rice-polishings fractions. S. S. Zilva (introduced by A. Harden) : The rate of destruction by heat of the peroxydase of milk. A. Harden and R. Rohison: A new phosphoric ester obtained by the aid of yeast juice. S. Walpole: Demonstration of cataphoresis apparatus — Her- mann's phenomenon. H. H. Dale and G. Barger: Liver-nitrogen in anaphylaxis. /. A. Gardner: Respiratory exchange of fish under low oxygen tension. December 8.2 Lister Inst., Chelsea Gardens, London, S. W. ( 5.30 P. M. ) . Demonstrations of " micro-methods " of analysis. G. Barger: Determination of molecular weights. H. Maclean: Estimation of glucose in blood. O. Rosenheim: Van Slyke's method of estimating amino nitrogen. C. Funk and J. C. Drummond: (a) Pregl's method of analysis of carbon, hydrogen and nitrogen. (b) Kjeldahl's method of esti- mating nitrogen (Bang). E. C. Grey: Analysis of aliphatic Compounds by moist com- bustion. March ii.^ (Annual general meeting). Medical Lecture iThe Society did not meet in August or September. See Biochemical Bulletin, 1914, iii, p. 452. 2 The Society did not meet in November. 3 The Society did not meet in January or February. The next meeting will be a Joint session, on May 5, with the Society of Public Analysts. It will be 174 igis] R. H. A. Flimmer 175 Theatre, St. Bartholomew's Hospital and Coli., London, E. C. (5.30 P. M.). S. Walpole: Counter diffusion in aqueous Solutions. B. Moore: Photosynthesis by inorganic catalysts. B. Moore: Forms of growth or deposit arising in metastable col- loidal Solutions. G. Graham and W. H. Hurtley: The effect of the vegetable-egg diet on severe diabetes. R. L. Mackensie Wallis: The estimation of the diastatic activity of the urine. G. Winfield: The fate of fatty acids in the survival processes of muscle. W. B. Bottomley: The formation of humus from organic sub- stances. At this meeting the following officers were elected : Hon. Treas- urer, /, A. Gardner; Hon. Secretary, R. H. A. Flimmer; Ordinary members of the Committee, W. A. Davis, T. A. Henry, and H. M. Vernon, to succeed F. G. Hopkins, E. J. Russell and /. S. Ford. University College, London. devoted to a discussion of " methods adopted in the estimation of the nitrogenous constituents of extracts derived from albuminous substances, such as meat ex- tracts and similar products, with special reference to the interpretation of the results." THE FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL BIOLOGY* Peace resolution The following resolution was unanimously adopted at the annual meeting held in Saint Louis, on the twenty-eighth of December, One thousand, nine hundred and f ourteen : Whereas, Various of the European nations with which many of our members are related by birth, descent, or intellectual friend- ship, are now at war ; Resolved, That we extend to the scientific men within these nations the hope of an early and enduring peace, which will leave the nations with no permanent cause of rancor towards each other, and which will insure to each the glories of scientific and humanita- rian achievement in accordance with its own conception of these ideals. The Physiological Society^ Walter B. Cannon, President; The Society of Biological Chemists, Graham Lusk, President; The Society für Pharmacolggy and Experimental Thera- PEUTICS, Torald Sollman, President; The Society for Experimental Pathology, Richard M. Pearce, President; Philip A'. S haffer, Secretary of the Federation, * The above is a copy of a formal Statement that was sent to every member of the Federation. — (Ed.) 176 PROCEEDINGS OF THE SECOND ANNUAL MEET- ING OF THE FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL BIOLOGY, IN ST. LOUIS, DEC. 28-30, 19 14 PAUL E. HOWE PrEPARED FROM REPORTS BY THE SECRETARIES OF THE CONSTITUENT SOCIETIES, A. J. CARLSON, P. A. SHAFFER, JOHN AUER and G. H. WHIPPLE Contents, (i) Federation of Amer. See. for Exp. Biol. : P. A. Shaffer, Sec'y of the Exec. Commit. of the Federation, 177; (H) Amer. Physiol. Soc. : A. J. Carlson, Sec'y, 180; (HI) Amer. Soc. of Biol. Chemists; P. A. Shaffer, Sec'y, 182; (IV) Amer. Soc. for Pharmacol. and Exp. Therap. : John Auer, Sec'y, 185; (V) Amer. Soc. for Exp. Pathol. : G. H. Whipple, Sec'y, 187; (Ad- dendum) Amer. Assoc. of Anatomists : Charles R. Stockard, Sec'y, 188. I. FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL BIOLOGY: SECOND ANNUAL MEETING P. A. Shaffer, Secretary of the Executive Committee for 1914 The second annual meeting of the Federation, comprising the Amer. Physiol. Soc, the Amer. Soc. of Biol. Chemists, the Amer. Soc. for Pharmacol. and Exp. Therapeutics, and the Amer. Soc. for Exp. Pathol., was held at St. Louis, Dec. 28-30, 1914, in the laboratories of the Washington Univ. Med. School. Dinners and smokers. This part of the program v^as in- augurated by a dinner given by the Local Commit., on Sunday evening, Dec. 27, to the officers and Councils of the constituent so- cieties of the Federation and of the Anatom. Soc. The customary and universally satisfactory informal subscrip- tion dinners and smokers were held on the evenings of Dec. 28-30 ; the first two at the Hotel Jefferson, the last one at the Hotel War- 177 178 Federation of 'American Biological Societies [March, wick. Perhaps the most enjoyable of these was the first, on Dec. 28. On this occasion a number of excellent speeches were deliv- ered, the Speakers being the guests of the evening, Mr. Brookings, and Drs. Graham Lusk, J. George Adami and G. Carl Huber. Scientific program. Three Joint sessions of the Federation were held, at which the following papers were presented. First Session. Monday, Dec. 28, 9.00 a. m. Presiding officer: President of the Biochem. Soc., and Chairman of the Exec. Commit. for 1914, Graham Lusk. Memorial addresses : S. Weir Mitchell, by E. T. Reichert (read by W. B. Cannon) ; Charles S. Minot, by Frederic S. Lee. W. B. Cannon, C. A. Binger and R. Litz: Experimental hyper- thyroidism. — David Marine: Further observations on the etiology of goitre in fish. — H. R. Basinger and A. L. Tatum: Studies on ex- perimental cretinism. — G. W. Crile, F. W. Hitchings and /. B. Austin: A research into the function of the thyroid. — 5". Simpson and R. L. Hill: The effect of repeated injections of pituitrin on milk secretion. — W. L. Gaines: The action of pituitrin on the mammary gland. — F. P. Knowlton and A. C. Silverman: On the mechanism of pituitrous diuresis. — George B. Roth: The several factors in- volved in the standardization of pituitary extracts. Second SESSION. TuESDAY, Dec. 29, 2 p. m. Presiding officer: President of the Pharmacol. Soc., Torald Sollman. J. R. Miirlin and B. Kramer: The influence of sodium carbonate on the glycosuria, hyperglycemia and the respiratory metabolism of depancreatized dogs. — /. S. Kleiner and S. J. Meltzer: The influ- ence of depancreatization upon the State of glycemia after intra- venous injections of dextrose in dogs. — /. /. R. Made od: The pos- sibility that some of the hepatic glycogen may become converted into other substances than dextrose. — R. T. Woodyatt: Narcotics in phlorhizin diabetes. — R. S. Hoskins: Adrenal deficiency. — H. Mc- Guigan: Hypoglycemia. — /. Auer and F. L. Gates: Some effects of adrenalin when injected into the respiratory tract. — G. W. Crile, F. W. Hitchings and /. B. Austin: The relation of the adrenals to the brain. — A. B. Macallum and /. B. Collip: Further observations of the origin of hydrochloric acid in the stomach. — C. C. Fowler, M. E. Rehfuss and P. B. Hawk: The effect of various fluids and igis] Paul E. Howe 17 9 cereals on gastric secretion. — R. W. Keeton and F. C. Koch: The distribution of gastrin in the body. — F. F. Rogers and L. L. Hardt: The relation of the digestion intractions to the hunger contractions of the stomach (dog, man). Third SESSION. Wednesday, Dec. 30, 9.00 a. m. Presiding officer: President of the Biochem. Soc., and Chairman of the Exec. Commit. for 19 14, Graham Lusk. F. D. Zeman, J. Kohn and P. E. Howe: Recuperation : Nitrogen metaboHsm of a man when ingesting successively a non-protein and a normal diet after a seven-day fast. — H. C. Bradley: Some studies in autolysis. — H. McGuigan and C. L. v. Hess: The diastase of the blood. — W. E. Bürge: The rate of oxidation of enzymes and their corresponding pro-enzymes. — C Voegtlin: The harmful effect of an exclusive vegetable diet. — C. L. Aisberg and C. S. Smith: The effect of long- continued feeding of saponin from the bark of Gnaiacum officinale. — E. L. Opie and L. B. Alford: Fat infiltration of the Hver and kid- ney induced by diet. — V. H. Mottram: On the nature of the hepatic fatty infiltration in late pregnancy and early lactation. — F. B. Kings- bnry and E. T. Bell: The synthesis of hippuric acid in experimental tartrate nephritis in the rabbit. Demonstrations. C. Brooks and A. B. Luckhardt: Blood- pressure method. — /. Erlanger and W. E. Garrey: Demonstration of a point-to-point method for analyzing induction shocks by means of the string galvanometer.— 5. M. Patten: A device for projecting a small spot of light suitable for exploring photosensitive areas. — S. Amberg and D. McClure: Demonstration of the effect of sodium iodoxy-benzoate on inflammation caused by mustard oil. — Worth Haie: An arrangement of the Porter clock to give three-time inter- vals at the same time.— F. L. Gates: A portable respiratory ma- chine furnishing continuous, intermittent and remittent streams ot air. — P. A. S ha ff er: The determination of blood sugar. Executive proceedings. The resolution printed on page 176 of this volume was unanimously adopted. Executive Committee for 1915. Chairman — Torald Soll- mann; Secretary — John Auer (Pharmacol. Soc); W. B. Cannon, C. W. Greene (Physiol. Soc.) ; Walter Jones, P. A. Shaffer (Bio- chem. Soc); Theobald Smith, Peyton Rons (Pathol. Soc). i8o Federation of American Biological Societies [March^ Next MEETING. The next meeting of the Federation will be held, 191 5, in Boston, at the Harvard Medical School. II. AMERICAN PHYSIOLOGICAL SOCIETY: TWENTY SEVENTH ANNUAL MEETING A. J. Carlson, Secretary The twenty-seventh annual meeting of the Physiol. Soc. was held in the Physiol. laboratories of the Washington Univ. Med. Seh., St. Louis, Mo., Dec. 28-31, 1914. Fifty-six of the Society's 208 members were present. Five scientific sessions were held, three of these being Joint meetings with the other societies of the Federation. At the two independent meetings the f ollowing papers were presented. Scientific program. First Session. Monday, Dec. 28, 2.00 p. m. F. S. Lee and D. J. Edwards: The action of certain atmos- pheric conditions on blood-pressure and heart-rate. — C. H. Dallwig, A. C. Rolls and A. S. Loevenhart: The relation between the eryth- rocytes and the hemoglobin to the oxygen of the respired air. — /. A. E. Eyster and W. J. Meek: The path of conduction for the cardiac impulse between the sino-auricular and the aurico-ventricular nodes. — C. Brooks and A. B. Luckhardt: An experimental and critical study of blood-pressure methods. — F. C. Becht and H. McGuiganr Mechanical factors in the flow of cerebro-spinal fluid. — J. F. Mc- Clendon: Oxidation in the erythrocytes of the goose (with note on a baro-thermostat). — Katherine R. Drinker and C. K. Drinker: The efifect of rapid and progressive hemorrhage upon the factors of co- agulation. — S. Simpson and A. T. Rasmussen: The effect of para- thyroidectomy on blood-coagulation time in the dog. — F. C. Mc- Lean: On the concentration of sodium chlorid in the serum and its relation to the rate of excretion in normal and diabetic men. — W. H. Spencer, M. E. Rehfuss and P. B. Hawk: Does regurgitation regu- late the acidity of gastric juice? Second SESSION. TuESDAY, Dec. 29, 9.00 a. m. T. S. Githens and S. J. Meltzer: Apnea vera without previous excess of respira- tion, and its dependence upon the vagus nerves. — M. L. Fleisher and Leo Loeb: The lytic action of tissues on blood coagulum. — Ida H. Hyde: The influence of light on the development of vorticella. 1915] Paul E. Howe 181 — 5". Tashiro: The metabolism o£ the resting nerve and its corre- lation to the direction and rate of the nerve impulse. — R. G. Pearce: Renal secretory nerve fibres. — A. L. Beifeld, H. Wheelon and C. R. Lovelette: The effect of pancreas extract on sympathetic irrita- biHty. — B. H. Schlomovitz, J. A. E. Eyster and W. J. Meek: Distri- bution of chromotropic vagus fibres within the sinoauricular node. — Ida H. Hyde: The relation of the nervous System to a tunicate larva. — /. F. McClendon: Some experiments on the oxidizing power of oxyhemoglobin. Papers read by title. E. G. Martin and P. G. Stiles: Some characteristics of vasomotor reflexes. — M. Dresbach: Experiments on transplantation of the pancreas. — W. J. Meek and /. A. E. Eyster: The action of adrenaHn in minimal doses. — E. G. Martin: The validity of inductorium calibrations. — A. J. Carlson: The al- leged action of the bitter tonics on the secretion of gastric juice in man and dog. — A. J. Carlson and H. Ginshurg: Blood transfusion in pancreatic diabetes. — A. J. Carlson: On the secretion of gastric juice in man. — /. F. McClendon: Increase of permeability of the frog tgg at the beginning of development, as determined with the nephelometer. In addition to eight papers that had been placed on the program to be read by title, sixteen Communications which were to have been orally reported were read by title only, because the authors were absent from the meeting. The failure of the authors of these sixteen papers to appear seriously marred the scientific program, The Sec'y hopes that this meeting will be the high-water mark of the bad habit of reporting papers to be read without going to the meeting to present them. In cases of unavoidable absence through sickness, the Sec'y should be notified, so that readjustment may be made even after the program is in print. As for those who asked to be placed on the program and then chose to stay away from the meeting, the Sec'y feels that the annual meetings of the society are too important to be made the subject of practical jokes of that type. Executive proceedings. Constitution. Some important changes in the Constitution were adopted. The importance of re- search as the qualification for election to membership in the society was more explicitly emphasized. Voting by mail or proxy was abolished. i82 Federation of American Biological Societies [March, Amer. Jour. of Physiol. The management of the 'American Journal of Physiology, owned and published by the Society, was entrusted to the Council. The Council was enlarged from five to seven members. In recognition of Dr. W. T. Porter's great service to physiology, in founding the Amer. Jour. of Physiol. and successfuUy Publishing it for many years, the Council was entrusted to arrange for the dedication, to Dr. Porter, of a volume of the Journal. (See p. 245.) New members: A. Arkin, Univ. West Va. ; A. T. Cameron, Univ. Manitoba; P. M. Dawson, Univ. Wis. ; C. M. Grnher, E. B. Krumhhaar, Univ. Pa. ; E. N. Harvey, Princeton Univ. ; H. L. Higgins, Nutrition Lab., Carnegie Inst. ; Jessie L. King, Goucher Coli. ; F. C. McLean, Rockefeiler Inst. ; 6". Morgiilis, E. L. Scott, Columbia Univ. ; G. B. Roth, Hygienic Lab., Wash. Officers-elect : President — W. B. Cannon; Secretary — C. W. Greene; Treasurer — /. Erlanger; Additional members of the Council— W/. H. Howell, J. J. R. Macleod, W. E. Garrey, W. J. Meek. Comment. Despite the unusual defaults in the matter of the scientific program, and the presence of only a few members from the Atlantic seaboard, the meeting was a success, due largely to the considerate efforts and the generous hospitality of the Local Com- mittee. The opportunity to inspect the new laboratories and hos- pitals of Washington Univ. Med. School itself justified the trip to St. Louis. It appears that this school has actually made an ad- vance beyond the " stone age " of American universities in general. In material equipment for medical research and teaching, Washing- ton Univ. Med. Seh. is second to none, if not superior to all other medical schools in this country. III. AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS : NINTH ANNUAL MEETING P. A. Shaffer, Secretary The ninth annual meeting of the Biochem. Soc. was held at St. Louis, on Dec. 28-30, 19 14, in the laboratories of the Washington Univ. Med. Seh. Three Joint sessions were held with the other 1915] Paul E. Howe 183 societies composing the Federation, in addition to two sessions con- ducted independently. The following Communications were pre- sented at the independent sessions. Scientific program. First Session. Monday, Dec. 28, 2.00 p. m. Graham Lusk: Presidential address, The influence of food on metaboHsm. — S. R. Benedict and E. Ost erber g: The retention of parenterally introduced creatin under various nutritive condi- tions in the dog. — Otto Polin and W. Denis: The occurrence of creatin in urine. — P. D. Zeman and P. E. Howe: The excretion of creatin during fasting. — /. L. Morris: The determination of creatin and Creatinin in urine; and the occurrence of creatin. — W. C. Rose: The influence of protein feeding on the ehmination of creatin in starvation. — P. A. Kober: The nephelometric estimation of purin bases, including uric acid, in blood and urine. — IV. H. Welker and Grover Tracy: The use of aluminium hydroxid in connection with nitrogen partition in urinary analysis. — H. R. Fishbach and P. B. Hawk: The fecal bacteria Output as influenced by dietary altera- tions. — N. Hendrickson, E. L. Connolly, B. M. Hendricks and M. E. Pennington: The dextrose content of the tgg of the common fowl. — H. J. Cor per: A method for determining and comparing the local toxicity of chemical Compounds. — E. A. Graham: The mech- anism of the toxicity of halogen narcotics. Second SESSION. TuESDAY, Dec. 29, 9.00 a. m. Olaf Berg- eim: Some influences affecting the action of phospho-nuclease. — ■ H. H. Bunsel: Biological oxidizabihty and chemical Constitution (II), — H. I. Mattill and H. A. Mattill: Digestive processes in Lim- ulus. — R. E. Swain: The action of alkaline hydrolytic agents on allantoin. — Arno Viehoever, C. 0. Johns and C. L. Aisberg: Cy- anogenesis in plants: (I) Studies on Sieglingia sesleroides. — R. T. Woodyatt: Experiments with c?-/-glyceric aldehyde. — /. /. R. Mac- leod and R. G. Pearce: The level of sugar in the blood flowing from the liver under laboratory conditions. — F. S. Lee and E. L. Scott: The action of certain atmospheric conditions on muscular work and blood-sugar.— P. A. Shaffcr and R. S. Hubbard: The level of blood- sugar in the dog. — C. C. Powler and P. B. Hawk: Sulfur partition as influenced by water drinking. — E. C. Kendall: A method for the decomposition of the proteins of the thyroid with a description of 184 Federation of "American Biological Societies [March, certain constituents. — F. D. Zeman, Jeronie Kohn and P. E. Howe: Variations in factors associated with acidity of human urine during a seven-day fast and during subsequent non-protein and normal feeding periods. Papers read by title. Jacob Rosenhloom: The effect of in- travenous injections of radium on the urinary nitrogen and sulfur partition. — Jacob Rosenhloom: The effect of external apphcation of radium on the metabohsm of a Cancer patient. — P. H. Mitchell: Carbohydrate metabohsm in the oyster. — Arnos W. Peters: Studies on the pathology of the feeble-minded : (I) The glycosuric reaction and its relation to their pathology. — G. W. Raisiss and H. Dubin: A method for the determination of benzoic acid in urine. — G. IV. Rai^iss and H. Dubin: The synthesis of hippuric acid in the animal body. Executive proceedings. New members : Olaf Bergeim, Jef- ferson Med. Coli.; Alex. T. Canieron, Univ. Manitoba; G. H. A. Clowes, Gratwick Lab., Buffalo, N. Y. ; B. M. Duggar, Missouri Botan. Garden; Cyrus H. Fiske, Harvard Med. Seh.; R. A. Hall, Univ. Minn. ; C. G. Imrie, Univ. Toronto; Benjamin Kramer, State Univ. Iowa; A. Bruce Macallum, Jr., Univ. Toronto; /. F. McClen- don, Univ. Minn. ; /. Luden Morris, Washington Univ. Med. Seh. ; Max Morse, Univ. Wis. ; V. H. Mottram, McGill Univ. ; C. F. Nel- son, Univ. Kansas ; E. L. Ross, Northwestern Univ. Med. Seh. ; E. C. Shorey, U. S. Dep't of Agric. Officers-elect : President — Walter Jones; Vice-president — Carl L. Aisberg; Secretary — P. A. S ha ff er; Treasurer — D. D. Van Slyke; Additional members of the Council — Otto Polin, Graham Lusk, L. B. Mendel; Nominating Commit. — /. /. Abel, S. R. Bene- dict, H. D. Dakin, P. B. Hawk, J. J. R. Macleod, E. V. McCollum V. C. Myers, T. B. Osborne, A. N. Richards. Attendance. J. G. Adami, S. Amberg, L. Baumann, H. C. Bradley, H. J. Corper, C. H. Fiske, W. E. Garrey, A. D. Hirsch- felder, R. A. Hall, P. E. Howe, E. C. Kendali, B. Kramer, A. S. Loevenhart, G. Lusk, J. J. R. Macleod, V. H. Mottram, H. Mc- Guigan, J. L. Morris, C. H. Neilson, E. W. Rockwood, P. A. Shaffer, T. Sollmann, C. Voegtlin, H. G. Wells, R. T. Woodyatt. 1915] Paul E. Howe 185 IV. AMERICAN SOCIETY FOR PHARMACOLOGY AND EXPERI- MENTAL THERAPEUTICS : SIXTH ANNUAL MEETING John Auer, Secretary. The sixth annual meeting of the Pharmacol. Soc. was held in St. Louis, at the Washington Univ. Med. Seh., on December 27-30, 1914. There were five scientific sessions, three of them being Joint meetings with the other members of the Federation. At the two in- dependent sessions the following papers were read. Scientific program. First Session. Monday, Dec. 28, 2.00 p. m. S. Amberg and H. F. Helmholts: The fatal dose of various substances on intravenous injection in the guinea-pig. — G. W. Crile: Experimental and cHnical research into alkalescence, acidity and anesthesia. — P. J. Hanzlik: Effects of cheHdonin on surviving Organs. — T. SoUmann^ W. L. Mendelhall and /. L, S tingle: The effect of temperature on the response of frogs to ouabain. — E. D. Brown: Artificial cerebral circulation after circulatory isolation of the mammalian brain. — Worth Haie: The uterine action of quinidin, cinchonin and cinchonidin. — C. D. Edmunds: Some vasomotor re- actions in the liver. — T. S. Githens and S. J. Meltser: Distribution of Solutions in cardiectomized frogs with destroyed or inactive lymph hearts. — F. L. Gates and S. J. Meltzer: The influence of intra-intes- tinal administration of magnesium sulfate upon fhe production of hyalin casts in dogs. Second SESSION. TuESDAY, Dec. 29, 9.00 a. m, W. deB. Mac- Nider: A study of the relative importance of the vascular mechanism of the kidney and of the epithelial element of the kidney in deter- mining the efficiency of various diuretics. — H. B. Myers: Cross- tolerance of drugs. — H. B. Myers and G. B. Wallace: Vascular re- actions in poisoning from diphtheria toxin. — A. D. Hirschfelder: The action of digitalis in experimental auricular fibrillation. — A. D. Hirschfelder: The effects of drugs upon the circulation in the Pia mater and the retinal vessels. — Clyde Brooks and /. D. Heard: The action of camphor on the circulation. — Don R. Joseph: The effect of carbon dioxid upon the convulsant action of acid fuchsin in frogs. — Carl Voegtlin: The mechanism of the toxic action of heavy i86 Federation of American Biological Societies [March, metals on the isolated heart. — C. W. Greene, L. R. Boutwell and /. O. Peeler: An analysis of the action of digitalin on the cardiac inhibitory centre and on the cardiac muscle. — W. H. Schultz: A com- parative study of the influence of the solvent upon the toxicity of thymol. — W. H. Schultz: The reaction of hookworm larvae to cer- tain chemicals. — A. E. Colin: A further Observation on the "T- wave " when digitalis is given. Executive proceedings. New members : F. C. Becht, Univ. Chicago; W. H. Brown, F. L. Gates, Rockefeller Inst. Officers-elect : President — Torald Sollmann; Secretary — John Auer; Treasurer — Wm. deB. MacNider; Additional members of the Council — Worth Haie, D. E. Jackson. Membership Commit. — 5". /, Meltser (term expires 1917). General comment. Among the topics discussed during the business meetings, was one especially which is of general interest. Several members expressed marked dissatisf action with the present arrangement of holding the annual meetings during the Christmas holidays. They suggested that practically any other time would be better. Their arguments, briefly, were as follows : The Christmas sessions always break the holidays as a family festival for members who live at some distance from the place of annual meeting; it has not been uncommon for members to spend Christmas day on the train. Secondly, if the meetings were held in June or July,^ more time would be available for the completion of work started at the beginning of the academic year, so that it could be reported to the Society. In the third place, a meeting in June or July would mean equable cHmatic conditions during the sessions, and the attending members would be less likely to experience in a few days, more or less unprepared, samples of all the seasons as at present during the Christmas holidays. There are, of course, objections to this suggested change. The gravest one, perhaps, is the fact that most of the Societies forming the Federation have clauses in their constitutions which fix the annual Session in the last week of December and the first week of January. Now, without losing time in deploring the tendency to regulate and direct every manifestation of life in a Society by constitutional pro- 1 The Easter holidays are not suitable as a meeting period because not all Colleges and universities give a vacation, nor do the vacations coincide in time. 1915] Paul E. Howe 187 visions, it may be remarked that even the necessity o£ a constitu- tional amendment should not permanently block an improvement. It must, however, be admitted that constitutions are not easily amended, that the Channels to this fortress are tortuous and often mined, so that the unwary navigator is frequently blown up with astonishing ease by the orthodox defenders of the citadel. This matter of altering the time of meeting has been mentioned here, not because of its novelty, for it has been discussed lightly on several occasions in the past, but in the hope that a majority of the Federation will take it under serious advisement Attendance. The attendance was excellent in general, but geographically it was ill-balanced, the eastern section of the country being represented by relatively few men. This absence, flatteringly enough for the Atlantic seaboard, caused a few subacid remarks. Entertainment. Trip around St. Louis. On Wednesday afternoon the Local Commit. arranged a series of enjoyable visits to the St. Louis Hospitals and laboratories, and also to the beautifully located and impressive buildings of Washington Univ. Vote of thanks. At the last executive session of the Phar- macol. Soc. a motion was passed unanimously to thank the author- ities of Washington Univ. for their hospitality and the Local Com- mit. for its broad and efficient efforts to render the stay of their guests in St. Louis as pleasant and profitable as possible. V. AMERICAN SOCIETY FOR EXPERIMENT AL PATHOLOGY: SECOND ANNUAL MEETING G. H. Whipple, Secretary At the second annual meeting of the Pathol. Soc, in addition to participation in the three sessions of the Federation, papers were presented at an independent session. Scientific program. Monday, Dec. 28, 2 p. m. E. C. Rose- now: Studies on Streptococci. — B. S. Kline and S. J. Meltzer: The efifect of previous intravenous injections of the pneumococcus upon experimental pneumonia by intra-bronchial insufflation of the same organism. — Ludvig Hektoen: Observations on the formation of anti-bodies. — Leo Loeh: Autoplastic and homoloplastic transplanta- tion of tissues. — H. T. Karsner: Further studies in nitrogen reten- tion and renal function. — H. G. Wells: Metastatic calcification.^ i88 Federation of American Biological Societies [March, G. H. Whipple and C. W. Hooper: Studies in bile pigment excre- tion. — C. W. Duval: Further studies upon the experimental produc- tion of leprosy in the lower animal. — E. L. Opie and L. B. Alford: The influenae of diet upon the progress of a bacterial infection. — G. W. Crile, F. W. Hitchings and /. B. Austin: Pathological lesions wrought by certain amino-acids, by skatol and indol, by iodin, foreign proteins, and certain organic acids, — and the control of the action of these agents by morphia. Executive proceedings. Officers-elect : President — Theo- bald Smith; Vice-President — G. H. Whipple; Secretary-treasurer — Peyton Rons; Councillor — R. M. Pearce vice Harvey Cushing, term expired. New Members: James B. Murphy, Rockefeiler Inst.; L. G. Rowntree, Johns Hopkins Hosp. ; Richard Strong, Harvard Med. Seh. ; M. C. Winternitz, Johns Hopkins Med. Seh. ADDENDUM The following papers of biochemical interest were read at the thirty-first meeting of the Amer. Assoc. of Anatomists, which was held at the Washington Univ. Med. Seh., in St. Louis, Mo., Dec. 28-30, 1914, in conjunction with the Federation of Amer. Soc. for Exp. Biology: C. M. Jackson: Effects of acute and chronic inanition upon the relative weights of the various organs and System of adult albino rats. — C. M. Jackson: Changes in young albino rats held at constant body-weight by under feeding for various periods. — R. M. Strong: Further observations on the origin of melanin pigments. — G. W. Bartelmes: Some efifects of mammalian thyroid and thymus glands upon the development of amphibian larvae. — Brest on Kyes: Morpho- logical evidence of intracellular destruction of red blood corpuscles. — Montrose T. Burrows: An attempted analysis of growth. — R. M. Strong: Microscopic slides showing feather germs with dermal pig- ment.— Eduard Uhlenhuth: Is function and functional Stimulus a factor in producing and preserving morphological structures? — E. I. Werber: Is defective and monstrous development due to paren- tal metabolic toxemia? — /. F. Gudernatsch: Feeding experiments on rats. Laboratory of Biological Chemistry of Columbia University. College of Physicians and Surgeons, New York. PAPERS OF BIOCHEMICAL INTEREST, PRESENTED BEFORE THE AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, AND AFFILIATED SOCIETIES, PHILA., DEC. 28, 1914-JAN. I, 1915 Selected by JOSEPH S. HEPBURN American Association for the Advancement of Science. General session. — E. B. Wilson: Some aspects of progress in mod- ern zoology (annual address of the retiring president). Section C (Chemistry). — P. A. Maignen: Chemical preserva- tion of manure. — C. P. Fox: Character of the glutinous contents of the fruit of the American mistletoe. Joint session of Sections C and K, and the Society of American Bacteriologists. — C. L. Aisher g: Theories of fermen- lation (vice-presidential address, Section C). — C. S. Hudson: En- zyme action. — A. I. Kendall: Role of microorganisms in the intes- tinal canal. — F. P. Gorham: Use of bacteria in the treatment of textile fibres. — C. E. Marshall: Micro-organisms in their application to agriculture. Section F (Zoology) and American Society of Zoologists. — E. P. Churchill: The absorption of fat by fresh-water mussels. — G. A. Baitsell: On a certain fibrin reaction which occurs in living cultures of frog tissues. — E. N. Harvey: Studies on the phospho- rescent substance of the fire-fly (p. 212). Section G (Botany) and the Botanical Society of Amer- ica.— /, C. Böse: Plant autographs. Section I (Social and Economic Science). — L. F. Bishop: The bearing of diet on efficiency in brain workers after forty. Section K (Physiology and Experimental Medicine). — l^heo. Hough: The Classification of nervous reactions (vice-presiden- tial address). Section K and Society of American Bacteriologists; Joint Session. Symposium on Ventilation. — A. C. Abbott: Air-borne 189 190 Papers of Biochemical Interest [March, diseases. — E. B. Phelps: Fundamental physical problems of Ventila- tion.— C.-E. A. Winslow: Standards of Ventilation — hygienic and esthetic. — D. D. Kimhall: Modern developments in air conditions. Section L (Education). — Louise S. Bryant: Blood pressure among feeble-minded people. American Society of Naturalists. G. G. Scott: Some indi- cations of the evolution of the osmotic pressure of the blood and other body fluids. Botanical Society of America. W. J. V. Osterhout: The chemical dynamics of living protoplasm; The nature of mechanical Stimulation; The nature of antagonism. — G. B. Reed: Studies on plant oxidases. — A. R. Davis: Enzymes of the marine algae. — C. O. Appleman: Concerning the measurement of diastase activity in plant extracts. — M. C. Merrill: Electrolytic determination of exos- mosis from the roots of anesthetized plants; Some relations of plants to distilled water and certain dilute toxic Solutions. — Mr. Kno: In- fluence of certain salts on nodule production in the vetch. — /. K. Wilson: Physiological studies of Bacillus radiciola of soy bean. — Lewis Knudson: Direct absorption and assimilation of carbohy- drates by green plants. — R. H. True and H. H. Bartlett: The ab- sorption and excretion of electrolytes by Lupinus albus in dilute simple Solutions of nutrient salts; The absorption and excretion of electrolytes by Lupinus albus in dilute Solutions containing mix- tures of nutrient salts. — H. S. Reed and H. S. Stahl: A preliminary study of the chlorophyl Compounds of the peach leaf. — L. A. Haw- kins: Some effects of the brown-rot fungus upon the composition of the peach. American Phytopathological Society. Caroline Rumbold: Some effects on chestnut trees of the injection of chemicals. Society for Horticultural Science. IV. H. C handler: Some Problems connected with killing by low temperatures. Society of American Bacteriologists. Jean Broadhurst: Some induced changes in Streptococci. — L J. Kligler: A study of the correlation of the agglutination and fermentation reactions among the Streptococci. — N. S. Ferry: The filterability oi B. bron- chisepticus, with an argument for a uniform method of iiltration. — ■ Zae Northrup: The influence of the concentration of the gelatin in gelatin media upon liquefying and non-liquefying bacteria. — M. R. iQisl Joseph S. Hephurn 191 Smirnow: Induced variations in chromogenesis ; induced variations in the cultural characters oi B. coli.—K. F. Keller man and N. R. Smith: Halophytic and lime-precipitating bacteria. — K. F. Keller- man and R. C. Wright: Relation of crop to bacterial transforma- tion of nitrogen in the soil. — F. W. Turner and L. V. Burton: A note on the occurrence and Classification of the gas formers in nature. —Charles Thom: The bacteriological work of the Bureau of Chem- istry and its possibilities. — R. S. Breed: The Standard method of determining nitrate reduction.— £. B. V edder: A culture medium for growing gonococci and tubercle bacilli. — S. A. Petra ff: A new and rapid method for the Isolation and cultivation of tubercle bacilli directly from the Sputum and feces, with the aid of sodium hydrate and gentian violet-egg-meat juice media. — R. G. Colwell: Com- parative tests of various peptones. — F. M. Scales: The preparation of cellulose for cellulose agar.— P. E. Brown: The Solution versus the soil method for the bacteriological examination of soils. — S. H. Ayers and Philip Rupp: The alkali forming bacteria found in milk. — C. W. Brown: Degradation of casein in the presence of salt by butter flora.— i?. E. Buchanan and B. W. Hammer: Bacteriology of slimy milk. — K. Peiser: Factors influencing the resistance of lac- tic acid bacteria to pasteurization. — Maud M. Obst: Bacteria in pre- served eggs. — C. G. Supplee: Efficiency of Endo's medium in detect- ing members of the colon group. — /. V and erleck: Bacteria which produce black colonies on aesculin-bile-salt agar plates and do not belong to the colon group. — C. Greathouse: Numbers and efficiency of Bacillus btdgaricus in commercial preparations from January to June, 19 14. — C. N. Hilliard: The death rate of bacteria upon drying. — L. F. Rettger and T. G. Hüll: The influence of milk and carbohydrate feeding on the bacteriology of the intestine. — John Weinzirl: A bacteriological method for determining manural pollu- tion of milk. — Thomas W. Melia: Some observations with the use of bile media. — A. J. Smith and M. T. Barrett: Oral endamebiasis. — Chas. Krumwiede, Jr. and Josephine Pratt: Methods of isolation and differentiation of the typhoid-paratyphoid-enteriditis group. — G. H. Smith: The production and detection of specific ferments for the typhoid-coli group. — /. F. Siler, P. E. Garrison and W. J. Mac- Neal: Recent studies of pellagra. — /. A. Kolmer and Emily Mosh- age: The Schick test for diphtheria.— /. B. Bronfenbrenner: The 192 Papers of Biochemical Interest [March, mechanism of the Abderhalden reaction (p. 87). — D. H. Bergey: Do bacteria produce pyrogenic poisons ? — E. C. L. Miller: How bacterial vaccines act. — P. B. Hadley: Reciprocal relations of virulent and avirulent cultures in active immunization. Philadelphia, Pa. SCIENTIFIC MEETINGS OF THE COLUMBIA UNI- VERSITY BIOCHEMICAL ASSOCIATION, AT THE COLLEGE OF PHYSICIANS AND SURGEONS, NEW YORK^ -PROCEEDINGS REPORTED BY THE SECRETARY, EDGAR G. MILLER, Jr. L EIGHTEENTH (FIFTH ANNUAL) MEETING The eighteenth scientific meeting of the Columbia Univ. Bio- chem. Assoc. was held in the Library of the Columbia Med. Seh., at 8:15 p. M., on June i, 1914. Abstracts of the papers are presented here (pages 194, 203) in two groups: {A) Abstracts of papers on research by non-resident members^ and (B) abstracts of papers fr am the Columbia Biochem. Dep't and affiliated laboratories. The ap- pended summary facilitates reference to the abstracts (i 33-1 51).' A SUMMARY OF THE NAMES OF THE AUTHORS AND OF THE TITLES OF THE SUCCEEDING ABSTRACTS (133-IS1). A J. Bronfenbrenner, W. T. Mitchell, J. Bronfenbrenner and J. Rockman. JR-. »"d M. J. Schlesinger. Studies A note on the use of purified anti- on so-called protective ferments. l. gen of Besredka in the serum diag- The sensitization of substratum for nosis of tuberculosis. (133) *^^ Abderhalden test. (136) J. Bronfenbrenner and J. Rockman. I- J- Kligler. Observations on the The diagnostic value of the Landau metabolism of Bacillus vulgaris. test for Syphilis. (134) (i37) J. Bronfenbrenner and J. Rockman. I. J. Kligler and V. E. Levine. The Further studies on Besredka tuber- Scheurlen-Klett selenium reaction in culin. (135) the diphtheria group. (138) 1 Scientific meetings are held regularly on the first Fridays of December, February, and April, and on the first Monday in June. Proceedings of the six- teenth and seventeenth meetings were published in the last number of the Biochemical Bulletin, 1914, iii, pp. 454 and 465. 2 Members of the Association who were not officially connected with the Columbia Biochemical Department when the researches were conducted. 3 Previous abstracts were published in the Biochemical Bulletin : 1-44, 1912, ii, p. 156; 45-62, 1913, ii, p. 285; 63-72, 1913, ii, p. 452; 73-85, 1913, ü, P- 462; 86-107, 1913, ii, P- 541; 108-119, 1914, iii, p. 302; 120-126, 1914, iii, p. 454; 127-132, 1914, iii, p. 465. See also pages 210, 224 and 228. 193 194 Proceedincjs Columbia Biochemical Association [March, Max Kahn and J. Subkis. On the presence of oleic and other unsatu- rated acids in the gastric contents. (139) Alwyn Knauer and Benjamin Horo- wiTZ. A Volumetrie determination of Sulfates in urine. (140) Sergius Morgulis. The respiratory exchange of fish. (141) Anton R. Rose and Katherine R. Coleman. A Standard for the de- termination of ammonia by means of Nessler Solution. (142) Anton R. Rose and Katherine R. Coleman. A micro-urease method for the determination of Urea. (143) Anton R. Rose and Arthur Knud- son. The influenae upon metabolism of feeding B. coli. (144) Matthew Steel. A further study of the influenae of electricity on metab- oHsm. (145) B O. C. Bowes. Studies in goat metab- olism. (146) Ruth S. Finch. On the precipitation of proteins with Solutions of Chro- mates. (147) Mark J. Gottlieb and Seymour Op- penheimer. Active immunization to hay fever. (148) Herman O. Mosenthal. Nitrogen metabolism in experimental uranium nephritis. (149) W. A. Perlzweig and William J. GiES. An alleged improvement of the ferric chlorid method for the determination of sulf ocyanate. ( 150) O. M. Schloss. Absorption of un- altered protein through the gastro- enteric tract in infants. (151) A. ABSTRACTS OF PAPERS BY NON-RESIDENT MEMBERS* 133. A note on the use of purified antigen of Besredka in the serum diagnosis of tuberculosis. J. Bronfenbrenner and J. RocKMAN. (Pathol. and Research Lab., West. Penn. Hosp., Pittsburgh, Pa.) Published in the preceding issue: Biochem. Bull., 1914, iii, p. 375. 134. The diagnostic value of the Landau test for syphiUs. J. Bronfenbrenner and J. Rockman. {Pathol. and Research Lab., West. Penn. Hosp., Pittsburgh, Pa.) Published in the pre- ceding issue: Biochem. Bull., 1914, iii, p. 377. 135. Further studies on Besredka tuberculin. J. Bronfen- brenner and J. Rockman. (Pathol. and Research Lab., West. Penn. Hosp., Pittsburgh, Pa.) Published in the preceding issue: Biochem. Bull., 1914, iii, p. 381. 136. Studies on so-called protective ferments. I. The sen- sitization of substratum for the Abderhalden test. J. Bronfen- brenner, W. T. Mitchell, Jr., and M. J. Schlesinger. {Pathol. and Research Lab., West. Penn. Hosp., Pittsburgh, Pa.) Published in the preceding issue: Biochem. Bull., 1914, iii, p. 386. * Members of the Association who were not officially connected with the Columbia Biochemical Department when the researches were conducted. 1915] Edgar G. Miller, Jr. 195 137. Observations on the metabolism of Bacillus vulgaris. I. J. Kligler. {Dep't of Public Health, Amer. Museum of Natural Hist., N. Y. City. ) B. vulgaris, ever since its discovery by Hauser, has been associated with putrefaction. Hauser, Tisier and Metchni- koff attribute to it putrefactive properties. Metchnikoff believes it is the etiologic factor in Infant diarrhea. Bienentock, and more re- cently Rettger, deny, however, that this organism has the power of initiating real putrefactive processes. Aside from these conflicting results, comparatively little is known of the metabolism of B. vul- garis and the various conditions influencing it. Glenn observed that glucose exerts an inhibiting influence on liquefaction, and attributed the Inhibition to the acid produced. De- tailed experiments, though as yet incomplete, indicate that, while the acid does inhibit the action of the liquefying enzyme, in the carbo- hydrate medium it is really the sugar that is directly responsible for the absence of liquefaction. The sugar has a decided sparing effect on the nitrogenous metabolism. The liquefying enzyme is either not secreted during the early stages of the carbohydrate metabolism or eise must be inactivated in some way by the acid. If the acid produced by the organism in a i percent sugar-gelatin sol. is neutral- ized, the medium shaken with toluene to kill the bacteria, and the tubes incubated, no liquefaction is obtained. This test was made with a number of strains after growing them for about three weeks on the sugar medium, which was thus rendered highly acid (5-6 percent N acid). Further evidence of the sparing action of the sugar is obtained from the end-products in a parallel series of gelatin with, and without, sugar. The former gave high acidity, no odor, and slight amounts of indol and ammonia; while the latter gave slight acid reaction, marked f ecal odor, with large amounts of ammonia and indol. The enzyme itself is active both in weak alkaline and acid Solutions ( — I percent N NaOH; -|- 2 percent A^ HCl), but is in- hibited by higher concentrations. The oxygen relation of the organism is very interesting. It is generally supposed to be a facultative anerobe. Preliminary experi- ments indicate, however, that only in the presence of a utilizable sugar can it grow in the absence of oxygen. The sugar molecule apparently supplies the oxygen. In the absence of sugar, when the protein has to be utilized for nutrition, oxygen in an available 196 Proceedings Columbia Biochemical Association [March, form is essential. It is noteworthy in this connection that lactose is not fermented under aerobic conditions but is broken down under anerobic conditions. My experiments on the putre facti ve properties of this species thus far bear out Rettger's conclusions. B. vulgaris does not de- compose coagulated meat and egg-albumen, either in the presence er absence of oxygen. B. vulgaris plus B. putrificus produce active putre faction under aerobic conditions, the vulgaris apparently using up the dissolved oxygen, thus enabHng the putrificus to act. That this is the case is indicated by the inhibition of decomposition for several days, in tubes containing added glucose (0.5 percent) . With sugar present, the nitrogenous metabohsm is very sHght and oxygen is not removed from the Solution. The results thus far show a very delicate physiologic adjustment to the food environment by the organism studied. Seven strains of B. vulgaris were used and, barring certain individual variations, they behaved uniformly in the essential points. 138. The Scheurlen-Klett selenium reaction in the diphtheria group. I. J. Kligler and V. E. Levine. (Dep't of Public Health, Amer. Museum of Natural Hist., N. Y. City.) Reduction is a property of living cells, including bacteria. Some forms of bacteria do not reduce as readily as others; some do not reduce at all. The phenomenon of reduction is utilized in differentiating these types, nitrates being generally used for the purpose. Recently selenium and tellurium have been employed in treatment of various pathological conditions ; and a number of workers, notably Scheurlen,. Klett, Gosio, Glöger and Maassen, having tested the effect of sele- nium or tellurium Compounds on bacteria, report reduction by most common forms, Gloger found, however, that diphtheria and pseu- do-diphtheria organisms among others do not reduce. We have tested the effect of this group of bacteria on selenium dioxid, and sodium selenite. Four strains of B. diphtheria, seven strains of B. pseudo-diph- theria, and three strains of diphtheroid organisms from Hodgkin's disease were used. These were grown on agar slants containing i part of selenium dioxid, or i part of sodium selenite, in 200,000, 100,000, 50,000, 25,000 and 10,000 parts of agar, respectively, Re- duction was induced by all organisms, but no action was observed 1915] Edgar G. Miller, Jr. 197 in dilutions above i : 100,000. Dilutions of i : 25,000 and i : 10,000 gave the best results. Reduction occurred only on the surface and the growth was colored brick red, due to the deposition of selenium par- ticles, which, under the microscope, appeared to be in the cell, indicat- ing that the reduction was intracellular. After a few days the color began to fade and a characteristic oder of volatile selenium was produced. None of the organisms was inhibited in growth by any of the dilutions used. There were no sharp distinctions in types of growth of the various strains, though the diphtheria bacilli gave characteristic discreet colonies, which differed markedly from those of the pseudo-diphtheria. This was especially evident in the higher concentrations after 12 to 18 hr. growth. It is uncertain whether the difference is sufficiently constant to be of diagnostic value. The experiments are in progress. 139. On the presence of oleic and other unsaturated acids in the gastric Contents. Max Kahn and J. Subkis. (Chem. Lab., Beth Israel Hosp., N. Y. City.) After administering to the patient a piece of bread and a glass of water, and withdrawing the gastric contents, the authors determined, by Hübl's method, the amount of iodin absorbed by the filtrate. Gastric contents of low acidity had a small iodin number; of high acidity, a relatively large iodin number. These results contradict Graf, who stated that the gastric juice of patients suffering from Carcinoma of the stomach contains an appreciable amount of oleic acid. 140. A Volumetrie determination of Sulfates in urine. Alwyn Knauer and Benjamin Horowitz. (Physiol. Lab., Fordham Univ. Med. Seh., N. Y. City.) Solutions required: (i) Barium chlorid, i cc. = 0.005 S^- of suKuric acid; (2) potassium Sulfate, I cc. = I cc. of sol. (i). I. Total {inorganic and ethereal) Sulfates: 50 cc. of urine and 5 cc. of hydrochloric acid sol. (sp. gr., 1.2) are poured into an Er- lenmeyer flask, and the mixture boiled for about 5 min. Barium chlorid sol., i, usually 10-15 cc, is then added and the mixture boiled 3-4 min. longer. Three small test tubes, of uniform bore and perfectly clean, are now placed side by side in a test tube rack, and labelled A, B and C. Small portions (not more than i cc.) of the sol. are filtered into each, using a very small funnel and a very small iilter paper for the purpose. A is kept as a control. To 5 3 drops 198 Proceedings Columbia Biochemical Association [March, of barium chlorid sol, and to C 3 drops of potassium sulfate sol., are added. If not enough barium chlorid sol. has been added, B will be cloudy; if barium chlorid is in excess, then cloudiness appears in C. In either case the contents of the test tubes are carefuUy poured back into the main sol, the test tubes and filter being thoroughly rinsed with dist. water, and i cc. of barium chlorid or potassium sulfate sol., depending upon which is in excess, is now added. The tests are repeated. If, in the first trial, test-tube B gave a cloudiness and, after the addition of another cc. of barium chlorid sol., it still continues to give a cloudiness (whereas C is clear or far less cloudy than B), there is evidently an excess of SO4 ions, and therefore more barium chlorid is added. If the further addition of barium chlorid causes the reverse to take place, namely, clearness or slight cloudiness in B and decided cloudiness in C, there is an excess of Cl ions, and therefore more potassium sulfate is added. It is evident that if, with a given vol., the results are the reverse of those seen with i cc. less, the end point must lie somewhere between these two volumes. If the determination need be approximate only, then the average of the volumes is taken. If not, further trials, with successive o.i cc, are made, until a point is reached where 3 drops each of barium chlorid and potassium sulfate sol. added to B and C give precisely the same cloudiness. To confirm this endpoint, take a f resh sample of urine, and add to it at once the total volume of barium chlorid sol. Samples of the filtrate should give equal cloudiness with ba- rium chlorid and potassium sulfate. IL Inorganic sulfates. The procedure is analogous to the above, except that acetic is substituted for hydrochloric acid, and the Solu- tion is not heated. I — II = Ethereal sulfates, III. Neutral sulfur. Here 50 cc. of urine are treated with 5 gm. of potassium nitrate and 7 gin. of sodium carbonate. The mixture is evaporated and then heated tili the carbonaceous mass is com- pletely oxidized. The residue is dissolved in water, the sol. poured into an Erlenmeyer flask, neutralized with hydrochloric acid and 5 cc. of hydrochloric acid sol. (sp. gr., 1.2) added. From here follow I. This will give total sulfur. If the total-sulfate sulfur (obtained in I) is subtracted from this, the result will be neutral sulfur. 141. The respiratory exchange of fish. Sergius Morgulis. 1915] Edgar G. Miller, Jr. 199 {U. S. Fisheries Biological Station, Woods Hole, Mass.) Apart from the technical difficulties involved in the investigation of the gaseous metabolism of aquatic animals, the inability to control their behavior is the strengest drawback in such studies. It is a well known fact that muscular exertion of any kind increases the gaseous exchange very considerably ; and unless a uniform base line, un- affected by bodily movements, can be established, the influenae of different factors cannot be determined with any degree of accuracy. To overcome this latter difficulty I have chosen, for experiment, the flounder, which normally does not move about but rests, some- times for hours, in the same position. The flounder, and a few other fish, have the habit of lying quietly on the bottom of the receptacle without changing their positions and, if not disturbed, move neither fins nor tail. These aquatic animals are therefore practically ideal subjects for metabolism investigations. Owing to lack of special apparatus for determining the carbon- dioxid Output and oxygen consumption, I was obliged to limit my research to the oxygen alone. Determination of the carbon dioxid in sea-water, by an easily available method, is still an unsolved problem. Oxygen, on the contrary, can be measured very ac- curately by means of the Winkler method. The latter is described in detail in special manuals and need not be given here. It will sufiice to say that it is an iodometric method and requires very little time. As an illustration of the accuracy of the method I may quote a few duplicate analyses of the oxygen content of sea-water in the Laboratory of the Bureau of Fisheries at Woods Hole. On different days the following results were obtained, expressed as CG. of oxygen per Hter: ABC S.58 5-95 5-52 5-54 5-95 5-55 The method of studying the oxygen consumption by the flounder was very simple. The fish, usually of small size, was put in a vessel of known volume filled with sea-water, of which a sample was analyzed for oxygen. The vessel was then tightly closed, the time recorded, and temperature of the water noted. From the percentage of oxygen the total quantity dissolved in the vessel 200 Proceedings Columbia Biochemical Association [March, could be ascertained. After a period varying from one to several hours, the vessel was opened and a sample of the water again ana- lyzed. The residual oxygen in the vessel could thus be calculated; and the difference between the first and second amounts gave the quantity of oxygen consumed by the flounder during the experiment. By this method it was found that the relative rate of oxygen consumption increased as the size of the flounder diminished. Thus, it was found that, with the body weights varying as i8:6: i, the oxygen consumption was in the ratio of i : 1.3 : 1.33. It was found, also, that the consumption of food invariably caused an increase in the oxygen intake by 25 to 30 percent. In the case of one small flounder, which weighed 3.75 gm,, it was found that the oxygen consumption per hour showed great regularity and a tendency to decrease in the course of a seven-day fast, as may be seen from the f ollowing data : Date IX 5 6 7 8 9 10 II 12 We observe a very abrupt drop in the oxygen consumption per hour, on the first day of fasting, which then remains fairly con- stant for the next three days. On the fourth and fifth days again a rather rapid decrease is seen, the oxygen intake per hour being now less than one half of that found 24 hr. after the last previous feeding. The entire experiment lasted 171.5 hr., of which the flounder spent fully one third in the respiration vessel. In that time it used up a total of 56.5 c.c. of oxygen, The loss in body weight for the same length of time was 0.32 gm. or 8.5 percent. It is noteworthy that the amount of substance which could be oxidized by 56.5 c.c. of oxygen is considerably less than the loss in body weight observed. The fact is significant, especially if we recall that Pütter^ has main- 5 Pütter : Zeitschr. f. allg. PhysioL, 1909, ix, p. 147. Weight, gn'a-iDS Oxygen per hour, cc, 375 0.492 — 0.359 — 0.368 — 0.398 — 0.271 — 0.207 — 0.226 3-43 0.230 iQisl Edgar G. Miller, Jr. 201 tained that the loss in weight is usually insufficient to account for the amount of oxidation and has postulated, therefore, the theory of a nutritive value for aquatic animals of substances dissolved in the water.® 142. A Standard for the determination of ammonia by means of Nessler Solution. Anton R. Rose and Katherine R. CoLEMAN. {Research Laboratory, Fenton B. Turck, M.D., Di- rector, N. Y. City.) Published in the preceding issue: Biochem. Bull., 1914, iii, p. 40?- 143. A micro-urease method for the determination of urea. Anton R. Rose and Katherine R. Coleman. {Research Labo- ratory, Fenton B. Turck, M.D., Director, N. Y. City.) Published in the preceding issue: Biochem. Bull., 1914, iii, p. 411. 144. The influenae upon metabolism of feeding B. coli. Anton R. Rose and Arthur Knudson. {Research Laboratory, Fenton B. Turck, M.D., Director, N. Y. City.) Bouillon inoculated with B. coli was fed to dogs in a basal ration of meat, cracker meal and lard. There was a somewhat pronounced change in the com- position of the urine during the first week, but later a gradual ten- dency towards the status of the normal periods. The amounts of sulfur and nitrogen ran parallel. The elimination of these elements in the urine was temporarily decreased. There was marked diminu- tion of neutral sulfur with an increase of sulfäte sulfur. The ether- eal sulfur rose immediately and then gradually subsided to the same plane as in the preliminary period. Feeding oi B. coli was followed by pronounced increase in indican in the urine, but this soon disap- peared, and protracted feeding of the bacteria did not bring it back. In general, the introduction of B. coli caused disturbance, but there was readjustment in the course of two or three weeks. 145. A further study of the influenae of electricity on metab- olism.'^ Matthew Steel. {Long Island College Hospital.) The present research consists of five experiments, of 9 to 12 days each. Four different kinds of electrical modalities were used. The subject was a normal healthy adult, and the diet was non-purin and uniform for each experiment. «Further results of this research have lately been published in detail in the Journal of Biological Chemistry, 1915, xx, p. 37.— (Ed.) 7 Steel : Biochem. Bull., 1914, üi, P- 309- 202 Proceedings Columbia Biochemical Association [March, Experiments I and IL Autocondensation current, with thick dielectric. Treatment: 500 m. amp. for 30 min. The following Symptoms were noted : Fall in blood-pressure, 4 to 10 mm. ; slight rise in pulse-rate; increase in the daily vol. of urine, 100 to 300 c.c; and increase of 5 to 6 gm. of total urinary solids per day. There were slight increases in the quantities of all the nitrogen constituents, the greatest increases being in urea and Creatinin nitrogens. Experiment III. Combination of direct d'arsonval current and the autocondensation current, with thin dielectric. Treatment through the feet, 1500 m. amp., 5 min.; through the hands and feet, 1750 m. amp., 15 min.; through the hands and feet, i960 m. amp., 6 min. ; through the hands and feet, 1600 m. amp., 4 min. The following Symptoms were noted : Fall in pulse-rate and blood-pres- sure; gentle warmth beginning at the wrists and gradually extend- ing over the entire body ; slight flushing of the capillaries, especially of the skin of the hands and wrists; rise in body temperature of 1° F.; decrease in the daily vol. of urine, 200 to 300 c.c; and in- crease in total urinary solids of about 2 gm. per day; slight increase in the quantities of all the nitrogenous constituents. Experiment IV. Static wave current. Treatment: a large metal plate was placed over the liver for 15 min., then over the kidneys for 15 min. The following Symptoms were noted: There was a small increase in the daily vol. of urine; increase in total urinary solids of 6 to 7 gm. per day; and increase of 0.82 gm. of total urinary nitrogen per day. The urea nitrogen was increased 0.76 gm.; the other nitrogen constituents were increased slightly. Experiment V. Galvanic sinusoidal current. Treatment: 70 m. amp., through the back and abdomen, for 30 min. This mo- dality caused decrease in the daily vol. of urine, 400 to 500 c.c. ; increase of about i gm. in total urinary solids per day; and small increase in the amounts of all the nitrogen constituents. In each of the above experiments the urine voided during the fore periods did not respond to the urorosein nor the nitrite tests, whereas the urine voided during the periods of electrical treat- ments responded strongly to each test. The urine voided during the after periods responded slightly.® 8 Steel : Loc. cit. IQISI Edgar G. Miller, Jr. 203 B. ABSTRACTS OF PAPERS FROM THE COLUMBIA BIOCHEM. DEP'T 146. Stiidies in goat metabolism. O. C. Bowes. The fol- lowing data relate to the preliminaries in a study of goat metab- olism. The animal was placed in a cage of the kind in regulär use in this laboratory in experiments on dogs. For the collection of the excreta certain modifications of the cage were made. The meshes of the wire platform, for example, on which the animal stood, were 7/8 in. Square. A special deep drip pan conveyed the excreta to a short chute with a screen of wire netting in the bottom, through which the urine passed into a Container, the feces rolling to the end of the chute into a separate receiver and thus affording a very satisfactory Separation of the latter from the former. A separate analysis was made of each feed in the ration, which included weighed quantities of hay, oats, bran, corn meal and lin- seed meal. The coefficient of digestibility, as recorded below, was for the entire ration. A small quantity of bone ash in the diet was found advantageous for hardening the feces and thus facilitating their collection. Aliquot portions of the daily feces were promptly dried and subjected to analysis in composite samples for the whole period. The results for 12 days of feeding the above mentioned rations, which followed a preliminary period of two weeks on the same diet, are summarized in the f ollowing table : Intake, grams Output in feces, grams Coefficient of digestibility, per cent Total nitrogen Total lipins Total carbohydrates Total ash 199.03 261.65 9,006.68 459-35 77.42 9467 3,698.20 353-92 61. 1 63.8 59-9 22.9 147. On the precipitation of proteins with Solutions of Chro- mates. Ruth S. Finch. Following the suggestions of Dr. Gies, I have continued some unpublished work by him and Dr. Wm. H. .Welker on the precipitation of proteins from acid Solutions by Chromates. In these preliminary experiments I have endeavored to determine the completeness of precipitation, the scope of applica- tion, and the possible practical uses, of this method. When 5 cc. of fresh watery extract of liver are treated with 5 204 Proceedings Columbia Biochemical Association [March, drops each of lo percent acetic acid and lo percent potassium Chro- mate sol., a heavy flocculent, yellow-brown precipitate is produced, which is easily separated by iiltration.^ Excess of Chromate may be removed from the clear filtrate with a few drops of lo percent barium chlorid sol., after nearly neutralizing the acid with ammo- nium hydroxid. When filtered through double quantitative filter paper, the clear filtrate gives no response to the biuret test, thus showing that precipitation of protein has been complete. Lithium, calcium, Strontium and f erric Chromates, and potassium bichromate, produce similar precipitates. Dilute mineral acids may be used in- stead of acetic, though boric acid is altogether too weak. Too much acid prevents clear filtration and too much Chromate seems to dis- solve the precipitate. Fresh extracts of most of the tissues of the body, as kidney, brain, lung, intestine, stomach, and heart give similar precipitates. Fresh blood must be diluted 1-5 or i-io, and treated with the proportions of 15 cc. of acid and 10 cc. of Chromate sol. per 100 cc. of diluted fluid, to give complete precipitation. Milk and dilute egg-white give very heavy precipitates but the filtrates are not clear unless the phosphates are first eliminated. Since the filtrate from egg-white always contained some protein, ovomucoid was pre- pared and purified in the usual way, and its precipitability tested. It was not precipitated by the acid-chromate combination. Of derived proteins, metaproteins obtained by acid extraction of meat were always completely precipitated. Amino acids, pep- tones and secondary proteoses give no precipitates. Primary pro- teoses, purified according to Kühne, yielded heavy yellow precipi- tates, but the filtrates responded weakly to the biuret test. Pos- sibly secondary proteoses were present despite the thoroughness of purification. Of related nitrogenous products, Adams' beef extract gives no precipitate, nor does an alcoholic extract of the total solids of urine. Of alkaloids, atropin yields an emulsion: narcein and brucin, char- acteristic crystals; narcotin, morphin, and apomorphin, dark floc- culent precipitates. 9 Gies : American Journal of Physiology, 1903, viii ; Proceedings of the American Physiological Society, p. xv. I9I51 Edgar G. Miller, Jr. 205 Chromates may evidently be used in many relations as a quali- tative protein reagent instead of potassium ferrocyanid, and they prove more advantageous under certain conditions because excess is easily eliminated from Solution. They should be useful as quantitative precipitants of various proteins. We have used the method successfully to remove protein from Solutions prior to the Isolation of such associated substances as ovomucoid and glycogen. It is our Intention to continue this study. 148. Active immunization to hay fever. Mark J. Gottlieb and Seymour Oppenheimer. Published in this issue : Biochem. Bull., 1915, iv, p 127. 149. Nitrogen metabolism in experimental uranium ne- phritis. Herman O. Mosenthal. Nitrogen metabolism in ex- perimental uranium nephritis bears certain resemblances to that in clinical nephritis. Urinary nitrogen may, in both of these condi- tions, be increased or diminished from the outset of the kidney in- volvement. It appears certain that retention of nitrogen is due to insufficient excretory powers of the kidney. Increased excretion is, in the experimental type of the disease at least, not due to pre- vious nitrogen retention but to increased protein catabolism. This is proved by the fact that in dogs poisoned with uranium, urinary nitrogen is present in excess as compared with the intake, while at the same time non-protein nitrogen of the blood is markedly in- ■creased in amount. A study of other phases of nitrogenous metabolism, when the blood and urine present the phenomena just stated, shows that the fecal nitrogen is unchanged in amount, and the quantity of nitrogen in the succus entericus, as determined by the Thiery-fistula method, tends to diminish. During the nephritic period, blood-pressure rises and remains above normal after all signs of renal disease dis- appear. Other urinary constituents — chlorids, sulfur and phos- phates — fail to parallel the nitrogen in excretion. Even individual urinary nitrogenous fractions, e. g., uric acid, may be retained while total nitrogen is increased. These facts indicate that uranium nephritis implicates the body as a whole and not the kidneys only. Furthermore, the various functions of the body do not necessarily Supplement each other by 2o6 Proceedings Columbia Biochemical Association [March, vicarious excretion, etc., as is often assumed, but each one to a' great extent follows its own independent laws. Human nephritis presents such a many-sided picture that probably many of the facts pertaining to experimental uranium nephritis are appHcable to it. However, these facts should be considered as nothing more than suggestions upon which to base further inquiry.^'' 150. An alleged improvement of the ferric chlorid method for the determination of sulfocyanate. W. A. Perlzweig and William J. Gies. Several years ago Bunting proposed the fol- lowing method for the quantitative determination of sulfocyanate in sahva :" " Pour 5 cc. of saliva into a thin curved watch-crystal about 3 in. in diam. Allow this to stand in the air or sunHght, or better still, set it on a slowly steaming water-bath, until the saliva has dried to the dish. To this add i or 2 drops of water and i or 2 drops of ferric chlorid sol. and stir with the residue to make a thick paste. To this add 5 cc. of ether, and stir the paste thor- oughly. When well mixed, hold the glass on a level with the eye and note the color of the sol." Compare, under similar conditions, with Standard colors representing known conc. of sulfocyanate. Some of the serious deficiencies of this method have been shown by Dr. Kahn and the senior author.^^ Bunting lately sought to eliminate the defects in the foregoing method by substituting for it the following procedure:^^ "Evap- orate 5 cc. of the sample (of saliva) to be tested, on a watch- crystal or small evaporating dish. To the dried film add ferric chlorid sol., drop by drop, spreading it gently with a glass rod; use just enough to moisten the whole film. Allow the mixture to stand for from i to 2 min., and then pour on 5 cc. of a mixture of amyl alcohol (5 parts) and ether {2 parts). Stir gently with a glass rod until all the color has been taken up by the ethereal sol. Decant the sol. into a test tube and compare with Standard colors representing known conc. of sulfocyanate." The Substitution of ether-amyl alcohol mixture, for ether, was 10 The results of this research have been published in detail in the Archives of Internal Medicine, 1914, xiv, p. 844. — (Ed.) 11 Bunting : Dental Cosmos, 1910, lii, p. 1346. 12 Gies and Kahn : Ibid., 1913, Iv, p. 40. 13 Bunting : Ibid., 1914, Ivi, p. 845. iQisl Edgar G. Miller, Jr. 207 intended to overcome the defects of the original method due to the use of ether alone, but Bunting merely evaded important deficien- cies of one kind to introduce serious imperfections of another. Bunting has not proved, by any experimental procedura, that his new method is more delicate quantitatively than the conventional f erric chlorid process ; he has merely indicated that the colorations obtained with his new method are more striking in some propor- tions — " more vivid " — than those observed with the f erric chlorid test as commonly applied. He has not shown that any given pro- portion of sulfocyanate which could not be detected by the conven- tional process would be revealed by his new method, which is the heart of the matter. He seems to have deluded himself into think- ing that, because the colorations obtained with his new process are, in certain selected proportions, more intense than those for the same selected proportions with the conventional ferric chlorid test, the new method itself is more distinct, therefore more accurate, and consequently more useful. H he had followed these compar- ative colorations step by step to their vanishing points for each test, with adequate controls, he would have avoided this fallacy in his Claims. With the aid of the conventional ferric chlorid process, it is not at all difficult to detect i part of sulfocyanate — potassium salt, Kahl- baum preparation — in 4,000,000 parts of water. Neither of us has been able to do so with Bunting's process. Bunting himself claims that "by careful technic a distinct color (the yellow of ferric chlorid?) may be obtained from a sol. which contains o.oooi per cent. — only i part in 1,000,000 — of KCNS." Our comparative tests with saliva have given us equally striking differences in favor of the conventional method. All our tests were suitably controlled, of course, and very slight though significant differences in color were easily observed as a consequence. [The senior author opened the discussion of Dr. Bunting's paper, on this " improved " method, af ter its original presentation at a meeting of the Academy of Stomatology of Philadelphia, March 28, 1914. The foregoing facts were pre- sented in that discussion. For further details see Dental Cosmos, 1914, Ivi, p. 856. In the printed version of his reply to the senior author's remarks, Bunting said that "no dilution of ferric chlorid is anything but yellow" (p. 866). This 2o8 Proceedinqs Columhia Biochemical Association [March, " break " shows how little Bunting knew about the disturbing influence on bis test of ferric chlorid itself. He evidently failed to compare bis results with those of control tests. Bunting was asked by Gies (p. 858) : " Does diacetic acid affect the new pro- cedure ; if so, is the disturbing effect more or less than that on the first process — possibly any contained diacetate would be decomposed by the preliminary desicca- tion ? " To these questions Bunting replied irrelevantly as follows (p. 866) : " He (Gies) then asks how the alcohol-ether method eliminates{!) the aceto-acetic acid, if present. Is it possible that he (Gies) does not know that the ferric salt of aceto-acetic acid is not soluble in ether ? " Bunting's äff ected surprise in this regard would be less amusing if he had f rankly answered the questions ; and his chemical ignorance would be less apparent if he had addressed himself to the possible influence, on his test, of diacetic acid (in saliva) through the ca- pacity of diacetic acid to affect the concentration of " soluble iron " in the final medium ; to which, of course, Gies' question directly referred, Bunting's inability, or unwillingness, to State correctly the simplest facts of the case were shown by his misuse of various remarks in his conclusion to the " discussion." Thus, Dr. Percy R. Howe, who participated in the discussion, said (p. 863) : " I have tried the test many times and in my hands the color is much deeper by Bunting's ether method (the first, rej ected by Bunting) than by the FeCU plus H2O." There is no indication in this remark, or in any other that Howe made, of any determination by him of the coloric influence of excess of ferric chlorid itself in the alcohol-ether test — no Suggestion of any comparison of the two tests at dilutions of sulfocyanate that might have been expected to develop the comparative values of the tests, yet Bunting says complacently and incorrectly of this remark (p. 867) : " Dr. Howe told us that he had tried the method and had found it trustworthy(.')" On page 867 Bunting states, in an- other connection : " Dr. Gies has given no evidence of having made an actual test of the validity of this Statement, but has contented himself with saying that it is not true." Yet the " evidence " was orally stated and appears on p. 857 — printer's proof of which was submitted to Bunting prior to the publication of his own Statement. (If the compliment has been returned by him, Bunting's over- sight in this connection would have been pointed out before it was too late for correction.) Again, Bunting presumed to state, inhis printed rej oinder, that Gies "says that dentists should not attempt problems which involve chemistry" (p. 867), and then worked up a ludicrous frenzy about "such a sentiment." Gies suggested, on the contrary, that " it is time for you (dentists) to eye with suspicion the expert dentist who persists in taking your time, and using space in your Journals, to discuss chemical research of douhtful validity and of dubious comprehension. Let us stick to our lasts " (p. 861). There was no Suggestion that dentists qualified to conduct chemical research should not do so. During the discussion, Gies said : " I publicly stated, recently, to some of your colleagues at a dental meeting in New York that I have taken the war- path against your pseudo-chemists, and was told, in reply, that I might never perform a better service for dentistry." In his printed rejoinder to this, Bunt- ing suggested that, should Gies continue in this direction, "he (Gies) will kill himself by his own misdirected efforts " (p. 868). Bunting is right — the more the senior author prepares himself for the execution of this purpose, especially 1915] Edgar G. Miller, Jr. 209 by reading Bunting's chemical comedies, the more he is likely to kill himself— kill himself laughing! Bunting's Suggestion that Gies' discussion of his (Bunting's) paper " reveals plainly the fact that he (Gies) has not done the work which he claims to have performed upon this method " leads us to propose that Gies be promptly inves- tigated. It is suggested that the junior author be called as the first witness and that Dr. A. P. Lothrop, who tested the validity of some of our conclusions, be called as the second.] 151. Absorption of unaltered protein through the gastro- enteric tract in infants. O. M. Schloss. The infants tested were given the whites of one to two eggs. The urine was collected for 6 hr. and used for precipitin tests. Urine, or protein precip- itated from urine by Saturation with ammonium sulfate, was in- jected into the peritoneal cavity of guinea-pigs and, 21 days later, similar injection of egg- white was made. The precipitin reaction was positive in very few normal infants, but was positive in a large percentage of those suffering from gastro-enteric disorders or mal- nutrition. In many instances the positive precipitin reaction was coincident with albuminuria (heat and acetic acid test) but, in a number of urines giving positive precipitin tests, no albumin was demonstrable. The anaphylactic reaction was positive in practi- cally all cases with both albuminuria and a positive precipitin re- action, but was uniformly negative when no albuminuria was present. The results indicate that unaltered, or but slightly altered, protein can be absorbed from the gastro-enteric tract of infants suffering from nutritional disorders. Tests were also made for protease in the blood-serum of infants. The dialytic technic of Abderhalden was used, and proteins from tgg and milk were employed. Proteases were present in a few normal infants and in a large percentage of those suffering from nutritional disorders. One of the characteristic anaphylactic reactions in the guinea- pig is a marked rise in the eosinophile blood-cells. It seemed of interest to determine whether, in the continued feeding of foreign protein, sufficient would be absorbed to cause such a reaction. Guinea-pigs were fed approximately 5 gm. of powdered egg-white a day. Four of the six animals thus fed showed marked increases in eosinophile blood-cells accompanied by leukocytosis. Control animals showed no such blood changes. 2IO Proceedinqs Columbia Biochemical Association [March, IL NINETEENTH MEETING The nineteenth scientific meeting of the Assoc. was held in the Biochemical Seminar Room, at the Columbia Med. Seh., at 4:15 P. M., on Dec. 4, 19 14. The appended summary facilitates refer- ence to the abstracts (152-170) of the papers presented, pages 211, 224. A SUMMARY OF THE NAMES OF THE AUTHORS AND OF THE TITLES OF THE SUCCEEDING ABSTRACTS (152-170) tion of agglutinative and fermenta- tive characters among the Strepto- cocci. (162) V. E. Levine. The reducing power of anerobes. (163) Sergius Morgulis. Body surf ace and metabolism of flounders. (164) }. Bronfenbrenner. The nature of the Abderhalden reaction. (152) J. Bronfenbrenner, W. J. Mitchell, Jr., and Paul Titus. The role of serum anti-trypsin in the Abder- halden test. (153) J. Bronfenbrenner, J. Rockman and W. J. Mitchell, Jr. Comparisons of urinary and serum findings in the diagnosis of tuberculosis. (154) J. Alexander Clarke, Jr., and Martin E. Rehfuss. (Communicated by P. B. Hawk.) The protein content of the gastric juice in normal and patho- logical States. (155) A. F. Blakeslee and Ross A. Gort- ner. Reaction of rabbits to intrave- nous injections of mould spores. (156) E. Newton Harvey. Studies on the photogen of luminous bacteria. (157) Alfred F. Hess and Max Kahn. Metabolism studies of two cases of hemophilia. (158) Max Kahn and Jacob Hoffmann. Calcium metabolism in normal and diabetic individuals. (159) Max Kahn and Isidore Jacobowitz. A modification of the Wulf-Junghans method for the diagnosis of gastric Cancer. (160) Max Kahn and William Spielberg. Condition of nutrition in nephrec- tomized patients. (161) I. J. KxiGLER. A study of the correla- B Frederic G. Goodridge and Max Kahn. The neutral-sulfur and colloidal- nitrogen tests in the diagnosis of Cancer. (165) V. E. Levine. Sodium selenite as a laboratory reagent for reducing sub- stance. (166) Alfred P. Lothrop and William J. GiES, with the collaboration of Henry W. Gillett, Charles C. Linton, Arthur H. Merritt and Herbert L. Wheeler. A further study of the effects of acid media on natural extracted teeth. (167) F. D. Zeman and Paul E. Howe. The excretion of creatin during a fast. (168) F. D. Zeman and Paul E. Howe. Re- cuperation : Nitrogen metabolism of a man when ingesting successively non-protein and normal diets after a seven-day fast. (169) F. D. Zeman, Jerome Kohn and Paul E. Howe. Variations in factors as- sociated with acidity of human urine, during a seven-day fast and during subsequent non-protein and normal feeding periods. (170) 1915] Edgar G. Miller, Jr. 2ii A. ABSTRACTS OF PAPERS BY NON-RESIDENT MEMBERS 152. The nature of the Abderhalden reaction. J. Bron- FENBRENNER. {Patfiol. Gfid ReseGTch Lab., West Penn. Hosp., Pittsburgh, Pa.) Published in this issue: Biochem. Bull., 1915, iv, p. 87. 153. The role of serum anti-trypsin in the Abderhalden test. J. Bronfenbrenner, W. J. Mitchell, Jr., and Paul Titus. (Pathol. and Research Lab., West Penn. Hosp., Pittsburgh, Pa.) Published in this issue: Biochem. Bull., 1915, iv, p. 86. 154. Comparisons of urinary and serum findings in the di- agnosis of tuberculosis. J. Bronfenbrenner, J. Rockman and W. J. Mitchell, Jr. {Pathol. and Research Lab., West Penn. Hosp., Pittsburgh, Pa.) Published in this issue: Biochem. Bull., 1915, iv, p. 80. 155. The protein content of the gastric juice in normal and pathological states. J. Alexander Clarke, Jr., and Martin E. Rehfuss. Communicated by P. B. Hawk. {Lab. of Physiol. Chem., Jefferson Med. Coli, Phila.) The protein content of the gastric juice was investigated by the method of Wolff, namely by successively diluting the gastric juice and adding phosphotungstic reagent. It was found that the normal gastric juice, per se, con- tained only traces of protein, never giving a reaction in dilutions greater than i : 40. The protein content of the specimens removed by the f ractional method, after the administration of Ewald meals, was also determined. The macerated Ewald meal in vitro never gave a reaction in a dilution greater than i : 40, Treated in the incubator with artificial gastric juice, the authors were able to dem- onstrate an increasing content due to the effect of the gastric juice on the proteins of the bread. If therefore material removed from the stomach at intervals develops a greater protein content than the theoretical content due to the action of the gastric juice on the bread proteins, we are able to say that it comes from other sources than bread. In functional states and in normal cases the protein curve followed approximately the curve for the action of the gastric juice on the Ewald meal in vitro. In pathological conditions, such as ulcer and Cancer, the curve was entirely different. The authors pointed out the importance of dififerentiating the presence of blood. 212 Proceedinqs Columbia Biochemical Association [March, infected Sputum rieh in proteins, and the possibility of protein rests due to deficient motility. In ulcer, a marked initial rise in the protein content was noted, which was out of proportion to the amount of acid secreted. This requires further study. In cancer the authors were able to confirm the increased protein content of cancerous achyHas and to show in practically all cases marked in- crease in protein concentration out of all proportion to the amount of acid present. In Cancer, this rises steadily and is usually most marked in the more advanced specimens. This disassociation be- tween the acidity and protein curves, the authors consider most important; it emphasi/:ed the steady rise in the protein content as digestion progresses. The high protein content cannot be due to the action of the secreted juice on the bread but must be a special elabo- ration from cancerous tissue. The value of this reaction, namely the disassociation in the protein and acidity curves, is of value in direct proportion as the acidity is low and as the protein continues to diverge. In inflammatory conditions, as contrasted with func- tional States, the authors find a greater protein content in the former and insist on the necessity of examining the entire digestive cycle owing to the possibility of undue protein concentration at certain periods. 156. Reaction of rabbits to intravenous injections of mould spores. A. F. Blakeslee and Ross A. Gortner. (Storrs Agric. Exp. Station, Storrs, Conn., and Carnegie Station for Exp. Ev., Cold Spring Harbor, N. Y.) Published in this issue: Biochem. Bull., 1915, iv, p. 45. 157. Studies on the photogen of luminous bacteria. E. Newton Harvey. (Physiol. Lab., Princeton Univ.) Masses of luminous bacteria were dried on glass wool in a vacuum over cal- cium Chlorid and ground to a powder. The powder will phosphor- esce if moistened with tap-water or sea-water. Since new colonies of bacteria can usually be obtained from the powder, if planted on a suitable culture medium, all the bacteria are evidently not killed by drying, but most of them are. If the dry powder is extracted with boiling ether for 10 hr., it phosphoresces as strongly as before, after the ether is removed and the powder moistened. Ether-treated material may give occasional I9I5] Edgar G. Miller, Jr. 213 new growths on agar-agar. We may therefore conclude that the living cell is not essential f or light production ; and, f urther, that the photogen is not a fat, or a lecithin, or any ether-soluble substance. Luminous bacteria require free oxygen in order to phosphoresce. If the bacteria could be broken up in an oxygen- free medium, any photogenic substance should dissolve in the medium and phosphor- esce when oxygen is again added. The bacteria were broken up (cytolyzed) by adding (a) toluene to a bacterial emulsion in oxygen- free sea- water and (b) oxygen- free distilled water to a dense mass of bacteria in a Space devoid of oxygen. After 10-15 ^ni"-» oxygen was admitted but in neither case did phosphorescence appear. We may therefore conclude that the photogen is unstable and breaks up without the production of light in water free from oxygen. Of course the photogen would rapidly burn up if any free oxygen were present. Exactly similar results were obtained with the dry powdered luminous organs of the fire-fly. (See Jour. Amer. Chem. Soc, 1915, xxxvii, p. 396.) 158. Metabolism studies of two cases of hemophilia. Al- fred F. Hess and Max Kahn. (Chem. Lab., Beth Israel Hosp., N. Y. City.) The intake and Output of nitrogen, sulfur, phos- phorus, chlorin, calcium, magnesium and fat were studied in two cases of hemophilia. It was found that in one of these cases (B. A.) there was a minus calcium balance which could be changed to a plus balance by administering calcium chlorid per os daily. It was found that this case also had a diminished calcium content in the blood. The other case (J.) was normal, so far as was shown by the metabolism experiments or calcium "content of the blood. In the first case, we used a sol. of calcium chlorid, in salin, of such strength that the addition of i drop of it to 10 drops of blood ex- actly supplied the amount of calcium that the blood seemed to lack. It was found that the coagulation time of blood so treated was reduced from about 45 to 50 min., to 10 or 12 min. 159. Calcium metabolism in normal and diabetic individ- uals. Max Kahn and Jacob Hoffmann. (Chem. Lab., Beth Israel Hosp., N. Y. City.) Diabetic patients who excreted sugar in the urine showed a distinct daily calcium loss. Administration 214 Proceedings Columbia Biochemical Association [March, of calcium chlorld caused a positive calcium balance. When the sugar excretion stopped, the calcium loss was much reduced. Cal- cium was determined by the McCrudden method. i6o. A modification of the Wulf -Junghans method for the diagnosis of gastric Cancer. Max Kahn and Isidore Jacob- owiTZ. {Chem. Lab., Beth Israel Hosp., N. Y. City.) The patient is given an Ewald test breakfast. The stomach is then thoroughly flushed, and the washings examined for nitrogen by the Kjeldahl method and for albumin by the Pfeiffer method. If the nitrogen is more than i8 mg. per loo cc. of gastric contents and, if the albumin is more than 0.5 part per thousand, malignancy is suggested. 161. Condition of nutrition in nephrectomized patients. Max Kahn and William Spielberg. {Chem. Lab., Beth Israel Hosp., N. Y. City.) Two cases of nephrectomy were studied. The various ordinary urinary constituents were normal in propor- tion, except that, in one case, neutral sulfur was much increased. 162. A study of the correlation of agglutinative and fer- mentative characters among the Streptococci. I. J. Kligler. (Dep't of Public Health, Amer. Museum of Natural Hist., N. Y. City.) Bacteria have evolved so little along gross structural lines that it is impossible to differentiate members of the same genus on a merely physical basis. Bacteriologists therefore resort to the more delicate criteria of protoplasmic structure and physiological activity, in which direction remarkable differentiation exists. Tests for the finer structural differences of these organisms are found in their behavior to differential stains, like the Gram stain; and to the im- mune substances induced by them in the animal body. Their phys- iological activity is measured by determining the end products of their metabolism. Bacteria generally have evolved in two main directions, one group possessing marked carbohydrate-splitting properties, the other having developed the property of digesting various protein substances. The Streptococci belong to the former division, showing but little tendency to effect proteolysis. It appears natural enough to assume that the biologic activities of a cell correspond with its protoplasmic Constitution. Yet such a correlation has not been worked out except in a few isolated cases. 1915] Edgar G. Miller, Jr. 215 Among the Streptococci such a correlation, if it exists, would be especially significant in that it would help to differentiate the vari- ous members of a genus that has puzzled many investigators. The agglutination, fermentation and hemolytic properties of sixty strains derived from various pathological conditions were studied, using four agglutinating sera having a titre of 800-1000; and six carbohydrates and other fermentable substances as f ollows : Disaccharides— /ac^o.?^_, sucrose; trisaccharide — raffinose; alcohol — mannite; glucoside — salicin; Polysaccharide — inulin. Only twenty-seven of the strains were agglutinated by the sera used. A definite correlation was, however, obtained between the agglutinative and fermentative characters. The serum produced by a strain of one fermentative group (the group that fermented salicin, for instance) agglutinated only cultures of its particular division and failed to agglutinate members of any of the other groups. No such correlation was obtained with the hemolytic property, members of one hemolytic group being agglutinated by the sera produced by strains from another hemolytic group. The results indicate that a Separation of the Streptococci ob- tained from various pathological conditions, into three fermentative types, would coincide most closely with their natural relationship. The groups suggested are : (A). Salicin fermenters only, generally hemolytic. — Str. pyogenes. (B). Raffinose fermenters; salicin usually fermented; man- nite always negative; generally produces a green colony on blood agar. — Str. salivarius. (C), Mannite fermenters; generally ferment salicin; rarely ferment raffinose; variable in their reaction to blood. — Str. fecalis. 163. The reducing power of anerobes. Victor E. Levine. (Dep't of Public Health, Amer. Museum of Natural Hist., N. Y. City.) It is a well established fact that anerobes reduce organic dyes, such as methylene blue (Cahen, Smith, Kitasato and Weyl). No difference on the ground of reduction may be claimed between aerobes and anerobes. Klett,^^ however, using sodium selenite as an indicator, found that the anerobes he examined lacked the power 1* Klett : Zeit. f. Hyg., 1900, xxxiii, pp. 135, 137. 2i6 Proceedinqs Columbia Biochemical Association [March, of reduction. He also observed that sodium selenite, even in very small conc, inhibited growth, whereas sodium sulfite favored it. In the 1910 editionof Kruse's Allgemeine Mikrobiologie, the Statement was made that anerobes do not seem to reduce sodium selenite, as indicated by the few preliminary findings of Klett. In Order to test the validity of this conclusion, experiments with sodium selenite were made with anerobes available in the bacterio- logical collection at the Museum of Natural History.^^ The follow- ing organisms were used: B. Welchi (four strains) ; B. sporo genes (three strains) ; B. Feseri (two strains) ; B. oedematis maligni (two strains) ; B. tetani (two strains) ; B. oedematis; B. hotidinis; B* putrificus. They were grown in media containing the following conc. of sodium selenite : i : 100,000; i : 50,000; i : 25,000; i : 10,000. The culture tubes were kept under anerobic conditions by means of alkalin pyrogallate. No appreciable inhibition of growth was observed except in conc. of 1 : 10,000. Reduction was found to have taken place within 24 to 48 hr. in conc. of i : 100,000, but the red selenium streak follow- ing the path of growth disappeared within a few days, so that there was no visible evidence of reduction. The higher selenite conc. showed excellent reduction but there was less tendency for the red precipitated selenium to disappear. At the end of 3 months the selenium streaks had completely disappeared in all the culture tubes except in the ones containing sodium selenite in conc. of i : 10,000. These experiments prove conclusively that aerobes and anerobes reduce sodium selenite equally well and that sodium selenite cannof be used as a reagent for differentiation between these two classes of micro-organisms. For practical demonstrations, conc. of i : 25,000 and 1 : 10,000 yield the best results. 164. Body surface and metabolism of flounders. Sergius MoRGULis (U. S. Fisheries Biological Station, Woods Hole, Mass.) In connection with various biological problems the impor- tance of the body surface has been frequently emphasized. Never- theless, owing to the difficulties involved in measuring the surface of an organism, knowledge on this score has been very fragmentary. The flounder is an unusually favorable object for an investigation of 15 Levine : Biochem. Bull., 1914, iii, p. 464. I9IS] Edgar G. Miller, Jr. 217 this matter. I have determined the surface of a large number of flounders ranging in size from about 4 to 25 cm.; and in weight, from about 0.5 to 150 gm. The surface can be computed from the formula S=K^IV^, wherein W is the weight of the animal. K, which has been found to vary within narrow hmits, is 13.44 for normal flounders. This value coincides closely with that found for higher organisms. Under normal circumstances the metaboHsm, as judged by the oxygen consumption, diminishes per unit of body surface as the latter increases. The relation of the metabolism to surface was well illustrated, in a series of experiments,where the surface was reduced 30-40 percent by the removal of the fins. The body weight was very little affected by the Operation, as the fins form only 2-3 percent of the weight. In this case the oxygen consumption remained un- changed, and must, therefore, have been dependent upon the mass of living substance. Furthermore, it is important to bear in mind that the value of K is constant under definite physiological conditions. In fasting flounders the value of K has been invariably much higher. This was due to the fact that the body weight diminished more rapidly than the surface, and probably, also, because the specific gravity of the organism was decreased. B. ABSTRACTS OF PAPERS FROM THE COLUMBIA BIOCHEM. DEP'T 165. The neutral-sulfur and colloidal-nitrogen tests in the diagnosis of cancer.^^ Frederic G. Goodridge and Max Kahn. Pubhshed in this issue: Biochem, Bull., 191 5, iv, p. 118. 166. Sodium selenite as a laboratory reagent for reducing substances. Victor E. Levine. Further experiments confirm the statement^^ that sodium selenite, in alkalin sol., can be used as an indicator for reducing substances, especially carbohydrates contain- ing free carbonyl groups. The following do not reduce sodium selenite (alkalin) : acetone, formaldehyde, tri-oxy methylene, acetaldehyde, furol, benzaldehyde, cinnamic aldehyde, salicyl aldehyde, piperonal, methyl alcohol, ethyl Iß Some of the work was done in the Beth Israel Hospital, N. Y. City. 17 Levine : Biochem. Bull., 1913, ii, p. 552. 21 8 Proceedings Columbia Biochemical Association [March, alcohol, glycerol, erythrol, mannite, inosite, phenol, the cresols, thy- mol, a-naphthol; acetic, butyric, ^S-oxybutyric, palmitic, stearic, tri- chloracetic, oxalic, tartaric, citric, oleic, mallc, cinnamic, and hippu- ric acids; glycocol, alanin, guanidin carbonate, leucin, urea, thio- urea, ammonium sulfocyanate, caffein, theobromin, uric acid, so- dium urate, Creatinin, lecithin, cholesterol, palmitin, Stearin, olein, serum proteins, blood fibrin, edestin, egg albumen, gelatin, peptone, proteoses, ovalbumen, collagen, osseomucoid, elastin, Saccharin, anti- pyrin, anthraquinone, sucrose, raffinose, cellulose, starch, dextrin, glycogen, inulin, esculin, amygdalin, and the f ollowing gums : arabic, tragacanth, guaiac, rosin, benzoin, kino, aloes, asafetida, myrrh, gam- bir. Alcoholic Solutions of gum benzoin, kino or aloes give a red- brown to cherry-red sol. without the addition of sodium selenite. The f ollowing reduce sodium selenite : amidol, arabinose, rham- nose, xylose, glucose, galactose, f ructose, maitose, lactose, hydroqui- none, phloroglucinol, pyrogallol ; hydroxylamin, Phenylhydrazin and benzidin hydrochlorids ; hydrazin hydrate; arsenious, hydrobromic, hydriodic, phosphorous, hypophosphorous and sulfurous acids; fer- rous Sulfate, stannous chlorid, sodium thiosulfate, zinc and hydro- chloric acid, hydrogen sulfid, acetylene; formic, gallic, lactic and tannic acids. Acetone, acetaldehyde, formaldehyde, aceto-acetic ester, ^-oxy- butyric acid, Creatinin, lactic acid, formic acid and inulin reduce in acid, but not in alkalin mixtures of sodium selenite. Methyl alcohol and ethyl alcohol reduce sodium selenite strongly acidified with sul- furic or with hydrochloric acid. Oxalic, citric, tartaric, malic and salicylic acids, benzaldehyde, cinnamic aldehyde and salicyl aldehyde, reduce neither acid nor alka- lin mixtures. The results show that monosaccharids readily reduce alkalin sol. of sodium selenite. Pentoses effect readier and more profuse re- duction than the hexoses and the reducing disaccharids. Of the pentoses, xylose causes the most striking reduction. Among the hexoses, fructose and galactose reduce more readily than glucose, and galactose less readily than fructose. Among the disaccharids only those having free carbonyl groups reduce. Maltose and lac- tose effect reduction, but sucrose does not ; raffinose, cellulose, starch, dextrin, glycogen, inulin do not reduce. 1915] Edgar G. Miller, Jr. 219 In Order to test the influence of acidity, or alkalinity, upon the reduction of sodium selenite, nineteen reagents were prepared. One consisted of sodium selenite neutralized with sulfuric acid. Ten were alkalin, the basicity being due to sodium selenite per se, to so- dium bicarbonate, sodium carbonate, sodium tetraborate, sodium Sili- cate, sodium hydroxid, di-sodium hydrogen phosphate, potassium hydroxid and Rochelle salt, or sodium carbonate and sodium citrate. Eight reagents were acidified by the addition of one of the follow- ing: potassium bi-sulfate, sodium di-hydrogen phosphate; hydro- chloric, nitric, sulfuric, phosphoric, citric, or tartaric acid. When these reagents were heated none reduced, even with complete evapo- ration, except those containing citric or tartaric acid. These two reagents deteriorated after standing several months. Experiments with the above-named reagents were conducted at 37.5° C. Solutions (0.5 percent) of arabinose, rhamnose, xylose, glucose, fructose, galactose, sucrose, maitose, lactose, glycogen, starch, dextrin, inulin, raffinose ; mucic, lactic and f ormic acids ; ace- tone, and formaldehyde, were tested. Three cc. of the sol. to be used were mixed with 2 cc. of the selenite reagent and toluene added. The tubes were kept at 37.5° C, and examined from time to time. Controls were run with Fehling and Fehling-Benedict reagents. The reagents containing sodium hydroxid and potassium hydroxid (selenite and Fehling) were the first to show reduction. Fehling reagent reduced more quickly than Fehling-Benedict. Glycogen, starch, dextrin, inulin and raffinose reduce acidified Solutions of so- dium selenite by the end of 4 days. Alkalin sol. were not affected. Formic acid, lactic acid, and formaldehyde reduce in acid sol. only. Acetone profusely reduces acid sol.; very faintly, some of the alka- lin sol. The reagent, acidified with nitric acid, showed no reduction, except in the case of acetone. Neutralized sodium selenite is a very ineffective indicator of reduction. The presence of sodium tetra- borate inhibits to a very striking extent the reduction of sodium selenite. A sol. containing 2 percent of sodium selenite, 10 percent of sodi- um citrate, and 10 percent of sodium carbonate, has been tested with reducing sugars ( 100° C). Reduction with this reagent takes place in one minute, or even less. At first a deep chlorin-yellow color is developed. After standing a minute or two, this color gives way to 220 Proceedings Columbia Biochemical Association [March, a light, wine-red, tint, then to a dense brick-red precipitate, which suffuses the volume of the liquid. A 0.02 percent sol. of glucose causes fair reduction; in o.oi percent sol, reduction is slight though perceptible. Sol. to be tested must be alkalin, and must not contain potassium Cyanid or oxidizing agents (e. g., free Halogen, hydrogen peroxid, potassium permanganate, potassium bichromate). Sugar- free urine gives a positive reaction when it is acidified with hydro- chloric acid. This positive reaction is probably due to acetone sub- stances and Creatinin, which reduce acidified sol. of sodium selenite. Minute amounts of selenium, in the form of selenite ion, can be detected by a procedure similar to that of the Marsh test for arsenic. One mg. of selenium dioxid yields a characteristic dull red mirror, soluble in oxidizing agents. 167. A further study of the effects of acid media on natural extracted teeth. Alfred P. Lothrop and William J. Gies^ with the collaboration of Henry W. Gillett, Charles C. Linton, Arthur H. Merritt and Herbert L. Wheeler.^® The fermen- tation of glucose on normal and filled teeth, with sound enamel " worn very little or not at all," in the presence of saliva, induced rapid decalcification of the enamel and speedy disintegration of some of the Allings. These effects were more pronounced on sali- vated teeth covered with muslin than on similarly salivated teeth that were not thus covered. Two daily brushings, for 4 months with tap water, and con- tinuous daily treatment under muslin Covers, during the intervening periods, with (a) water, (b) water containing carbon dioxid, and (c) water holding an abundance of salivary mucin (mucin not muci- nate), failed to induce injurious effects on teeth with sound enamel " worn through and exposing dentin," and with enamel " worn very little or not at all." Two daily brushings, for 4 months with tap water, and con- tinuous daily treatment under muslin Covers during the intervening periods, with (a) 0.25 percent Solution of mono-sodium di-hydrogen phosphate (NaH2P04), and with (b) aqueous Suspension of sahv- ary mucin (not mucinate) plus 0.25 percent Solution of mono- sodium di-hydrogen phosphate, failed to induce injurious effects on either the enamel or the fillings in teeth having sound enamel that ^^ Journal of the Allied Dental Societies, 1914, ix, p. 554. I9I5] Edgar G. Miller, Jr. 221 was " worn very little or not at all," or which was " considerably worn without exposure of dentin." Unfilled teeth, with sound enamel that was (a) " considerably worn without exposure of dentin," (b) "worn very little or not at all," or (c) "worn through and exposing dentin," when subjected to two daily brushings, in comparative tests, f or 8 months, with ( i ) dilute vinegar (i : i), a (2) common tooth powder or a (3) com- mon tooth paste, with intervening salivation, were uninjured. No change of any kind could be detected as a result of the vinegar treat- ment. Two daily brushings of teeth similar to those referred to in the preceding paragraph (some of them filled), with dilute vinegar (1:1), for 8 months, 9 months and 17 months, were free from injurious influences on both the enamel and on most of the fillings,^^ whether the teeth were salivated (covered or uncovered) during the intervening periods or not. 168. The excretion of creatin during a fast.^o F. D. Zeman and Paul E. Howe. Recent criticism^^ of results obtained with Folin's method for the determination of creatin in urine, in the pres- ence of acetone and aceto-acetic acid, has thrown doubt upon the presence of creatin in the urine of fasting man. We have deter- mined creatin in the urine of a fasting man throughout a 7-day fast. The method of Graham and Poulton was employed for the removal of acetone and aceto-acetic acid; quantitative determinations were made of these substances, together, and of ^-hydroxy butyric acid. Control experiments were made with untreated urine. Determina- tions before and after the appearance of the interfering substances showed the method to be accurate in their absence. Creatin was ex- creted on each fasting day in amounts equal, in most cases, with those obtained in previous fasts under similar conditions. 169. Recuperation : Nitrogen metabolism of a man when ingesting successively non-protein and normal diets after a 19 The fillings consisted of the foUowing materials : malleted gold, gold in- lays, gutta percha, Arnes' black copper cement, Stanle/s red copper cement, Arnes' pearl white inlay cement, Arnes' berylite, fellowship alloy, Silicate cement, oxyphosphate of zinc, synthetic porcelain, alloy, amalgam. 20 Most of the work was done in the Biochemical Laboratory at Teachers College. 21 Graham and Poulton : Proc. Roy. Soc, 1914, Ixxxvii, B, p. 205. 222 Proceedings Columbia Biochemical Association [March, seven-day fast.^^ F. D. Zeman and Paul E. Howe. The third^^ of a series of experiments on changes in metabolism of man following the Ingestion of food after a fast. In the recuperation periods (4 days) of this experiment, non-protein and normal diets were fed; the preliminary and final diets were the same. The non- protein diet consisted of sucrose, clarified butter, alkalin salt mixture, and agar-agar, having an approximate daily fuel value of 3500 cal. Determinations were made of the body weight, and the excretion of urinary water, total nitrogen, urea, ammonia, creatin and Creatinin.^* The excretion of the urinary constituents followed the usual course during the fast; the total-nitrogen excretion on the 7th day was approximately 10 gm. and creatin appeared daily. The inges- tion of a calorically sufficient, non-protein, diet resulted in decrease of the nitrogen excretion, which became constant on the ßd and 4th days. Minimum values obtained on the 2nd day of feeding, were as follows: Total N, 3.56 gm.; urea-N, 1.59 gm.; ammonia-N, 0.54 gm.; creatinin-N, 0.61 gm.; creatin-N, 0.05 gm. A relatively high ammonia-N excretion (0.72 gm., 17.4 percent of the total N) oc- curred on the 3d day. Normal conditions tended to return in the final period while the subject was retaining nitrogen. Lowered ab- solute and relative ammonia-N excretions were observed. The daily excretion of fecal nitrogen during the non-protein period was 0.50 gm. A comparison of the changes in body weight, and in the nitrogen balances, shows an increase in body weight during the non-protein feeding period, accompanied by a loss of nitrogen; the reverse oc- curred in the final period. The initial increase in weight after the ingestion of food was the result, chiefly, of the retention of water and to a smaller degree of non-nitrogenous food substances. 170. Variations in factors associated with acidity of human urine, during a seven-day fast and during subsequent non-pro- 22 Most of the work was done in the Biochemical Laboratory at Teachers College. 23 The first two experiments were reported by Howe, Mattill and Hawk: Jour. Amer. Chem. Soc, 191 1, xxxiii, p. 568, and Howe and Hawk: Proc. Amer. Soc. Biol. Chem., 1912, ii, p. 65 ; Jour. Biol. Chem., 1912, xi, p. xxxi. 2* Variations in factors associated with changes in the urinary acidity are referred to in the succeeding abstract. 1915I Edgar G. Miller, Jr. 223 tein and normal feeding periods.^^ F. D. Zeman, Jerome Kohn and Paul E. Howe, A study was made of the variations in acidity (true and titratable) of human urine, with relation to modifying factors present during fasting and recuperätion. The ränge of var- iations of the acidity extended from a fairly acid urine, Ph 5-I (3^^ day of fast) to an alkalin urine, P3 8.0 (last day of the final period). The diet of the preliminary and final feeding periods was the same, in nature, as that used in previous experiments.^^ In the non-pro- tein period, sucrose, clarified butter, salts (alkalin mixture) and agar-agar were ingested. Determinations were made of the H^ ion conc. (indicators) : titratable acidity or alkalinity (with Phenolphtha- lein, neutral red and methyl orange); phosphates; ammonia; ace- tone-aceto-acetic acid ; and /3-hydroxy butyric acid. In the absence of exogenous phosphorus (fasting) we found the acidity (true and titratable), phosphates, acetone-aceto-acetic acid and total nitrogen, varied together. During the non-protein, post- fasting, period there was an increased H^ ion conc. and acidity, with- out accompanying increase in nitrogen excretion ; acetone and aceto- acetic acid were absent. The increased excretion of ammonia in fasting is correlated with that of i8-hydroxy butyric acid ; when not influenced by this factor, as in the preliminary, non-protein, and final feeding periods, the ammonia excretion fluctuated with the H"*" ion conc. and the acidity. The low ammonia excretion in the final period showed that the low H"^ ion conc. and titratable acidity re- sulted from a loss of fixed base. This phenomenon is apparently characteristic of recuperätion (nitrogen retention). It seems probable that increased nitrogen excretion during the early days of a fast in a human individual is related to metabolic processes that result in the excretion of aceto-acetic acid. III. TWENTIETH MEETING The twentieth scientific meeting of the Assoc. was held in the Biochemical Seminar Room, at the Columbia Med. Seh., at 4:^5 25 Most of the work was done in the Biochemical Laboratory at Teachers College. 26 Howe, Mattill and Hawk: Jour. Anier. Chem. Soc, igii, xxxiii, p. 568. Howe and Hawk: Proc. Amer. Soc. Biol. Chem., 1912, xii, p. 65; Jour. Biol. Chem., 1912, xi, p. xxxi. 224 Proceedinqs Columbia Biochemical Association [March, P. M. on Feb. 5, 191 5. The appended summary facilitates refer- ence to the abstracts (171-176) of the papers presented, pages 224, 227. A SUMMARY OF THE NAMES OF THE AUTHORS AND OF THE TITLES OF THE SUCCEEDING ABSTRACTS (171-176) A Edwin D. Watkins. Studies of some Allan C. Eustis. The detoxicating Compounds of cinchona alkaloids, effect of the Hver of Cathartcs certain metals and phosphoric acid. aura upon Solutions of /3-imidazo- (174) lylethylamin. (171) V. E. Levine and Herman Yahr. B Reductions with Compounds of the F. G. Goodridge. Biochemical studies rarer Clements: I. Ammonium mo- of mercaptan. (175) lybdate. (172) M. K. Thornton. Efforts to precipi- Max Morse. Autolysis and nuclear täte pepsin and erepsin with safra- relations. (173) nin. (176) A. ABSTRACTS OF PAPERS BY NON-RESIDENT MEMBERS 171. The detoxicating effect of the liver of Cathartes aura upon Solutions of iS-imidazolylethylamin. Allan C. Eustis. (Dep't of Dietetics and Nutrition, Coli, of Med., Tulane Univ.,-. New Orleans.) Published in this issue : Biochem. Bull., 1915, iv, p. 97. 172. Reductions with Compounds of the rarer elements. I. Ammonium molybdate. Victor E. Levine and Herman M. Yahr. {Lah. of Organic Chem., Fordham Univ. Med. Coli., N. Y. City.) Ammonium molybdate, in acid sol, heated with many organic Compounds, gives rise to a green, greenish-blue, or blue coloration. That this effect is due to reduction of the ammonium molybdate may be concluded from the following observations. Gaseous hydrogen produces the characteristic color, when led through a sol. of the molybdate acidified with hydrochloric or pref- erably with sulfuric. Acetylene, sulfur dioxid and hydrogen sulfid react similarly. Carbon mon-oxid gives negative results. Potas- sium iodid treated cold, and sodium bromid treated hot, with am- monium molybdate acidified with sulfuric acid, also produce a blue color. Ferrous and stannous Compounds can be distinguished from ferric and stannic, the former two giving the color reaction; the latter, not. Arsenious oxid yields a dark green color. Oxidizing 1915] Edgar G. Miller, Jr. 225 agents (hydrogen peroxid, sodium peroxid, nitric acld, potassium nitrate, manganese dioxid, potassium chlorate, potassium di-chro- mate, potassium permanganate) destroy the color or inhibit its formation. Furthermore, the color sometimes fades when the colored sol. exposed to the air is allowed to stand. Many organic substances reduce acidified sol. of ammonium molybdate. Sulfuric acid gives far better results than hydrochloric acid; phosphoric and nitric acids should not be used. To deter- mine the influence of the conc. of sulfuric acid upon the intensity of the reduction three reagents were prepared. Reagent A con- sisted of 30 gm. of ammonium molybdate, and 25 cc. of conc. sul- furic acid in i 1. of dist. water. Reagent B contained the same amount of molybdate, but 50 cc. of the acid per 1. Reagent C con- tained 100 cc. of the acid per 1. The sol. to be tested were heated in a water-bath for f rom 5 to 30 min. It was found that Reagent A yielded the most intense reductions, Reagent B gave weaker colors, while Reagent C gave negative results in most cases. The greater the conc. of sulfuric acid, the less sensitive the reagent. Many Compounds gave strikingly beautiful reactions. These are fructose and the carbohydrates that yield it by hydrolysis (su- crose, raffinose, inulin), rhamnose, arabinose, xylose, maitose, starch, glycogen, dextrin, agar-agar, amygdalin, salicin, esculin; the gums — benzoin, tragacanth, gambir, asafetida, guaiac, myrrh, kino and acacia; mucic acid, formaldehyde, trioxymethylene, ace- taldehyde, acetaldehyde-ammonia, reduced oxalic acid, erythro!, resorcinol, tricresol, hydroquinon, orcinol, tartaric acid, malic acid, tannic acid, amidol; Phenylhydrazin, (cold), /7-phenylene-diamin, benzidin and hydroxylamin hydrochlorids ; hydrazin sulfate, casein- ogen, osseomucoid and thiourea. Less intense reduction was observed with glucose, arbutin, mannite, aceto-acetic ester, chloral, benzal- dehyd, p-nitrobenzaldehyd, di-methyl aminobenzaldehyd, cinnamic aldehyd, salicylaldehyd, Cumarin, acetone, /?-amidoacetophenon, methyl alcohol, ethyl alcohol, glycerol, phenol, m- and />-cresols, thymol, <2-naphthol, phloroglucinol ; oxalic, citric, gallic, lactic, and uric acids; caffein, salicylic acid, asparagin, leucin, edestin, egg albumen, fibrin, collagen, gelatin, proteoses, serum protein, mucoid, ovalbumin, Creatinin zinc chlorid, nitrobenzol, lecithin, choles- 2 26 Proceedinqs Columbia Biochemical Association [March, terol, olive oil, olein, palmitin, Stearin. Still weaker reductions were obtained with vanillin, hippuric acid, /^-amino aceto-phenol, chloretone and camphor oxim. Benzene sodium sulfonate, potas- sium ethyl sulfate, palmitic and stearic acids, and urea, gave negative results. Potassium sulfocyanate yielded a red coloration with Reagent A. On diluting with water the liquid became greenish blue. When minute amounts of the sulfocyanate were used, the character- istic green or blue color, indicative of reduction, was observed at once. Miller and Taylor^'' found that acetone, acetaldehyd, benzal- dehyd, vanillin, glycerol, phenol, thymol, orcinol, phloroglucinol, salicylic acid, uric acid and tannic acid failed to reduce ammonium molybdate. They observed that although ketones and aldehydes did not reduce, ketone and aldehyde sugars did reduce. Our findings are not in accord with those of Miller and Taylor quoted above. Aldehydes, ketones, monosaccharids, disaccharids, Polysaccharids, gums, glucosides and glucoproteins and also other proteins reduced acidified sol. of ammonium molybdate. Egg albumen reduced even in the cold. Glucose reduced slightly in comparison with fructose. Lecithin, olein, palmitin and Stearin reduced, owing, probably, to the presence of the glyceryl radical, glycerol itself causing reduc- tion. Phenol reduced even in cold acetic or sulfuric acid mixture of ammonium molybdate. Uric acid gave positive results. The reactivity of ammonium molybdate in this respect is too general to be of value as a differential test. The study is in progress. 173. Autolysis and nuclear relations. Max Morse. {Dep't of PhysioL, Univ. of Wis., Madison.) There is a more or less direct relation between the character of an organ with respect to its nuclear content, from the histological Standpoint, and its rate of autolysis, whereby those organs in which there are relatively greater masses of nuclei to that of matrices (cytoplasm, interstitial sub- stance, protoplasmic differentiation in the form of fibres, etc.), such as glands, show greater rates of Salkowskian autolysis. Ac- cordingly, the hypothesis might be formulated that some connection exists between the distinctive chemical component, nucleic acid, and the rate of tissue-enzyme action. This hypothesis was tested by 27 Miller and Taylor : Jour. Biol. Chem., 1914, xvii, p. 531. I9IS1 Edgar G. Miller, Jr. 227 adding given amounts of liver, spieen and thymus nucleic acid^^ to pig liver hrei, and f ollowing the rate of autolysis by estimations of total nitrogen on tannic acid filtrates. As the accompanying table shows, there was no apparent modi- fication of rate of enzyme action, Doubtless the relation between nuclear component and rate of autolysis concerns the activity of Organs which are relatively richer in nuclei, such organs being in a more active State, metabolically. Table showing relation between percentage of thymus nucleic acid and rate of autolysis in pig liver brei as measured in terms of c.c. of n/5 NH3 for 25 c.c. aliquot portions of tannic acid filtrates Percent of sodium nucleate Initial 24 hr. 2 days 6 days 9 days 12 days Control 1.2 3-2 3-4 4.2 4-5 S-I o-S I.I 3-3 3-7 4.0 4.4 4.4 I.O 1.3 3-5 4-5 4.9 5-3 5-7 2.0 1-7 2.9 4.0 4.1 4-7 4-5 50 2.3 2.3 4.0 4.6 S-i 50 174. Studies of some Compounds of cinchona alkaloids, certain metals and phosphoric acid. Edwin D. Watkins. {Univ. of Tenn., Memphis.) Published in this issue: Biochem. Bull., 1915, iv, p. 94. B. ABSTRACTS OF PAPERS FROM THE COLUMBIA BIOCHEM. DEP'T 175. Biochemical studies of mercaptan. F. G. Goodridge. Mercaptan, when given subcutaneously to either cold or warm blooded animals, has marked anesthetic effects. The first result of the administration is irritation, and then follow promptly abolished reflexes and loss of consciousness. Respiration is at first increased and then slowed. The heart is rapid and feeble and, in wann blooded animals, the temperature is much reduced, and the color of the blood is changed to a dark brown. If the elimination by means of the breath is not prompt and thorough, the kidneys become im- paired, and acute parenchymatous nephritis supervenes. This con- dition causes death after an interval of from one to five days. When death follows promptly after the administration, it is prob- ably due to respiratory depression. 28 Jones and others have shown that all animal nucleic acids are undoubtedly identical, chemically. V 228 Proceedinqs Columbia Biochemical Association [March, Inhalation of mercaptan causes rapid and overwhelming results. Anesthesia is complete in less than a minute and, if the animal is not promptly exposed to the air, death follows quickly from re- spiratory depression. The administration of the drug per os causes nausea, vomiting and increased peristalsis. There is irritation and impairment of the kidneys, and these organs are rendered more permeable to the passage of glucose. This damage, as shown by the urinary find- ings, rapidly passes off and the kidneys return to normal. 176. Efforts to precipitate pepsin and erepsin with safranin. M. K. Thornton. Neither pepsin nor erepsin (unlike trypsin) was precipitated by safranin from gastric and intestinal extracts; er, if they were precipitated under the conditions of the tests, the products were inactive. The experiments are in progress. OFFICERS ELECTED AT THE EIGHTEENTH MEETINGS» HoNORARY OFFICERS. President — Prof. Alfred P. Lothrop, Queens University, Kingston, Ont. Vice-presidents — Prof. John S. Adriance, Williams College; Prof. Josephine T. Berry, University of Minnesota; Dr. E. Newton Harvey, Princeton University; Prof. Burton E. Livingston, Johns Hopkins University; Mrs. Jessie Moore Rahe, Cornell University Medical College. Active officers. President — Prof. A. J. Goldfarh; vice-pres- iderit, Dr. Alfred F. Hess; secretary, Dr. Edgar G. Miller, Jr.f"^ treasurer, Prof. William J. Gies. Additional members of the exec- utive committee — Dr. F. G. Goodridge, Prof. Paul E. Howe, Dr. William Weinherger. 29 See page 193. so Elected at the nineteenth meeting. See page 210. Biochemical Laboratory of Columbia University, College of Physicians and Surgeons, > New York. BIOCHEMICAL BIBLIOGRAPHY AND INDEX 8-10. Third and fourth quarters, 1914 (July-Dec); and first quarter, 1915 (Jan.-Mar.) WILLIAM A. PERLZWEIG and WILLIAM J. GIES (Biochemical Laboratory of Columbia University, at the College of Physicians and Surgeons, New York) Change in the plan of presentation. Publication of the current portions of our " biochemical bibliography and index "^ has been inter- rupted because of unavoidable delay in the issuance of this number of the Biochemical Bulletin (page 270). The European war has also afJected the bibliography and index, by reducing materially the Output of papers in, and numbers of, the European publications on our Journal list. This delay in publication, and the simultaneous curtailment of content, have suggested an improved plan for the presentation of the bibliography and index, which we inaugurate herewith, The chief improvement consists in the arrangement of titles in subject-groups rather than, as heretofore, in the order of the place- ment of the papers in the successive numbers of the respective Journals. Each title is placed under the subject-head that is suggested by the main feature of the content of the corresponding paper. Thus, papers consisting primarly of descriptions of methods are indicated coUectively under " Methods." This arrangement follows the general style of that proposed by the senior author some years ago for the Biochemical Department of Chemical Ahstracts, and which is still in vogue. The advantages of this arrangement are so obvious that a mere Statement of its adoption here is sufiEicient to suggest its purpose and its merits. Considerable space is saved by the use of small letters to indicate particular volumes of the listed Journals. The position and signif- icance of these letters are indicated below. The names of authors follow, in this new plan, the titles of the papers, whereas, in the preceding issues, the reverse order was the rule. The bibliography and index remain practically unchanged other- wise, both in style and scope. iQies: Biochem. Bull., 1913, ", PP- 298, 470, 559; Perlzweig: Ibid., 1913, iii, pp. 103, 315, ^75. 229 230 Biochemical Bibliography and Index [March, [Volumes. BZ: a = 65; b = 66; 0 = 67; d==68. ZpC : g = 92; h = 93. JBC: m = i8; n=i9; 0 = 20. BJ: s = 8. JACS: w = 36; 7: = 37. BB : y=:3. (See page 230.)] Explanation of abbreviations, arrangement, notation, etc. Bibliography, Titles of papcrs are f reely shortened, minor words ignored, common terms con- veniently abbreviated or chemical Symbols substituted ; surnames of collabora- tors are connected by hyphens ; most punctuation marks are omitted — all for the sake of condensation. Names of authors are printed in italics. Bibliographie items are separated by em dashes, and are preceded by numerals indicating, for index purposes, sequence in the bibliography. When two or more papers by the same author occur together, they are regularly numbered and separated by semicolons ; but follow one em dash. The numeral at the end of each item, separated f rom the name of the author or authors by a comma and a small letter, indicates initial page of the corresponding paper. The small letters between the names of the authors and the page numerals, at the ends of the items, represent specific volumes of the listed Journals, as indicated below: Biochemische Zeitschrift (BZ)^: a^65; b = 66; 0^=67; d = 68. Zeitschrift für physiologische Chemie (ZpC) : g = 92; h = 93. Journal of Biological Chemistry (JBC) : m = 18; n= 19; 0 = 20, Biochemical Journal (BJ) : s= 8. Journal of the American Chemical Society (JACS) 3; ^ = 36; x = 37. Biochemical Bulletin (BB) : y = 3. Index (subjects). The numerals in the index (page 239) correspond with the numbered items in the bibliography. Pages are not indicated. Numerals held in groups by hyphens are piain abbreviations in accord with the indications of the first numeral of each such series (see footnote, p. 239). Abbreviations of w^ords in the index are similar to those in the bibliography. Each group of index references is terminated by a semicolon ; commas mark off subdivisions of a general index subject. Names of authors are not indexed. Practical use of the bibliography. The bibliography is useful from sev- eral Standpoints. Thus, if it is desired to ascertain whether the Journals included in the bibliography contain papers (during the indicated period) on a particular subject, e. g., alcohol, find the key word in its alphabetic place in the index and turn to the items in the bibliographic sequence indicated by the index numerals [in this case 178, 180, 181, 183, 186, 205, 342, 351, 365]. The abbreviated items thus identified give the names of authors and suggest the nature of the corre- sponding papers (nine papers in the case selected for Illustration), and help the reader to decide w^hether to examine the original publications. When the index gives a negative answer to an inquiry, a large mass of literature in that connec- tion is removed from further consideration. During the intervals between pub- lication of the indexes of Journals, Zentralblättcr and year books, this running bibliography directs the reader to the main tracks through most of the current literature on the leading biochemical subjects. 2 Abbreviations employed at the tops of the pages. 3 Included in this bibliography for the first time. The section in the Journ. Amer. Chem. Soc. regularly devoted to biological chemistry is steadily growing in interest and importance. 1915] William A. Perkweig and William J. Gies 231 [Volumes. BZ: a = 65; b = 66; 0 = 67; d = 68. ZpC : g = 92; h = 93. JBC: m = i8; n=i9; 0 = 20. BJ : s = 8. JACS: w = 36; x = 37. BB : y = 3. (See page 230.)] Apparatus.— iBericht'g: Mik'resp'nsapp,i(?Hi^,m263.— 38Quant determ album urin,Fo/iM-Z>^m'j,m273.— 39V0I m tot S urin,i?airm-i?M&iXni297.— 4oPurif a melt point sat aliphat ac\d,Levene-lV e st, m463.—4iSimp m determ tot N co- lorim,GM/ic;fe,m54i.— 42Dry urin chem anal,Brömaw,nios.— 43Prep Ureas, determ urea,FaH5'Zy)^^-CM//^n,n2ii.— 44IS0I subst butter fat exerts stim infl grow,Mc- Co//Mm-I>az;w,n245.— 45Determ I connect thyr'd activ,i^en(fa//,n25i.— 46Determ sugar bl'd,5"/ia#(?r,n285.— 47Catal react bl'd :(i)Factors benzid test occult bl'd, Lyle-Curtman-Marshall,n445.—48Est fat feces,G^/'/iarf-Oo«^a,n52i.— 49Precip serum-alb a glutin by alk'oid reag,Han^/i^oi3.— SoFolin-Farmer m colorim estim N,Bock-Benedict,o47.—5iM sugar small quant hVd,Lewis-Benedict,o6i.— 52M inorg PO4 tissue a food product,C;ta/'üt-Powtc^,o97.— 53Est benzoic acid urin,i?at^m-Z)Mfcm,oi25.— 54Use col'd Fe determ lactos milk,Hi7/,oi75-— SSCo- lorim m am'acid N, Har ding -MacLe an, 0217. —s6U groups in P-lipin,Foj/^r,o403- — 57Correct: M inorg PO« tissue a food product,C/ja/'i»-PowJC^,o46i.— 58M est sugar bl'd,Gar(incr-MacZ^o»,s39i.— 59Produc w-hydrox-J-meth'furfurald fr carbohydrat a infli on est pentosan a meth'-pentosan,CMHHm5/tam-I>orcc,s438-— 232 Biochemical Bibliography and Index [March, [Volumes. BZ: a = 65; b = 66; 0 = 67; d = 68. ZpC: g = 92; h = 93. JBC: m = i8; n = i9; 0 = 20. BJ : s = 8. JACS: w = 36; x = 37. BB : y = 3. (See page 230.)] 6oQuant est aspart a glutam acid, prod prot hydrolForematiM^S- — öiRegulat mixt, recent md'icator (2), Walpole, 5628. — 62Est allantoin urin presence glucos, Plimmer-Skelton,s64\. — 63Grav est minute quant 'P,Raper,s6A9- — 64Determ As org mat,Fmo5rrad,wiS48. — 65lnvers sucros invertas :(8)M prepar streng invert sol fr top or bot yeast,//Mdjon,wi566. — 66Adsorp glucos bone-black,Mor^on, W1832. — 67Nat'l in6.\C2itov,Brubaker,viig2S. — 68Prep rsLBnos,Hudson-Harding, W2II0. — ögCompar m determ proteol activ pancr prep,Long-Barto7i,w2isi. — 70 Nat'l indicator,5rMfcaÄ'(?r,w2385.— 71IS0I cryst d/-glycer ald fr syrup by oxid g\yceTo\,Witsemann,w222S. — 72Quant extr diastas fr plant t{ssue,Thatcher-Koch, W2542. — 73Sep constit nat'l gas fr which gasolin condenst,Burrell-Scibert,x3g2. — 74Determ small quant HCN, Vie ho ever-Johns,x6oi.— 7 sQuant determ S pepton, Redfield-Huckle,x6o7.—76Amy\3iS : (9) Purif malt amy\,Sherman-Schlesingcr,x643. — 77Determ S cult med detec bacter produc HnS,Rcdfield-Hucklc,y.6i2. — 78Ab- derhalden serum test pTegn,Rosenbloo7n,y373. — 79Purif antigen Besredka serum diag tuherc,Bronfenbrenncr-RockmaH,y37S. — SoDiag value Landau test syph, Bronfenbrenner-Rockman,y377. — 8iSo-called protect ferm :(i)Sensitiz substr Abderhalden testBronfenbrenner-Mitchell-Schlesinger, ysSö. — 82Stand determ NH3 Kessler sol,i?oJc-Co/(?OTaH,y407.— 83Mic'-ureas determ urea, Rose-Coleman, y4ii. Colloids (general).—84Fibrin: (5) Bezieh Fragen Biol u Kordchem,//^fewm, 3311. — SsAdsorp, Quell u osmot Druck Kord,Po/anyj",b258. — 86Verteil u Schicks kol'd Ag Säugetierkörp(3),I/ot>^d477.— 87Temp'abhäng'k Au-zahl u Viskos kol'd Lös,Lichtwits-RenHer,gii2.—SSAdsorp Elek'lyt u kol'd Körp dur Casein, Palme,gi77. Salts and ions — 89Wirk Moderator (Puffern) b Verschieb Säu'-Bas'- gleichgew biol Flüs'keit,Ä'o/'/'e/-5/'2>o,a409. — 9oIon'perm u Membranpotent,i?o- hotiyi,h2Si. — pilrreziprok Charak Antagon zwisch Anion u Kation,Loeb,h277. — 92IS antag act salts due oppos charg ions,Loe&,n43i. — 93Antag betw acid a salt, Osterhout,nsi7. Carbohydrates (glucosids). — 94Säu'dis'konst KohVhyd,Michaelis,z36o. — 95 Zn-hydrox'-Ammon auf Rha.mnos,lVindaus-Ullrich,g276. — 96S0I polysac low fungi:(i)Mycodextran, new polysac P expans,Dox-Neidig,mi67. — g7ldem:(2) Mycogalactan, new polysac,Do.r-A''£?tdi5r,n235. — 98Mutarot phen'-osazon pentos a hex.os,Levene-LaForge,o42g. — 99Gelatinis pectin in sol alkali a alk earth,Haynes, S553. — lOoStruc maltos a oxid prod w alkal 'H.^,02,Lewis-Buckborough,w228s. — lOiP cont starch,T/io»?aj,y403. Lipins. — io2Pilzsterin:(i)Sterinähnl Subst Lykoperd gemmat, Ikeguchi, g257. — i03Lipoid mensch Serum u Meth Lip'bestim,Ä'/^m-I?iH^i/i,g302. — 104 Abbauprod Cholesterin tier Organ {QdWszu) {7), Lif schütz, gz^Z- — lOsOxyd Cho- lesterin d Blut(8),Li7jc/iMf^,h209. — io6Sphingomyelin,Lez'^M^,m453. — i07Cere- bron 2iCid,Levene-West,m477. — io8Mode oxid fat acid w brauch chain,i^a/'er,s320. — i09Acetone-sol phos'tid,MarL£'an,s453. — iioCompar betw molec weight Prota- gon, a Phosphatid and cerebrosid obtain fr \\.,Pearson,?,6i6. — iiiOil Ocym pilos roxb, Bhadiiri,wi772. — 112V0I oil Calycanth florid,Miller-Taylor-Eskew,w2i82. 1915] William A. Perhweig and William J. des 233 [Volumes. BZ: a = 65; b = 66; 0=167; d=:68. ZpC : g = 92; h = 93. JBC: m = i8; r\ = ig; 0 = 20. BJ: s = 8. JACS: w = 36; yi = 2,7. BB : y = 3. (See page 230.)] — 113H numb essen oil a product :(i)Oil Sassafras, anis, fennel, clove, pimenta, Albright, w2i88. — ii4Brain cephalin :(i)Distr hydrol prod ceph MacArthur,w22g7. Proteins (amino acids). — iisFix Metal d Prot,Benedicenti-RebelloAlves, Z107. — ii6Physik-chem Eig'schaf alko'lös Prot Weizen u Roggen,Gröh-Friedl, bi54. — iijEinw alko'l Sau auf Ei\veiss,Hersig-Landsteiner,c334. — iiBBericht'g: Am'säu dur H^SOi-hydrol Pf erdefleisch, Gayc?a,c504. — ii9Pepton(2, 3),Bernardi- Fabris,d436^ 441. — i2oMyxommucin,0.rwaW gi44. — 121 Am'säu ( i ) ,Geake-Nieren- stein,gi4g. — I220rgan S-verb, aus Prot Behand m HN03,Mö>n£?r,hi7S. — 123 Cystin-NOa u Cystin-HCl, Mörner,h203. — i24liistid,Kossel-Edlbacher,h2^. — 125 Vicin,L^z/^n^,m305. — i26Coag album pressure,Br{dgnian,nSii- — 127P in casein, Bosworth-P'anSlyke,n67. — i28Action diazometh on c3.stmog,Geake-Nierenstein. 5287. — i29Fibrin,5o^«;or;/j,09i. — i3oCond casein a salts in milk,VanSlyke-Bos- worth,oi3S- — i3iAppar form euglob fr pseudo-glob ; relat betw these prot,Chick, S404. — i32Transf glutam acid into /-pyrrolidonecarbox acid in aq so\,Foreman, S481. — i33Comp diff prot ox a horse serum, meth VanSlyke,//aW/^y^s54l. — 134 Histidin-like subst pituit (post. lobe),^/JnV/i,x203. Enzymes. — i35Wirk'beding Fepsm,Michaelis-Mendelssohn,zi. — i36Enzym Chelidon'sam (2) , Bournot, 2^140. — i37Erepsin, Kobzarenko ^344. — i38Hef ereduk- tas,Lz/o#,b440. — i39Ungleich Verbal Carboxylas u " Zymas " zu antisept Mittel, Netiberg-Iwano ff, ci. — i4oCarboxylas Saft obergär 'Htit,Ncuberg-Czapski,cg. — 141 Nucleas Serum Gravid u '^tp]\rit\k,Kotschneff,zi63. — i42Pflanz'enzym :(2)Amy- las KartoffelknoIle,I>o&y,ci66. — i43Enzym Versuch B. Delbrück, Euler-Cramer, 0203. — 144 Perm- u Antiferm-wirk S&r\xm,Hälsen,c277. — i45Einfl Säur auf dial Maltas, Kopacsewski,c2gg. — i46Bericht'g :Pflanz'enzym (2) , Do&y,c504. — i47Einw Serum u Am'säu auf Ureas,/aco&y-t7me(fa,d23. — i48Pflanz'enzym :(3)Pathol Veränd Kartoffelamylas,£'o&y--5od;idr,di9i. — i49Proteol Fermen,H^r^/^W,d402. — iSoChymos- u Pepsinwirk :(i)Gerin'geschw als Mass Chymos'meng.Hawjnar- sten,giig. — 151 Diasat wirks Subst aus Milchzuck ( 12), PaJ!irer,h3i6. — i52Dias- taspräp aus versch Kohrhydr(i3),Fan5:^r,h339. — i53Purin enzym orangutan (Sim satyr), chimpanzee (Atithropopith troglodyt),Wclls-Caldwell,miS7- — IS4 Mode action ureas a enzym in gen'l,Fow5"/y^^-CM//^n,ni4i. — issPurin enzym Opossum (Didelph virgin),Caldwell-Wells,n27g. — isöAct rennin on casein,i?oj- ivorth,n3g7. — i57Eff H-ion conc a inhib subst on ureas,VanSlyke-Zachar{as,ni8i. — iSSEnzym A. terricola,Scales, n4Sg. — iSgSpectr invest reduct Hb by tissue reductas,Harris-Creighton,oi7g. — löoHydrol glycog by dias enzym : (3) Factors infl endpoint hydrol,A''om^,S42i. — löiUreas cont Indian secd,Annett,s44g. — 162 Activ trypsinog,Ft^rnon,s494. — i63Action poisons on reductas ; attempt isol it, Harris-Crcighton,ssS5. — i64Rate inactiv by heat peroxidas milk(i),Zj7z^o,s656. — löslnvers sucros by invertas :(9)Is react revers,HMrfjon-Pam^,wi57i. — i66Enzym act : ( 1 1 ) Cast-bean uveas,Falk-Sugiiira,vf2i66. — i67Enzym alfalfa : (5), /oco&joh- H olmes, W2170. — i68Enzym act :( 12) Esteras a Hpas cast-bean,Fa/^-5'M5'mra,x2i7. — i69Amylas:(8)Infl acid a salt act malt a.myl3iS,Sherman-Thomas,x623. — 170 Enzym act : (i3)Lipas soy bean,Fo/^,x649. — i7iEnzym cent nerv syst,English- MacArthur,x6s3. 234 Biochemical Bibliography and Index [March, [Volumes. BZ: a = 65; b = 66; c = 67; d = 68. ZpC: g = 92; h = 93. JBC: m = i8; n=i9; 0 = 20. BJ : s = 8. JACS: w = 36; ^ = 37. BB : y = 3. (See page 230.)] Fermentation. — i72Durch abgetöt Hefe hervorgeruf Oxydat u Redukt auf Kost W3iSseT,Palladin-Lo'wtschinowskaja,ai2g. — i73Gärwirk frisch Hefe Gegenw Antipsept,A''eM&^r5r-A/'orrf,ci2.— i74Varaldeh- u Am'alkoholgär Meth'äth'brenz- tTauh'säu,Neuberg-Peterson,c32.—i75E{nü biol wicht Sau (Brenztraub'säu, Milchsäu, Äpfelsäu, Weinsäu) auf Vergär Trauh'zuck,Neuberg-Csapski,cSi.— i76Verhal a-Ketosäu z Mikroorg(i, 2),Neuberg,cgo, 122.— i77Reduk Zimtaldeh dur Hefe; Vergär Benz'brenztraub'säu, i?ona,ci37.— i78Alko'lgär :(7)Verarb Acetaldeh dur Hefe versch Vtrh:i\i,Kostytsche'W,g^02.—i7gFtvm HjO.-zersetz {e,),Waentig-Steche)^222,.—i?>6QrtnztTdLnh'sivi Aktiv alko'l Gä.r,Oppenheimcr, h235.— i8iBild Milchsäu bei alko'l GäiT{2),0ppenheimer)ci262.—id>2E\nvi Schim'- pilz-auf Alk'dgeh Opium,FnVc/nc/ij,h276.— i83lnhib autol b zXco'iWells-Caldwell, n57.— 184S0I polysac low fungi:(3)Infl autol mycodextran cont A. niger,Dox, 083. — i85Behav bacter purif anim a veg ^To\.tm,Sperry-Rettger,Oi\4S. Bacteria (fungi).— i86Einw Alko'l u Os auf bind Grup Bikttr, Thor seh, b486.— i87Vermehr B. Delbrück in lactos-bzw. glucoshalt Nährlös,Fa/wr,c209.— i88Einfl Temp auf Invert'gehal 'at{G^{3),Meisenheimer-Semper,c36^.—i^hss\m COj u N-verb dur Schim'pilz,Ä"oj5owtV^,c39i.— igoVerh Hefe u Schim'pilz zu Nitrat(i),Ä'o^Jowtc«,c400.— i9iGleichzeit Veränd Gehalt Glykog, N u Enzym in leb Hefe,ifM//&^rfir,g340.— i92Bemerk:/d^m,5a/*otü^^t,h336.— i93Form hexon a purin base autol Glomerella,Reed,n2S7.—i9A'M.tcha.n agglut bacter by specif sera, T«//oc/t,s293.— i95Chem nature bacter hemolysin,/if^m,s328.— i96Bacter metab (31-37), Kendall-Day-WaIker,wig37.—ig7ldem(38), Kendall-Day-W alker, vjig62. Food (drink).— i9SHundemilch,Gnwwi^r,d3ii.— i99Genn Milch, Löslichk Gerinnsel in Salzwass,OWa-/^»j^;f,h283.— 20oOrg PO, comp wheat-bran(2),^»- d^rjon,m42S.— 20i/rfew;(3)Inosit monophosph, new org P04,^"dc''^on,m44i. — 202Cause acid fresh milk, meth determ d.ciA'yVanSlyke-Bosworth,n73.—203 Relat Vitamin lipoid,Coo/'cr,s347.— 204Phosphotung precip fr rice-polish.DrMW- >nond-FM«;^,s598.— 205Methyl alco'l in corn silage,//ar^LaM!&,w2ii4.— 2o6Chem chang dur silage form,A''^id/5r,w240i.— 207Digestib maize consumed swine,GM(?rn- sey-Evvard,y36g. — 2o8Bleach flour,//a/fy,y440. Nutrition (metabolism) normal.— 209Energ'ums Marscharb: (2) Marsch- vers auf ansteig Bahn,5rcano-Aro/jn^r,ai6.—2io/(fem; (3) Gesetz Marsch auf ansteig Bahn,5r^^ma-i?^icÄ(?/,a35.— 211 Spezi f'dynam Wirk 'R'ihrstCserna-Kele- »new,b63.— 2i2Enthäl norm Harn frei Glycerin,Far^aj,bii5.— 2i3Verh Eiweiss- speich Leber bei ent u parent Zuführ versch Eiweissabbauprod,Ca/z«-5roH>ier, b289.— 2i4Kommen in Harn Kresol isom Subst, "Urogol" u " Urogon " vor, A^^ufe^r^f-C^a/' jiti,c28.— 2i5Glykol (4) ,Lö&,d368.— 2i6Chem Determ'ant Wachs» Funk-MacaUum,gi3.—2i7Qnt\ Thiosulfat Kannin'harn,5"a/^owjfet,g89.— 2i8Her- kunf Kreatin tier Organis : (2) Verhal e-Guanid-, e-Ureid- und e-Am'-n-capron- säu im Organis Kaninch,T/io«za^-Go^rn^,gi63.— 2i9Vorkom Carnosin,Meth'gua- nid u Carnitin Schaffleisch,5"OTorodw^ew,g22i.— 22oSchildrüs :(6)I-speich und I-bind im Organis,5/«m-GrMfi:rHer,g36o.— 22iRate disapp NH3 fr bl'd norm a thyroidect anim,/aro&JOH,mi33.— 222Basal gas metab norm men and women, Benedict-Emynes-Roth-Smith,mi3g.—223Con\.x\h bacter to feces aft diets free 191 5] William A. PerUweig and William J. Gies 235 [Volumes. BZ: a = 65; b = 66; 0 = 67; d = 68. ZpC : g = 92; h = 93, JBC: m = i8; 11 = 19; 0 = 20. BJ : s = 8. JACS: w = 36; k = 27- BB : y == 3. ( See page 230.) ] indigest compon,Osborne-Mendel-Hogan-Ferry,mi77. — ^224Creatin a creat'in me- tab:(3)0rig urin creatinß enedict-0sterberg,mig5. — 225Liver urea-form fr am'- add,Fiske-Sumner,m285. — 226Compar bioch purin metab : ( i ) Excr purin catab'it urin marsupial, rodent, ca.rmv,Hunter,m287. — 227 1 dem: (2) Excr purin catab'it urin ungul3ite,Hunter-Givens-Hill-Oberle,m403. — 228Behav sugar a lact acid bl'd flow fr liver, after tempor occlus hepat pedic,Macleod-lVedd,m447. — 229Act tissue meth glucosid, tet'-meth glucosid a nat'l disacch,Levene-Meycr,m46g. — 23oInfl prot intak form ur acid,Taylor-Rose,m5ig. — 23i\Jncol,Taylor-Adolph, m52i. — 232Creatin a creat'in,5"/faj^^r,m52S. — 233Brd fat:(i) Variat fat cont bl'd, norm condit,5/oor,ni. — 234Urea a tot non-prot N norm human bl'd : Relat conc to rate e\im,McLean-Selling,n2i. — 235Metab isom xanth, a isom meth'xanth, Goldschmidt, r)&2,- — 236Excr creat'in norm viomtn,Tracy-Clark,niis. — 237Carbo- hydr metab: (3) Infi subcut injec dextros a Ca-lact on bl'd sugar cont, a on tet aft thyr'parathyr'dec,C7nd20.— 27oVerbren Zucker Pankr'diabet (3) , Vercdr-Krauss,h48.—27iIdem : (4) ,Krauss-S einer, hsö.— 272ldem(5),Versär,h75.—273E'mü Ca-arm Nähr auf Zusam'setz wachs Knoch, W eiser, hgs—274Ca- u Mg-St'wechs b Hyp'chlorhydr,5'^<:cÄt,ci53.— 275Einseit 236 Biochemical Bihliography and Index [March, [Volumes. BZ: a = 65; b = 66; cz=67', d = 68. ZpC : g = 92; h = 93. JBC: m = i8; n = i9; 0 = 20. BJ: s = 8. JACS: w = 36; x = 37. BB : y = 3. (See page 230.)] Ernähr: (2) H„0-verteil u Ödembild b Salzzuf,Tac/iaM,c338.— 276Einfl mech Zer- stör Zellstruk a versch 0-proz Tiergeweb,5af;^//j-5"?^m,c443.— 277Cholest'- inst-wechs Fall angebor hämolyt Gelbsucht mit S^\tnomtg,McKelvey-Rosen- bloom,d7S.—27%Y orkom kryst nicht koag Eiweis Harn Magencarcinom,5"c/tMmm- Kimmcrle,gi.—27gN-v&ttn\. b Zufuhr NH^-salz od Harnstoi,H enriques-Andersen, g2i.— 28oPerman-intraven Injek Pepton u genuin 'Pto\.,H enriques-Andersen, gi94.— 28iAcetessigsäu'bild aus Glykolsäu Leber,Lo^fc,h270.— 282Chem glucon'- genesis:(9)Form glucos fr dioxyaceton diabet OTga.ms,Ringer-Frayikel,ra22,Z.— 283Metab relat aceton subst, Marriott, m24i. — 284Metab Bence-Jones proteinur, Fo/m-£>£?nw,m277.— 285Pentosur,L£'z;^M^-LaFor5r^,m3i9.— 286Eff acute destruc lesion liver on effic reduct NH3 cont hV6.,Fiske-Karsner,m2,B,i.—22>7¥a.rtnt util disacch sugar,//o5^an,m48s.— 288Brd acidos fr quant standp't,Mamo/?,m507.— 289Acidos omniv a herbiv, relat prot storage,Steenbock-Nclson-Hart,n3gg. — 29oExcr creat'in human individ prolong creatin-fr diet,Ringer-Raiziss,n4S7. — 291 Synth hippur acid exper tartr nephrit räb'ts,Kingsbury-Bell,o73. — 292Fat in bl'd hpemia,/?nnV,o87.— 293Theor diabet : (4) Paral betw effec pancreas a metal hydrox on sugar,fFood:va^^oi29.— 294Non-prot N-comp'd bl'd nephrit, spec ref creat'in a ur acid,Mycrs-Fine,o39i.—2gsRe\ amounts /3-hydroxybut acid a aceto- acet acid excr acetonur,X'^nHoway,s355.— 296Infl excess H^O ingest prot metab, 0?'r,s530.— 297Fast stud:(i4)Elim urin indic dur 2 fast over 100 days each, Sherwin-Hawk,y4i6. Physiology.— 298Wirk Prinz Hypoph,G'M(7£feHÄ^em,ai89.— 299Verh Serum geg nativ Placentaze\l,Wilhelm-Szandics,a2ig. — 30oHämocon,H^e//OTawn,a440. — 30iVork gerin'hem'd Subst weib Geschl'org u Placent,FM;«;b368.— 302Wirk Organextr auf iieTz:(i),Bürgi-vTraczewski,h4i7.—303Ergänz; Bloch Vagus- prob: (2), //^WOTe;^r,b437.—304Unters physik Hautwas'abgabe,Lo£'Wi>,c243.— 305 Luftballonfahr,ikfo/ir-Ä'MAn,c3o6.— 3o6Entsteh'weis Loew PupiVphön,Loewy-Ro- senberg,c323.—207Perspirat insensib norm u pathol Bedmg,Galeotti-Macri,c472. — 3o8Bericht'g : Wirk Prinz Hypoph,G'M£/5r^»Ac/wf,c504.— 309Physiol Blutgerin : (4),Hirschfeld-Klinger,di63.—3ioNeutvaVreg gravid Organis,Hasselbalch-Gam- meltoft,d2o6.—3iiPhysio\ Höhenklim : (2, 3),Hasselbalch-Lindhard,d26s, 295.— 3i2Temp'abhäng Quell Muskel u 'i^i&v,Lichtwits-Renner,gio4.—3i3Gt^aar Glu- kur'säu:(3)Spalt Orcin- u Phlorogluc'glukur'säu dur Organsäft,5'^ra,g26i.— 314 Verh Harnsäu zu Organextr,La«(fwaHW,g4i6. — 3i5Milchsäu- u PO^-bildg, Mus- ]<.t\'pv'sait,Embden-Griesbach-Schmitz,\ii.—3i6Yer\\ Milchsäu u POi-säu im Uteruspr'saft,CoÄM-M^:y^r,h46.— 3i7Einw Uteruspr'saft auf Hexos-PO^-säur, //a5r£'wa«n,h54.— 3i8Abbau Hexos-PO^-säu u Lactacidogen dur Organpr'säft, Embden-Griesbach-Laquer,hi24. — 3i9Muskelschwel, ivöröjy,hi54.— 32oNicht-Ex- ist "Uroleucinsäu",OjwaW,h307.— 32iConjug-H2S04 fr tendomucoid,Let^ene- LaForge,m237. — 322Bioch I:(i)Distr I plant a anim tiss,CameroH,m335.— 323 Size a compos thym gland.Ff^ßr^r.oiis. — 324Black pigm skin Austral black, Young,s46o. — 325Viscos bile,5Mrfon-0/'iV^,y35i. Immunity. — 326Bezieh Bind zur Wirk Komplem b Hämol, W^(?t7,a332. — 327 Abderhalden Dial'verfahr u Anaphylax,5'a/M^,a38i. — 328Antianaphyl Erschein b I9I5] William A. PerUweig and William J. Gies 237 [Volumes. BZ: a = 65; b = 66; 0 = 67; d = 68. ZpC : g=:=92; h = 93- JBC: m = i8; n=i9; 0 = 20. BJ : s = 8. JACS: w = 36; x = 37. BB : y = 3. (See page 230.)] homol u heterol Antihammelser Kaninch,For^jman-F^jr,b3o8.— 329Sensibil Wirk Porphyrin,//aMJmawn,c309.— 33oAntigen Eigensch tier Eiweis,5"a/MJ,c357-— 33 il^ie 3 Kompon Komplement, TAor Jc;i,d67.—332Ambocep u Recep : Geschw'immun : ( i ) , Mor^^nro^/i-5iV/inö',d85.— 333Anaphylac reac,5rad/iV;nc/i,dii8.— 352Bericht'g:Physiol Wirk prot'ogen Amin, Fanyje^,d350.— 353 Schick Papaverin tier Organis,Za/in,d444.— 354Verh Phen'hydrox'amin u Nitro- soderivimOrganis,S'iV&Mr£?,g33i.— 355Physiol Tag'schwank Blutzuck'geh Mensch unt Beeinfl dur Äth- u Chlor'narkos,J/jVjc/f,h355.— 356Synth hippur acid anim : (2), Synth a rate elim hipp acid aft benzoat ingest man,L^wi^,m225.— 357/'-Hy- droxyphen'eth'amin, press comp'd Amer mistletoe,Craw/ord-rFafana&^,n303.— 3S8Distr As human body,l/«d£'r/ji7/,n5i3.—359l^istr Hg acute HgCl^ pois'g,Rosen- fc/oow,oi23.— 36oCarbohyd metab :(8),Infl hydrazin utiHz dextros,Underhill- Hogan,o203.—36ildem:(9),lni^ hydrazin glyoxalas act \iver,Underhill-Hogan, 0211.— 362New physiol act deriv cholin,£wi«J,s366.— 363Bit'r princ com ragweed, A/'e/^oH-Craw/ord,w2536.— 364Cicutoxin: Pois princ water hemlock (Cicufa), Jacobson,x.gi6. Botany.5— 365Alko'l Gär höh Pflanz,Mt«en^ow,b467.— 366Phytoch Redukt iS),Neuberg-Welde,ci8.—367ldem:{6, 7),Neubcrg-Nord,c24.—36SIdem: (8, 9), Ar^M&fr5r-{^c/(f^,ci04.— 369Zichorie,Gra/?,di.— 37oN-halt Bestand Pflanz'gall : (i) ,Nierenstein,gS3.—37iWirk Chl'form auf Chlor'phylIassim,A'oröjy,hi4S.— 372 Col matter in seed-coat,^&r /;r^caior,Sarfear,s28i.— 3730ccur a signif Mn coat various seeds,Ai'cHar5rMe,w2532.— 374V0I oil genus Solidago,Miller-Eskew,w2538. — 375Tot am'-N in seedl Alaska pea,T/fOMi/'^o»,x230.— 376Chlorophyl,PFi7/jfä7f^r, X323.— 377EfT salicyl aldehy plants, soil and sol cult,5'/;t»n£7r,y390. Zoology.— 378Abhäng Gaswechs u Oxyd'geschw v 0-geh umgebend Medium beim Frosch,L(?j^er,a400.— 379Zusam'setz u physik Eigensch Ent- u Hühn'harn, Szalägyi-Kriwuscha,hi22.—38^Yevh Am'-säur Stoff wechs yögel,Scaldgyi-Kri- WMjcÄa,bi39.— 38iReakt ruh u arb Froschmusk,F(?cÄj^^fu,di40.— 382Fähigk norm 4 See also "Nutrition (abnormal)" and " Immunity." 5 See also "Fermentation" and " Bacteria (fungi)." 23^ Biochemical Bibliography and Index [March, [Volumes. BZ: a = 6s; b = 66; 0 = 67; d = 68. ZpC : g = 92; hc=93. JBC: ni=i8; n=i9; 0 = 20. BJ: s = 8. JACS: w = 36; x = 37. BB : y = 3. (See page 230.)] u fet Degenerat überleb Froschleber, Zuck zu bild,5"ra^di,d320.— 383Hautsekr Fisch :(i),Aalschleim,MM//£'r-i?,oi53.— 432Chondroit-H2S04 : (4),Levene-LaForge,o433. — 433 4-Br-6-ni- tro-w-cresol a (ienv,Raiford-Leavcll,wi4g8. — 434Partit N plant, yeast, a meat extr,Coo^,wiS5i.—435Hydantoin: (28), Synth 1,3,4-trisubs hydantoin fr dieth'- 1915] William A. Perhweig and William J. Gies 239 [Volumes. BZ: a = 65; b = 66; 0 = 67; d = 68. ZpC: g = 92; h = 93- JBC: m = i8; n = i9; 0 = 20. B J : s = 8. JACS: w = 36; x = 37. BB : y = 3. (See page 230.)] anilinmalonate,/oÄ?wow-5'/fc/'arrf,wi735.— 436Pyrimidin : (71 ), Synth pyrimid nu- cleosid, 4-hydroxyineth'urac./oÄ«JO»-C/ierno#,wi742.— 437Phosph 2,3-distearin, Renshazv-Stevens,wi770. — 438Pyrimidin: (72), Synth 4-hex'urac a relat urac'- glucosid,7o/i>uon,wi89i. — 439Synth prep rf/-glycer 3i\deh,lVitsemann,vfigoS. — 440 Addit comp org subst with H.SOi,Kendall-Carpenter,w24gS.—44iOpt rotat power, chem constit,lVesson,yv2S22. — 442Hydantoin :(29),Geom homjohnson- Harf/^y,xi7i.— 443Pyrimidin : (73) ,AlkyIat 2-mtrcsiptopynmidJohnson-Haggard, ■K177. — 444ldem : (74) , Synth 4-pheny\cytosm, Johnson-Hemingway,x378. — 445Hy- dantoin : (30) Stereoisom modif benzalhydant,/oÄ«^on-5a<^j,x383. — 446Infl temp on acid catal,Tay/or,x55i.— 4470rigin petrol,Afa&-hydroxyphen'-eth'amin357 ; hypoph298,3o8 ; hyp'chlor- hyd274; hyperglyc267. Immunity326to335 ; Indian-seedi6i ; indic'ors6i-7,70 ; in- dican24,297 ; indox'-H2S0424 ; inosit-monophosph20i ; intest243,putrefac258 ; in- volut390; invertas65, 165-^8,349; 145,220-46,322-37-41; ion89to93 ; Fe54,384; iso- electr4ii; isom442. Jaund277. Keton-acidi76; Kjeldahl-fl6; kidni2,3i2-36; Kronecker448. Lacmoid7; lacmosol7; lactacidogen32,3i8; lactat237; lact-acid 22,i75-8i,228,3i5-6-85-<5; lactos54,i5i-87; lamps4; lampyrid398; Landau-testSo ; larva390-6; Pb26; lecith2o; Iesion286; Limul 387-8-91 ; lipasi68-7o; lipem292; lipinsiO2toii4,247,lip'di03,203 ; liver2i3-25-- *. ENGE AND MEDICAL MEN* •''-^^■ S. J. MELTZER a4.W--W* The Chief aim of my remarks is to point out the unique Posi- tion which medical sciences and medical men occupy in the horrible war which is going on now between civiHzed nations. Interna- tional moraHty may possibly derive some permanent benefit from a conscious knowledge of this position. However, in order to make my point clear, I shall introduce it by a general discussion of some aspects of ethics. Moral philosophy assumes for granted that ethical relations of civilized men are safely established; it concerns itself merely with the question regarding the nature of the origin of ethical precepts. In general, it may be admitted that the vast majority of civilized men indeed do not question the correctness of ethical demands. But writers on moral philosophy fail to distinguish between intra^ national and int ermt'ionsil ethics. Hence, we find frequently that international occurrences are discussed from the point of view of intranational principles; international occurrences are brought be- fore the forum of a supreme court of the world for judgment, but the merits and demerits of the cases argued from the point of view of ethics which obtain in intranational moral relations. But the truth is that there is an abyss between the two domains of morality. * Address delivered at the fourth annual dinner of the Columbia University Biochemical Association, March 26, 191 5. 279 28o Intranational and International Ethics [June-September Let US first look at the Status of intranational morality. The ethical relations among civilized fellow-men, united by bonds of race, nation or country, are firmly established. Justice and duty are deeply rooted conceptions, the compelHng force of which is spontaneously recognized by all normal members of the individual Community; the small fraction of dissenters consists of defectives and criminals. Sympathy, kindness, altruism and self-sacrifice are not enforceable human virtues, but are nevertheless profoundly appreciated and admired by the individuals of all civiHzed nations. Honesty is an indispensable virtue. In parenthesis I may, how- ever, say here that to my knowledge "honor" is not among the general precepts of ethics. It is an artifact; it is mostly an arti- ficial virtue of a class which considers itself as being above the simple requirements of justice and duty. It is not an unusual occurrence that in the name of honor a man may slay with relative impunity a fellow-man whose home life he has dishonored. From Sokrates to our day students of moral philosophy offered various theories concerning the nature of the principles underly- ing the "science of conduct." I shall not discuss the merits of the theories of Hedonism or Utilitarianism, the Law of God or the Categorical Imperative; they do not concern us here. But I have to refer to one theory which was not received with great favor and which had only a short life of populär existence. In the latter half of the last Century, under the powerful influence of Darwin's theory of natural selection in the domain of biology, a systematic attempt was made by some philosophers (Herbert Spencer and others) to look upon ethics as a purely biological phenomenon. Family ties of lower animals, it was thought, developed into the ethics of civilized nations. Whether on account of the feverish social and altruistic activities which have been going on in the last decade or two and for which a biologic theory of ethics could hardly have served as a sufficient Stimulus ; or whether on account of the general decadence in populär enthusiasm for the theory of natural selection in general, the fact is that the theory of biologic origin of ethics seems to have been generally abandoned in recent years. But whatever we may think philosophically regarding the nature of fundamental origin of ethics, we can not deny that morality 1915] S' J- Meltzer 281 is subject to evolutionary influences; it has undergone and is contin- ually undergoing development. Morality manifests a continuous growth. The development of savage races into cultured, ethical nations is a matter of historical record. In fact, the progressive widening which conceptions like justice or duty are continually undergoing within the confines of a nation is practically a matter of direct Observation during an individual's lifetime. I shall dwell here especially on two elements which are operative in this process. The foremost factor in the evolutionary progress of intranational morals is to be f ound undoubtedly in the intellectual activities peculiar to man. The growth and development of the sciences, of arts, music, poetry, literature and religion, from their rudimentary phases into their present high states, elevated the spe- cific human character and favored the widening and deepening of morality of any individual nation or rather the morality of the individuals of which these nations are composed. The human in- tellect may or may not be the primary cause of morality; but the unfolding of human intelligence and the growth of intellectual activ- ities specifically human, are undoubtedly important elements in the growth and development of specific human morality. This con- nection between intelligence and morality is practically a matter of direct Observation. On this basis the further assumption is justified, that even the conscious primitive morality of primitive man did not make its ap- pearance abruptly. It developed very slowly, parallel, to a certain degree, with the development of man in the animal stage into man with rudimentary intelligence. I presume, then, that conscious morality did not begin abruptly, but developed very slowly, parallel with and assisted by the develop- ment and growth of human intelligence. However, important as the human intelligence may be, evidently it is not the only Controlling factor of morality. We see animals acting towards their fellow- creatures in a manner which, if seen in human beings, we would consider as highly ethical. We all know how animals care for their offspring. We see dogs licking the wounds of their fellow- dogs — an act resembling a samaritan service. We see altruistic activities in the communities of the bees and the ants. We desig- 282 Intranational and International Ethics [June-September nate these animal activities as instincts and we have indeed no evi- dence that a conscious morality is at the bottom of these phenomena. We have, however, to keep in mind that the harmonious relations between animals are observed only among individuals of the same species or race, or the same drove or swarm, whether they are pre- sided over by a bell-wether, a queen or any other single leader, or have a democratic form of government with several contending leaders. Animals belonging to different species, races or strains get frequently into ferocious fights as soon as they meet, or as soon as there is a coUision of interests and instincts. There are therefore sufficient reasons for assuming that the purely animal, instinctive dement is involved to a considerable degree in the moral relations between individuals of the same group of human beings which have some efficient bond in common. Now let US look at the moral aspects which international rela- tions present. The history of nations, civilized or uncivilized, con- sists chiefly of a tale of more or less ferocious wars interrupted by periods of peace. War is nothing but wholesale murder; but the men of one tribe or nation who are murdering men of another tribe or nation have no idea that they are committing crimes ; on the con- trary, the more civilized individuals among the fighters are honestly possessed by the conviction that they are performing a moral duty. It is true that in times of peace Citizens of one country enjoy in another country most of the Privileges enjoyed by the Citizens of that country. This is guaranteed by treaties. There are also inter- national laws which even presume to prescribe the mode of warf are among the signatory powers. In time of peace a sincere friendly intercourse frequently prevails between the individuals of various nations. There are numerous international reunions for the pur- pose of furthering human knowledge and general human interests in all lines of human endeavor. All these facts may give us the right to speak of international morality. Nevertheless, even peace, especially peace in modern times and among civilized people, is prac- tically nothing more than a truce during which nations are fever- ishly active in preparing for the next war, preparing to slaughter their apparent friends of to-day and to lead or to drive their own men to be slaughtered. During peace the leaders of nations are 1915] S. J. Meltzer 283 engaged in their military quarters or in their chancelleries in spying upon and intriguing against the nations with whom they exchange international amenities. In international dealings cunning and deceit are essential factors in success; it is diplomacy. Honesty has hardly a place in these dealings. Only honor is the big word which is loudly used hy those who speak for nations as units, that shain virtue in the name of which crimes are committed hy the privileged classes within each nation and in the name of which hundreds of thousands of honest and innocent Citizens of various nations are murdered or crippled for life in the groundless and senseless strife of nations, brought dbout by the amhitions of unprincipled leaders. Furthermore, inter- national relations in time of peace, which have an ethical appearance, are held together by flimsy ties. International peace Conferences, international law, and peace treaties are merely scraps of paper which are torn to shreds at first sight of a bone of contention be- tween nations. In a previous section I insisted, and I believe rightly, that intel- lectual growth and activity are most important factors in the devel- opment and growth of intranationa.\ morals. What is the value and influence of intellectual growth and activity in international morals? Highly intellectual, civilized nations fight one another with a rage, a ferocity and with an intent to kill as probably did their animal ancestors of different strains or races, hundreds of thousands of years ago. But different species of another type of animals, let us US say dogs and cats, are probably fighting to-day as their ancestors fought thousands of years ago, that is, tooth and nail, the only weapons at their disposal ; their physical agility, their promptly act- ing reflexes, the finer developed senses and their remarkable instincts did not help them in developing new weapons or new ways of fight- ing ; they had no human intellect. But the human race ? We need not go back thousands of years. It suffices to compare warfares separated only by a hundred years. I need not enter upon a com- parison of the rage, brutality and barbarity with which the wars are conducted ; in this regard the present war is surely not behind its predecessors, and none of the cultured belligerent nations is ahead of or behind the others. But as to destructiveness of human 284 Intranational and International Ethics [June-September life, that cardinal aim in the war of nations, the progress made in this comparatively short span of human history is immense; it reads like a fairy tale. From high in the air a human bird directs you to turn a micrometer screw one miUimeter or two and a huge shell annihilates hundreds or thousands of your enemy. A small group of human fishes bubble up in the vicinity of a huge leviathan, a dreadnought, and in less than ten minutes hundreds of men and mil- lions of dollars are fcrever at the bottom of the sea. In a Stretch of hundreds of miles, hundreds of thousands of soldiers are moved rapidly without a hitch from one place to another where they are needed most The success is wonderful. In barely eight months milHons of people were killed or crippled, perhaps as many more were made homeless and driven into starvation, and billions of dol- lars borrowed and wasted. And that astounding result was not accomplished as in olden times, merely by extraordinary physical force or endurance or by that virtue in which wild beasts greatly excel men, the virtue of physical courage; it was accomplished by specific human ingenuity. Mathematics, physics, chemistry and other theoretical and practical sciences have made these awful re- sults possible. In fact, practically every kind of intellectual activity took and takes a profound part in the bitter struggle which now goes on among highly civilized nations. Historians, philosophers, literary men and others are busy contributing offensive and ven- omous literature about their fellow-men of nations with whom their country is at war, whose friends they were and whose honors they enjoyed. Poets sing the song of profound hatred and musicians write the melody to it, or compose war marches and songs. Re- ligion offers an extraordinarily sad spectacle. Nations having the same religion and believing in the same God, pray to Him that He may help them destroy their enemy. Think of the robber and mur- derer who on his most godless errand prays to God for aid and guidance ! But here I must call your attention to a paradoxical but remark- able fact. Beastly as international morality is, when nations are at war, war nevertheless unquestionably elevates the intranational morality. The majority of Citizens in every country are not ideal- ists; in time of peace they comply with the laws of their country 1915] ^- J- Meltser 285 and fulfill their simple dtities, not more and not less. But when their country is at war, a new spirit comes over them ; they become altruists, they are ready to bring sacrifices, to lose their lives or to become cripples for life. Whether a country is right or wrong with regard to the merits of a particular war in the eyes of an outsider, a neutral, this has no bearing upon the moral Status of the man in his own country. That Status is unquestionably elevated during war, and even after the war his relations to his countrymen remain on a higher moral plane. Now let me recapitulate briefly. Human morality, whatever the nature of its origin may be, was and is subject to evolutionary influ- ences. It began in the pre-savage State of men. Its development has been and is a very slow process. In its present State we must sharply distinguish between intranational and international ethics; there is an abyss between them. Intranational morals attained a high State. Intellectual activities of all kinds were and are most important factors in its growth. The morality in international re- lations, on the other band, is generally low, and is frightfully bad when these relations are interrupted by war. War is an animal method of settling differences between two contending vicious spe- cies, and human intellectual activities greatly intensify the deadliness of the procedure. The efforts to create international laws for the purposeof restraining the ferocityof international struggles proved of little avail. We have cultured, civilized Germans, Frenchmen, Englishmen, and so on, hiit the world is not yet inhabited by cultured civilized men. Apparently biological processes are operative in these horrible differences between the intranational and international states of morality. Intellectual activity is capable of efficiently assisting in the development of morality among individuals which are allied by some organic and social bonds ; thus little or no resistance is offered to the beneficent intellectual influence. But individuals of different strains, with natural divergences and antagonisms, sustained by differences in education, customs, forms of law, etc., offer great resistance to the unifying influences of intellectual activity. Accordingly, biological traits common to all animals, while some of them may exert a favorable influence upon the evolution, rate of 286 Intranational and International Ethics [June-September growth and the direction of human morality, are surely not the main factors of its creation and development. On the contrary, in inter- racial and international relations many biological traits are pro- foundly inimical to a development of proper moral ideals. Struggle for existence, physical strength and dexterity, love of fight, hate, rage, bravery, etc., are traits which the human race has in common with wild beasts, and an uncontrolled cultivation of these traits may often prove disastrous to all human morality. On the other hand, intelligence and intellectual activities are traits which distinguish man from beasi. Their intense cultivation by civilized men has been the main cause of the high State of morality which prevails and is visibly progressing within the confines of civilized countries — the intra- national ethics. But now let us turn again to mf^rnational ethics. We have Seen that there is an abyss between international and intranational morality. We have seen further that war between civilized coun- tries brings in modern times incomparably more frightful results than in previous ages, which is undoubtedly due to the astounding discoveries and inventions brought to light by the intense intellectual activities in the various cultured countries. Are discoveries and inventions, are even apparently sound intellectual activities, danger- ous to international morality? Is this morality rather regressive instead of being progressive? And what can we do to make it progressive or to accelerate the imperceptible progress? The last question is the more important one, since it presents a practical and not merely an academic problem. In the following I intend to dis- cuss some factors which may contribute in some modest way to its Solution. I am fully aware, as all of you are, of the immensity of the problem, and I am aware, more than you, of the microscopical dimensions, metaphorically speaking, of your guest of the even- ing. But I shall act now as I always acted, upon the principle that it is neither good nor wise to possess less courage or more modesty than that drop of water which innocently and cheerfully undertakes to drill a hole in a rock. As one who swore allegiance to the medical tribe, I shall begin by saying that the case of international morals is very bad indeed, but it is by no means hopeless; that only hopeful men are capable 1915] 'S". /. Meltzer 287 of attaining desirable results; that a remedy which promises to bring some help, be it ever so small. is not to be despised, and that a sum of such reniedies mav save even a bad case. It seems to me quite probable that interracial and international morals are also subject to evolutionary influences and are undergo- ing a developmental process ; but the progress is extremely slow be- cause it has to struggle against the beastly nature of man. Even the development of intranational morality is a slow process; it must have taken many thousands of years before it reached its present stage. The present condition of international ethics would perhaps appear to us even quite high, if we had the means to compare it with its Status of hundreds of thousands of years ago. This recognition, namely, that interracial and international morals are undergoing a progressive development, but that their progress is necessarily very slow, seems to me to be a very useful one; because it encourages us to try to accelerate this progress, be the rate of the possible increase in the acceleration ever so small and be the means at our disposal for accomplishing it ever so meager. I do not consider it as my province to try to discuss here all sorts of means which possibly may serve to increase progress in inter- national morality. My chief purpose is, as stated at the beginning, to Dring torward the value of medical sciences and medical men EvS efficient factors in furthering the progress of international morality. However, before Coming to it, I wish to call attention briefly to a point or two to which reference has been made before. I believe, in the first place, that it is of prime educational importance to point impressivety to the fact- that th^re -is a gulf between national morality, on the one band, and interracial and international moral- ity, on the other band. A confusion between the two sets of ethics may härm the former and retard the possible progress of the latter. Citizens in neutral countries at a^I times, and Citizens of all coun- tries in times of peace, should know, should feel it deeply in their hearts, that war has not the slightest feature of morality, that it is simply a mode of settling differences between two or more strains of the human race in the fashion of wild beasts, increased in dead- liness and ugliness by the activities of human intelligence. Here is an incontestable fact which gives pain and distress to the moral man ; 288 Intranational and International Ethics [June-September humanity, as a whole, shows that its moral conduct is not above that of vicious animals of various species. The discussion of the ques- tion as to who began the war and who prevents its conclusion is far f rom the mark ; it is purely academic and is borrowed f rom the point of view of intranational morals. Justice and law had little to de with the beginning of the war and will have very little to say with its settlement. War is carried on by brüte force and is settled by it with the aid of exhaustion and starvation. The many circum- stances which lead to the numerous wars are mere incidents, but not the real cause of them. There is only one cause for all the wars and that is the possession by human beings of ferocious qualities pe- culiar to wild beasts, often entirely unrestrained and sometimes even directly cultivated to a higher degree. In teaching intranational morality it ought to he made clear that physical strength, coiirage, dexterity and efficiency, tiseful and de- sirahle as they are for the success in the life of the individiials and the nation they compose, are not moral principles. On the contrary, they may greatly magnify the evil residts when used for unethical principles. Bravery and efficiency, which are most highly valued qualities in war, are qualities which are most destructive to your s\o-called enemy of to-day and perhaps your friend of yesterday and, moreover, perhaps of your friend of a day after to-morrow. I now come to the chief point I wish to discuss. Short as the discussion will be, it is nevertheless the chief object of my entire dis- course. I have stated above that the striking feature of this war, the great destructiveness of human life, owes its success to the em- ployment of scientific results in carrying on the war. All sciences which may contain some practical dement are contributing in some way or another to the wholesale destruction of human life. And not only the scientific results, but the scientists themselves are active at the front in laboratories improvised in large automobiles to search for new inventions and discoveries which may be of some immediate practical use or to predict the nature of the weather to be expected at different points, etc. And those who can not assist in such a direct way try to contribute to the spirit of war by spread- ing enthusiasm, by abusing the enemy, and by implanting hatrea against it. I9I5] 6^. /. Meltzer 289 But there is one most inspiring exception to this sorrowful rule. It is the utilization of the medical sciences and the behavior of med- ical men in the war. The results of medical investigations of the last few decades and the activities of medical men are of immense practical importance to modern warfare. In some of the former wars perhaps as many soldiers were wiped out in consequence of disease as were killed by the bullet or bavonet. The combined modern studies in pathology, bacteriology, hygiene, surgery, medi- cine, pharmacology, preparation of antiseptics, etc., have immensely reduced the ra vages of war as far as sickness and injuries are con- cerned. Medical sciences and medical men are part and parcel of wars. But what is their ethical Status with reference to strife of nations in comparison with other sciences, with other men of sci- ence, men of culture and education ? Here is the answer. None of the numerous important discoveries made in the med- ical sciences was ever used for the destruction of life or harming the enemy in modern civilised warfare. Any discovery or invention made in the sciences or the practise of medicine, made in one of the warring countries, is freely given to the mf^dical fraternity of a belligerent country — unless it involves a business relation over which medical men have no power. It i<5 illuminating to read a review in an English medical Journal of med- ical reports made at a German medical meeting held on a battlefield. On the battlef.eld, on ihe firing line, perhaps in the midst of a hail of bullets and fragments of shrapnel, physicians and surgeons, some of thcm volunteers, pick up wounded soldiers without regard to nationality, and treai friend and foe alike. It is practically of no moment to the sick and wounded soldier to which of the hospitals of the civilised belligerent nations he will he taken for treatment. The physician, as a ptiysician, knows no difference between races and nations, between friend and foe. And withal physicians in every one of the warring countries are as good patriots, and are as ready to sacrifice their lives in their country's struggle, as any other patriotic Citizen of his beloved country, with the only difference that he, the physician, is merely ready to die, or to he crippled for life, in the service for his country, 290 Intranational and International Ethics [June-September but he is not engaged in killing or harming any one helonging to another nation or country. There might be a few exceptions — it would be miracnlous in- deed if there would be none; any large group has its exceptions. But such few exceptions can not be held up against this wonderful picture which medical men present in war. And wonderful indeed this picture is. We have seen how low international morality is at all times; we see how infamously bad it is in time of war and especially in the present ferocious war of cultured nations. And in the midst of this inferno we perceive a group of sciences which are in intimate contact with life and with war, and which nevertheless never contribute to the degradation of interracial or international morality. We perceive, furthermore, in every belligerent nation among the combatants a group of patriotic men, brave and ready for every self-sacrifice, who do nothing but render help to those who need it, who render it as members of their particular country, but render it to foe and friend alike. Here are representatives of humanity, as a whole, here is a most encouraging example of an elevated international morality. This wonderful fact is not my discovery; it is a fact well estab- lished, and well known to everybody, at least ought to be known by everybody. But the calling of this fact to fidl consciousness in the members of our profession may render a great service to the prog- ress of international morality. In the dawn of history, the medical man was also the exponent of philosophy and morals. In the middle ages when knowledge became specialized, medical men more and more devoted their ac- tivity exclusively to medical practise. On account of the inefficiency of medicine at that time, medicine lost its prestige. Recently, how- ever, medicine became a science and one marvelous discovery fol- lows another; and the efficiency of medical practise increases rapidly. Medicine makes inhabitable to man hitherto uninhabitable parts of the world. It prevents disease, and with increased effi- ciency it learns to eure it. Medical sciences and medical men have steadily risen in the estimate of discriminating civilized mankind. Could medtcal sciences and medical men not become again the standard-bearers of morals^ especially of international morality? 1915] ^- J' Meltser 291 In the furious struggle which is going on at present amongst civil- ized nations international morals lost its friends; religion, sciences, and the brotherhood of mankind proclaimed by the followers of socialism failed it; medicine alone did not desert it. In times of peace and for the purpose of furthering useful knowledge medical sciences and medical prart'ses are working in separate groups, ac- cording to their specific aims. But all medical men of various shades and groupings ought to unite for this one high aim, oiight to establish a Medical Brotherhood for the Purpose of Upholdinq and Accelerating the Progress of International Morality. Every one of the scientific and practical men in medicine in our large country ought to join with enthusiasm such a missionary enterprise. The initiative ought to be taken by our large neutral country, but we may appeal to our neutral brethren in other neutral countries to join our Crusade. However, we must not approach our medical confreres in the belligerent nations as long as the war lasts, lest it may be interpreted as an attempt to weaken their patriotism and their enthusiasm for the cause of the particular countries of which they are integral parts. Rockefeller Institute for Medical Research, New York City. MEDICAL BROTHERHOOD FOR THE FURTHERANCE OF INTERNATIONAL MORALITY Fraternitas medicorum F. M. OrIGIN, ORGANIZATION, PROCEEDINGS, REPORTED BY THE FiRST SecRETARY, WILLIAM J. GIES At the fourth annual dinner of the Columbia University Bio- chemical Association (March 26, 1915), which was attended by about 200 members and guests of the Biochemical Association, and at which he was the guest of honor, Dr. S. J. Meltzer deHvered the address that is published on the opening pages of this issue of the Biochemical Bulletin. In that address Dr. Meltzer proposed that " all medical men of various shades and groupings . . . ought to establish a Medical Brotherhood for the Purpose of Upholding and Accelerating the Progress of International MoraUty." At the conclusion of Dr. Meltzer's address the large assembly was invited to express its opinion, by a rising vote, on the desirability and feas- ibility of organizing such a Brotherhood. The vote was unani- mously in the affimiative. (See pages 263 and 267 of this volume of the Biochemical Bulletin.) Dr. Meltzer's address was published in Science (April 9, 1915; p. 515). Shortly thereafter Dr. Meltzer communicated with some of his colleagues regarding their personal willingness to participate in the Organization of the proposed Brotherhood and was heartily encouraged in his plans to effect its establishment. On July 3, 1915, Dr. Meltzer issued a statement, in part, as f ollows : 292 1915] William J. Gies 293 Dear Doctor : It gives me pleasure to in form you that the matter o£ establish- ing a Medical Brotherhood for the Furtherance of International Morality has now reached a satisfactory stage. I am, therefore, taking steps to form a definite Organization. More than 140 med- ical men, among them many of the most prominent and influential men in this country, have agreed to serve on the Committee that will issue an Appeal to the medical profession of this country. I enclose the list of the members of the Committee; it is probable that the final list will comprise 150 names. In response to an appeal by me, the Executive Committee of the Carnegie Endowment for Inter- national Peace has appropriated $1500 for carrying out the prelim- inary work, and it is hoped that other contributions will later be received. I am sending you a copy of the Appeal that will be sent to a large number of physicians in this country. Will you kindly read it and suggest any improvements in its wording that may occur to you. In Order to effect a promptly active Organization, I suggest that you authorize me to invite New York colleagues to serve on a pro- visional Executive Committee of 15 members. Please let me have your suggestions of names of such colleagues within the next ten days. At the expiration of this period we shall count the votes and the fifteen who receive a majority will be consid- ered as constituting the Executive Committee. A meeting of these men will then be called, at which a permanent Organization will be effected. Sincerely yours, S. J. Meltzer. On July 18, Dr. Meltzer forwarded to all of the members- elect of the provisional Executive Committee a note inviting them to meet him at the New York Academy of Medicine for the pur- pose set forth in the foregoing letter. Pursuant to Dr. Meltzer's call, as stated above, there assembled, in the Council room of the N, Y. Academy of Medicine, on Tues- day, July 20, 191 5, at 4.15 p. m., Drs. S. Josephine Baker, John W. Brannan, Harlow Brooks, Rufus Cole, John A. Fordyce, Nellis 294 Medical Brotherhood [June-September B. Fester, William J. Gies, Samuel J. Meltzer and Robert T, Morris. Dr. Meltzer took the chair, called the meeting to order and re- quested Dr. Gies to serve as temporary secretary. Dr. Meltzer stated the object o£ the meeting to be the Organization of the Medical Brotherhood for the Furtherance of International Morality, in har- mony with formal authorization to that end on behalf of the 150 men and women whose votes designated the membership of - the executive committee assembled at this meeting. Dr. Meltzer then stated that the colleagues named below, who favored the Organization of the Medical Brotherhood, had been designated, by pluralities of the votes cast in accordance with the terms of his invitation dated July 3, 191 5 (as copied above), to serve as an Executive Committee and to proceed with provisional Organization of the proposed Medical Brotherhood : Robert Abbe J. A. Fordyce Graham Lusk S. Josephine Baker Nellis B. Foster Samuel J. Meltzer J. W. Brannan Wilham J. Gies Robert T. Morris Harlow Brooks S. S. Goldwater William H. Park Ruf US Cole Abraham Jacobi John A. Wyeth It was voted that a provisional Organization be effected by the election of officers to serve for a term of one year, or until the elec- tion of their successors. The following officers were then elected : I. EXECUTIVE COMMITTEE (Residents of the City of New York) A. Active Officers PRESIDENT: S. J. Meltzer, Member, Rockef eller Institute. VICE-PRESIDENTS : Rufus Cole, Director, Rockef eller Hospital; S. Josephine Baker, Director, Bureau of Child Hygiene, De- partment of Health. FIRST SECRETARY : Wm. J. Gies, Professor of Biological Chem- istry, Columbia University. SECOND SECRETARY: Harlow Brooks, Professor of Clinical Medicine, University and Bellevue Hospital Medical College. TREASURER : Robert T. Morris, Professor of Surgery, Post-Grad- uate Medical School. igi5] William J. Gies 295 B. Councillors Abraham Jacobi, Professor of Diseases of Children, Emeritus, Colum- bia University. Robert Abbe, Surgeon, St. Luke's Hospital. John Winters Brannan, President, Board of Trustees of Bellevue and Allied Hospitals. J. A. Fordyce, Professor of Dermatology, College of Physicians and Surgeons. Nellis B. Foster, Assistant Professor of Medicine, Cornell University Medical School. S. S. Goldwater, Commissioner, Department of Health. Graham Lusk, Professor of Physiology, Cornell University Medical School. William H. Park, Professor of Bacteriology, University and Bellevue Medical College. John Allan Wyeth, President, Polyclinic Hospital. IL ADVISORY COMMITTEE A. Honorary Presidents Rupert Blue, Surgeon General, U. S. Public Health Service, Washing- ton, D. C. W. C. Braisted, Surgeon General, U. S. Navy, Washington, D. C. Russell H. Chittenden, Director, Sheffield Scientific School, Yale University. W. T. Councilman, Professor of Pathology, Harvard Medical School. W. C. Gorgas, Surgeon-General, U. S. Army, Washington, D. C. W. S. Halsted, Professor of Surgery, Johns Hopkins Medical School. W. H. Howell, Professor of Physiology, Johns Hopkins Medical School. Abraham Jacobi, Professor of Diseases of Children, Emeritus, Colum- bia University. W. W. Keen, President, International Surgical Congress; President, American Philosophical Society, Philadelphia. Edward L. Trudeau, Saranac Lake, New York. James Tyson, Professor of Medicine, Emeritus, University of Penn- sylvania. Victor C. Vaughan, Professor of Hygiene and Physiological Chem- istry, University of Michigan. WiUiam H. Welch, President, National Academy of Sciences; Pro- fessor of Patholog}^ Johns Hopkins Medical School. 296 Medical Brotherhood [June-September B. Honorary Vice-Presidents J. J. Abel, Professor of Pharmacology, Johns Hopkins Medical School. Herman M. Biggs, Commissioner, State Board of Health, New York City. Frank Billings, Professor of Medicine and Dean, Rush Medical College. Clarence John Blake, Professor of Otology, Emeritus, Harvard Medi- cal School. W. B. Cannon, Professor of Physiology, Harvard Medical School. W. H. Carmalt, Professor of Surgery, Emeritus, Yale Medical School. George Dock, Professor of Medicine, Washington Medical School, St. Louis. James Ewing, Professor of Pathology, Cornell Medical School. Wilfred T. Grenfell, Medical Missionary. Alice Hamilton, Expert on Occupational Diseases, Federal Bureau of Labor Statistics, Washington, D, C. Ludvig Hektoen, Professor of Pathology, Rush Medical College. Howard A. Kelly, Professor of Gynecology, Johns Hopkins Medical School. George M, Kober, Dean, Georgetown School of Medicine, Washing- ton, D. C. Robert G. LeConte, President, American Surgical Association, Phila- delphia. Rudolph Matas, Professor of Surgery, Tulane University. William J. Mayo, Rochester, Minnesota. Chas. K. Mills, Professor of Neurology, University of Pennsylvania. John B. Murphy, Professor of Surgery, Northwestern Medical School. E. L. Opie, Professor of Pathology and Dean, Washington Medical School, St. Louis. Charles A. Powers, Professor of Clinical Surgery, Emeritus, Uni- versity of Colorado. W. L. Rodman, President, American Medical Association, Phila- delphia. G. E. deSchwelnItz, Professor of Ophthalmology, University of Penn- sylvania. Henry Sewall, President, Association of American Physicians, Denver. F. C. Shattuck, Professor of Medicine, Emeritus, Harvard Medical School. igis] William J. des 297 C. Additional members of the Advisory Committee Isaac Adler, Consulting Physician, German Hospital, New York City. Fred H. Albee, Professor of Orthopedic Surgery, New York City. Carl L. Aisberg, Chief, Bureau of Chemistry, Washington, D. C. James M. Anders, Professor of Medicine, Medico-Chirurgical College, Philadelphia. John F. Anderson, Director, Hygienic Laboratory, Washington, D. C. John Auer, Associate Member, Rockefeller Institute. Edward R. Baldwin, Saranac Lake, New York. Helen Baldwin, Attending Physician, New York Infirmary for Women and Children, New York City. J. C. Bloodgood, Associate Professor of Surgery, Johns Hopkins Medical School. George Blumer, Professor of Medicine and Dean, Yale Aledical School. Joseph A. Capps, Associate Professor of Medicine, Rush Medical College. A. J. Carlson, Professor of Physiology, University of Chicago. Henry Dwight Chapin, Professor of Medicine, New York Polyclinic Hospital. Henry A. Christian, Professor of Medicine, Harvard Medical School. Frank S. Churchill, Associate Professor of Pediatrics, Rush Medical College. C. G. Coakley, Professor of Laryngology, N. Y. University and Belle- vue Hospital Medical College. S. Solis Cohen, Professor of Clinical Medicine, Jefferson Medical College. Warren Coleman, Professor of CHnical Medicine, Comell Medical School, Joseph Collins, Neurologist, Neurological Institute, New York City. T. S. Cullen, Associate Professor of Gynecology, Johns Hopkins Medical School. Elizabeth M. Cushier, Montclair, N. J. F. X. Dercum, Professor of Nervous and Mental Diseases, Jefferson Medical College. Theodore Diller, Associate Professor of Clinical Neurology, Uni- versity of Pittsburgh. Samuel G. Dixon, Commissioner, State Board of Health, Philadelphia. Isadore Dyer, Dean, Tulane ]\Iedical School, New Orleans. D. L. Edsall, Professor of Clinical Medicine, Harvard Medical School. 298 Medical Brotherhood [June-September C. A. Eisberg, Surgeon, Mt. Sinai Hospital, New York City. Henry L. Eisner, Professor of Medicine, University of Syracuse. Joseph Erlanger, Professor of Physiology, Washington Medical School, St. Louis. Harold C. Ernst, Professor of Bacteriology, Harvard Medical School. Henry B. Favill, Professor of Clinical Medicine, Rush Medical College. Charles H. Frazier, Professor of Clinical Surgery, University of Pennsylvania. T. B. Futcher, Associate Professor of Medicine, Johns Hopkins Medical School. Fielding H. Garrison, Editor, Index Medicus, Washington, D. C. Arpad G. Gerster, Professor of Clinical Surgery, Columbia University. Virgil P. Gibney, Professor of Orthopedic Surgery, Columbia Uni- versity. Joel E. Goldthwait, Orthopedist, Boston. H. A. Hare, Professor of Therapeutics and Diagnosis, Jefferson Medical College. R. A. Hatcher, Professor of Pharmacology, Cornell Medical School. I. Minis Hays, Secretary, American Philosophical Society, Phila- delphia. Yandell Henderson, Professor of Physiology, Yale Medical School. F. P. Henry, Professor of Medicine, Woman's Medical College of Pennsylvania, Philadelphia. James B. Herrick, CHnical Professor of Medicine, Rush Medical College. A. W. Hewlett, Professor of Medicine, University of Michigan. J. W. Holland, Professor of Physiological Chemistry, Emeritus, Jef- ferson Medical College. C. F. Hoover, Professor of Medicine, Western Reserve University. John Howland, Professor of Pediatrics, Johns Hopkins Medical School. Reid Hunt, Professor of Pharmacology, Harvard Medical School. Woods Hutchinson, New York City. Holmes C. Jackson, Professor of Physiology, N. Y. University and Bellevue Hospital Medical College. George W. Jacoby, President, Neurological Society, New York City. Theodore C. Janeway, Professor of Medicine, Johns Hopkins Medical School. Philip Mills Jones, Editor, CaHfornia State Medical Journal, San Francisco. 1915] William J. Gies 299 Frederic Kammerer, Professor of Clinical Surgery, Columbia Uni- versity. Frederic S. Lee, Professor of Physiology, Columbia University. E. Libman, Physician, Mt. Sinai Hospital, New York City. Howard Lilenthal, Surgeon, Mt. Sinai Hospital, New York City. Hanau W. Loeb, Dean, St. Louis University Medical School. Warfield T. Longcope, Professor of Medicine, Columbia University. F. J. Lutz, Professor of Clinical Surgery, Washington Medical School, St. Louis. W. G. MacCallum, Professor of Pathology, Columbia University. Ward J. MacNeal, Director of Laboratories, Post-Graduate Medical School, New York City. James F. McKernon, President, Post-Graduate Medical School, New York City. Albert P. Mathews, Professor of Physiological Chemistry, University of Chicago. Chas. H. Mayo, Rochester, Minnesota. L. B. Mendel, Professor of Physiological Chemistry, Yale University. Walter Mendelson, Trustee, Columbia University, Joseph L. Miller, Associate Professor of Medicine, Rush Medical College. Rosalie S. Morton, New York City. F. G. Novy, Professor of Bacteriology, University of Michigan. Albert J. Ochsner, Professor of Surgery, University of Illinois. Stewart Paton, Neurologist, Princeton University. Richard M. Pearce, Professor of Experimental Medicine, University of Pennsylvania. Howell T. Pershing, Professor of Neurology, University of Colorado. G. M. Piersol, Editor, American Journal of the Medical Sciences, Philadelphia. W. M. Polk, Director, Cornell Medical School. Joseph H. Pratt, Instructor in Medicine, Harvard Medical School. W. A. Pusey, Professor of Dermatology, University of Illinois. J. J. Putnam, Professor of Neurology, Emeritus, Harvard Medical School. David Riesman, Professor of Clinical Medicine, University of Penn- sylvania. Beverley Robinson, Professor of Medicine, Emeritus, N. Y. University and Bellevue Hospital Medical College. M. J. Rosenau, Professor of Preventive Medicine, Harvard Medical School. 300 Medical Brotherhood [June-September B. Sachs, Neurologist, Mt. Sinai Hospital, New York City. W. T. Sedgwick, Professor of Biology, Massachusetts Institute of Technology. P. A. Shaffer, Professor of Biological Chemistry, Washington Medical School, St. Louis. Frank F. Simpson, Pittsburgh, Pa. Allen J. Smith, Professor of Pathology, University of Pennsylvania. Winford H. Smith, Superintendent, Johns Hopkins Hospital. Torald Sollmann^ Professor of Pharmacology, Western Reserve Uni- versity. Thomas L. Stedman, Editor, Medical Record, New York City. C. G. Stockton, Professor of Medicine, Buffalo Medical College. Mabel Ulrich, Minneapolis, Minnesota. A. Morgan Vance, Louisville, Kentucky. Albert Vanderveer, Vice-Chancellor, University of the State of New York, Albany, N. Y. George B. Wallace, Professor of Pharmacology, N. Y. University and Bellevue Hospital Medical College. Richard Weil, Assistant Professor of Experimental Therapeutics, Cornell Medical School. H. Gideon Wells, Professor of Pathology, University of Chicago. J. Whitridge Williams, Dean, Johns Hopkins Medical School. Ray L. Wilbur, Professor of Medicine, Dean, Leland Stanford Junior University. J. C. Wilson, Professor of Medicine, Emeritus, Jefferson Medical College. Martha Wollstein, Associate, Rockefeiler Institute. Hiram Woods, Professor of Ophthalmologe, University of Maryland. Jonathan Wright, New York City. The officers were instructed to proceed, with all necessary or desirable measures, to enlarge the membership of the Brotherhood and to distribute, to that end, copies of the following appeal, as originally circulated by Dr. Meltzer: An Appeal to the Men and Women Engaged in Medical Prac- tice and the Advancement of Medical Sciences The present horrible war among civilized nations has brought out impressively certain sad f acts : that although there are civilized 1915] William J. des 301 individual nations, we are still very far from having a civilized humanity — there is an abyss betvveen mfranational and international morality ; that, no matter how cultured and enlightened nations may be, they still settle their international differences by brüte force, by maiming and killing their adversaries ; and, finally, that the present high development of science and invention in individual nations only serves to make the results of this war more destructive than any other in history. The war has demonstrated, however, one encouraging fact; namely, that among all the sciences and professions, the medical Sciences and medical practice occupy an almost unique relationship to warfare, and that among all the Citizens of a country at war, medical men and women occupy a peculiar and distinctive position. No discovery in medical science has been utilized for the pur- pose of destroying or harming the enemy. Medical men in each of the warring countries are as courageous, as patriotic, as any other Citizens, and are as ready to die or to be crippled for life in the Service of their country as any other class of their fellow country- men. Their Services, however, consist in ministering to the sick ancl to the injured, and in attending to the sanitary needs. Further- more, they often risk their lives by venturing into the firing line to bring the injured to places of safety and to attend to their immedi- ate needs. hi these heroic and humanitarian acts friend and foe are treated alike. Finally, the majority of the members of the medical profession and of the medical Journals of the neutral as well as of the warring countries, abstain from public utterances that might be grossly offensive to any of the belligerent nations. These facts — this advanced moral position in international rela- tions which medicine and its followers are permitted to occupy in all civilized nations — ought to be brought to the füll consciousness of the men and women engaged in the medical sciences or in medical practice. Such a realization could not fail to have an elevating in- fluence upon the medical profession itself, and would probably exert a favorable influence upon the development of international morality in general. At the dawn of history, medical men were frequently also the exponents of philosophy and morals. In the middle ages, when 302 Medical Brotherhood [June-September knowledge became specialized, medical men more and more devoted their activity exclusively to medical practice. Because of its in- efficiency at that time medicine lost its prestige. In recent times, however, medicine is becoming an effective science ; one marvelous discovery has followed another, and the efficiency o£ medical prac- tice has been rapidly increasing. Medicine makes habitable to man hitherto uninhabitable parts of the world. It prevents disease ; and, with increasing theoretical and practical efficiency, medicine is learn- ing to alleviate and eure disease and prevent injuries. Medical sci- ences and medical men have steadily risen in the esteem of civilized mankind. May not the medical sciences and medical men become again the Standard bearers of morality, especiaUy of international morals? To accomplish these objects, it is proposed to organize as large and effective an Association as may be possible, of men and women engaged in the medical sciences or in medical practice, under the name of THE MEDICAL BROTHERHOOD Für the Furtherance of International Morality It is obvious that such a Brotherhood could not exercise an im- portant influence at once. But our modest expectation for prompt results should not prevent us from attempting now to take the first Step in the riglit direction. Many important results have often had small beginnings. A committee of physicians and medical investigators regnest you herewith to enroll as a member, and to declare your ivillingness to endorse and siipport the moral Standard which the medical profes- sion generaUy upholds when called lipon to perform its patriotic duties in international strife. It should be expressly understood that it is not the object of the proposed Brotherhood to influence the feelings and views of anyone regarding the problems involved in the present war. It is desired merely to bring to the füll consciousness of the members of the medical profession the exceptional moral position which all civilized nations, even while at war, perniit and expect medical men to occupy, at least so long as they remain in the medical profession and act in this capacity. This consciousness cannot fail to elevate 1915] William J. des 3^3 the moral Standards of physicians. Furthermore, after the close o£ the present war, the Brptherhood could without doubt f acilitate the reunion of the members of the medical profession of all the nations which are now at war and increase good feeling among them. A humanitarian body such as this proposed Brotherhood, if already in existence and ready for service, might and could be of the greatest usefulness in many ways. The foregoing Appeal, signed by the members of the Executive and Advisory Committees, as listed above, has been widely circu- lated among physicians and others engaged in the advancement of medical sciences. Any reader of this Statement of the objects and proceedings of the Medical Brotherhood, who may be eligible for election to mem- bership and who, not having enrolled as a member, desires to join the Brotherhood, is hereby invited to communicate with the Presi- dent, or the Secretary, or any other officer. Biochemical Laboratory of Columbia University, College of Physicians and Surgeons, New York. RESULTS OF STUDIES ON VITAMINES AND DE- FICIENCY DISEASES, DURING THE YEARS 1913-1915* CASIMIR FUNK CONTENTS Page I. Introduction 304 IL Beriberi 308 III. Scurvy and infantile scurvy (milk problem) 323 IV. Relationship between beriberi, scurvy and Pellagra 326 V. Pellagra 327 VI. Sprue 333 VII. Rickets 334 VIII. Chemistry of cod-liver oil 336 IX. Osteomalacia, spasmophilia, eclampsia 33^ X. Chemistry and physiology of growth 339 XL Growth in plants 348 XII. Influence of diet on the growth of tumors 34^ XIII. Concluding general considerations 35^ XIV. Bibliography 354 I. INTRODUCTION In my book on vitamines and deficiency diseases, which appeared early in 1914, and in several other articles püblished about the same time, these subjects were reviewed very extensively. The present ar- ticle is written with the purpose of reviewing the progress of the work in this field since the pubHcation of my book. For the benefit of any who may not be famihar with this particular branch of science, however, the chief data obtained prior to 1914 will also be briefly summarized. The subject of vitamines owes its existence to results of the study of beriberi, a disease occurring in oriental countries where rice is used as staple food. This disease was first regarded either as an * An abstract of this review constituted an address by the author, at the 22nd meeting of the Columbia University Biochemical Association, at the Columbia Medical School, April 9, 1915 (Biochem. Bull., 1915, iv, p. 266). A review of further developments in the study of vitamines, since April, 1915, will be püb- lished by the author in a late issue of Volume V of the Biochemical Bulletin. 304 1915] Casmiir Funk 305 intoxication or an Infection. The remarkable increase in the fre- quency of its incidence during the past twenty-five years suggested to several excellent workers in tropical medicine that the greater number of recent cases has been due to the introduction of modern machinery for the decortication of rice. It was shown that in cer- tain parts of the Malay States and India, where either hand-milled rice or parboiled rice (rice steamed previous to decortication) is used, the incidence of beriberi is much less frequent than in parts where machine-milled rice is used as food. A suspicion arose that with the removal of the superficial layers of the rice grain, an in- gredient is lost which is essential for the maintenance of life on a rice diet. This lost constituent was regarded, at first, as a kind of antidote, or antitoxin, against hypothetical intestinal poisons pro- duced during the digestion of rice. Uncertainty continued until 1897, when Eijkman observed that fowls, which had been fed on residues of food supplied in a hospital with beriberi patients, developed a disease that closely resembled human beriberi. This condition in fowls was called Polyneuritis gallinarum. This very important discovery paved the way for the experimental investigation of beriberi. Eijkman found, also, that alcoholic extracts of rice-polishings cured experimental Polyneuritis in chickens. He believed the effects of such extracts were due to the presence of an antidote. Schau- mann, in 1910, advanced another theory, which was based on the alleged fact that foodstuffs able to eure Polyneuritis contain high percentages of organic phosphorus. He concluded, therefore, that beriberi is due to lack of organo-phosphorus Compounds in the food. In accord with this view, he found that yeast is an excellent curative agent. The phosphorus-deficiency theory was advanced at a time when there was a widespread belief that lipoids had im- portant physiological and pharmacological properties. Recently it has been shown that the animal organism is able to synthesize lipoids and other organo-phosphorus Compounds f rom phosphoric acid, pro- vided the remaining constituents (radicals) are available. We now know, also, that the importance hitherto attributed to lipoids was dependent very largely upon substances of basic nature which occurred incidentally in lipoid f ractions. The phosphorus-deficiency 3o6 Vitamines and Deficiency Diseases [June-September theory of Schaumann had to be abandoned when it was shown, in my early experiments with yeast, that, after hydrolysis for 24 hr., with 20 percent sulfuric acid sol., yeast retained its curative prop- erties. The experimental data indicated that the active substance was comparatively simple in chemical nature, more or less basic in character and, to some extent, thermostable in acid Solution. On the assumption that the active substance contains nitrogen, the alcoholic extracts of different foodstuffs were fractioned by means of the usual methods for the Separation of organic bases. Chemical attention, however, was chiefly devoted to extracts froni yeast and rice-polishings. It was found that vitamine was precipi- tated with phosphotungstic acid, partially with mercuric chlorid in alcoholic sol., and with silver nitrate and baryta, the latter precipita- tion proving to be the best for the Isolation of vitamine. The cura- tive fraction obtained in this way was very small — between 3-5 gm. from 100 k. of dry yeast, or locx) k. of rice polishings. This fraction, administered orally or subcutaneously to beriberi pigeons, exhibited the following effects : The animals recovered very speedily, often in 2-3 hr., but it was found impossible to keep them permanently on polished rice even when injections were repeated every few days. By further f ractioning the curative material from yeast, three substances were obtained. One was definitely identified as nicotinic acid. The second substance, when completely purified, proved to be inactive but represents without doübt a new chemical substance; this is now undergoing a complete investigation. The third substance was obtained only in traces. None of these three products, given either separately or together, showed anything like the action of the original fraction. Thus far rice-polishings, as we shall see later in this resume, have yielded nothing in this connection but nicotinic acid. It does not seem improbable that this substance is a decomposition product of an unstable vitamine. It is not surprising that little has been achieved in the elucidation of the chemical structure of these puzzling substances. In their study unusual experimental difficulties are encountered which will be discussed below. Even a relatively simple problem like the chemical structure of adrenalin required a series of years for Solution. 1915I Casimir Funk 307 These preliminary results had an importance that exitended be- yond their application to beriberi. The substances isolated from yeast or rice-polishings, were regarded of vital importance, hence the name given to them, vitamines, a conception that found its com- plete justification in the ntimber of pubHcations, on this subject, dur- ing the last three years. Vitamines have proved to be constant con- stituents of the diet, eqtial in importance to proteins, carbohydrates, fats and salts, and not replaceable by any of these. Plants are evidently able to synthesize vitamines. The animal organism possesses considerable synthetic capacity, as has recently been shown, but itssupply of vitamines is obtained from the vege- table kingdom, a rule from which there are no known exceptions. Every animal so far investigated in this respect, when kept on an artificial but chemically definite diet, or on a purified one, exhausts its störe of vitamine and gradually declines. Beriberi is not the only disease due to dietary deficiency of vitam- ines. Many other diseases, such as scurvy, infantile scurvy, Pel- lagra, rickets, sprue, several nutritional disturbances in infants,* a metabolism disease in cattle called " lamziekte," etc., are due to deficiency of vitamines, and have been grouped under the name of "deficiency diseases" or avitaminoses. These diseases have one character in common : they are due to partial or entire deficiency of vitamines in the food. Such deficiency causes very profound changes in the functions of the central nervous System. All other Symptoms, such as gastric symptoms, changes in the bones, influence on the heart and skin, etc., are only the results of the changes in the central nervous system. It is easy tö realize, there fore, that these diseases are more easily prevented than cured. The changed nerves (fatty degeneration) cannot be restored to the normal condition after a certain stage of the disease is passed. This is an important point for attention in the study of " deficiency diseases." We come now to our task of reviewing the whole subject as it developed during the years 1913 to 1915, inclusive (to date).* The distribution of the chapters accords with the arrangement in my book on vitamines, and brings the latter work up to date. * Received for publication, May 24, 191 5. The author will review, for publi- cation in the October, 1916, issue of the Biochemical Bulletin, the further developments in vitamine research to October, 1916. [Ed.] 3o8 Vitamines and Deficiency Diseases [June-September IL BERIBERI In spite o£ numerous proofs that beriberi is a " deficiency dis- ease," a number of papers deal with the subject from a different point of view. Most of these papers have no scientific value and may be disregarded; only those will be reviewed which are based on sound Observation or on experimental evidence. Human beriberi. Now and then cases of beriberi, and even epidemics of this disease, are reported in which the point is empha- sized that no polished rice was eaten. This fact is cited against the Vitamine theory. We know, however, that rice as such is not the cause of the disease. White bread, sago, and, in general, any food that is naturally poor in vitamines, or is rendered deficient in them by cooking (either too prolonged or under pressure) or by extrac- tion, is apt to cause beriberi. This point must be clearly understood. The most prominent paper opposed to the vitamine theory of the etiology of beriberi is that of Caspari and Moszkowski (i), who consider beriberi a disease of toxic origin. They based their opinion chiefly on the results of experiments on animals. They found that addition of egg to the diet prevents the onset of the disease in ani- mals kept on polished rice, but increase in the quantity of rice pro- voked the disease despite the addition of egg. They seem to believe that eggs contain an antidote f or poison formed from rice, but, as we shall see in the section on the "physiology of vitamines," their re- sults can be explained on the basis of the vitamine theory. Similar opinion was expressed by Abderhalden and Lampe (2), who found that pigeons on a diet of cooked rice developed beriberi later than those kept on raw rice. Their explanation is that during the cook- ing a poison was eliminated from the rice. Although the Observa- tion of these authors was confirmed in my experiments, their con- clusion is quite wrong, as we shall see. Practically all the evidence in support of the view that beriberi is a "deficiency" disease was obtained in studies on pigeons and fowls. Several authors have tried to prove that although avian beri- beri (Polyneuritis) is a "deficiency" disease, it has nothing in com- mon with human beriberi. This opinion has been frequently stated, especially by Japanese authors. Thus, Shibayama (3) believes that the protective substance from rice polishings is very much less effec- iQisl Casimir Funk 3^9 tive in human beriberi. Tasawa (4) also concludes that vitamine has no effect in human cases. Segawa (5) considers the avian dis- ease identical with the human, but regards both as due to an intoxi- cation. Why vitamine was found by Japanese authors to be in- effective in human cases is difficult to understand. Possibly this failure was due to selection of cases for treatment that were too advanced in their anatomical changes to be curable. Vedder and WilHams (6) prepared a vitamine-fraction from rice poHshings, following my early method, and report very good results in human cases, especially of "dry" beriberi. They re- mark that for the wet form of beriberi, the results were not so conclusive. They assume, therefore, the existence of several vitam- ines, in this connection, a conception which to my mind is pre- mature, since both forms of the disease may be very different in the severity of the Symptoms. It is interesting to note the exact composition of diets which have caused the outbreak of beriberi. Dubois and Corin (7) de- scribe a small epidemic in the Belgian Congo, which began 4-5 months after a rice diet was instituted. The population there received a weekly addition of f resh meat to the rice diet, in a quantity which apparently was insufficient to prevent the disease. Very instructive was the outbreak of alleged beriberi on the converted cruiser " Kron- prinz Wilhelm," a large number of the crew having taken the dis- ease, although apparently they subsisted on a normal diet. A very large amount of frozen meat was available, which had been taken from captured ships from Argentine. It seems stränge, at first thought, that beriberi occurred under these conditions. No cases of beriberi were observed, however, among the officers of the ship. Careful inquiry showed that the officers received daily, in addition to the ordinary food, a certain amount of fresh fruit. This latter fact classifies the disease, it seems to me, as so-called ship-heriheri, a disease on the borderline between beriberi and scurvy, but more like the latter, and occurs on ships where sufficient quantities of fresh provisions are not available. Avian beriberi (Polyneuritis gallinarum) . A series of papers dealt with the pathology of avian beriberi. Vedder and Clark (8) de- scribed an excellent study of Polyneuritis gallinarum. Schnyder (9) 3IO Vitamines and Deficiency Diseases [June-September reported that very little nerve degenerätion occurred in birds, and that most avian cases were cured by rice-polishings, showing, in his opinion, that the paresis is not due to degenerative changes in the nerves. Segawa (5) described two distinct forms of avian beriberi; one, a simple Polyneuritis; a second, more like inanition with marked aversion to a rice diet. He finds, however, that in 66 per- cent of the cases both forms occur together. This investigation was conducted on fowls, some of which remained in good health for 219 days on a polished-rice diet, a result that was due, in my opinion, to the probäbility that the animals picked up other food than rice. The most marked pathological changes occurred in the peripheral nerves, in accord with previous findings. Intestinal catarrh, as a secondary Symptom, and degenerätion in the parenchymatous organs, were also observed. The same results were obtained with pigeons. Tasawa (4) found that the Symptoms of starvation in birds can be eliminated by adding egg to a polished-rice diet; in this case a picture of pure Polyneuritis is obtained. He confirmed my results showing that cane sugar is able to produce beriberi in birds ; that rice- polishings heated to 120° C. lose their protective power, and that potatoes exert very marked prophylactic action. Eijkman (10) Claims that the disease in fowls :s different f rom that in pigeons, and considers that only fowls develop typical Polyneuritis, He injected a mixture of one part of sodium chlorid and three parts of potassium chlorid (20-40 mg.) into chickens and pigeons, and observed eures in pigeons but not in chickens. I have repeated these experiments with pigeons (Funk 11) and, as one would expect, no eures followed this treatment. Cooper (12) has continued his studies of the amounts of food- stuffs which, in addition to polished rice, are able to prevent the onset of the disease. In addition to his previous work on the protec- tive power of muscle, sheep-brain, fish-meat, egg-yolk, lentils and bar- ley, he investigated the foodstuffs named in the summary on p. 311. Portions of these foodstuffs were added to the daily diet. On the basis of the results of these experiments, Cooper concluded that Vitamine alone is not sufficient to induce maintenance of body weight, a conclusion that, as we shall see in the section on the "physiology of vitamines," seems to be entirely erroneous. Gib- 1915] Casimir Funk 311 FoodstuSFs Amount necessary for prevention Wet (natural) Dry grams grams Ox cerebrum 6 1.2 Ox cerebellum 12 2.4 Ox liver 3 0.9 Cow milk > 35 > 3-5 Nuts (husked filberts) — 2.0 Cheese > 8 > 5-6 son (13) finds that human milk is less protective than cow milk; also, that compensated salt mixtures (often o£ calcium and sodium tartrate) delayed the onset of Symptoms and rendered the degeneration of nerves less pronounced. Ohler (14) and Weill and Mouriquand (15) confirmed the findings of Hill and Flack (16) who found that altho white bread caused beriberi in fowls, tk3 latter remained healthy on whole wheat bread. Ohler finds, also, that fowls remain well on whole corn but not on hominy (the inside of the corn kernel) ; in the latter case they develop the same condition as that on white bread, a very interesting Observation to be discussed further under pellagra. Merklen (17) describes a peculiar disease in ducklings (3-4 weeks old) with Symptoms of cramps and paral- ysis of the legs. These Symptoms disappeared when the diet was variable. Possibly the disease was beriberi. AUeged beriberi and beriberi-like diseases in other animals, especially mammals. The question whether other animals than birds and man are suitable for the study of beriberi is still open to discussion. It is undoubtedly true that no animal is able to live on polished rice or any vitamine-free food, but it seems probable that the condition described in monkeys, dogs, mice, rats, rabbits and guinea-pigs is either scurvy or a condition of general weakness. None of the conditions described (even the fatty degeneration of nerves) classifies the disease as beriberi. The symptom usually ob- served is a peculiar weakness of the bind legs which can hardly be interpreted as beriberi. Schnyder (9) found that the disease in mice, dogs and cats is etiologically and chemically the same as in birds, but not pathologically, for there is very little nerve degeneration. Empirically, I have divided animals into two groups : those in which the terminal purin metabolism results in allantoin; those in which uric acid is the final product. In the first group are monkeys, 312 Vitamines and Deficiency Diseases [June-September dogs, cats, goats, rabbits, guinea-pigs, mice, rats and horses and cows. In these animals beriberi has not yet been produced — it occurs only in animals in which uric acid is the terminal product of purin metabolism (e. g., birds and man). It is undoubtedly true that "deficiency" diseases of great practical importance occur in cattle. These diseases, which are chiefly found in cows and are called "lamziekte" (and perhaps "stijfziekte") in South Africa, rickets in Australia and Stallmangel in Germany, resemble rickets and osteomalacia more closely than beriberi. The problem of " lamziekte " is so important f or South Africa that a special research laboratory was created a few years ago, under the direction of Dr. Theiler, to investigate this disease from the view point of avitaminosis. A clinical description of the disease appears in my book. A disease similar to " lamziekte " was described by Scheunert, Schattke and Lötsch (i8), and by Lötsch (19), in horses and cattle in poor districts of the mountainous region in Saxony, where in winter only very poor and restricted diets are available for these animals. The disease called "Stall- mangel/' which closely resembles rickets and osteomalacia, is a very serious metabolic disease, with pathological changes in the bones. The fodder fed to these animals was very deficient in calcium, mag- nesium and phosphorus, but Scheunert is inclined to consider that this disease is due to deficiency of vitamines Oseki (20), in Hofmeister's laboratory, investigated the food value of different restricted diets in mice. When put on wheat bread or on barley, a cereal frequently used in infants' food, the mice died in 20 days, without special Symptoms and without any sign of fatty degeneration in the nerves. On maize flour they lived 60 days. Whole meal rj^e bread was excellent food for mice, whereas oat meal, and meal prepared from peas and beans, were not satisfactory. When rye meal was extracted with water, the total extract very rapidly improved the condition of diseased mice, but the ash from the extract had only a slight beneficial effect. Extraction of rye meal with alcohol or ether had less deleterious efifect on its food value than extraction with water; extraction with acetone was also without effect. By fractionation of milk, the protective substances were found in the buttermilk but not in the butter. Tachau (21), iQiS] Casimir Funk 3 1 3 in the same laboratory, investigated also the effects of restricted diets on mice, but from a slightly different Standpoint, his experi- ments resembling, in certain respects, some of my own (to be de- scribed later) on the influence of additions of different food con- stituents. As a Standard diet whole-meal bread was chosen, to which either salts, fats, or carbohydrates, were added. The mice died very soon. Addition of sodium chlorid or other salts caused very marked edema, much like this condition in infants. After adding cane sugar neither edema nor diarrhea was observed. The same applies to palmitin, but in this instance the food intake was markedly smaller. Tachau explains this phenomenon as due either to an aversion of these animals for such kinds of food, or to im- paired resorptive power of the intestine. He suggests that the utilization of sugar, for instance, is dependent on the intake of proteins; in other words, that the constituents of the diet must be well balanced. Chemistry of vitamines. Rice-polishings. Several Japa- nese authors have tried unsuccessfully to isolate the active substance from rice-polishings. Such attempts were described, for instance, by Mural (22) and Kondo (23). A very exhaustive study was published by Vedder and Williams (6). They repeated the frac- tionation of alcoholic extracts of rice-polishings, following my early method, with silver nitrate and baryta, and obtained a crystallin curative fraction. Some of the other conclusions of these authors are very interesting and confirm my Statements with regard to yeast, namely that alcoholic extraction removes the Vitam- ine only very incompletely, and that the vitamine is not very stable in the presence of fixed alkali. They found, also, that hydrolyzed extracts are very much more active than non-hydrolyzed ones. Drummond and I (24) made a very systematic investigation of both hydrolyzed and non-hydrolyzed alcoholic extracts of rice- polishings. This work was undertaken less with the view of isolat- ing Vitamine than with the aim of separating new cleavage prod- ucts of the latter. We also attempted to isolate the substances described by Suzuki, Shimamura and Odake. The latter attempt failed entirely; vitamine could not be precipitated with picric acid, and no substances were found of the type of the a- and )S-acids de- 3^4 Vitamines and Deficiency Diseases [June-September scribed by these authors. Among the substances detected with certainty were betain, nicotinic acid, cholin, guanin and adenin. Guanidin was probably also present. We also isolated a product which, in spite of several recrystallizations, had a constant melting point and was either a mixture or a very unstable Compound of betain and nicotinic acid. This combination could not be isolated by crystallization but was obtained either by extraction with hot alcöhol or by precipitation with copper acetate. It was also found that the substance isolated by me from the vitamine fraction, to which the formula C26H20O9N4 had been ascribed, and which on analysis showed the composition of nicotinic acid, was really nicotinic acid as suggested by Barger (25). Spinal cord. Voegtlin and Towles (26) prepared extracts from spinal cord and made the very interesting Observation that alcoholic extract of fresh cord has less effect than that from cord which, previous to extraction, is allowed to autolyze f or 2 days. One cc. of extract corresponding to 4 gm. of dry cord was enough to eure pigeons but insufficient to maintain their body weight. Yeast. Schaumann (2y) fractioned yeast with methods used for the Separation of Phosphatids. Yeast was extracted with 96 percent alcohol. The residue after evaporation of the alcohol was partially soluble in ether. The ethereal sol. was precipitated with acetone. The precipitate was curative. It was incomplete, how- ever, some of the vitamine remaining in Solution. The vitamine, according to Schaumann, is present in yeast in several different un- stable combinations. In the same paper Schaumann describes his tests of seeds of Phase olus radiatus. It was found that they lose their protective power with storage without undergoing any visible microscopic changes. Based on my hydrolytic study of yeast, which proved that com- pletely hydrolyzed yeast retains its curative power, Cooper (28) performed some experiments on autolysis of yeast. Fresh brewer's yeast was left for 36 hr. at 35° C. The autolyzed product was as strongly curative as the original yeast. To this mixture 95 percent alcohol was added and the liquid filtered. The filtrate was precipi- tated with basic lead acetate. Practically all the vitamine was found in the filtrate. The drying of yeast at 20° C, previous to autolysis. igis] Casimir Funk 315 did not diminish the curative power; and no toxic effect was db- served from the administration of the autolytic products, even in ten times the curative dose. During the past year I have continued the investigation of yeast, the chief aim having been the improvement of available methods. After many more or less unsuccessful attempts to isolate pure Vitam- ine from such a complicated mixture as that in the alcoholic ex- tract of yeast, it seemed desirable to devise a method that would separate the bulk of the impurities in one Operation, at a stage where the Vitamine is present in stable combination, and would also avoid the use of alkali, which destroys most of the vitamine. The experi- ments were conducted on the alcoholic extract of yeast and on auto- lyzed yeast. "Alcoholic extracts." On treating, with acetone, the phospho- tungstate precipitate from alcoholic extract of yeast, about 10 per- cent of the total precipitate remained insoluble. The soluble frac- tion, which formed about 90 percent of the total, was entirely free from vitamine. The insoluble fraction, after decomposition with neutral lead acetate, was very active. The liquid, freed from ex- cess of lead and evaporated in vacuo, left an entirely crystallin residue. With this preparation curative tests were performed on pigeons, with the results recorded below in the section on " physiol- ogy of vitamines." This preparation is still a mixture of sub- stances, however, the bulk of it being adenin. After the Sepa- ration of adenin some inactive, crystallin substances were eliminated with platinic chlorid and picrolonic acid. Finally the vitamine frac- tion was obtained by means of mercuric chlorid in alcoholic sol. Autolysed yeast. As already recorded in one of my early papers, alcoholic extraction presents the disadvantage of being very incomplete, most of the vitamine remaining in the residue. On the other hand it offers the advantage that a large proportion of the impurities remain behind, Fractionations were therefore conducted with autolyzed yeast which, according to Cooper, contains the same amount of vitamine as original yeast. Here, also, the acetone- method in its original form was applied, but then another difficulty arose. The acetone-insoluble residue from the phosphotungstate precipitate amounted not to 10 percent, as in the case of the alco- 3IÖ Vitamines and Deficiency Diseases [June-September holic extract, but to 34 percent. This was a more compllcated mix- ture. I then took advantage of an Observation which was made in collaboration with Mr. Drummond. We prepared a series of phos- photungstates f rom several natural, chemically pure, bases : cholin, betain, nicotinic acid, stachydrin, guanin, adenin, guanidin and Creatinin. The solubility of these phosphotungstates, estimated in mixtures of acetone and water of various concentrations, differed considerably, indicating that the method may be of use for their Separation. Solubility did not increase, however, as one would expect, with increase in the proportion of acetone, but increased in the direction of the arrows in the f ollowing sequence : 25% -» 100% -> 50% -^ 75 %. By applying these findings to the fractionation of autolyzed yeast, i. e., by treating the phosphotungstates obtained with the successive concentrations of acetone mentioned above, the f ollowing percentage results were obtained, expressed in terms of the total precipitate : Percentage of acetone as solvent .. ..25 100 50 75 Residue Percentage of dissolved matter 46.4 16.8 13.7 9.6 13.3 We see, then, that by applying this modified acetone-method to autolyzed yeast, the residue is only slightly greater than that for the alcohol-method. The results so f ar obtained indicate that most of the Vitamine, even in the case of autolyzed yeast, is contained in the insoluble residue. The results are very promising since, even in the case of autolyzed yeast; 86.7 percent of the impurities can be re- moved in a single Operation, altho the Separation does not seem to be as complete as it is in the case of alcoholic extract. Physiology of vitamines. Relationship of vitamines to LIPOIDS. One of the chief promoters of the idea that lipoids are indispensable for life was Stepp. This is a misconception based on the Observation that food extracted with alcohol is rendered inade- quate for life. Although the Observation was quite correct in itself, the mistake in the conclusion was due to the fact that, at that time, everything soluble in so-called lipoidal solvents was assumed to be lipoid. It has been very difficult to overcome the influence of this generally accepted though erroneous idea. Lately, however. Stepp iQiSi Casimir Funk 3^7 (29) has changed his attitude ; he agrees, now, that he called the life- important substance, Hpoid, for the sake o£ convenience. He has f ound that purified lecithin, cholesterol, kephahn, cerebrin, and phytin are not able to replace the substances extracted from food with alcohol Food extracted with ether has füll nutritive value, but not after extraction with alcohol. The important substance was also insoluble in acetone. In a second paper Stepp (30) described further extraction ex- periments. He found that if food is extracted first with acetone and then with alcohol, both extracts, either separately or combined, are inactive ; but a primary alcoholic extract is active. In these results he sees proof that several substances are necessary for life, one of which is Vitamine. Another explanation may, however, be offered for these results, namely, that vitamine was destroyed by acetone in these particular experiments. Marshall (31) also concluded that the organo-phosphorus Compounds have no more therapeutic value than the inorganic ones. Direct proof that vitamine occurs only by accident in lipoid frac- tions was furnished by Cooper (32), who has fully confirmed my results. He found that the vitamine of voluntary muscle can be separated from the alcoholic extract by means of ether. Ether did not precipitate the vitamine in the case of brain; acetone did. The brain Phosphatides, including protagon, kephalin, cholesterol and cerebron (phrenosin), were entirely inactive. Vitamine can be com- pletely extracted from brain with 95 percent alcohol. In view of all these results we can safely say that vitamine is not lipoidal in character; and if lipoid products eure beriberi, it seems certain that they contain vitamine as impurity. Problem of vitamines and deficiency of salts in food. A number of papers dealing with the influence of a diet poor in salts, especially of phosphorus, calcium and magnesium, are based on the wrong assumption that the authors were really working with diets deficient in inorganic but not in organic constituents. Thus, food poor in phosphorus, polished rice and sago, have frequently been chosen, and the results obtained with them regarded as being due to deficiency of phosphorus or various salts. It must be insisted, however, that if the effect of a diet poor in a particular substance is 3i8 Vitamines and Deficiency Diseases [June-September to be studied, an artificial diet ought to be used that is " complete " in all constituents except the one wliose " deficiency-influence " is to be investigated. The workers who have been concerned, thus far, with the prob- lem of salt-deficiency are not yet aware of the fact that, when Vitam- ine is deficient, the balance of most inorganic constituents also becomes negative. Schaumann (27) performed metabolism experi- ments with maize and rice preparations on rabbits and pigeons. The nitrogen-balance was negative, and was followed immediately by a negative phosphorus-balance. The work in connection with the need for certain salts was chiefly performed in studies of rickets, osteomalacia, and growth of young animals, and will be described in the succeeding sections. Since the publication of my book, two papers of a general character on this subject have appeared and can appropriately be reviewed here. One of these papers, by Hornemann (33), deals with the salt-content in our ordinary food, which, in the opinion of several authors, is deficient especially in calcium and iron. Hornemann was not able to confirm these Statements. The other paper was by Heubner (34), who has already published some work on the im- portance of phosphorus in the diet. In his last paper, he apparently is more inclined to accept the importance of vitamines. He finds that deficiency of phosphorus in the diet only gradually reduces the phosphorus-content in the body. In a case of combined phosphorus- and vitamine-deficiencies the quantity of lipoids in muscle was abnormally low. The addition of phosphates decreased the amount of phosphorus in the central nervous System, but had no influence on the bones or muscles. Beriberi produced by a synthetic vitamine-free diet. I have found (35) that pigeons develop typical beriberi, on a diet consisting of casein, starch, lard, sugar and salts, as rapidly as on polished rice. The onset of the Symptoms is hastened if, prior to its use in this way, the casein is purified by extraction with hot alcohol or water, as we see f rom the appended summary : Uncookf d diet Cooked diet Onset of beriberi Death Onset of beriberi Deatb 37 days 40 days 27 days 31 days I9I5] Casimir Funk 3^9 By using casein extracted with hot alcohol, the animals developed beriberi in 26 days, and died in 28 days; much earlier than with unpurified casein. A very convenient technic was used for these experiments. The diet was transformed by means of pill machines into pills and aliquot parts of the initial mixture were forcibly fed. RoLE OF viTAMiNES IN METABOLisM. An investigation was suggested to me by the paper of Abderhalden and Lampe (2), who found that pigeons, on boiled rice, develop beriberi later than those on raw rice. This result was attributed by these authors to the elimination from the rice of a poison during the process of cooking. They doubted, for this reason, the existence of vitamines. For a long time I could not make out where the mistake of Abderhalden and Lampe's had occurred until I repeated their experiments (35). The mistake was at once apparent and was a very simple one. To each of two sets of pigeons I proposed to feed 30 gm. of either raw or cooked rice, daily. But it was soon found impossible to feed 30 gm. of cooked rice, daily, because of its enormous bulk, the weight of 30 gm. of rice after cooking being between 150 and 200 gm. This preliminary result suggested that the results of Abderhalden and Lampe were due to the use of unequal amounts of rice. The original Statement of these authors was found to be correct: Cooked rice Raw rice Onset of beriberi Death Onset of beriberi Death 44 days 44 days 25 days 26 days These figures represent average results. By giving to each pigeon exactly 10 gm. of rice, cooked or raw, daily, the following results were obtained : Cooked rice Raw rice Onset of beriberi Death Onset of beriberi Death 27 days 29 days 28 days 30 days After it had been ascertained that the onset of Symptoms of beriberi had some connection with the quantity of food consumed, an experiment was performed with different amounts of raw rice. The following results were obtained (36) : Pigeons were fed 0.5 gm., 5 gm., IG gm. or 20 gm., daily. The animals on 0.5 gm. a day died with starvation-symptoms, a fact which does not agree with the State- ment of Chamberlain, Bloombergh and Kilbourne, who claim to 320 Vitamines and Deficiency Diseases [June-September have observed beriberi in starving fowls. On the contrary, it seems certain that if no food is metabolized beriberi does not occur. The results with other quantities of raw rice are recorded below : Raw rice 5 gm- 10 gm. 20 gm. Onset of beriberi (days) 39 42 36 38 22 Death (däys) 22 These results made it necessary to ascertain which of the regulär food-constituents possesses this quickening action on the onset of beriberi. This problem could be solved by varying the amounts of the different constituents in an artificial diet. Four such diets were prepared. The composition of the diet, and the results of the feed- ing tests, are recorded below : Diet Saks, gm. Casein, gm. Sugar, gm. Fat, gm. Starch, gm. Onset of beri- beri, days A B C D 4 4 4 4 60 12 12 12 12 12 12 60 12 60 12 12 12 12 60 12 30 40 24 28 From the results of this experiment the conclusion is justified that the metabolism of carbohydrates, starch particularly, requires and uses up the larges.t amount of available vitamine. To complete these findings Funk and v. Schönborn (37) studied the influence of the f oregoing diets, with ref erence to the content of glycogen in the liver and of sugar in the blood. We aimed to deter- mine in which stage of carbohydrate metabolism vitamines play an active röle. The results so far obtained are not very clear, but they suggest that vitamine is involved in the synthesis of glycogen in the liver. We obtained the following results for pigeons. Normal diet: Glycogen (liver), 1.17%; sugar (blood), 0.1%. Füll artificial diet (percent — casein 12, starch 28, fat 28, sugar 28, salts 4) : Glycogen, 0.48% ; sugar, 0.15%. Carbohydrate-free diet (percent — casein 12, fat 42, salts 4, made up to 100 with french chalk) : Glycogen, 0.33% ; sugar, 0.21%. Starch-free diet (percent — casein 12, sugar 42, fat 42, salts 4) : Glycogen, 0.68%; sugar, 0.21%. Fat-free diet (percent — casein 12, sugar 42, starch 42, salts 4) : Glycogen, 4.3% ; sugar, 0.15%. 1915] Casimir Funk 321 Sugar-free diet (percent — casein 12, starch 42, fat 42, salts 4) : Glycogen, o; sugar, 0.26%. The latter diet, with addition of vitamlne: Glycogen, 0.6%; sugar, 0.19%. We see, from the results of the above experiment, that, on vitamine-free diets, marked hyperglycemia developed, with partial or entire disappearance of hepatic glycogen. This result is especially marked in the case of carbohydrate-free diets, and also on starch- free diets, but is most pronounced on sugar-free diets. These results seem to suggest that, in the absence of vitamine, synthesis of glycogen from protein and fat is greatly diminished. The result on a fat-free diet further shows that the presence of fat in the diet prevents the formation of glycogen in the complete absence of Vitamine. The addition of vitamine had a good effect in diminish- ing the hyperglycemia and increasing the glycogen-content in the liver. These studies will be extended to the action of adrenalin, phlorhizin and thyroid-extract in animals on vitamine-free diets. Braddon and Cooper (38) confirmed my results regarding the utilization of vitamine in carbohydrate metabolism. It was a Problem of great importance to determine whether polished rice and vitamine form a " complete " diet. Although many investigators accepted vitamine as a substance that is able to prevent beriberi, some considered, however, that, for the maintenance of body-weight, other complicated phosphorus Compounds are neces- sary. By using the fraction I obtained recently from yeast, with the acetone method, I was able (39) to eure very quickly, and to induce an actual gain in weight, on a diet of polished rice. The animals were kept in good health for a month, on repeated injec- tions, with marked appetite for polished rice, so that the usual forced feeding was unnecessary. The experiment had to be dis- continued, after that length of time, because of lack of material and also because of infections, as the vitamine Solution could not be sterilized without risk of destroying its curative properties. Cooper found (12) that vitamine, when given in the form of normal food, is not completely resorbed but a part of it appears in the feces. Schaumann (27) learned that vitamine passes into the circulation of some animals. It seems to me that this problem could J 22 Vitamines and Deficiency Diseases [June-September be investigated by comparing two pigeons, one f ed on polished rice and the other on a normal diet. A similar experiment was performed by Morpurgo and Satta (40), who kept two mice in parabiosis for long periods, one of which was fed sucrose alone, the other a normal diet. The former received vitamine and other necessary constitu- ents from the general blood-supply. I have shown (35) that pigeons, during attacks of beriberi, are able to utilize only a part of their bodily stock of vitamine — the part contained in the less vital tissues, e. g., muscle. Beriberi-pigeons lose about 25-40 per- cent of their body-weight; but, if such a pigeon is extracted with alcohol and the evaporated alcoholic extract given per os to another beriberi-pigeon, the latter quickly recovers. No toxic effect of these extracts was observed. Beriberi and glands of internal secretion. The products of glands of internal secretion show, in their chemical character, a certain degree of resemblance to vitamines. Vitamines might be the precursors of these substances. It is too early to attack this problem chemically, but Douglas and I (41) compared the pathological changes produced in this relation in beriberi pigeons. We examined pituitary, thyroid, suprarenals, ovary, testes, kidney, liver, pancreas and Spleen. The glands examined .were diminished in size in every case; microscopicaliy there were marked degenerative changes in the most important cells, only the framework remaining. The most marked change was the entire disappearance of the thymus, a Symptom which must be regarded, however, as due more likely to inanition than to beriberi. I found later, however, that the thymus appears very quickly when vitamine is administered. Douglas (42), at my suggestion and by using the animals from my feeding experiments, investigated the pathological changes in thyroid under the influence of various diets, particularly of diets of polished rice. No special changes were found, but it was observed that the colloid of the vesicles had a tendency to disappear. The most interesting glands of internal secretion that remain to be investigated are the parathyroids, the removal of which induces Symptoms somewhat similar to those of beriberi. ipis] Casimir Funk 323 III. SCURVY AND INFANTILE SCURVY (MILK PROBLEM) Progress in the chemistry of scurvy-vitamine has naturally been very much slower than in that of beriberi-vitamine, the former sub- stance being even less stable than the latter. Holst and Fröhlich (43) have continued their studies on the extracts of various vege- tables. They found that cabbage, dried at 37° C. and kept in a desic- cator, retains its curative properties for fifteen months. They also prepared an active alcoholic-glycerol extract from cabbage. Dif- ferent vegetables can be successfully extracted for vitamine either with water alone or with 80 percent alcohol containing 0.5 percent of citric acid. Freudenberg (44) found that antiscorbutic sub- stance can be extracted from various vegetables by means of alcohol, a 'behaviour which shows remarkable analogies with the beriberi- vitamine. This finding was confirmed by Freise (45) who prepared an active alcoholic extract from turnips. As regards the etiology of scurvy it seems worth while to record the composition of the kind of diet that occasions scurvy in adults. In an editorial in the Bulletin of Tropical Diseases (46) there is described a diet that occasioned a number of cases of scurvy, with very marked fever, in the Burma Prison. It was found that addi- tion of vegetables, milk, meat, or fish, had practically no influence, but the addition of sweet potatoes was effective. The composition of the daily diet was as follows : Oz. Oz. Rice (husked) 24 Condiments 0.125 Beans 4 Fish paste 0.5 Vegetables 10 Salt 0.25 Oil (vegetable) 0.5 In this connection it is also interesting to note the occurrence of several hundred cases of ship-beriberi among the sailors of the commerce-raider " Kronprinz Wilhelm," which recently arrived in a port of the United States. The sailors had a liberal diet of frozen meat but an inadequate supply of fresh vegetables and fruit. A report on the composition of the diets of sailors on the different steamship lines can be found in a paper by Markl (47). Extensive epidemics of scurvy occurred in the mines of Southern Rhodesia, as reported by Fleming, Macaulay and Clark (48). The diet in these mines had the composition indicated on p. 324. 324 Vitamines and Deficiency Diseases [June-September Mealie meal (milled Beans 2 Ib. weekly maize) 2 Ib, daily Monkey nuts 1.5-2 Ib. weekly Meat I Ib. weekly Salt ad. lib. Here we find cases o£ scurvy on a diet of milled maize, The compo- sition of milled maize will be indicated in the section on pellagra (V), with some interesting reflections upon the influence of diet on resistance to infectious diseases. Darling (49) described scurvy cases in the Rand due to overmilled maize. He finds, in these cases, hypertrophy and dilatation of the right heart, fatty degeneration of the heart muscle, severe degeneration of the vagus, which show the relationship of beriberi to scurvy Scurvy in animals. A description of a very complete investi- gation of scurvy in animals appears in a book on Infantile Scurvy, by Hart and Lessing (50). They studied various animals, subject to scurvy, in which allantoin is an end-product of purin metabolism. From their account it is apparent that the most suitable animal f or the study of scurvy is the monkey, the pathological lesions correspond- ing with those of human scurvy. In other animals the foUowing changes were noted on dififerent diets : Food Animals Lesions Oats Rabbits Fragility of bones Guinea-pigs Scurvy Mice, rats, cats No lesions Pigs Scurvy and beriberi Maize Guinea-pigs Scurvy Sterilized milk Calves Fragility of bones Young rats Arrest of growth « « « « The last fact above will be discussed in the chapter on growth. Infantile scurvy. The identity of infantile scurvy and adult scurvy does not require further discussion. It is also universally admitted that infantile scurvy is caused by overheated milk. Hess and Fish (51) have found that scurvy develops in infants fed on milk pasteurized at 145° F. for 30 min. The cases could be cured either with raw milk, fruit-juices or by addition to milk of potato- water. Cod liver oil proved to be inactive in cases of scurvy. The resistance of blood vessels was found to be weaker than in normal individuals. Another interesting finding by these authors was the fact that orange-peel extract was just as effective as orange juice iQisl Casimir Funk . 325 itself. A sample of such an extract was kindly given to me. The extract contained 0.027 gm. of nitrogen per 100 cc. — it consisted, as we see, not only of essential oils, but also of nitrogenous matter. The acidity of the extract was slight, corresponding to that of 8 cc. of n/io sodium hydroxid sol. per 100 cc. of extract, with phenoltha- lein the indicator. Metabolism experiments on children with Barlow's disease were performed by Lust and Klocman (52) who found, in active stages of the disease, a positive balance of nitrogen, chlorin, ash, calcium and phosphorus; in recovery, a negative balance of the same. Bahrdt and Edelstein (53) have analyzed various organs in Bar- low's disease. They found that bone marrow contained only 0.2-0.3 of the normal calcium content and 0.2-0.25 of the normal propor- tion of phosphorus, as in rickets ; the marrow was also poor in dry substance. Muscle was poor in calcium. It is now admitted by leading pediatrists that, in cases where highly pasteurized milk is given, additions of fruit or potato-juice as an antiscorbuticum is absolutely essential. It is admitted, then, that the boiling or heating of milk changes very markedly its food value. This fact has considerably changed, since the appearance of my book, the aspect of the milk prohlem, especially with regard to the accredited value of boiled milk in Infant nutrition. Beriberi-vitam- ine is stable enough to sustain a reasonable degree of pasteuriza- tion, but not sterilization ; the antiscorbutic substance can be added after pasteurization. But the question whether scurvy-vitamine is the only thermolabile substance of vital importance is still open. In her first report on the value of boiled and raw milk, Lane-Claypon (54) concluded that there was no difference in value between them. She changed her opinion, to some extent, in her second report, where she considered the question of vitamines in relation to the heating of milk. Sittler (55) described results obtained in the milk kitchen in the Children's Clinic of Marburg University. There milk is heated for 5 min. in a water bath. No cases of scurvy were observed with such milk. A very interesting and practically unique experiment with twins is described by this author. One of the twins was breast- fed and served as a control; the other infant received the milk from 326 Vitamines and Deficiency Diseases [June-September the same wet nurse — in the first period, heated to 60° C. in a water bath; in the second period, boiled for 3-4 min. At the end of the experiment the second child weighed 400 gm. less, and was weaker than the control. On the other hand Dennett (56) found that prolonged use of boiled milk does not necessarily cause rickets. Scurvy can be avoided by adding antiscorbutica. He found that boiled milk is just as digestible as raw milk but is more liable to cause indigestion. He expressed the opinion, however, that further study of this subject was desirable. Dennett's paper was read before the American Medical Association. In the ensuing discussion, Neff said that he noted the development of scurvy on a diet of raw milk, a condition which in my opinion must be extremely rare and due, very likely, to natural deficiency of scurvy-vitamine in the particular milk in- volved. Lowenberg cured scurvy with raw milk; in his own cases he had not seen scurvy develop with a diet of boiled milk to which had been added meat broth and vegetable broth. Scott expressed the opinion that the boiling of milk diminishes its food value. Graves saw a case of scurvy which resulted from a diet of boiled milk and thinks additions of antiscorbutica to boiled milk are neces- sary to prevent scurvy under such conditions. IV. RELATIONSHIP BETWEEN BERIBERI, SCURVY AND PELLAGRA These diseases show to a careful observer some connecting links. Darling described scurvy that developed on a diet of overmilled maize. Ohler was able to produce beriberi with maize-meal in f owls. I was not able to produce any disturbance in pigeons, however, by feeding them on highly milled maize. Stannus (57) described 131 cases of pellagra, on a rice diet, in Central Prison, in Zomba (Nyasa- land). The diet there consisted of i^^ Ib. of rice daily, with salt. Vegetables, fish or meat were given only occasionally — about once in a fortnight. The question arises : What are the differences among diets that occasion the outbreak of these entirely different though closely allied diseases. Few words of explanation are necessary here. Beriberi occurs on diets which consist chiefly of starchy food, e. g., polished rice, white bread, tapioca, sago and other vitamine-free 1915] Casimir Funk 327 or vitamine-poor foods. In the latter case an increase in the quan- tity of cereals may induce beriberi, for the quantity of vitamine is insufficient. Small additions of meat, once a week, or vegetables, are not sufficient to prevent beriberi, but since such additions usu- ally are eaten, no scurvy results. In the case of beriberi the etiology is clear — it occurs on starchy food with a negative or insufficient supply of beriberi-vitamine. We must remember that in accord with the results of my experiments on pigeons, beriberi does not occur on a maize diet. The etiology of scurvy has also been entirely cleared up. The disease breaks out v^hen dry food, in most cases such as has been stored for a long time, is eaten or when sterilized food (canned foods) is eaten. Beriberi does not occur in such cases because the beriberi- vitamine is stable enough to resist storage for a long time; but the beriberi-vitamine is incapable of preventing scurvy. The Pellagra cases of Stannus had pellagra and not beriberi be- cause the rice was only partially decorticated. In my opinion these cases are very important, for they give us a clue to the etiology of Pellagra. These cases suffered from pellagra, and not from beriberi and scurvy, because the food contained enough beriberi-vitamine to prevent the outbreak of beriberi, and there were apparently enough fresh vegetables and fruit to prevent an outbreak of acute scurvy. As a general conclusion, and at the same time as a working hypothesis, I regard pellagra not as a separate disease but as a very chronic disorder due to partial insufficiency of beriberi- and scurvy- vitamines. V. PELLAGRA Pathology. The problem of pellagra, for this country and for other maize-eating countries, is one of the utmost importance. Lavinder (58) gives the number of pellagrins in the United States, between 1907 and 1912, as 19,915 with a 40 percent mortality. Of these cases, nearly 55 percent were reported in Oklahoma, Arkansas and Texas. Devoto (59) made an excellent clinical study of pel- lagra. He described the diet of Italian peasants in pellagra districts, which in winter consisted only of maize. Acute erythema appeared early in March. The first Symptoms of the disease are weariness, lassitude, loss of weight, anorexia, paresis of the legs. In this stage 328 Vitamines and Deficiency Diseases [June-September the disease can be stopped by a change of diet; and, usually, the cases improve in July and August when the diet is better. Devoto often found alimentary glycosuria in bis cases, an Obser- vation that corresponds with my results on pigeons. Bardin (60) found an increase in lymphocytes, large and small, at the expense of the polymorphonuclear neutrophiles, a fact that I have noted in experimental beriberi in pigeons. Beeson (61) found, in 25 cases of Pellagra, complications with disease of the thyroid gland. In one of these cases, with enlarged thyroid, pellagra disappeared when the thyroid trouble ceased. Nicolaidi (62) found that, in acute pel- lagra, there are enormous losses of all kinds of nutritive constitu- ents, mostly thrpugh the feces; losses far greater than those ob- served in chronic enteritis. In chronic pellagra a negative balance, chiefly through loss in the feces, was found concerning salts, phos- phorus, magnesium, sodium and chlorin. Albertoni and Tullio (63) reported a negative nitrogen-balance on a maize diet, which became positive after addition of meat. Myers and Fine (64) performed metabolism experiments on pellagrins, put not on their original diet but on normal ordinary f ood. The utilization of various food-stuffs was very slightly lower than in the controls. There were low creatin and Creatinin excretions, and a high indicanuria combined with an increase in the amount of ethereal sulfates. Siler and Garrison (65) found the disease prevalent among women. Thus, in Spartanburg, S. C, out of 282 cases the incidence of the disease was 3 females to i male. It is very frequent after childbirth. Grimm (66) observed that pellagra occurred in women twice as often as in men. As predisposing causes he considers preg- nancy, lactation, puerperal fever and eclampsia. Weston (67) de- scribed 15 cases of infantile pellagra in one of which the mother was pellagrous. As a remedy he recommends weaning and the main- tenance of the best hygienic surroundings. The cases of infantile pellagra complete the analogy of this disease with infantile beriberi and scurvy. Etiology. Since the appearance of my book many authors see in the diet the cause of pellagra. Weiss (68) reported cases in Rovereto, in Austrian Tyrol, where the number diminished from 8,053 'ri 1904 to 3,503 in 1912, as a result of improved dietary con- 191 5] Casimir Funk 329 ditions. The peasants there now eat polenta two to three times a day, but with additional food. They are encouraged by the authori- ties to keep poultry and to use garden products as food. Perez (69) reported on the diet in the Canary Islands, which consists chiefly of gofio, a mixture in equal parts of wheat and maize, first roasted and then ground. This finding would be of practical importance, as no cases of pellagra occur there, provided the observations were correct, that is to say, if no fresh food was eaten, which is hardly possible. McDonald (70) described cases of pellagra, in Antigua, that de- veloped on diets of corn-meal and dried fish, a typical scurvy-pro- ducing combination. The disease occurs in Antigua only in blacks, although the white population eats maize as a part of a mixed diet. The author is inclined to accept the " deficiency " theory f or pellagra. In this country the food theory, if not the vitamine theory, is now generally accepted. Among the champions of this view we may count the following authors. Grimm (66) stated that most pel- lagrins live on corn-bread and hominy. He found the disease preva- lent among paupers — in 258 cases out of 323, and chiefly among whites. Goldberger (71) found that attendants, nurses, and doc- tors, in pellagra asylums, never suffer f rom the disease because they receive an adequate diet. He advocated fresh meat, eggs and milk instead of the cereals and canned meat, so largely consumed in Southern States. Siler, Garrison and MacNeal (72) described 847 cases in Spartanburg county, S. C. They do not endorse the " deficiency " theory, but they admit that the result of dietary treat- ment was good. They state, however, that the patients relapsed when returned to their original environment. These authors should not have been surprised at this outcome, for the patients probably returned, also, to their previous imperfect diets. It is interesting here to note that, as early as 1835, Rayer (73) expressed the view that pellagrins should change their habits and occupation, which, in his opinion, alleviated the Symptoms of the disease. Finally, I wish to quote the opinion of Voegtlin (74), who admits the importance of the diet as an etiological factor and con- siders the following three possibilities : , 330 Vitamines and Deficiency Diseases [June-September 1. A chronic intoxication by soluble aluminium Compounds; 2. Lack o£ vitamines (Casimir Funk) ; 3. Lack of certain amino-acids. Treatment. In some parts of Italy the government initiated very energetic measures against Pellagra. Alpago-Novello (75) has summarized the work undertaken in the province of Belluno, where Pellagra diminished very rapidly when the cultivation of beet-root and potatoes was substituted for that of maize. The pellagra com- mission of the district of Mariano, near Triest, has distributed, among the population, seeds of sweet potato {76). In Southern Tyrol Kleiminger (yy) cured 13 cases of lunacy, on the basis of Pellagra, by a change of diet even without addition of fresh vege- tables and meat, Allison (78), Sylvester (79), and Elebash (80) recommend fruit or fruit-juices, milk, eggs, and vegetables. Lorenz (81) treated 27 cases of pellagra with what he calls an excessive diet for eight weeks, with the following result: 7 cases died, 3 re- mained stationary, 13 cases improved, and 4 recovered completely, even in respect to the mental Symptoms. Quite recently Blosser (82) observed 133 cases of pellagra, all of which, except 3, ate cane products freely. Exclusion of all partially refined sugars and sirups was followed by a eure in 121 cases and improvement in 8 cases; 4 died. It would be interesting to know the percentage of cane products in the original food of these patients. If they repre- sented the bulk of the nutritive substances, the results of this ex- periment would bear striking analogy to those of my experiments on pigeons, in which beriberi was produced by a diet of sugar alone. As a practical conclusion to this section on pellagra, I wish to draw attention to the following point. Pellagra is, beyond doubt, a disease peculiar to districts inhabited by poor people, i. e., in dis- tricts where the population lacks the financial means to Supplement the routine diet with imported foods. We know of many examples that illustrate the limitation of pellagra to areas devoted to maize- culture. The peasants of Poland and Russia live exclusively on soup consisting of potato and cabbage, and whole-rye bread. One could not imagine a cheaper and less varying food. We can safely State, however, that deficiency diseases are practically unknown in these countries, with the sole exception that during periods of bad harvests, scurvy epidemics sometimes appear. As, however, we 1915] Casimir Funk 33 ^ approach the zone where the cultivation of potato ceases and maize plantations appear, as in South Russia, on the Roumanian border, or in Western GaHcia, we encounter cases of pellagra. It seems, theref ore, that the United States government could eradicate pellagra entirely by an introduction of potato culture in pellagra districts, as I have already suggested. It is obvious that such plans could not be executed at once but it seems advisable that such an experiment should be tried on a large scale. We may now consider another point of practical importance, namely, the question of the preparation or milling of maize, pre- vious to its use as a food. Milling of maize. On studying the data for the mortality from pellagra in different countries I was surprised to note the discrep- ancies among the figures. Thus, for example, in Italy and Egypt the mortality is very low, attaining only 4 percent, whereas in the United States the mortality is as high as 20-25 percent. The dif- ferences in mortality may be due to several causes as, for example, more or less exclusive diet of maize. But among the possibilities one factor requires particular attention, i. c, the question of the preparation of maize. My attention was drawn to this point by Dr. Macaulay, of Cape Colony, who was one of the members of the commission for the investigation of scurvy in the mines. He noticed that the maize used there as a staple food undergoes a severe milling process, during which 14 percent of the grain is lost for human use and employed for feeding cattle. The population there suffered severely from scurvy, as has already been stated. Macaulay tried to introduce legislative measures prohibiting such excessive milling but encountered considerable Opposition from the farmers, who had, in the wastage of the milling process, a very cheap and nourish- ing food for cattle. Finally, however, he succeeded in placing on the market maize which had been subjected to less extensive milling and which represented 97 percent of the total grain. Samples of this maize sent to me were analyzed. The results are given in the table (84) on p. 332. We see, from the accompanying diagram, that the chief nutritive substances are localized, in the South African maize, in the aleurone layer of the kernel near the surface of the grain, and in the germ. 332 Vitamines and Deficiency Diseases [June-September Data pertaining to milled maize (^97 percent of the total grain). See page 331. 1. Whole maize grain. 2. Highly milled meal, 86% 3. First milling from No. 2 4. Second milling from No. 2 5. Slightly milled meal, 97% 9. Bran from 5, above u tr, < Nitrogen c5 tfi 3 0 J3 0. CA J= E 3 0 s c C > 12.71 1.56 1-73 1.74 0.I4I0.99 0.56 12.63 1.48 1.67 1-73 0.13 0.95 0.36 10.48 2.09 1.23 — 0.14 0.61 0.30 10.71 4.04 2.31 2.34 0.16 1.40 1-43 12.41 1.60 1-73 1.84 0.12 I.OO 0.54 10.55 1.40 0.65 — 0.07 0.30 0.23 4-5 3-87 4.66 12.79 4.21 1.86 a c9 3-88 336 4.18 11.09 o U o Ph 'S a 0.247 0.22 0.357 0.438 0.0154 0.0098 0.0164 0.0353 Color- reaction: Polin and Macallum Alcoholic extract Hot 3.63I0. 233:0.0153 i.58|o.27i]o.oo84 0.35 0.23 i.oo 0.70 0.30 0.35 Cold 0.55 0.4s 1.00 0.60 0.45 0.70 During the process of milling, a large part of these layers is milled away, as is shown by the figures in the above table. Pigeons were fed this highly milled maize for several weeks, but they remained in perfect health and differed totally in their be- DlAGRAM OF A SeCTION OF A MaIZE GrAIN 5": Skhi. E: Endosperm. A: Aleurone layers. G: Germ havior from fowls which, according to Ohler, develop, on such a diet, a disease that he considered identical with beriberi, Driscoll (85) observed that chickens, fed on maize with the outer layer re- moved, developed erythema on the legs, and acquired a disease simi- 1915] Casimir Funk 333 lar to human pellagra. The Symptoms disappeared in three weeks, when the chickens were fed on whole corn. In four cases of Pel- lagra in man the same author saw good results on a diet of corn- meal to which bran had been added. The work o£ Driscoll requires confirmation, before his conclusions can be accepted; but the prob- lem of the influenae of the mode of milling of maize on the incidence and severity of the Symptoms can not be disregarded, especially in view of the observations of Macaulay and Darling on the occur- rence of scurvy on overmilled maize. Analyses of various samples of maize have been made by Juritz (86) and MacCrae {^7), the latter having determined especially the phosphorus-content in maize milled to different degrees, and con- firmed fully my results. Poppe (88), who described the diet of the working class in Belgian Congo (i k. of maize meal, 1.4 k. of corn, 0.14 k. of meat and 14 gm. of salt, daily), stated that the water from cooked maize should not be thrown away, for 36.2 percent of the nutritive ingredients would thus be rejected (89). Weiss (90) found that pellagra is prevalent, in Tyrol, in dis- tricts where fine maize, without the husk, is used as food. Night- ingale (91) gives a similar account but of much greater value, as his conclusions are based on 12 10 cases. He described the diet in Southern Rhodesia, where mealie meal is passed through a sieve that retains the husk. The husk proved to be an excellent food for cows, increasing greatly the yield of milk. This maize preparation caused a disease he called zeism, which is nothing but pellagra of a mild type. On the other hand, the hand-milled maize (rapoko) proved to be preventive and curative. Cod-liver oil was also of great value, in his hands, for the treatment of pellagra. In summarizing the results obtained and the views expressed by various authors, it seems to me that we already possess all the knowledge necessary success fully to combat pellagra. VI. SPRUE Our knowledge of sprue advances with our knowledge of pel- lagra. This disease occurs less frequently than pellagra, however, and less work has been published on the subject. The disease results directly from deficient diets. Cantlie (92), who has done 334 Vitamines and Deficiency Diseases [June-September much work on this disease, continues to regard sprue as due to in- fection, but the data f rom his own paper indicate that it is dietary in origin. He saw, in many sprue cases, tetany with anemia, often accompanied by unconsciousness and fever up to 103° F. Continued fever indicates the last and fatal stages, results (very likely) of secondary infection. For treatment he recommends strawberries, ripe raw gooseberries and the juice of blackberries. Bahr (93) found that the disease is more common in women (analogy to Pel- lagra). His post mortem findings were: great waste of internal Organs and inflamed intestines. Leede (94) also recommended straw- berries as a eure; but he made an interesting Observation from our point of view, namely, that preserved strawberries are not equal in curative power to fresh fruit. He suggested that the beneficial prin- ciple is destroyed by heat. Mühlens (95) believes that sprue is sometimes complicated by dysentery, when, besides ingestion of strawberries, the administration of emetine is indicated. He is in- clined to accept the deficiency theory. Werner (96) observed scurvy Symptoms (petechise) in a case of sprue. VII. RICKETS Our knowledge of rickets has not increased sufficiently, in this connection, to advance us beyond the stage of the very promising hypothesis that the disease is due to deficiency of vitamines. Ad- vance has been slow because there have been many cases where the differential diagnosis of rickets and bone porosity was impossible. Bone porosity {Fragilitas ossium) is not improved by treatment with calcium salts, phosphorus or cod-liver oil, according to Ostheimer (97), but by good food. Ostheimer considers this dis- ease a result of faulty metabolism during pregnancy (congenital fault of metabolism) and that it has nothing to do with rickets or osteomalacia. On the other band we have to differentiate, clinically, between cases that improve on dietary additions of calcium and phosphorus only, and those that do not improve after the further addition of vitamine to the diet. The latter types of cases must not be regarded as rickets. As we have seen in the preceding chapters, special attention must be given to the fact that, in experiments with so called calcium- and 1915] Casimir Funk 335 phosphorus-poor diets, the latter are usually also deficient in vitam- ines, This fact applies, for instance, to the work of Schmorl (98) who investigated effects in puppies fed with Heubner's diet. Schmorl admits, however, a possible deficiency of vitamines in this diet. He found diminished formation of bone, a decreased number of osteoblasts and an increased amount of osteoclasts. The bones were very soft despite a sufficient supply of calcium salts in the food. The lesions in the bones resembled those of Barlow's disease; but there were small hemorrhages in the sub-cartilaginous zones, as in human rickets. Schmorl sees a possible connection between a de- ficiency of vitamines and a disturbance of internal secretion. Weiss (99) treated ten rickety children with hypophysochrom of Klotz and obtained a favorable impression as the result. Investigators who believe there is a definite connection between rickets and disturbance of internal secretion meet the truth half way, as it is possible that certain vitamines, which are necessary for the prevention of this disease, may be used in the organism as " start- ing material" for the activity of the glands of internal secretion, and in this way may play a role in the prevention of rickets. We may accordingly disregard the results obtained by Rominger (100) with the aid of Abderhalden's dialysis method, which led him to conclude that there is no evidence of a disturbance of internal secre- tions in rickets. As to the cause of rickets, very few data have been obtained recently. Sittler (55) saw cases of rickets on sterilized commercial butter-milk, to which starch in the form of cereals had been added. He was able to eure these cases with raw milk. More work was done on the therapeutic action of cod-liver oil, either as such or in conjuction with phosphorus. Schloss (loi) described the effect of cod-liver oil with phosphorus on three breast-fed rickety children and found negative calcium- and phosphorus-balances. An addition of calcium acetate rendered the calcium-balance positive. He believed there was a definite connection between the calcium- and magnesium-balance, which showed opposite tendencies, one being positive when the other was negative, and vice versa. Frank and Schloss ( 102) could not find a difference between cod-liver oil alone, or in combination with phosphorus, as regards effects on protein 33^ Vitamines and Deficiency Diseases [June-September metabolism. It seems that the phosphorus could be dropped from the combination without loss of advantage. Regarding the influence of cod-liver oil on calcium metabolism, we find in the literature opinions that disagree with those of Schloss, as in the case of Kurt Meyer (103), for example. In Osteogenesis imperfecta (congenital porosity of bones) good results with cod- liver oil were observed by Borkman (104), and with cod-liver oil and hypophysochrom by Schabad (105). The discordant results of various authors can be explained, perhaps, as we shall see later, by the use of dissimilar preparations. The most important objection any one has advanced against the vitamine-deficiency theory of the origin of rickets is the relative frequency in the occurrence of rickets in breast-fed infants. This fact seems to me to be one of the best arguments in favor of this theory, as is indicated in the succeeding section. VIII. CHEMISTRY OF COD-LIVER OIL Exceedingly little work has been done on this subject. In 1888, Gautier and Mourgues (106) isolated, by extraction and by distilla- tion at a high temperature, two nitrogenous bases called by them morrhuin and aselin. Judging from. the character of the methods used, these substances may be regarded as secondary products result- ing from the distillation, or as decomposition products of some more complicated substances. A series of papers on this subject were published by Iscovesco (107), who, working with the usual methods for the preparation of Phosphatids, isolated a lipoid either from cod- liver oil or from the liver of codfish. He claimed to have obtained, with this preparation, a specific action on the growth of rabbbits and guinea-pigs, which could not be duplicated with any other lipoid. We shall consider, in the section on growth, the effects claimed for this preparation. During the year 1914 I took up the subject. As a basis for my investigation I relied upon the Observation, of several specialists in children diseases, that more pronounced therapeutic effects have been obtained with crude cod-liver oil than with the highly refined pharmaceutic preparations. My experiments were made on young chickens which, when kept under laboratory conditions, develop a 1915] Casimir Funk 337 disease that shows in every respect the greatest analogy i£ not identity with rickets in children. One observes in these animals every kind o£ deformation o£ various bones, chiefly the sternum, deformities which may proceed so far that the intake o£ food is rendered impossible. The mortaHty of these animals was very high, sometimes as high as 80 percent. (I have observed several cases of typical rickets in young growing rats which had been kept on artificial diets.) I tried to diminish mortaHty in the chickens by changing the food, by adding yeast, fresh grass and green vegetables, but only with very sHght effect. It seems, therefore, that young chickens can be raised without rickets only when they are kept in the open air where they have opportunity to pick up worms. Two samples of cod-liver oil were at my disposal : a sHghtly refined oil of brown color and a crude oil. By adding the two kinds of oil to the ordinary diet a marked difference was observed, the crude oil being very much more efficient in preventing the rickety condition and diminishing the mortaHty of the chickens. Extrac- tion of these two samples proved that the crude oil contained a much higher proportion of nitrogenous extractives than the slightly puri- fied oil. As my paper on this subject appears in this issue of the BiocHEM. Bull. (p. 365), I shall refrain from mentioning here the details of the work (108), except to say that the extraction products will be tested clinically, as there is no reason to suppose, from our present knowledge, that there is any particular value in the adminis- tration of the oil as such. In studying the effects of these oils in infantile rickets, or even in experimental rickets, we encounter great difficulties. In clinical cases signs of rickets persist after the actual disturbance due to a de- ficiency of vitamine has ceased. Marked effect of our preparation could not be expected in such cases. Even in florid cases of rickets, Observation had to be extended over long periods because effects could not be expected to occur as promptly as they do after the administration of beriberi- or scurvy-vitamine. In concluding this section I wish to express the belief that cur- rent differences of opinion regarding the therapeutic action of cod- liver oil are due to the use of highly refined oils by the clinicians. 33^ Vitamines and Deficiency Diseases [June-September IX. OSTEOMALACIA, SPASMOPHILIA, ECLAMPSIA Osteomalacia may be considered as a rickety condition in adults. This disease affects only women in pregnancy and Puerperium. A slight degree of this affection occurs so frequently that we see, in von Noorden's book on the Pathology of Metabolism, an expression of the view that there may be a " physiological " osteomalacia (109) in pregnant women. I have already referred to an analogous affec- tion in cattle, called " Stalhnangel." Antoine (iio) described typi- cal osteomalacia in dogs. In this review I have occasionally pointed out the frequency in the occurrence of deficiency of vitamines dur- ing pregnancy and lactation. Let me remind the reader of the occurrence of infantile scurvy and beriberi in breast-fed infants where the Symptoms of the diseases may or may not be shown by the mothers. In the latter cases the mothers possess just enough Vitamine to keep them free from these diseases but there is not enough vitamine in their milk to protect their infants. In the same way the infantile form of pellagra can be explained and also the occurrence of rickets in breast-fed babies. The occurrence of rickets in breast-fed infants is often con- sidered an argument against the vitamine-deficiency theory of the etiology of rickets. These cases occur chiefly among poor people, and the food of the mothers should undergo careful inquiry. But even among wealthy people a deficiency of vitamines is not impos- sible in pregnancy and lactation, for the appetite in such cases is very often not up to the demand considering the condition of the organism. Study of the nutrition in pregnancy suggests, so far as vitamines are concerned, that the quantity of these substances which would suffice under ordinary physiological conditions is often in- adequate in pregnancy and lactation. I went into this field with special interest in the mysterious eti- ology of eclampsia. Every available theory of its origin was tested but without the slightest success. This condition occurs in the late stages of pregnancy and also in the puerperium. I wish to draw the attention of the clinicians to the possibility of a deficiency of vitam- ines in this condition and to suggest research in this direction. We shall better understand this possibility if we compare eclampsia with beriberi, especially the spastic, tetanic form. 191 5] Casimir Funk 339 Very similar Symptoms occur in spasmo philia in children and also in a condition which the German authors call "Mehlnähr- schaden" Spasmophilia occurs in children fed on large proportions of farinaceous foods and can be safely regarded as infantile beri- beri. Takasu (m), for instance, describes spasmophilic dyspepsia in breast-fed babies in Japan as beriberi. Liefmann (112) was able to detect, in normal children between 10 days and 2% years of age, a daily excretion of 1-5 mg. of acetone. In a spasmophilic attack this excretion increases up to 93 mg. This Observation, and also the fact that spasmophilic children show increased irritability of the muscles to the electric current, can be used successfully for the diagnosis of this condition and also of other avitaminoses in children. Freudenberg and Klocman (113) recommend the use of oxy- cod-liver oil (oil treated with hydrogen peroxide) for the treat- ment of spasmophilia. It seems, however, that treatment with the original oil, or a concentrated extract as described under rickets, is more promising. Brüning (114) described a condition in new born white rats, when separated from their mother and fed artificially on a diet rieh in carbohydrates, which he considers identical with " Mehlnährschaden." No pathological changes in the bones were found. In summarizing this section I conclude that it seems highly de- sirable to apply the vitamine therapy to osteomalacia, spasmophilia, Mehlnährschaden and eclampsia. We understand in this connection chiefly the therapeutic use of yeast preparations and cod-liver oil, the latter in accord with the Statements in .the previous sections. If these diseases are rightly regarded by me as avitaminoses, the application of this therapy not only would bring about practically instantaneous eures but also would serve very well for the purpose of testing quickly the value of the clinical diagnosis. X. CHEMISTRY AND PHYSIOLOGY OF GROWTH This subject has received special attention during the past two years but no definite conclusion has as yet been attained. At present there are several theories on the chemical nature of the substance that promotes growth in young animals. The most important of 340 Vitamines and Deficiency Diseases [June-September them relate to (a) Inorganic salts, especially of phosphorus; (b) lipoids, or in reality Phosphatids; (c) fats or oils of peculiar Consti- tution; (d) certain amino-acids and (e) vitamines. Which of these theories has the greatest justification the reader will be able to judge for himself from the succeeding discussion. Theory of inorganic salts. Masslow (115), in his experi- ments on the growth of young dogs, studied the influence of wliat he thought was a diet poor in phosphorus. The diet he used had the following composition : Rice, 100 parts; egg albumen, 50 parts, with addition of potassium, sodium and calcium; sugar, 40 parts, with addition of magnesium and iron; starch, 50 parts. The dogs re- mained in good condition for a month, on this diet, then a great wastage of flesh, loss of appetite and death resulted. This diet was typical vitamine-free food; and the addition of phosphates and glycerophosphates had no effect, whereas the administration of " Lecithine Merck " resulted only in temporary improvement. In spite of these results Masslow regards these failures as due to lack of phosphorus. In a second paper, on the same subject, Masslow (115) gave the results of analysis of the organs of the dogs kept on the above- mentioned diet. There was diminished content of phosphorus and ferments. Masslow (116) found that the bones underwent marked changes, with small hemorrhages resembling those of infantile scurvy. Durlach (117) carried out, on a similar diet, some experi- ments on dogs, but the animals did not grow on vitamine-free food. Röhmann (118) fed mice on artificial diets. The duration of the experiments was extended to the second generation. In the second generation, however, the diet did not suffice. Although Röhmann concluded that an artificial diet does not have the value of ordinary food, he refused to accept the existence of any unknown chemical factors. The composition of the diets in his experiments were the following : First Second 12 gm. casein 14 gm. casein 4 gm. chicken protein 4 gm. chicken protein 4 gm. nucleoprotein from liver 4 gm. vitellin 180 gm. potato starch i ^o gm. potato starch ^ ^ t 120 gm. wheat starch 12 gm. margarine ig gm. margarine 4 gm. Salt mixture 4 gm. salt mixture 191 5] Casimir Funk 34i The Salt mixture contained lo gm. of calcium phosphate, 40 gm. of potassium bi-phosphate, 20 gm. of sodium chlorid, 15 gm. of sodium citrate, 8 gm. of magnesium citrate, and 8 gm. of calcium lactate. This diet was inadequate for mice; they grew less rapidly than on a diet of bread and milk. The Substitution of vitellin for the nucleoprotein did not yield better results. No pathological alter- ations were seen in these animals and, on changing this diet to a normal one, quick recoveries followed. The second diet gave good results in old mice, but in young ones no growth was observed. The Substitution of a part of the vitellin in the second diet by chicken protein, or edestin, failed to affect growth. The same was true of meat-extract and meat-powder. On the other band, addition of malt-extract or nucleoprotein from liver stimulated growth. Egg- yolk fat had an unfavorable effect on adult mice. In young mice egg-yolk prolonged life but did not stimulate growth. Very good results were also obtained when the food was mixed with yeast and baked ; lecithin was without effect. Hart and McCollum (119) performed some experiments on swine. No growth was observed either on wheat alone, or on corn meal and gluten feed. Growth occurred, however, when to the above mentioned food, potassium phosphate, potassium citrate and calcium lactate were added. Under these conditions the growth on corn meal was more successful. Wheeler (120), in Mendel's laboratory, extended to mice, the rat studies of Osborne and Mendel, and found that mice are also well adapted for experiments on growth. Wheeler's work was under- taken at a time when Osborne and Mendel believed in a special relation between milk-salts and growth, but the results indicated the necessity of adding organic milk food, even in larger relative quan- tity than for rats, to induce complete growth, in which case mice grew more rapidly than rats. Osborne and Mendel (121) have amended their statement regarding the beneficial effect of protein- free milk, and of artificial protein-milk, on the growth of rats. Hart and McCollum (122), in continuation of their studies on growth in swine, concluded that even after an addition of salts to corn- or wheat-feed only partial growth can be obtained, the pigs showing signs of paralysis with ultimate decline. 342 Vitamines and Deficiency Diseases [June-September As a conclusion from these experiments we notice the complete failure to demonstrate any special value of inorganic salts in the process of growth; that even the addition of mixtures of salts to artificial diets was unable to replace the important constituents of natural diets. Value of lipoids and Phosphatids in growth. Iscovesco (107) performed some experiments on growing animals with lipoid.iso- lated by him from cod-liver oil or from the liver of codfish. This lipoid could not be replaced in its action by any other lipoid. For instance, for young rabbits he obtained the following figures for increase in weight: Controls, 33 percent; cod-liver oil, 55 percent; the same, without the lipoid, 37 percent; ohve oil, 33 percent; olive oil, with the lipoid, 56 percent Desani (123) fed white mice with starch and casein that had been extracted with alcohol and ether. This extraction was under- taken in order to get a food free from cholesterol. The animals died after 18-19 days with a loss of weight of 41 percent. McCollum and Davis (124) investigated the value of hpoids from boiled eggs. The ether- and petroleum-extracts were effective growth stimulants, the acetone extract only to a slight extent. Lander (125), who worked with a carefully purified diet, found that rats live on such a diet about 14 days, the addition of choles- terol, cholesterol esters and lecithin having no effect on growth. MacArthur and Luckett (126) proved that lecithin, cephalin, cerebrosides and cholesterol are not vital dietary constituents for mice; but they State that a fraction of egg-yolk insoluble in ether and soluble in alcohol, probably vitamine (thermolabile), is neces- sary to make a complete food. The above mentioned papers disprove the theory that lipoids are necessary for the growth and maintenance of animals. Importance of pecuhar fats and oils for growth. A new impulse in the study of the growth problem was given by the very important discovery of McCollum and Davis (127) that butter-fat or fat from egg-yolk is able to stimulate growth of rats which have declined on artificial diets. This Observation was confirmed by Osborne and Mendel (128) and extended by them (129). They separated butter by centrifugation into three fractions, solid detritus, 1915] Casimir Funk 343 fat fraction and butter-milk. The fat fraction which, according to these authors, contained no nitrogen and phosphorus had apparently the same effect on growing rats as the original butter; rats which were in a bad nutritive condition quickly recovered. Especially, an infectious disease of the eyes was promptly cured. The beHef that this butter fraction contained no nitrogen, was used as an argument against the vitamine-theory of growth, and induced me to investi- gate the butter problem more closely. Macallum and I (130) fractioned butter, by the method of Os- borne and Mendel but further purified the butter-fat fraction in the following way. The fat was dissolved in acetone and shaken with a weak Solution of hydrochloric acid. From 12 k. of butter 23.4 mg. of nitrogen were obtained by the Dumas method, and slightly less by the Kjeldahl method. The butter-fat, after its subjection to this process of extraction, was hydrolyzed with weak acid and again 22 mg. of nitrogen were obtained. These iigures are certainly low and would suggest that the butter-fat was prac- tically nitrogen-free, if there were not the possibility that some of the contained nitrogenous substances were volatilized during con- centration in vacuo. This possibility must be tested. McCollum and Davis (131) also questioned the absence of nitrogen and phosphorus from Osborne and Mendel's butter-fat. Osborne and Wakeman (132) again tested the purity of their butter preparation and found only traces of nitrogen and phosphorus. Aron (133) has also observed beneficial effects of butter on growth. Osborne and Mendel (134) have found that old stunted rats resumed growth when the inadequate diet was changed to a " complete " one. The possibility of a Stimulation of growth with nitrogen-free butter seems to me so highly improbable that I am unable to accept the Statements to that effect without further proofs. Macallum and I performed some comparative experiments with crude butter and purified butter, following our method on rats. The percentage com- position of the diet was as follows: Casein, 20; starch, 41; sugar (cane), 21; butter crude or purified, 12.4; salts, 2.6. The casein was extracted for several days with boiling alcohol. Lactose was avoided as it contains a small quantity of nitrogen — according to 344 Vitamines and Deficiency Diseases [June-September McCollum and Davis (135), 0.02-0.034 percent. Salts were used in the same kinds and proportions as those for the experiments by Osborne and Mendel, but no protein-free milk was added. We did not succeed in keeping cur rats longer than two months on this diet. We were unable to see any good effects of the addition of the butter. Slightly better results were obtained, how- ever, with crude butter. Male rats increased in weight, after 42 days, 54 percent; females, 41 percent. At that time, in the purified butter series, several rats had already died. After 24 days the males in this group increased their weight by 45 percent, the females by 31 percent; whereas, at the same time (24 days) in the crude butter experiments, the weights of the males increased by 66 percent, of the females by 48 percent. Both butter preparations were fed in an artificial diet to pigeons, the latter developing beriberi in the usual time in both series. In neither butter samples could any appreciable amount of beriberi- vitamine be detected by this method. I would not call our experiments entirely conclusive, especially as further studies are in progress, but I received the Impression that the remarkable results obtained by Osborne and Mendel are due, to a great extent, to the use of protein-free milk. It seems to me that in the experiments of Osborne and Mendel the excellent results are due to the addition of vitamine with casein, lactose, protein-free milk and butter. One of the last papers of McCollum and Davis (135) gives a less enthusiastic account of the influence of butter on growth. They believe that even the butter diet lacks something es- sential which is present in natural food for rats. The study on fats was extended to cod-liver oil (Iscovesco, 107) by Osborne and Mendel (136), which was found to have the same action as butter, whereas almond oil and tri-glycerides of the fatty acids had no effect. McCollum and Davis (135) found lard, olive oil, tallow and cotton-seed oil inactive, but the ether extract of dried, ripe cod testicles and pig kidney were very active. Recently Osborne and Mendel (137) have fractioned beef- and butter-fat more extensively. Abdominal fat of cattle and butter were dissolved in alcohol at 40° C. and allowed to crystallize. The crystallin fraction was ineffective. The oily part, which was 1915] Casimir F-unk 345 greater in quantity in the case of butter, was very effective. This fact strengthens my belief that the stimulating action of butter-fat may be due to vitamine. It is regrettable that Osborne and Mendel did not determine whether the oily f raction contained nitrogen. On summarizing this section we see that the growth-promoting substance was found only in materials from sources which are known to contain vitamines, e. g., milk, cod-liver oil, beef-fat, etc. Question of thermo-stability of the growth-promoting sub- stance. Here we also find great divergence of opinion as in the case of beriberi-vitamine. Thus, Hart and Lessing (50) observed complete arrest of growth in young rats fed on sterilized milk. On the other band McCollum and Davis (124) were able to obtain an active extract from boiled eggs. They went even further: butter- fat was hydrolyzed (131) in petroleum ether, at room temperature, with an alcoholic potassium hydroxid Solution. After neutraliza- tion the soaps were extracted with olive-oil and the growth-promot- ing factor was found intact. Osborne and Mendel (136) also con- sider the growth-promoting substance thermo-stable. Role of amino-acids in growth. This problem was studied extensively by Osborne and Mendel, and by McCollum. The former authors usually arranged their experiments as they did those men- tioned above. In the artificial diet, containing butter-fat and protein- free milk, the casein was replaced by protein known to be deficient or poor in certain amino-acids. It was found by Osborne and Mendel (138) that gliadin without addition of lysin was inadequate for growth. The same applies to zein without addition of trypto- phan. In another paper (139) they. stated that the addition of cystin and lysin made an inadequate supply of casein suitable for growth. Lactalbumin was found to promote growth very strik- ingly. This protein was found to yield more tryptophan than that from any other protein and they attribute the influence on growth to the presence of this amino-acid. In this inference there is the fal- lacy, however, that lactalbumin may contain vitamine from milk. These authors found, also, that a deficiency in any particular in- gredient of the diet did not induce a corresponding compensatory increase in the food intake (140)- McCollum (141) found that the proteins of milk are superlor to 346 Vitamines and Deficiency Diseases [June-September any other proteins so far as growth is concerned. Street (142) has compared the value of sanatogen and casein for growing rats but could not detect a difference between these two f ood-stuffs. Hektoen (143) found that the production of anti-bodies was normal in rats fed with pure vegetable proteins (Osborne and Mendel's diet). The experiments on the influenae of isolated proteins, with addi- tions of certain amino-acids, show what had previously been demon- strated : that amino-acids, like trypophan, tyrosin, lysin and cystin, are indispensable components of a complete diet. In experiments on growth it is obviously essential that the protein in the diet should yield all the necessary amino-acids in adequate amounts. Importance of vitamines for growth. Until recently the growth Problem had been studied only on rats, mice, dogs and pigs. I have extended these studies to chickens and, to my surprise, found that practically all the preparations which stimulated growth in rats failed to do so in young chickens. It was also found that polished rice as well as unpolished rice was entirely inadequate to stimulate growth in these animals. The best results ( 144) were obtained by adding yeast to an unpolished-rice diet. On these diets, however, all the chickens died in several weeks. On adding cod-liver oil to an unpolished-rice diet the animals remained in fairly good health for several months but no growth resulted (130). The addition of tumor-tissue (Rous's sarcoma) also had a stimulating effect. In another series of experiments I tried the effect, on the growth of chickens, of an addition of phosphotungstic precipitate, and of phosphotungstic filtrate, to an unpolished-rice diet. No growth was obtained (145). It seems, therefore, that growth in chickens is de- pendent on such substances as are contained in living worms, for even additions of fresh grass and salad to the inadequate diets were without effect. Experiments were also carried out on young chickens with both germinated and ungerminated oats, rice and barley, but these diets proved to be unsuitable for young chickens. The experiments will be repeated on chickens and rats. In conjunction with Macallum (130) experiments were per- formed on rats in the diets for which various portions of the starch were replaced with unpolished rice or with polished rice. The per- IQIS] Casimir Funk 347 centage composition of each of the diets is indicated in the appended summary : Biet I Diet II Diet III 22 22 22 37 29 20.5 4 12 20.5 21 21 21 12.4 12.4 12.4 2.6 2.6 2.6 Diet IV Casein Starch Unpolished rice. Polished rice. . . Sugar Lard Salt mixture . . . 22 29 12 21 12.4 2.6 The experiment with polished rice terminated very early — in 29 days, all the animals dying without any sign of growth. The unpolished-rice experiment continued for 61 days. The best results were obtained with Diet III, which contained the largest amount of unpolished rice. If we take, for convenience of comparison, a period of 28 days we find the following average figures in the records of these experiments. On Diet I the rats show a weight-loss of 3 percent; on Diet II, there was a gain of 7 percent; on Diet III, a gain of 16 percent. The chief chemical difference between pol- ished rice and unpolished rice, so far as we know, is the diflference in the proportion of vitamine. These results suggest very strongly that vitamine plays an important if not a decisive role in the experi- ments on growth. An experiment similar to the one described above was per- formed by McCollum and Davis (146). They compared the bio- logical properties of corn-meal, wheat, wheat-embryo, rye, and rolled oats with dry pig-heart or kidney. The experiments, which were carried out on rats, demonstrated the beneficial effect, on growth, of corn-meal and wheat-embryo, the addition of rye had a slight favorable effect, but whole wheat-grain and rolled oats had practically no effect. The addition of pig-heart had less effect than the addition of kidney. These experiments show that food-stuffs known to contain a relatively large amount of vitamine stimulate growth, in rats, better than do those that contain relatively little vitamine. As the results so far obtained sufficiently demonstrate the im- portance of vitamines for the growth of rats, Macallum and I have Started new experiments with diets including different fractions from yeast. These experiments are now in progress. 34^ Vitamines and Deficiency Diseases [June-September XL GROWTH IN PLANTS Bottomley ( 147, 148) is the only one who claims to have demon- strated the importance of vitamines for the growth of plants. He was able to isolate, from bacterized peat, a substance which proved to be a powerful stimulant of the growth of plants. This substance was obtained from a fraction which corresponds entirely to the Vitamine- fraction. XII. INFLUENCE OF DIET ON THE GROWTH OF TUMORS The results obtained thus far by applying the Osborne-Mendel diet, or any other similar vitamine-free diet, to animals bearing tumors have not justified the hope we had for this method. One is able partially to arrest the growth of a tumor on such a diet, but the avidity of the tumor for food is so great that its growth proceeds on a diet which is entirely inadequate for the growth of the animal. Some of the recent papers illustrate this point very well. I was able to show (144) that in chickens, on normal diets and inoculated with Rous's spindle-cell sarcoma, there were higher percentages of " takes " and larger tumors than in chickens f ed on unpolished rice even after addition of yeast or sarcoma extract. Different results were obtained with diets of polished rice. Here the tumor did not "take" at all; whereas, in birds fed on polished rice plus yeast, tumors developed in a large percentage of cases. On these restricted diets no metastases were ever noticed. Food containing vitamines undoubtedly had marked influence on the growth of the tumors. Rous (149) has also studied the influence of simple underfeeding and of Osborne-Mendel diets on rats. Flexner-Jobling Carcinoma was not afifected by underfeeding after the tumor had been growing for some time. Spontaneous tumors of mice were affected by re- stricted diets if the feeding of such diets was started before the inoculation. Two explanations for these results are available: When the organism is weakened the tumor does not grow as well as it does when the organism is more vigorous; in the case of a vitamine-free diet, the tumor does not receive essential specific nutrients. A great difficulty in such experiments is the fact that it is practically impos- sible to find a vitamine-free diet or a vitamine-poor diet on which 1915] Casimir Funk 349 the animals can live in good condition. A second serious difficulty is the avidity of the tumor for food and the difficulty of rendering the animal itself vitamine-free before the tumor is actually inocu- lated. We saw that the tissues of pigeons that died from beriberi contained a certain amount of vitamine. However, it seems worth while to try the diet of unpolished rice, which is a complete diet, in human Cancer, especially in rapidly growing inoperable cases. Centanni (150) conducted experiments similar to those just referred to. He used what he called an aviride diet (seeds, dry fruit), in which the products of cellular activity are at a latent stage. This diet had a marked inhibitory effect on adenoma in mice, a slight degree of Inhibition persisting even when fresh food was given. Van Alstyne and Beebe (151) have reported results of experiments with non-carbohydrate diets on the growth of sarcomas in rats. They found that the sarcoma grew very much less on a diet of casein and lard than on a diet of casein, lard and lactose. The foregoing results led Benedict and Lewis (152) to investi- gate tumor-growth in rats with phlorhizin glycosuria. In such cases the growth of the tumor was markedly inhibited. A similar but not so striking a result was also obtained with a few human cases. At present a satisfactory explanation for these results is not available. Further proof of the importance of specific food supplies for the growth of tumors is afforded by the results of experiments relating to transplantation of tumors into foreign species. That this is impossible, as a rule may be learned from recent papers by Rondoni (153) and Nassetti (154). The transplantation of tumors into foreign species has been conducted successfully, thus far, by one of the following three methods (A-C). (A) Murphy (155) was able to implant rat tumor into a chicken embryo, which demonstrates either that the organism at a growing stage possesses the necessary specific substance for the growth of a tumor, even from a foreign species, or that resistance to tumor growth is less at embryonic stages than at later ones. That both factors play important roles we shall see presently. (B) Murphy and Morton were able (156) to induce tumors to grow in foreign species by reducing the activity of the leucocytes with X-rays. (C) 350 Vitamines and Deficiency Diseases [June-September I have shown (157) that mouse tumor can be successfully implanted in rats, for three generations, if the rats are fed on mouse-tumor tissue, The latter result demonstrates the necessity of a specific food supply for tumor growth. The fact is demonstrated, also, by the inhibition of tumor-growth in pregnant animals, as has lately been shown by v. Graff (158). That there are substances which stimulate growth we know from the activity of glands of internal secretion, especially of the anterior lobe of the pituitary. Robertson and Burnett (159) have found that emulsions of anterior lobe of this gland stimulate very markedly the growth of primary Carcinoma in rats, whereas liver emulsion had not the slightest effect. I should like to point out, here, the necessity of further research regarding the activity of pituitary gland (anterior lobe) on Cancer. We know that this gland is im- portant in regulating the growth of the organism. When the an- terior portion of the gland is extirpated from animals, they remain stunted. This is also true, of course, of other glands of internal secretion but it is not so marked as in the case of the pituitary. On the other band, the occurrence of adenomas of the anterior lobe is not infrequent. As a consequence of this abnormality, there is in- creased activity of the anterior portion of the pituitary. In children such abnormality produces Symmetrie exaggerated growth of the individual (gigantism). Between the age of 20-30 such general growth is no longer possible and the adenoma produces exaggerated growth of the bones of the extremities and the skull (acromegaly). It is conceivable that, in later life, when gigantism and acromegaly are no longer possible, increased activity of the pituitary gland may be responsible for the production of tumors. When one considers that the symmetrical orientation of organs in a growing embryo must be accomplished by chemical substances and that growth in a seed is brought about by a renewal of enzymic activity, which transforms inactive substances (possibly vitamines) into new substances which stimulate cell division, one is not far from a chemical conception of Cancer etiology. The idea that the etiology of tumors can possibly be explained by the existence of specific growth-promoting substance received a new impetus from Rous's discovery of tumors, in fowls, that could igisl Casimir Funk 35 1 be propagated by cell-free filtrates. There seems to be a tendency to regard such tumors as entirely different from the known tumors in mice, rats and men. This may be true f or the tumors in mice and rats, from which there are rarely metastases and which, as a rule, are entirely encapsulated. But we do not know whether very mahgnant human tumors, which are metastatic, can be propagated by means of a cell-free filtrate. On the contrary, the tumors in fowls seem to resemble human rather than any other experimental tumors. As to the etiology of avian tumors two possibilities exist at the present time. They are either caused by a " filter-passer," as pointed out by Rous, or they are caused by a very unstable chemical sub- stance, of a nature similar to that of vitamine. At present there is only a faint hope for a demonstration of the existence of such an unstable substance, but the problem was attacked by me, in collabo- ration with my late assistant, Mr. Drummond, in an investigation of the chemical composition of tumor extracts. I am also aware of the fact that no comparison can be made between the composition of the tumor, which consists of connective tissue, and of other tissue. Normal breast muscle of the fowl was taken as a control. At pres- ent I am comparing the chemical composition of spindle-cell sar- coma and osteochondroma, with the hope that new chemical sub- stances can be isolated which will prove to be fragments of the hypothetic active substance. I have recently found (i6o) that the blood of tumor- fowls (spindle-cell sarcoma) shows a very marked diminution in total nitrogen; as a rule, about 20-30 percent. This difference is not due to a change in the concentration of the serum, for one gets much less blood from tumor animals. It is surprising that this chemical change occurs a few days after the inoculation of the tumor, at a time when there is not the slightest trace of a tumor. At present I am investigating which fraction of the serum-protein is affected. Whether these chemical changes will throw any light on the etiology of the avian tumors we do not know, but they already constitute a perfect method for the diagnosis of these tumors. In human cases, however, such changes in the protein con- tent of the serum could not be detected. This negative result is very likely due to the fact that the chemical changes in the serum 352 Vitamines and Deficiency Diseases [June-September can be at best only very slight, since human tumors are very chronic. In f owls the whole blood was drawn and analyzed ; and, as the duration of the disease is only a few weeks, very much more marked changes would be expected for fowl tumors than for tumors of human origin. XIII. CONCLUDING GENERAL CONSIDERATIONS We see, from the present review, that the estabHshment of the existence of a new group of important substances, the vitamines, has stimulated a great deal of work in this connection. As the numerous papers are scattered among many different Journals, only a certain amount of the available data could be reviev^^ed. I have been severely criticized along two lines. In the first place objection has been taken to the fact that a name was given to these substances before they were isolated in a pure chemical con- dition. To this objection I answer that, besides the fact that we already know something about the vitamines from the chemical point of view, it has always been customary in physiological chem- istry to give names to substances which exercise definite chemical influences, whether their chemical Constitution is understood or not. Ferments, hormones and products of internal secretion, are among "substances" the chemical nature of which has not as yet been ascertained. As a matter of fact three-fourths of biological chem- istry deals with this kind of " substances." As I use the term " vitamines," it indicates merely a group of chemical substances which possess every analogy to the already well-known class of nitrogenous bases, the members of which are precipitated by phos- photungstic acid or similar reagents, and which are thrown down, in the mercuric chloride fraction, and by silver nitrate and baryta. So far no other reagents have proved of any value for their Iso- lation. Detailed knowledge of vitamines is now only a question of time and improved methods. A second important objection to my views in this connection is the fact that many diseases have been included amongst the " avi- taminoses," for which there may be other etiologies than deficiency of vitamines in the diet. There may be other etiologies for some of these diseases, it is true, but as the followers of other ideas have 1915] Casimir Funk 353 entirely failed to throw any light on the etiology of pellagra, sprue, rickets, or spasmophilia, it seems advisable to direct them into another far more promising direction. My hypothesis on pellagra has already met with practica! success; and rickets will very likely follow, when some of the clinicians test the new hypothesis. The only Position that may have been unwarranted, I admit, was that of ascribing to Cancer a possible chemical origin. My justification here is the fact that we are not making any headway in our knowl- edge of Cancer by working with any of the existing hypotheses My ideas have been found, by several authors, to be of value in general nutrition. It would be impossible to quote here all the available references but I should like to mention especially the papers by Melocchi (161), concerning general nutrition; by Fried- enthal (162), regarding the feeding of infants; by Sternberg (163), on the vitamines in connection with appetite; and by Kunert (164), on the influence of deficient food on teeth. Every observer who has studied experimental avitaminoses has noticed the diminished resistance of the animals to bacterial infec- tions. Hüssy (165) has reported very good results, with a prep- aration from rice-polishings, in the treatment of weakness and inanition in women. Reach (166) investigated the resistance of mice, on different diets, against picrotoxin, a poison which produces spasms. Regarding the action of this drug on the central nervous System, the animals were found to be more resistant on a meat diet than on a bread diet. Reach accepts the existence of unknown favorable substances in the food. Every investigator who has studied experimental deficiency- diseases has noticed the diminished resistance of the treated animals to bacterial infections. Some data are already available for men. Peiser (167) pointed out the importance of different fats and oils in Infant nutrition and their protective power against infections. Morrison (168) sees great danger in restricting the diet of typhoid patients. Thomas (169) found, clinically, a diminished degree of immunity in children on chronic deficient nourishment. Renon (170) suggested that there is diminished immunity to tuberculosis on diets containing insufficient quantities of vitamines. He pro- posed to investigate the influence of whole-meal bread and of 354 Vitamines and Deficiency Diseases [June-September white-bread, and other vitamine-containing and vitamine-poor diets, on cases of human tuberculosis of the lungs and on experimental tiiberculosis in animals. The chief investigation on this phase of the general subject was carried out, in the mines in the Rand, by Macaulay (48) who found that the occurrence there of an epidemic of scurvy (which was due, in his opinion, to the use of over-milled maize as staple foo.d) had, as sequelae, large epidemics of pneumonia and meningitis, and increases in the number of cases of tuberculosis. Especially in pneumonia the administration of pneumococcic Vaccine was applied without success. Macaulay then suggested that the maize should be less extensively milled. The execution of this Suggestion proved to be a great success, for not only did scurvy disappear but also with it the occurrence of pneumonia. These data possess a great deal of importance since the results were obtained for a large number of cases: 2251 cases of pneumonia were reported in the year 1908, with 686 deaths, which gave an incidence of 72.93 per 1000 and a death rate of 22.23 P^'" thousand workmen. The incidence of scurvy in one mine among the natives, for instance, was over 100 cases out of 700 natives, XIV. BIBLIOGRAPHY Beriberi 1. Caspari and Moszkowski: Beriberifrage; Deut. med. Wochschr., 39, 1479, 1529, 1913- Weiteres zur Beriberifrage; Berl. klin. Wochschr., 50, 1515, 1913. 2. Abderhalden and Lampe: Gibt es lebenswichtige, bisher unbe- kannte Nahrungstoffe; Z. ges. exp. Med., i, 296, 1913. 3. Shibayama : The present State of the study of beriberi in Japan ; Trans. XVII Congr. of Med., London, Sect. 21, Part II, p. 87, 1913. 4. Tasawa: Experimentelle Polyneuritis besonders bei Vögeln, im Vergleich zur Beriberi des Menschen; Z. f. exp. Path. u. Ther., 17, 27, 191 5. 5. Segawa: Ingestion of polished rice by chickens and pigeons; Mitt. med. Ges., Tokyo, 27, No. 7, 1914. Wesen der experi- mentellen Polyneuritis der Hühner und der Tauben und ihre Beziehungen zu Beriberi des Menschen; Virchow's Archiv, 215, 404, 19 14. 1915] Casimir Funk 355 6. Vedder and Williams : Concerning the beriberi-preventing sub- stances or vitamines contained in rice polishings; Philipp. J. Sei., Sect. B, 8, 175, 1913. 7. DuBOis and Corin : Rapport sur une petite epidemie de beriberi ä Bokala (Congo Beige) ; Bull. soc. path. expt., 7, 402, 1914. 8. Vedder and Clark: A study of Polyneuritis gallinarum, a fifth contribution to the etiology of beriberi ; Philipp. J. Sei., Sect. B, 37. 423, 1912. 9. Schnyder: Pathologisch-anatomische Untersuchungen bei ex- perimentellen Beriberi (Reispolyneuritis) ; Arch. Verdau- ung skr ankh., 20, 147, 1914. 10. EijKMAN : Ueber die Natur und Wirkungsweise der gegen ex- perimentelle Polyneuritis wirksamen Substanzen; Arch. Sehiffs- u. Tropenhyg., 17, 328, 1913. 11. Funk: Fortschritte der experimentellen Beriberiforschung in den Jahren 1911-1913; Müneh. med. Woehsehr., No. 36, 1913. 12. Cooper: On the protective and curative properties of certain foodstuffs against Polyneuritis induced in birds by a diet of polished rice; /. Hyg., 14, 12, 1914. 13. Gibson: The protective power of normal human milk against Polyneuritis gallinarum (beriberi) ; Philipp. J. Sei., Sect. B, 8, 469, 1913. The influence of compensated salt mixtures on the development of Polyneuritis gallinarum and beriberi; Ibid., 351, 1913. 14. Ohler: Experimental Polyneuritis — Effects of exclusive diet of wheat flour, in form of ordinary bread, on fowls; /. Med. Res., 31, 239, 1914. 15. Weill and Mouriquand: Troubles provoques par une alimenta- tion exclusive; Ann. de med. et chir. infant, 18, May 15, 1914. 16. Hill and Flack: Brit. Med. J., Sept. 16, 1311, 1911. 17. Merklen: Modification of diet saves duckhngs from epidemie disease; Bull, de la soc. de ped., 16, April, 1914. 18. Scheunert, Schattke and Lötsch : CaO-, MgO-, P2O5- Gehalt von Heu und Hafer, nach deren Verfütterung Pferde an Osteomalazie erkrankten; Biochem. Z., 36, 240, 191 1. 19. Lötsch : Ueber den Stallmangel, eine eigenartige Rinderkrank- heit im sächsischen Erzgebirge; Z. Infekt, der Haustiere, 12, 205, 1912. 20. Oseki : Untersuchungen über qualitative unzureichende Ernäh- rung; Biochem. Z., 65, 158, 1914. 356 Vitamines and Deficiency Diseases [June-September 21. Tachau: Versuche über einseitige Ernährung. (I.) ; Biochem. Z; 65» 253, 1914. (IL) Wasserverteilung und Oedem- bildung; Ihid., 67, 338, 1914. 22. MuRAi : A new method f or the extraction of the efficacious con- stituents of rice bran; /. Pharm. Soc. Japan, No. 386, 1914. 23. KoNDO and Gomi: Efficacious constituents of rice bran; Ibid., No. 391, 1914. 24. Drum MOND and Funk: The chemical investigation of the phos- photungstate precipitate f rom rice-pohshings ; Biochem. J., 8, 598, 1914. 25. Barger : The simpler natural bases. Longmans, Green and Co., London, 1914. 26. VoEGTLiN and Towles : The treatment of experimental beriberi with extracts of spinal cord ; /. Pharmacol. and Exp. Therap., 5, ^7, 1913- 27. Schaumann : Aetiologie der Beriberi (II) ; Arch. Schiffs- u. Tropenhyg. (Beih. 6), 18, 359, 1914. 28. Cooper: The curative action of autolysed yeast against avian Polyneuritis ; Biochem. J., 8, 250, 1914. 29. Stepp: Fortgesetzte Untersuchungen über die Unentbehrlichkeit der Lipoide für das Leben; Z. BioL, 62, 405, 1913. 30. : Ueber lipoidfreie Ernährung und ihre Beziehungen zu Beriberi und Skorbut; Deut. med. Wochschr., 40, 892, 1914. 31. Marshall: Therapeutic value of organic phosphorus Com- pounds; /. Am. Med. Assoc, 64, 573, 191 5. 32. Cooper : The relationship of vitamines to lipoids ; Biochem. J., 8, 347. 1914- 33. Hornemann: Zur Kenntnis des Salzgehaltes der täglichen Nahrung des Menschen ; Z. Hyg. u. Infekt., 75, 553, 1913. 34. Heubner: Phosphorstoffwechsel— (VII). Der P-gehalt ver- schiedener Organe unter gewissen Nahrungsbedingungen; Arch. exp. Path. Pharm., 78, 24, 1914. 35. Funk: Studies on beriberi. The probable role of vitamines in the process of digestion and utilization of f ood ; Proc. Physiol. Soc, Dec. 13, 1913. Studien über Beriberi— (X). Experi- mentelle Beweise gegen die toxische Theorie der Beriberi; Z. physiol. Chem., 89, 373, 1914. 36. : Studien über Beriberi— (XI). Die Rolle der Vitamine beim Kohlenhydrat-Stoffwechsel ; Ibid., 378, 1914. i9is] Casimir Funk 357 37. Funk and v. Schönborn : The influence of a vitamine-free diet on the carbohydrate metabolism; /. Physiol., 48, 328, 1914. 38. Braddon and Cooper: The influence of the total fuel-value of a dietary upon the quantity of vitamine required to prevent beriberi ; Brit. Med. J., June 20, 1914. The influence of meta- boHc factors in beri-beri ; /. of Hyg., 14, 331, 1914. 39. Funk : Is polished rice plus vitamine a complete f ood ; /, Physiol., 48, 228, 1914. 40. MoRPURGO and Satta: Sugli scambi dei sostanze nutrienti fra topi in parabiosi ; Arch. di fisioL, 11, 360, 1914. 41. Funk and Douglas: Studies on beriberi — (VIII). The rela- tionship of beriberi to glands of internal secretion ; 7. Physiol., 47, 475, 1914. 42. Douglas : The histology of the thyroid in animals fed on various diets; /. Path. Bact., 19, 341, 1915. Scurvy and infantile scurvy (milk problem) 43. Holst and Fröhlich: Ueber experimentellen Skorbut — (II). Weitere Untersuchungen über das Konservieren und Extra- hieren der spezifischen Bestandteile der antiskorbutischen Nahrungsmitteln; Z. f. Hyg. u. Infektionsk., 75, 334, 1913. 44. Freudenburg : Beitrag zur Frage des Barlow-Schutzstoff es ; M. f. Kindcrh., 13, 141, 1914. 45. Freise : Der Alkoholextrakt aus Vegetabilien als Träger barlow- heilender Stoffe ; Ibid., 12, 687, 191 4. 46. : Fixed diet in prisons; Bull. Tropical Dis., 4^ 446, 1914. 47. Markl: Nutrition of sailors; Arch. Schiffs, u. Tropenhyg., 18, 583, 1914. 48. Fleming, Macaulay and Clark : Report on the prevalence and prevention of scurvy and pneumonia in Southern Rhodesia amongst native laborers, 1910. 49. Darling: The pathological afifinities of beriberi and scurvy; /. Amer. Med. Ass., 63, 1290, 1914. 50. Hart and Lessing: Der Skorbut der kleinen Kinder, Stuttgart, 1913- 51. Hess and Fish: Infantile scurvy; Am. J. of Dis. of Children, 8, No. 6, 1914. 52. Lust and Klocman: Metabolism study of Barlow's disease; Jahrb. f. Kinderh., 75, 663, 1914. 53. Bahrdt and Edelstein : Analyse der Organe in Barlow'schen Krankheit.; Z. /. Kinderh., 9, 415, 1914. 358 Vitamines and Deficiency Diseases [June-September 54. Lane-Claypon : Report to the Local Government Board upon the "biological properties " of milk, both of the human species, and of cows, considered in special relation to the feeding- of infants ; New Series No. 76, London, 1913. 55. Sittler : Das Säuglingsheim der medizinischen Universitätsklinik zu Marburg während der ersten Jahre seines Bestehens, 1905- 1908; Z. f. S02. Med., ( Säuglings fürsorge), 4, i, 1909. 56. Dennett: The use of boiled milk in infant feeding, with a dis- cussion by Neff, Lowenberg, Scott and Graves; /. Am. Med. Assoc, 63, 1991, 1914. Beriberi, scurvy and pellagra 57. Stannus: Pellagra in Nyasaland. Annual Med. Rep. on the Health of the Nyasaland, No. 78, 1913; (II) Trans. Soc. Trop. Med. and Hyg., 7, 32, 1913; Btdl. Trop. Dis., 4, 444, 1914. 58. Lavinder: Pellagra. Prevalence and geographica! distribution in Arkansas, Oklahoma and Texas ; U. S. Pub. Health Rep., 28, 1555, 1913. 59. Devoto: Aetiologie und Klinik der Pellagra; Wien. med. klin. Woch., No. I, 1913. 60. Bardin : Note on the dififerentiated blood-counts in three cases of Pellagra; Old Domin. J. of Med. and Sitrg., 17, July, 1913. 61. Beeson: The thyroid gland in pellagra; /. Am. Med. Assoc, 63, 2129, 1914. 62. NicoLAiDi : Untersuchungen über die Ernährungs-bilanz der Pellagrosen; Rev. Stiinzie Med., 9, No. 6, 1913. 63. Albertoni and Tullio: L'alimentazione maidica nel sano e nel pallagroso ; R. Acad. Science, Ist. di Bologna, iith Jan., 1914. 64. Myers and Fine : Metabolism in pellagra ; Am. J. Med. Sei., 145, 705, 1913- 65. SiLER and Garrison : An interesting study of the epidemiology of pellagra; Ibid., 146, 42 and 238, 1913. 66. Grimm: Pellagra: some facts in its epidemiology; /. Am. Med. Assoc, 90, 1423, 1913. 67. Weston : Pellagra in early childhood ; Am. J. Dis. of Children, 7, 124, 1914. 68. Weiss: Die Pellagra in Südtirol und die staatliche Bekämp- fungsaktion ; Oester. Sanitätswes., 26, 309, 1914. 69. Perez : Pellagra ; Brit. Med. J., March 14, 624, 1914. 1915] Casimir Funk 359 70. McDonald: Pellagra in Antigua; Lancet, 188, 127, 1915. 71. Goldberger: The etiology of pellagra. The significance of cer- tain epidemiological observations with respect thereto. U. S. Pub. Health Rep., 29, 1683 and 2354, 1914. y2. SiLER, Garrison and MacNeal : Further studies of the Thomp- son-McFadden Pellagra Commission. A summary of the second progress report; /. Am. Med. Assoc, 63, 1090, 1914. 73. Rayer: Pellagra; New Orleans Med. Surg. J., 66, 718, 1914. 74. VoEGTLiN : The treatment of pellagra ; /. Am. Med. Assoc, 63, 1094, 1914. 75. Alpago-Novello : Commissione Pellagrologica Provincia di Bel- luno. Riv. Pellagr. Italiana, 13, 67, 191 3. yS, : Comm. pellagr. di Mariano, February, 1914. yy. Kleiminger: Pellagra; Z. ges. Neur. 11. Psych., 16, Heft 5, 1913. 78. Allison : Report of cases of pellagra ; Texas State J. Med., 10, 123, 1914. 79. Sylvester: Pellagra; South. Med. J., 7, 449, 1914- 80. Elebash : Treating the gastro-intestinal tract in pellagra ; Ibid., 7, 447, 1914. 81. Lorenz: The treatment of pellagra. CHnical notes on pellagnns receiving an excessive diet ; U. S. Pub. Health Rep., 29, 2357, 1914. 82. Blosser : Sugar-cane products as a cause of pellagra in the South ; /. Am. Med. Assoc, 64, 543, 191 5. 83. Funk: Prophylaxe und Therapie der Pellagra im Lichte der Vitaminlehre; No. 13, 698, 1914. 84. : Studies on pellagra. The influence of the milling of maize on the chemical composition and the nutritive value of maize-meal; /. Physiol, 47, 389, 191 3. 85. Driscoll: A theory of the etiology of pellagra; South. Med. J., 6, 400, 1913. 86. JuRiTz: The chemical composition of South-African maize and other cereals; Union Agricultural J., Sept., 495, 1913. 87. MacCrae : The phosphoric oxide content of maize flours : J. Hyg., 14, 395, 1914. 88. Poppe: Bemerkungen über die Ernährung in Katanga; Bull, boc chim. Belg., 27, 23, 191 3. 89. : Wanderung der Bestandteile des Maiskornes in Wasser und in wässerigen Lösungen ; Ibid., 27, 103, 1913- 360 Vitamines and Deficiency Diseases [June-September 90. Weiss : La pellagra nel Tirolo meridionale e l'azione del Governo contro la stessa; Riv. Pellagr., 13, 90, 1913. 91. Nightingale: Zeism or pellagra?; Brit. Med. J ., 300, 1914. Sprue 92. Cantlie: Some recent observations on sprue; Brit. Med. J., 1296, 1913. 93. Bahr: Researches on Sprue; Trans. Soc. Trop. Med. and Hyg., 7, 161, 1914. 94. Leede : Ein Fall von Sprue durch Erdbeeren gebessert ; Zeitschr. f. Hyg., u. Infektionsk., 75, 578, 191 3. 95. MÜHLENS : Behandlung bedrohlicher Zustände bei Tropenkrank- heiten ; Deut. med. Wochschr., 40, 1249, 1914. 96. Werner: Skorbutsymptome bei Sprue; Arch. f. Schiffs- u. Trophenhyg., No. 7, 191 4. Rickets and cod-liver eil 97. Ostheimer: Fragilitas ossium ; /. Am. Med. Ass., 63, 1997, 1914- 98. Schmore: Ueber die Beeinflussung des Knochenwachstums durch phosphorarme Ernährung; Arch. f. exp. Path. Pharm., 73, 313, 1913- 99. Weiss : Zur Frage der Hypophysentherapie der Rachitis ; Therap. Monatsh., 7, 490, 1913. 100. Rominger: Rachitis und Interne Sekretion; Z. f. Kinderh., 11, August, No. 5-6, 1914. loi. ScHLOSs: Zur Behandlung der Rachitis mit Lebertran, Kalk und Phosphor auf Grund von Stoffwechselversuchen ; Deut. med. Wochschr., 39, 1505, 1913. Therapie der Rachitis; Jahrb. Kinderheilk., 78, No. 6, 1914. 102. Frank and Schloss: Zur Therapie der Rachitis. IV. Ver- gleichende Untersuchungen über die Wirkung von Lebertran und Phosphorlebertran beim künstlich ernährten rachitischen Kinde. Jahrb. Kinderheilk., 79, 539, 1914. 103. Kurt Meyer: Mineral metabolism in rachitis; Ibid., 77, 28, 1914. 104. BoRKMAN : The metabolism in Osteogenesis imperfecta, with special reference to calcium; Am. J. of Dis. of Children, 7, 436, 1914. 105. Schabad: Der Stoffwechsel bei angeborener Knochenbrüchigkeit (Osteogenesis imperfecta). Auf Grund gemeinsam mit Sorochowetz ausgeführten Stoffwechseluntersuchungen; Z. Kinderheilk., 11, 230, 1914. I9IS] Casimir Funk 3^1 io6. Gautier and Mourgues : La composition chimique de l'huile de foie de morue; Compt. rend. de l'acad. d. sc, 107, iio, 626, 1188. 107. IscovESCo: Lecithides contenus dans l'huile de foie de morue; Compt. rend. Soc. de Biol, 76, 34, 74, 117, 1913, loth and I7th Jan., 1914. 108. Funk: Biochemistry of cod-liver oil; Biochemical Bulletin, 4, 365. 1915- Osteomalacia, spasmophilia, eclampsia 109. V. Noorden: Textbook on Pathology of Metabolism, 2, 859 (chapter on rickets). HO. Antoine: Sur l'osteomalacie du chien; Bidl. acad. de Belg., 28, 213, 1914. 111. Takasu: Spasmophile Dyspepsie bei natürlicher Ernährung. Säuglingskakke ; Jahr. Kinderheilk., 80, No. 5, 1914- 112. Liefmann: Die Aceton Ausscheidung im Harne von Gesunden und Spasmophilen Kindern ; Ibid., 77, 125, 1914. 113. Freudenberg and Klocman : Spasmophiha; Ibid., 79, 700, 1914. 114. Brüning: Untersuchungen über das Wachstum von Tieren jenseits der Säuglingsperiode bei verschiedenartiger künst- licher Ernährung; Ibid., 79, 305, 1914. Die Entwicklung der Neugeborener weissen Ratten, getrennt von der Mutter, bei künstlicher Ernährung; Ibid., 80, No. i, 1914- Growth 115. Masslow: Ueber die biologische Bedeutung des Phosphors für den wachsenden Organismus; Biochem. Z., 55, 45, 1913; Ibid., 56, 174, 1913. 116. : Zur Frage nach den Zerrütungen des Knochensystems durch phosphorarme Ernährung (III) ; Biochem. Z., 64, 106, 1914. 117. Durlach : Untersuchungen über die Bedeutung des Phosphors in der Nahrung wachsender Hunde. Diss. Göttingen, 1913. 118. Röhmann : Ueber die Ernährung von Mäusen mit einer aus einfachen Nahrungstoffen zusammengesetzter Nahrung; Biochem. Z., 64, 30, 1914- 119. Hart and McCollum : The influence of restricted rations on growth; /. Biol. Chem., 17, 2, 1914. 120. Wheeler: Feeding experiments with mice; /. Exp. ZooL, 15, 209, 1913. 302 Vitamincs and Deficiency Diseases [June-September 121. OsBORNE and Mendel: The relation of growth to the chemical constituents of the diet; /. Biol. Chem., 15, 311, 1913. 122. Hart and McCollum : Influence on growth of rations restricted to the corn or wheat-grain ; /. Biol. Chem., 19, 373, 1914. 123. Desani: Untersuchungen über die Genese des Cholesterins; Chem. Zentr., 2, 1764, 1913. 124. McCollum and Davis: Further observations on the physiological properties of the Hpins of the egg-yolk ; Proc. Soc. Exp. Biol. Med., II, loi, 1914. 125. Lander : On the cholesterol content of the tissues of growing rats when under various diets ; Biochem. J ., 9, 78, 1915. 126. MacArthur and Luckett: Lipins in nutrition; /. Biol. Chem., 20, 161, 1915. 127. McCollum and Davis: The necessity of certain lipins in the diet during growth; Ibid., 15, 167, 1913. 128. Osborne and Mendel: The rclationship of growth to the chemical constituents of the diet; Ibid., 15, 311, 1913. 129. : The influence of butter-fat on growth; Ibid., 16, 423, 1913. 130. Funk and Macallum : Die chemischen Determinanten des Wachstums; Z. pJiysiol. Chem., 92, 13, 1914. 131. McCollum and Davis: Observations on the isolation of the sub- stance in butter-fat which exerts a stimulating influence on growth; /. Biol. Chem., 19, 245, 1914. 132. Osborne and Wakeman : Does butter-fat contain nitrogen and phosphorus?; Ibid., 21, 91, 1915. 133. Aron : Untersuchvmgcn über die Beeinflussung des Wachstums durch Ernährung; Berliner klin. Woch., No. 21, 1914. 134. Osborne and Mendel: The suppression of growth and the process of growth ; /. Biol. Chem., 18, 95, 1914. 135. McCollum and Davis: Nutrition with purified food substances; Ibid., 20, 641, 191 5. 136. Osborne and Mendel: The influence of cod-liver oil and some other fats on growth; Ibid., 17, 401, 1914. 127. : Further observations on the influence of natural fats upon growth; Ibid., 20, 379, 1915. 138. : Some problems of growth; Ibid., 17, 2, 1914, 139. : Amino-acids in nutrition and growth; Ibid., 17, 325, 1914. 140. : The comparative nutritive values of certain proteins I9I51 Casimir Funk 3^3 in growth, and the problem of protein minimum; Ibid., 20, 351, 1915. 141. McCollum: The value of the proteins of the cereal grains and of milk for growth of the pig, and the influence of the plane of protein intake on growth; Ibid., 19, 329, 1914. 142. Street: The feeding value of sanatogen compared with com- mercial casein with respect to maintenance and growth; /. Am. Med. Assoc, 63, 1831, 1914. 143. Hektoen: The formation of antibodies in rats fed on pure vegetable proteins. (Osbome-Mendel stunting diet) ; /. In- fect. Dis., 15, 278, 1914. 144. Funk : Studien über das Wachstum. Das Wachstum auf vitam- inhaltiger und vitaminfreier Nahrung; Z. physiol. Chem., 88, 352, 1913. 145. : Studies on growth: The influence of diet on growth, normal and malignant; Lancet, Jan. loth, 1914. 146. McCollum and Davis: The influence of certain vegetable fats on growth; /. Biol. Chem., 21, 179, 1915. 147. Bottomley: Some accessory factors in plant growth and nutri- tion; Proc. Roy. Soc. (B), 88, 237, 1914. 148. : The significance of certain food substances for plant growth; Ann. Botany, 28, 531, 1914- Tumors 149. Rous: The influence of diet on transplanted and spontaneous mouse tumors ; /. Exp. Med., 20, 433, 1914. 150. Centanni: La dieta aviride per lo sviluppo dei tumori speri- mentali; Tumori, 2, 466, 1914. 151. Van Alstyne and Beebe: The effect of non-carbohydrate diet upon the growth of sarcoma in rats; /. Med. Res., 29, 219, 1913- 152. Benedict and Lewis: The influence of induced diabetes on malignant tumors (including a report of a case of human phlorhizin glycosuria) ; Proc. Soc. Exp. Biol. Med., 11, 134, 1914. 153. RoNDONi: Esperienze sui tumori. VIII; Rinne. Soc. Ital. di Patol. Pisa, Mar., 1913. 154. Nassetti: Innesti eterogenei dei tumori; Tumori, 3, Nov.-Dec., 1913. 155. Murphy: Transplantation of tissues to the embryo of foreign species; /. Exp. Med., 17, 482, 1913. 364 Vitamines and Deficiency Diseases [June-September 156. Murphy and Morton: The lymphocyte in natural and induced resistance to transplanted cancer, /. Exp. Med., 22, 204, 1915- 157. Funk: The transplantation of tumors to foreign species; /. Exp. Med., 21, 571, 1915. 158. V. Graff: Der Einfluss der Schwangerschaft auf das Wachstum maligner Tumoren; Wiener klin. Wochschr., 27, 7, 1914. 159. Robertson and Burnett: Influence of the anterior lobe of the pituitary body upon the growth of carcinomata ; /. Exp. Med., 21, 280, 1915. 160. Funk : Serum diagnosis of Rous's chicken sarcoma, based on chemical methods; Biochem. Bull., 4, 24, 1915. General nutrition 161. Melocchi: Nuovi orizzonti suH'alimentazione dell'uomo, Napoli, 1914. 162. Friedenthal: Ueber Säuglingsernährung nach physiologischen Grundsätzen mit Friedenthal'schen Kindermilch und Ge- müsepulvern; Berl. klin. Wochschr., No. 16, 1914. 163. Sternberg: Diät und diätetische Behandlung vom Standpunkte der Vitaminlehre und vom Standpunkte der Lehre der diätetischen Küche; Arch. Verdauungs-krankh., 20, 200, 1914. 164. Kunert: Unsere heutige falsche Ernährung; Breslau, 1914. 165. HüssY : Zur klinischen Bedeutung der Vitamine ; Münch. med. Wochschr., 61, 18, 1914. 166. Reach : Studien über die Nebenwirkungen der Nahrungstoffe; Ber. Wien. Akad., Abt. III, i, 1913. 167. Peiser: Fett in Kindernahrung; Berl. klin. Wochschr., 51, 1065, 1914. 168. Morrison : Some of the dangers of too greatly restricted diet in typhoid; Kentucky Med. J ., 12, Nov. I5th, 1914. 169. Thomas : Beziehungen der chronischen Unterernährung zur In- fektion und zu klinischen Zeichen der verminderten Im- munität; Z. Kinderh., July, 1914. 170. Renon : La tuberculose et les vitamines; Soc. de therapeut. de Paris, 24th June, 1914. 21S Manhattan Avenue, New York City. BIOCHEMISTRY OF COD-LIVER OIL. (Preliminary note) CASIMIR FUNK Introduction. It is very surprising that the chemistry and physiology of cod-liver oil have been neglected to such a degree that the author was able to find only a few papers deaUng with the subject. In 1888, Gautier and Mourgues (i) showed that even the refined yellow oil contains a small quantity of organic bases (which were then considered to be ptomaines or alkaloids). Their technic was as f ollows : 100 k. of yellow cod-liver oil were extracted with the same volume of 33 percent alcohol, to which 4 gm. of oxalic acid per 1. were added. The aqueous-alcoholic extracts were saturated with calcium hydroxid, filtered and evaporated in vacuo, at 45° C. Toward the end of the distillation, precipitated calcium carbonate and calcium hydroxid were added, the mixture evaporated to dry- ness, and the residue extracted with 90 percent alcohol. From this alcoholic extract the alcohol was removed in vacuo, water and strong caustic potash were added to the residue, and the alkaline mixture was extracted with ether. To the ethereal extract, a sol. of oxalic acid in ether was added; this precipitated the bases as Oxalates. The yield was 52-65 gm. of Oxalates from 100 k. of cod-liver oil. The Oxalates were dissolved in dil. caustic potash sol., and the free bases separated, as an oil, on the surface of the liquid The oil was removed and dried over freshly calcinated potash. In this way 0.35-0.5 gm. of dry substance was obtained. Subjected to fractional distillation, products were obtained as f ollows : (a) Between 87-90° : Butylamin (6) Between 96-98° : Amylamin {c) Under 100° : Hexylamin {d) Between 198-200° : Hydrotoluidin 365 366 Biochemistry of Cod-Liver Oil [June-September The distillation was continued to 215°. After cooling, the dark brownish residue was extracted with ether. The ethereal extract was evaporated and the residue dissolved in dil. hydrochloric acid sol. To this sol., platinic chlorid was added, The resuhing precipitate was the chloro-platinate of aselin, an alkaloid of the composition indi- cated by the formula C25H32N4, present in the original oil in small quantity. From the mother-liquor the chloro-platinate of morrhuin was obtained, to which the formula C19H27N3 was ascribed. It is possible that these two substances were secondary products of the distillation, . A second paper dealing with this subject was published by Isco- vesco (2), who Claims to have isolated a lipoid from cod liver that possesses all the known therapeutic properties of cod-liver oil. The writer has been working on this subject during the past year in the hope of isolating vitamine-like substances that might account for the action of the oil in curing rickets and accelerating growth. The results of the preliminary work are given below. Regarding the action of cod-liver oil there are two distinct views: that of the writer (3), who attributes the action to the presence in the oil of a vitamine-like substance; and of Osborne and Mendel (4) who regard the action as due to the special nature of the fats in the oil. The work of Gautier and Mourgues, and that of the writer, show that cod-liver oil contains a certain amount of organic bases, a fact which must be taken into account. If the writer's view is correct, it might be advisable to admin- ister, in rickets, cod-liver oil that is less purified than that used at present. Also, it should be possible to administer an extract of the organic bases without the oil. Such products have been obtained and will be tested on animals at the earliest opportunity. As ex- perimental animals, chickens will be used, which develop in captivity, on a uniform diet, a condition resembling rickets. Some of the results obtained with cod-liver oil have already been published else- where (3). The cod-liver oil subjected to fractionation was a very dark crude oil. A second sample of crude oil, though lighter in color, gave much less extractive material than the darker one. In the first case the oil was extracted by a method very similar to thal I9I5] Casimir Funk 3^7 used by Gautier and Mourgues ; and the extract was worked up by ordinary methods for the Separation of organic bases. In the sec- ond case the oil was extracted with dilute sulfuric acid sol., and the phosphotungstate precipitate obtained was worked up by the acetone method— which has been used successfully in our work on yeast (5) . Substances were separated in each fraction, but the quantity of each product was so small that the work will have to be repeated with more material. ExPERiMENTAL. I. The cod-Hver oil was entirely soluble in ether, acetone, ligroin and Chloroform, and partially so in benzene. With alcohol alone, or with alcoholic mercuric chlorid sol., a slight precipitate was noticed. About 23.5 k. of the oil were used. Each k. was extracted with a sol. of 660 cc. of abs. alcohol and 50 cc. of conc. hydrochloric acid made up to 2 1. with water. Each k. was extracted three times, a third part of the sol. having been used in each extraction. The extracts were isolated in a separatory funnel, and evaporated in vaciio. The residue, which had an agreeable smell, was dissolved in alcohol, filtered from a precipitate which consisted mainly of inorganic salts, and the sol. evaporated again. Oil separated, which was twice washed with water and then hydro- lyzed with 5 percent sulfuric acid sol. The watery extract was precipitated with 5 percent sulfuric acid sol. containing 50 percent of phosphotungstic acid. The precipitate was filtered, washed with dilute sulfuric acid sol., and dried. It weighed 969 gm. when nearly dry. Treatment of the phosphotungstic acid precipitate. The precip- itate was decomposed with 2 k. of baryta in a mortar. The phos- photungstate of barium was suspended in water and shaken. The combined filtrates were freed from baryta with sulfuric acid and evaporated in vacuo. The residue did not give a precipitate with alcoholic mercuric chlorid sol. No precipitate was obtained with silver nitrate and baryta. The sol. was freed from silver and baryta, and was reprecipitated with phosphotungstic acid. The resulting precipitate amounted to only 304 gm., consequently con- siderable decomposition of the nitrogenous substances must have taken place. This view is strengthened by the fact that the precip- itate, decomposed with neutral lead acetate, gave a very heavy pre- 368 Biochemistry of Cod-Liver Oil [June-September clpitate with mercuric chlorid. Before using this reagent, others were tried, but without success. The decomposed phosphotung- state precipitate had a very pronounced smell of nitrogenous bases. The final sol. of free bases was precipitated with mercuric chlorid in alcoholic sol. Both the precipitate and the filtrate gave crystalline Chlorids after elimination of mercury and evaporation. Treatment of the phosphotimgstic acid filtrate. The filtrate was freed from phosphotungstic and sulfuric acids by means of neutral lead acetate. The filtrate, freed from lead with hydrogen sulfid, was conc. in vacuo. It was thought likely that it contained a large amount of amino-acids derived from liver tissue. The whole sol. contained 4.5 gm. of nitrogen and only 0.24 gm. of amino-nitrogen, as determined by Van Slyke's method. The residue, after evapora- tion, was entirely soluble in alcohol with the exception of a small quantity of inorganic material. The liquid was freed from chlorid with silver acetate, and the silver removed with hydrogen sulfid. The residue, when dissolved in alcohol and slowly evaporated, gave a crystalline substance. This will be investigated in the near future. Treatment of the fatty residue obtained from the evaporated alcohoUc-aqiieous extracts. This oil was hydrolyzed with 5 percent sulfuric acid sol. for 2 hr. The filtered liquid, which smelt like herring, was precipitated with phosphotungstic acid; 19.5 gm. of dry precipitate were obtained. Treatment of oil extracted with dilute alcohol. The extracted oil was hydrolyzed for 2 hr. with 5 percent sulfuric acid sol. The acid extracts were precipitated with phosphotungstic acid; 186 gm. of precipitate were obtained. 2. About 25 k. of the same supply of dark oil were extracted in portions of 2 k. each with 2 1. of 10 percent sulfuric acid sol. for 2 hr. on a shaking machine, and then lef t over night. The extracts were isolated in a separatory funnel and precipitated with phosphotung- stic acid. The resulting precipitate (dry), which weighed 877 gm., was extracted with acetone and 57.2 gm. of insoluble fraction ob- tained. A second extraction of the oil, as above, yielded 240 gm. of phosphotungstate but only 4 gm. of the acetone-insoluble fraction. Treatment of the acetione-insoluble fraction. The 61.2 gm. of material insoluble in acetone, obtained by the above mentioned treat- 1915] Casimir Funk 3^9 ment, were treated in a mortar with 150 gm. o£ neutral lead acetate and shaken on a machine for i hr, Alcohol was added to render the precipitate more insoluble ; the liquid was filtered. The filtrate was freed from excess of lead and evaporated. The resulting white residue was dissolved in water, and alcohol added. Gelatinous material separated out, which was filtered off; 1.3 gm. was obtained. The aqueous filtrate from this substance was evaporated in vacuo, the residue dissolved in alcohol and precipitated with alcoholic mer- curic chlorid sol. The resulting precipitate was decomposed with hydrogen sulfid and the filtrate evaporated to dryness. The residue was redissolved in water, and the liquid freed from chlorid by means of silver acetate. The filtrate, freed from silver, gave 3 gm. of substance, which is now being carefully investigated. The mercuric chlorid filtrate was freed from mercury with hydrogen sulfid, and evaporated ; the residue was dissolved in water and freed from chlorid with silver acetate. The filtrate from the silver sulfid gave, after evaporation, o. i gm. of substance. Treatment of the acetone-soluble fraction. The acetone sol., obtained by the above mentioned treatment, was diluted with water, decomposed with 2 k. of neutral lead acetate, shaken for i hr., and filtered. The precipitate was again suspended in 30 percent acetone and filtered. The combined filtrates were freed from excess of lead and evaporated in vacuo. The residue, which did not crystallize, was dissolved in water and precipitated with alcoholic mercuric chlorid sol.: 99 gm. of precipitate (dry) were obtained. Both the precipitate and the filtrate were freed from mercury and evaporated ; both yielded small amounts of crystalline hydrochlorids. The fil- trates from the hydrochlorids were fractioned in the usual manner with silver nitrate, and with silver nitrate and baryta. These f rac- tions, which gave only exceedingly small amounts of different sub- stances, will be investigated later when larger quantities of material are available. SuMMARY. With the idea that the therapeutic action of cod- liver oil is not due to peculiar fatty constituents in the oil, but to the presence of nitrogenous substances, a Separation of the latter from the oil was effected. The raw material used was crude cod-liver oil, since this is richer in organic bases than the purified variety. 370 Biochemistry of Cod-Liver Oil [June-September Bihliography 1. Gautier and Mourgues: Compt. rend. Acad. des Sciences. 1888, cvii, pp. HO and 626. 2. IscovESCo: Compt. rend. Soc. de BioL, 1913, Ixxvi, pp. 34, 74 and 117; 1914, loth and I7th January. 3. Funk and Macallum : Zeitschr. f. physiol. Chem., 1913, xcii, p. 13- 4. OsBORNE and Mendel: Journ. BioL Chem., 1914, xvii, p. 401. 5. Funk: Biochemical Bulletin, 1916, v. (Accepted for publica- tion, June i, 1915 ; to be the opening paper in Vol. v.) 213 Manhattan Avenue, New York City. THE PROBLEM OF REJUVENESCENCE IN PROTOZOA* LORANDE LOSS WOODRUFF It is a pleasure to accept the invitation to present a summary of the results derived from our genetic cultures of Paramcecium at Yale, with special reference to the bearing of this and other work on the problem of rejuvenescence in protozoa; for, it seems to me, the problem has now passed successfully through the periods of youth and adolescence, and is approaching that of maturity, when we may confidently expect the production of some conclusions of general significance. Although the problems of protoplasmic senescence and the func- tion of conjugation have afforded the Stimulus for investigations on the life history of infusoria since Ehrenberg, nearly a Century ago, theorized on the potential ' immortality ' of those forms, we may take, as the point of departure for our present brief review of the subject, the classical experimental studies of Maupas. As is well known Maupas' studies ajfforded a wealth of data, all of which indicated that continued reproduction by division results in degen- eration and death, and seemed to place the conclusion, that conju- gation is a sine qua non for the life of infusoria, upon a firm em- pirical basis. A series of important Investigations by Hertwig and Calkins confirmed Maupas' general conclusion that infusoria, after a more or less definite number of divisions, degenerate and finally die if con- jugation is prevented. Calkins, however, made the significant dis- covery that artificial Stimuli of different kinds may, for a time, be substituted with success for conjugation since, by the opportune use of artificial Stimulation, he was able to prolong the life of one cul- ture of Paramcocium caudahim to the 742d generation. * Presented at the Symposium on protozoology. Amer. Ass'n. Adv. Science, Berkeley, Cal., August 5, 1915. For a review of the earlier work on this subject, at Yale, see Biochemical Bulletin, 1912, i, p. 396, 371 372 Problem of Rejuvenescence in Protozoa [June-September Enriques studied the same general problem and reached the con- clusion that the degeneration and death of infusorian cultures was due to bacterial poisons, because he succeeded in breeding Glaucoma scintillans for 683 generations without signs of degeneration when he took measures to ehminate this factor. Whether this was the crucial factor in his method is open to question, but the significant fact remains that his animals survived nearly twice as long as thöse of earlier workers, without conjugation or artificial Stimulation, thus suggesting that, if suitable conditions are supplied, reproduction by division can proceed indefinitely. At this point I took up the problem and first investigated the possibility that the degeneration observed in the previous investiga- tions was induced by too great uniformity in the conditions of cul- ture, or bythe culture-medium being deficient in something essential for the continued well-being of the organisms. A race of Para- mcecmm aurelia was isolated in 1907 and bred on infusions of vari- ous materials found in the natural environment of the animal, while a sub-culture was subjected to the relatively constant hay-infusion culture-conditions generally employed. The result was that the cells bred in the constant hay-infusion medium died out after a typical Calkins cycle, while those bred on the ' varied-environment ' medium did not pass through periods of marked physiological depression or show morphological changes which could be interpreted as abnormal. This race is still, after more than eight years in culture, in a normal condition, having attained over 5250 generations without conjuga- tion or the use of artificial Stimuli. The success with the varied culture medium naturally led to the question whether the longevity of Paramcscium, on a varied environ- ment, is dependent upon intrinsic Stimuli from the frequent changes of the medium, or whether a constant medium of hay-infusion is unfavorable because it lacks some Clements which are essential for the continued existence of the organism. Accordingly,^ a sub-cul- ture of this race was bred for a period of nine months on a constant culture-medium of beef-extract. The continued health of the or- ganisms on this constant medium throughout the experiment, which was continued sufficiently long to include a Calkins cycle, if such 1 Woodruff and Baitsell: Journ. of Exp. ZooL, 1911. 1915] Lorande Loss Woodruff 373 was inherent, indicated that it is the composition of the medium, rather than the changes in the medium, which is conducive to the unhmited development of this race without the necessity of conju- gation or artificial Stimulation, From a study of various species of hypotrichous infusoria, as well as the main culture of ParamcEcium aurelia, it was found that minor periodic rises and falls of the division-rate occur, from which recovery is autonomous. These fluctuations were termed * rhythms ' and contrasted with the so-called cycle, which comprises a varying number of rhythms and, according to Maupas and Calkins, ends in the death of the race, if conjugation or artificial Stimulation is not resorted to. The problem of rhythms was then studied intensively.^ It was found that the subjection of the culture to the most constant en- vironmental conditions failed to eliminate the rhythms and thus to resolve the graph of the multiplication rate into an approximately straight line; but, instead, the rhythms appeared slightly more pro- nounced. It was also found, from a study of the temperature co- efficient^ of the rate of reproduction of the culture, that this is influ- enced by temperature at a velocity similar to that for a chemical reaction, except when the rhythms interfere. Thus, it is apparent that there are inherent rhythmical changes in the phenomena of the cell which produce slight fluctuations in the division-rate. The results, then, from the study of this pedigreed race of Paramcociwn aurelia led us to conclude that this organism, when subjected to suitable culture conditions, has the power of unlimited reproduction by division without conjugation or artificial Stimula- tion; the only necessary Variation in the rate of reproduction being the normal minor periodic rise and fall of the division-rate, due to some unknown factor in cell phenomena, from which recovery is autonomous (rhythm). Calkins,^ however, did not share this optimism and sought the explanation, of the diametrically opposite results derived from his and from our cultures of Param<^cium, in variations in the tendency 2 Woodruff and Baitsell: Journ. of Exp. Zool., 1911, 3 Woodruff and Baitsell: Am. Journ. Physiology, 191 1. * Calkins: Journ, of Exp. Zool., 1914. 374 Problem of Rejuvenescence in Protosoa [June-September to conjugate, which he and Jennings had found to exist in different races of this organism. Thus, Calkins emphasized the fact that he could readily induce conjugation in his culture, whereas experi- ments to secure conjugation in our cultures were without effect. He, therefore, stated that "the two races cannot be compared in regard to vitahty, since normal conjugation was prevented in the conjugating race, whereas in the non-conjugating race there "has been no artificial prevention of a normal process." With this issue raised, it was essential to determine whether our race was actually non-conjugating. Accordingly, a more extensive series of mass cultures were started from it, with the result that conjugants were finally secured, thus demonstrating that this race is a conjugating race when the proper conditions for conjugation are realized. Therefore, there is no evidence extant that a non-conju- gating race of Paramceciiim exists. In a recent paper, Calkins^ states that possibly his terms " con- jugating " and " non-conjugating " were not happily chosen, and that he merely meant to indicate that some races are more prone to con- jugate than others. Admitting this Interpretation of his terms, they express a fact. But this interpretation begs the question which his Suggestion was advanced to explain. With this theory eliminated, the results derived from this cul- ture demonstrate, we believe, that the very limited periods in which Maupas, Calkins, and others observed degeneration, have no sig- nificance for the question as to whether degeneration and death are inevitable results of reproduction without conjugation. In other words, this one positive result from this race outweighs all the negative evidence derived from work on the infusoria, and justifies the Statement that these organisms can live indefinitely, when sub- jected to favorable environmental conditions, without conjugation or artificial Stimulation. With conjugation eliminated as a necessary factor in the life history, obviously the next point to be elucidated, if possible, was the underlying factor inherent in the cell, the physiological expression of which is the rhythm. Although morphological or physiological variations that could be interpreted as the result of degeneration 5 Calkins : Am. Naturalist, 1915. 1915] Lorande Loss Woodruff 375 were never observed in this race of Paranicucmm, we early noted " that various nuclear changes which are not at present recognized occur normally in the life history of Paramceciiim" ; and we sug- gested that possibly, when conjugation is prevented, a reorganization of the nuclear apparatus within the individual cell occurs.® Erd- mann independently reached an essentially similar position from a consideration of the ptiblished data on this culture and a critical study of infusorian life histories; and further, in an experimental study of Amceba diploidea, suggested that a relation exists between sexual phenomena and rhythms.'^ Accordingly, we collaborated in a study of the daily cytological changes of this race of Paramcs- cium during a period of six months, and discovered that the rhythms in the division-rate are the physiological expression of internal phenomena which involve the formation of a complete new nuclear apparatus, by a definite sequence of normal morphological changes that simulate conjugation.^ This nuclear reorganization, which we term endomixis, consists, in essence, of a gradual disintegration and absorption of the macronucleus in the cytoplasm. Simultane- ously, a multiplication of the micronuclei is in progress. Certain of the resulting micronuclei degenerate while the remaining one (or two) form the new macronuclear and micronuclear apparatus. This results in the reorganization of the cell without the fusion of two animals. An essential morphological difference between endomixis and conjugation is the absence of the third micronuclear division, which, in conjugation, forms the stationary and migratory micronuclei; and, of necessity, the non-formation of a syncaryon. After conju- gation the reorganized cell has a new macronuclear and micronu- clear apparatus, composed of combined material from the conju- gants, while, after endomixis, the reorganized cell has a new macronuclear and micronuclear apparatus composed of material from its own micronuclei. In a word, the essential distinctive features of endomixis are the absence of the third micronuclear division and ^ Woodruff : Amer. Naturalist, 1908. ■^ Erdmann: Ergeb. d. Anat. u. Ent., 1908; Archiv, f. Protistenk., 1913. 8 Woodruff and Erdmann: Proc. Soc. Exp. Biol. and Med., 1914; Journ. Exper. Zool., 1914; also Erdmann and Woodruff: Biol. Cent., 1914. 3/6 Problem of Rejuvenescence in Protozoa [June-September the absence of the introduction of foreign nuclear (and cytoplasmic) material into the cell. For reasons advanced elsewhere, we hold that endomixis is not parthenogenesis, but it is not necessary at this time to enter into a more or less academic discussion in regard to the exact Classifica- tion of endomixis among Entwicklungserregimg phenomena. In the light of the discovery of the details of endomixis, by the daily study of pedigreed cells of this race, a survey of the cells, which had been preserved at intervals during the previous seven years of its life, revealed a number of the crucial stages of endo- mixis, thus showing that the process has been in progress ever since the race has been bred, and is not, as Hertwig^ suggests, a develop- ment during long subjection to culture. That it is not even a pecu- liarity of this race is evident from the fact that we have found endomixis in four other distinct races selected at random — three from America and one from Germany. Further, Hertwig, in 1889, incidentally noted in a mass culture, in which conjugation had not been observed for a long time, certain animals whose nuclear structure apparently indicated isolated stages of the process which have been elucidated in our cultures. Therefore, it seems well established that endomixis is of general and probably universal occurrence in Paramcecinm aurelia. It also occurs, with essentially similar features, in all the races of Paramcocium caudatum which we have studied.^*^ Now, in regard to the significance of endomixis from the stand- point of our subject — rejuvenescence in protozoa: It is clear that the cycle emphasized by Maupas, Calkins and others, is merely a phantom which has continually receded as each successive investi- gator has approached the problem with improved culture methods, until it has vanished with this eight-year-old culture. What remains then is the rhythm and in the light of endomixis — the underlying cytological phenomenon of which the rhythm is an outward physio- logical expression — the whole problem takes on a new aspect. The cell automatically reorganizes itself periodically by a process which, in its main features, simulates conjugation, but without a contribu- tion of nuclear material from another cell. »Hartwig: Biol. Cent., 1914. 10 Erdmann and Woodruff : Journ. Exp. Zool., 1916. iQis] Lorande Loss Woodruff 377 At the present stage of our knowledge, the rhythm may perhaps ' be considered as an expression of a sort of temporary " senescence," for which is provided an internal, automatically working, antidote in the form of endomixis. But, if one considers this as a "senes- cence "-phenomenon and endomixis a " rejuvenation "-phenomenon, then it is equally pemiissible so to consider the momentary fluctuat- ing periods of anaboHc and catabolic ascendency in the metab- oHsm of the cell. But I would point out that it is a case of trying to f orce new wine into old bottles in order to save an idea, and that this line of reasoning, pushed a little farther, approaches perilously near a reductio ad absurdum. One certainly must grant that the prevalent idea of "senescence " in infusoria is far removed from this subtle, automatically eliminated type which rhythms may indicate. The work at Yale in the past has been to determine whether con- jugation is a necessary factor in the life of infusoria; and we believe that 5250 generations without conjugation is strong evidence in the negative. But, obviously, because conjugation is not necessary in the life of Paramcocium under favorable environmental conditions, it does not follow that conjugation is not necessary under other conditions, or that it does not have a "rejuvenating" function. There is nothing mutually exclusive in the fact that conjugation is not necessary and the idea that conjugation has a dynamic function when it occurs. In fact we have ahmys leaned toward and defi- nitely stated the view that conjugation probably has a dynamic function, which is important when the organisms are subjected to unfavorable conditions. Calkins has secured some evidence which indicates that, after conjugation, all the processes of the cell includ- ing reproduction proceed with greater vigor ; and thus he substan- tiates the view of Bütschli, Maupas, Hertwig, and others. Jen- nings, on the other band, definitely states that there is no evidence from his work that conjugation in the infusoria increases the repro- ductive power of, or rejuvenates, the organism physiologically in any way and puts all the emphasis on the side of Variation and heredity. However, since the cytological phenomena of conjugation, with the exception of syncaryon f ormation, are so similar in their broad features with those of endomixis, and since accelerated vital activities 37^ Problem of Rejuvenescence in Prot 020a [June-September including reproduction do follow endomixis, it seems reasonable to believe that accelerated vital phenomena follow conjugation — that is, that both processes, broadly speaking, " rejuvenate " the organism physiologically. Both processes afford opportunity f or a rearrange- ment of the molecular Constitution of the cell, conjugation afford- ing amphimixis and endomixis affording ^wc^omixis. To recent contentions that our conclusions were wrong, in reg-ard to conjugation not being a necessity for the continued reproduction of infusoria, we would reply that endomixis is not conjugation; and no one had any other phenomenon than conjugation, involving syncaryon formation, in mind until the discovery of endomixis, in which a syncarj'^on is not formed. To say that endomixis fills essen- tially the same röle as conjugation in the infusorian life-history is to beg the entire question. In a word, the whole aspect of the prob- lem of senescence and rejuvenescence in protozoa has changed with our knowledge of endomixis. The question is now not whether conjugation is necessary — for it is not — ^but whether endomixis is necessary. If endomixis is necessary, as it may well be, and if one feels justified in considering the physiological phenomena which are synchronous with the start of endomixis as evidence of " se- nescence " and those synchronous with the end of endomixis as in- dicating " rejuvenation " — then, this is a radically new phase of the old idea of protoplasmic senescence and rejuvenescence in the in- fusoria. Osborn Zoological Laboratory, Yale University. THE CHEMICAL CONSTITUTION OF STARCH A review ARTHUR W. THOMAS The size and configuration of the starch molecule have been the subject of much chemical research since about 1836, when Payen^ announced the chemical composition to be CgHioOg. This is the empirical formula generally accepted at the present time. The real molecular structure is known to be many times this empirical unit, however. The highly colloidal nature of starch, the ease with which it can be removed f rom its Solutions by merely f orcing them through porous earthenware, and the extremely small influence which it exerts on the freezing point of its solvent, are facts that indicate a high degree of molecular complexity, which probably approaches that of the proteins. The (CgHioOs)» structure indicates that starch is an anhydride condensation product of glucose or maitose. This view is borne out by the fact that hydrolysis by acid or diastase yields glucose or maitose, respectively, as the end products. The contributions to our knowledge of the molecular weight have all, or nearly all, de- pended upon the study of the hydrolysis of starch by infusions of malt, a type of study which has naturally been fostered by the great fermentation industries. In 1860 Musculus^ noted that it was difficult, in fact quite im- possible, completely to hydrolyze starch to maitose by means of malt. After much experimenting he announced, in 1878 (Muscu- lus and Grueber),^ his view that starch must be a Polysaccharide of a molecular size indicated by the formula (Ci2H2oOio)5-6- In 1879 O'Sullivan* announced that the starch molecule is as large as 1 Payen : Ami. Chim., 1836, [II] Ixi, p. 355 ; 1837, [II] Ixv, p. 225. 2 Musculus: Attn. Chim., 1860, [III] Ix, p. 203; 1865, [IV] vi, p. 177. 3 Musculus and Grueber : Bull. soc. chim., 1878, xxx, p. 54. * O'Sullivan : Jour. Chem. Soc, 1879, xxxv, p. 770. 379 380 Chemical Constitution of Starch [June-September the molecular size (Ci2H2oOio)6' thus corroborating the work of Musculus. Herzfeld,^ while not contributing directly to the knowledge of the size of the starch molecule, started a new line of thought, in which he pointed out that the hydrolysis progressed through a series of dextrins of diminishing complexity before, or in the course of, the conversion of starch to sugar, i. e., starch, to soluble starch, to erythrodextrin, to achroodextrin, to maltodextrin, finally to maitose. To maltodextrin he assigned the formula C18H36O16. The discovery of maltodextrin was quite a significant step, for all subsequent work has depended on the study of just such substances — substances which combine the properties of sugar and of dextrin. Brown and Heron® found that the hydrolysis of starch by malt stopped when four fifths of its weight of maitose was formed, the remaining one fifth consisting of a dextrin. They accepted the theory of Musculus and Grueber, and of Herzfeld, that starch was hydrolyzed in successive steps to dextrin and to sugar. From their experiments with malt diastase they concluded, most naturally, that the starch molecule must be at least five times the size of the residual dextrin, and proposed (Ci2HooOio)io as the formula for starch, the simplest dextrin molecule being thought to be (Ci2H2oOio)2- In 1885, Brown and Morris^ reported that a dextrin was always present as one of the hydrolytic products of starch, which, while not identical with the maltodextrin öf Herzfeld, bore a resemblance to it. They assigned to this Compound the formula (Ci2H2oOio)2* C12H22O11. Inasmuch as this dextrin was difficult to hydrolyze they gave starch the formula 5(Ci2H2oOio)3, in order to make it agree with the principles evolved from their earlier work. The (Ci2H2oOio)3 group was called amylin, the starch molecule con- sisting of four such groups arranged symmetrically about a fifth. Upon hydrolysis an amylin group was thought to split off as malto- dextrin, leaving the other four as a more complex dextrin, the maltodextrin in turn Splitting directly into maitose. Brown and Morris^ determined the molecular weight of amylin 5 Herzfeld : Berichte, 1879, xii, p. 2120. ß Brown and Heron : Arm. Chem., 1879, cxcix, p. 165. "^ Brown and Morris : Ann. Chcm., 1885, ccxxxi, p. 72. 8 Brown and Morris: Jour. Chcm. Soc, 1889, Iv, p. 96; Berichte, 1891, xxiv, p. 723- 1915] Arthur W. Thomas 381 by means of its depression of the freezing point of water. Their experimental figure for the molecular weight was 6221, which agreed most closely with the formula (Ci2H2oOio)2o> the molecular weight of which is 6480. They adhered to their theory that starch was composed of five amylin groups and represented the hydrolysis by these equations : First, (Ci2H2oOio)20 + HsO^ Ci2H220u- (Ci2H2oOio)i9 Amylin Last, (Ci2H2oOio)20 + 19^20-^ (Ci2H220ii)i9-Ci2H2oOio Maltodextrin Brown and Morris concluded, from the results of their later work, that the maltodextrins split into smaller substances of varied composition. Two different maltodextrins were isolated by the authors, one with the formula (Ci2H2oOio)2*Ci2H220ii (malto- dextrin) and another with the formula (Ci2H2oOio)6"Ci2H220ii (amylodextrin). Scheibler and Mittlemeier,^ in 1890, discussed the hydrolytic products of starch and dextrin. One noteworthy feature of their paper was the preparation of the hydrazone of a commercial dex- trin which, upon analysis, was found to have a composition indi- cated by the formula, C96H162OS0N2HC6H5. This corresponds with (C6Hio05)i6. which is somewhat similar to the amylodextrin reported by Brown and Morris. Lintner and DuelF^ claimed that, in its hydrolysis, the complex starch molecule split first into amylodextrin (better known at the present time as soluble starch), and that this soluble starch then broke down into three molecules of erythrodextrin, which in turn split into three molecules of achroodextrin, the latter Splitting into iso-maltose, iso-maltose changing to maltose. They determined the molecular weight of these substances by means of the freezing- point method of Raoult, with the f ollowing results : Soluble starch 17,496 (CiaHaoOjo)« Erythrodextrin 5,850 ( Ci^HooGic) « • H2O Achroodextrin 1,962 {Ci2^2

CH n W tri B ' 2 o tq' n u w .w ö — 0- — a — 0 5 CO -n 0- 0 — 0- 0 0- Fig. 2. Proposed structural f ormula for starch (Johnson). 386 Chemical Constitution of Starch [June-September To use Johnson's own words, — "As is seen, two dextrose molecules condense in a secondary group (HC. OH HO .CH) whilst the maitose molecules condense in the primary group (H2C.OIH HOI.CH2), the former becoming HC.O — CH and the latter HgC.O — CH2. The two aldehyde groups of the original dextrose molecules condense in the amylin groups as is shown above. This explains the non-reducing character of the starch." The suggested condensation of "primary" and "secondary" OH groups in each sugar molecule has no experimental justifica- tion. In my opinion it does not seem necessary to assume poly- merization of the aldehyde groups to explain the non-reducing character of starch. The disaccharide sucrose is not a reducing sugar, yet we do not assume that the aldehyde groups of the con- stituent monoses polymerize with one another. The latest attempt to construct a starch molecule, and probably the most painstaking of any previous hypothesis, was brought out by Synkiewski^'^ in 1902. His work consisted in studying the products of hydrolysis of starch by infusions of malt, with exhaust- ive investigation of the properties of the several dextrins isolated therefrom. The starting point in his work was the isolation of two dextrins, one called by him "protodextrin I" which was formed by hydrolyzing starch at ordinary temperature ; and another, called " protodextrin H," which was formed by the hydrolysis of starch at 78° C. The preparation of protodextrin H was accomplished by allow- ing malt extract to act on starch at about 78° C. until all starch was hydrolyzed. The dextrin was then separated by evaporation of the water, extraction with dilute alcohol to remove soluble carbo- hydrate, and final precipitation with strong alcohol. The product was a white amorphous powder which, after ultimate analysis and by determination of the depression of the freezing point of water, showed a molecular weight and structure equivalent to CgeHgaOsi or (C6Hio05)6 + H20. This is similar to the formula for the 1^ S3mkiewski : Ann. Chem., 1902, cccxxiv, p. 212. 1915] Arthur W. Thomas 387 maltodextrin-a of Ling and Baker/ ^ the achroodextrin II of Lint- ner/^ and the maltodextrin of Brown and Morris. ^'^. This proto- dextrin was f urther hydrolyzed by malt extract ; and, by a similar process, another dextrin was isolated from the hydrolytic products. This dextrin was termed y-maltodextrin. By analysis and depres- sion-of-the-freezing-point-measurements, its elementary constitu- tional formula was determined to be C24H42O21. Synkiewski Claims that this dextrin is identical with the maltodextrin-jS of Ling and Baker, and the achroodextrin of Prior.^^ Its formation from protodextrin II is represented by the equation ^ZQ^Q2^Z1 ~r H2Ü~^ ^24^42^21 "T" ^12ll22^11 Hydrolysis of this maltodextrin yields the isomaltose of Lintner. Protodextrin I was prepared and isolated in a manner similar to that for protodextrin II, with the exception that the malt extract acted in the cold. The molecular formula was found to be C72H124O62 or just twice that of protodextrin II. Hydrolysis of this substance by malt yielded a sugar with the same formula as maitose, but, because of its higher optical rotation and lower re- ducing power than maitose, it was believed to be a polymer of the latter sugar and received the name " dextrinose." The next part of this author's investigations consisted in the study of soluble starch (prepared by heating starch paste in an auto- clave under pressure) or, as he called it, amylodextrin. He deter- mined its formula tobe (C54H90O44)» + I" H2O; and, by means of acetylation, found it to contain thirty hydroxyl groups. Years ago Schützenberger and Naudin^^ analyzed an acetyl derivative of ordi- nary starch, with results, according to Synkiewski, that demonstrated the presence in ordinary starch of twenty-seven hydroxyl groups. Since the formation of maitose from this thirty-hydroxyl amylo- dextrin is complete, and since the hydrolysis of ordinary starch stops before all of it is converted to maitose, he concludes that the amylogen residues of the starch molecule (each of which contain ^8 Ling and Baker: Jour, Chem. Soc, 1897, Ixxi, p. 517. 19 Lintner : Zeitsch. f. d. ges. Branw., 1894, p. 339. 20 Brown and Morris : Jour. Chem. Soc, 1885, xlviii, p. 527. 21 Prior: Bayr. Bierbr., 1896, p. 157. 22 Schützenberger and Naudin : Ann. Chem., 1871, clx, p. 77. 388 Chemical Constitution of Starch [June-September three maitose residues), under the influence of malt extract, first split off these maitose residues as maitose molecules, provided they have been previously provided with hydroxyl groups. For each hydroxyl that the amylogen nucleus takes on, one maitose residue is ready to be split off by the diastase. Upon the configuration of the amylogen residues rests the structure of the starch molecule. Synkiewski holds that the amyl- ogen residue contains three different kinds of carbonyl linkings : the one which is readily hydrolyzed by malt, and yields maitose and protodextrin I, is called an a bond; the second, which is broken only by long action of malt diastase and finally yields glucose f rom the protodextrin, is called ß ; and the third, which connects the glu- cose residues in maitose, is called y. According to this scheme the amylogen nucleus may be represented by (Ce) -^- (Ce) -y- (Ce) ß (Ce) -a- (Ce) -y- (C«) ß (Ce) -a- (Ce) -y- (C«) Since starch consists of n amylogen residues connected by an- hydride carbinol linkings, then, when an a-carbonyl hydrolysis takes place, 3n molecules of maitose are produced and n molecules of the protodextrin I. A j8-carbonyl hydrolysis splits the starch so that each amylogen complex is divided into three similar portions, which can be termed protodextrin-residues IL These residues each consist of three glucose molecules. Since protodextrin II con- tains six glucose molecules, it must consist of two protodextrin- residues II, and its Constitution can be schematically arranged as f ollows : (Ce) -y- (Ce) -^- (Ce) - {C,)-a-{C,) —y- (C,) From this formula it is apparent that the molecule of this sub- stance contains two intact y bonds and two maitose groups con- 1915] Arthur W. Thomas 3^9 nected to a Cg — Cg radical by means of a linkings. Since a link- ings are easily attacked it can readily be seen why protodextrin II is so easily converted to sugar. This saccharification can be repre- sented in two stages by the equations 1. (Ce) — y— (Ce) -a— (Ce) — (C«) — a— (Ce) — y— (Cß) Protodextrin 11 -> (Ce) -y- (Q) -^- (Ce) — (Ce) + (C«) -y- (Ce) 7-maltodextrin maitose 2. (Cc)-y-(Ce)-^-(Ce)-(Ce) ->(Ce)-y-(Ce) +(Ce)-(Ce) maltodextrin maitose dextrinose Synkiewski points out that C54, the number of carbon atoms in the amylogen group, is not a multiple of C36, the number of carbon atoms in protodextrin II, hence the molecule of protodextrin II cannot be f ormed f rom a single amylogen group. Both amylogen, and the last named dextrin, have in common the Cig group, so that C54 = 3(Cis) and C36 = 2(Ci8). Since, however, these sub- stances come from the starch molecule quantitatively he feels that there can be no doubt that the 2(Ci8) residues of protodextrin II originate from two amylogen groups according to the expression, 2[3(Q8)]=3[2(C,s)] er, since the starch molecule consists of « amylogens, w[3(Q8)] = t [2(Ci8)] From the fact that the protodextrin-II molecule is made up of two different amylogens bound together by only a carbinol linking, it follows that the linking which joins the two Cjg groups of proto- dextrin II is a carbinol bond (the bond in the dextrinose molecule). The amylogen nucleus consists of nine glucose groups, contain- ing nine carbonyl radicals; but, since amylogen does not reduce Fehling Solution, all the carbonyl groups must take part in uniting these glucose groups. Synkiewski thinks that there are two possible ways in which these glucose residues may be joined together: between the nine glucose molecules there are eight carbonyl bonds, which causes the 390 Chemical Constitution of Starch [June-September assumption of one dl-carbonyl linklng; or, there are nine mono- carbonyl bonds, which necessitates the assumption of a ring structure. Let US consider the first possibility, for which a di-carbonyl group is assumed. It is evident that none of the y bonds is a car- bonyl (there is no di-carbonyl bond in maitose). It is also easy to see that none of the ß bonds in the dextrin residue is di-carbonyl. Free protodextrin I reduces Fehling Solution, which indicates that it contains a free carbonyl radical. At the two linkings between the three glucose molecules of this dextrin, only two carbonyl radi- cals are f ormed upon hydrolysis : these are mono-carbonyl. If a di-carbonyl linking were present in the amylogen, it could be only one of the a. bonds. By use of the sign ( < ) to denote a carbonyl bond, and putting di-carbonyl bonds in the place of a link- ings, the formula of amylogen may be written as follows : (Co) > (C,2) A (Ce) > (QO A (Ce)><(Ci2) Under this scheme, hydrolysis would give no maitose; but, on the other band, two different kinds of dextrin, one possessing no reducing power. This is not the case and, therefore, there is no di-carbonyl bond in the amylogen nucleus. This conclusion naturally suggests the other possibility. The nature of the hydrolytic products of amylogen that have been istudied would make it difficult to imagine an a or a y linking in a ring structure. Such a structure, Synkiewski claims, may be easily made up of ß bonds, and there are two possibilities : [-(Ce)— a— (Ce)— 7— (Ce) I ß I (A) ß (Ce)— a— (Ce)- 7— (Ce) ß -(Ce)-a-(Ce)-T-(Ce) iQis] Arthur W. Thomas 39^ (Ce) \ (Ce) ■ \ ^(C6)-/3-(C6)-«-(C6)-7-(C6) \ / (B) \/ (Ce) / a / (Ce) / (Ce) As shown before, the rational formula for the amylogen group is some multiple of Ci8H270i2"03' (Ci2H230ii)3 and by making use of Fischer's formula for maitose, Synkiewski writes the graphic formula for amylogen as f ollows : ^^\CH(CHOH)4CH2— O— CH(CH0H)rCHCH0HCH20H / "^\CH(CH0H)4CH2— O— CH(CHOH)2CHCHOHCH20H C18H27O12 O^ \^Q>CH(CHOH)4CH2— 0-CH(CHOH)2CHCHOHCH20H Synkiewski suggested that this formula represents a half acetal of the ten-hydroxyl alcohol of maitose, which is analogous to the alco- holates of chloral ; and that it might be named " tri-malto tri-glu- cosate," if the not impossible but as yet undiscovered triglucose sugar were known to exist. In Order to combine all facts, including a ring combination of the three glucose molecules, Synkiewski shows that the formula for the amylogen nucleus could be written in accord with the arrange- ment shown in Fig. 3, on p. 392. 392 Chemical Constitution of Starch [June-September ffi X X o o O X C4 X M K u u U ffi ffi X o o O X ffi X u u U X X X /V /9 /9 1 ä /^ / ^ / ^ X O X o 9 E \5 9 d: 9 X • cn \ u 1 o o o bo o 1» . e? r^ >^ i ffi X ffi u u u 4_« ^ ■* •^ X""^ Sh ffi K X ^ o O o ffi X X cd 3 B U u X X X o u u u CO / \ / \ / \ i / V / \ / \ CO o o o o o o u X \ .K \ X \ ^ \x \x . \x c^ ^u •u 'U i • • X X tö o o o X X X MH u u u ffi X X /^ /^ y rV /9 ?2 \ ? K X o \ 9 o \ \ K O \ X o \x O \v \\ 5 ^g I9I5] Arthur W. Thomas 393 The nature of the linkings between the amy logen groups in the starch molecule is deduced through the fact that, in the formation of soluble starch or amylodextrin, hydrolysis takes place without the formation of reducing Compounds, from which it is concluded that a carbinol hydrolysis has taken place; hence the amylogens must be connected one to another in an ether-like or carbinol manner. The next step was the construction of the constitutional f ormula for the starch molecule and incidentally to set limits by geometrical means for the possible sizes which the starch molecule may assume. The fact that after i8-hydrolysis, molecules of only a single dextrin of two Ci8-groups are obtained is proof that the carbinol bonds contained in these molecules, and the carbinol hydroxyls of the amylogen residue which bring about the union, are of equal value. These three hydroxyls must occupy similar places in the amylogen molecules. If we connect the middle points of these places we un- consciously form an equilateral triangle on each corner of which is located the dextrin carbinol bond. Since the three dextrin-carbinol bonds of each amylogen must be equivalent to similar bonds of all other amylogens, we assume that this condition will find expression in the parallel position of these bonds in the starch molecule. The triangles, which we have assumed to be the configurations of the amylogens, must be ar- ranged in the starch molecule so that every two neighboring groups will have similar, opposite, positions. On the ground of the above argument, in the construction of the starch molecule, only so many amylogens take part as the num- ber of equilateral triangles that can come together in a closed fig- ure in which every two of them shall always have an adjacent angle and similar opposite positions. There are only four geometrical constructions, according to Synkiewski, which can accommodate these premises. Place two triangles atop each other and we have two superim- posed triangles. Place a triangle at each edge, and have all tri- angles possess a side in common, and we arrive at a tetrahedron. Again, by similar processes we may arrive at an octahedron ; finally at an icosahedron. This hypothesis allows only four alternatives 394 Chemical Constitution of Starch [June-September to choose from, a molecule consisting of two, four, eight or twenty amylogen groups. Synkiewski points out that in forming amylodextrin only the maitose carbinol bonds were dissolved, the dextrin-carbinol bonds remaining intact. These dextrin-carbinol bonds were also not attacked by malt at 78° C. By a /^-hydrolysis, protodextrin II is formed, in which these linkings persist. By Solution of the a- bonds, maitose is split off and dextrinose is left, in which the dex- trin-carbinol bond remains. Dextrinose is also obtained from pro- todextrin I, which shows that this substance has the bond intact; and, since it comes from amylodextrin, it must have just as many dextrin groups as amylodextrin and therefore just as many as amylogen. From the molecular weight of protodextrin I it follows that it consists of four protodextrin I residues; therefore, the starch molecule is composed of four amylogen residues and its empirical formula must be CsieHgßoOiso- For the sake of simplicity, the amylogen residues are repre- sented in triangulär form : (C,s) /\ (Cx8)-(Ci8) and the starch molecule may then be represented on a plane surface as follows : JCisl Fig. 4, I9I5] Arthur W. Thomas 395 The elaborate work of Synklewski ends with this hypothetical configuration f or the starch molecule, the size of which(C6Hio05)36, is only about one sixth as large as that proposed by Brown and Morris, (C6Hio05)2oo' The experimental work of Brown and Morris is not nearly so extensively appHed to the question at issue as that of Synkiewski, and on that account one might be incHned to give preference to Synkiewski's suggested molecule; but it must be borne in mind that Synkiewski's hypothesis rests entirely on two dextrins, protodextrin I and protodextrin II, the one formed by malt diastase in cold Solution and the other in hot Solution. He assumes that these dextrins of different molecular weight are dif- ferent individuals and that both are present in the starch molecule simultaneously. This assumption is open to doubt, however, since malt diastase does not appear to have any different influence in hot and cold Solutions other than that due to difference in rate of hydrolytic action. Since Synkiewski's work appeared there have been several papers of minor Import dealing with this same topic. During the same year there was published by Hale-^ a review of work pre- viously reported about the Constitution of starch; also an account of his own ideas and the results of a study of starch iodides. He constructed a molecular formula embodying all the good features of former workers. From his own work on starch iodide he con- cluded that the size of the starch molecule might be at least (CcHio05)i8, because he had obtained an iodide of starch contain- ing 4.38 percent of iodine; expressing the formula (C6Hio05)i8l. This formula does not merit much consideration because, with our present knowledge of colloidal chemistry, we are prone to regard the blue complex formed by starch and iodine as a special colloidal phe- nomenon. In 1904, Kladiaschwili^* treated starch with formic acid and obtained a mono-formyl derivative which seemed to indicate by the freezing-point-depression-method in chloracetic acid and phenol Solutions the formula (C7Hio06)6. There is danger, however, that this derivative is a hydrolytic product of starch. 23 Haie: School of Mines Quarterly, 1902, xxiv, p. 125. 24 Kladiaschwili : /. Russ. Phys. Chem. Soc, 1904, xxxvi, p. 905. 39^ Chemical Constitution of Starch [June-September In 1905, Skraup,^^ working along similar lines, acetylated dex- trins and starch derivatives under the influenae of hydrochloric acid gas at — 20° C. From a study of some of the chloracetyl derivatives he announced his behef that starch has a molecular weight of (CcHio05)46 to (C6Hio05)5o. A method altogether different in principle from those preceding was pubHshed in 1909 by Wacker. ^^ The procedure consisted in mixing the substance to be investigated with Phenylhydrazine sul- fonic acid and alkaH, when a red color was formed, the depth of color depending upon the molecular weight. This method worked very well with carbohydrates and Compounds having alcohol and aldehyde groups. Wacker's conclusion, however, that starch is made up of two substances, one (CgHioOg)« and another "of a more cellulose character," (CeHioOg)^ to (CgHio05)s, indicates that his method does not hold in the case of starch Solutions. It is very stränge that many modern chemists, or chemists liv- ing in modern times, still seem to think that starch has a low molec- ular weight. The large amount of trustworthy experimental evi- dence which has been reported in the last fifteen years ought to dispel such a notion. Nevertheless, in a recent review, Frank- forter^''' seriously considered such formulas as (C6Hio05)2, (^eHioOg)^, and (C6Hio05)6, giving his preference to the "hexa- polymer." He cited Wacker's experiments, drawing from them the conclusion that " the starch molecule is at least of the size (CßHi 005)6 or probably greater." He included a graphical for- mula, made up of six glucose groups united by oxygen linkings in a continuous structure. Such a simple structure seems quite impossible. Malt hydrol- ysis of starch results in the formation of maitose and of dextrin of great complexity. We feel confident that malt diastase as ordi- narily used is a hydrolyzing and not a constructive agent. Where could the complex dextrinous substances come from if the starch molecule were a simple hexapolymer of glucose? Does not also the colloidal nature of starch in Solution suggest a high molecular weight ? 25Skraup: Monatsh. Chem., 1905, xxvi, p. 1415. 26 Wacker : Berichte, 1909, xlii, p. 2695. ^'^ Frankfurter : Proc. 8th Intern. Congr. Appl. Chem., 1912, viii, p. 133. iQis] Arthur W. Thomas 397 From 1912 to 1915 the work of Pringsheim^^ has helped to es- tablish more firmly than ever the theory of the great molecular com- plexity of starch. By fermentation and hydrolysis of starch, under different conditions, he obtained several dextrins which were tetra- and hexa-polymers of the simple dextrin molectile and similar to those isolated by Schardinger i^^ [(CeH,o05)2]4 + 4CÄOH [(CoHio05)2]3+?CAOH [(C6Hio05)2]2 + 2CÄOH which, by acetolysis and saponi- fication of acetyl derivatives, yield " diamylose," (C6Hio05)2+2H20;or yields " triamylose," "] yields triamylose, [(CeHio05)3]2 + 9H20 I (CeHio05)3+4H20 From his experimental results he concludes that these dextrins or " amyloses " have a ring structure, such as 1 — ° — I /CH • [CH(OH)]2CH • CH(OH) ■ CHj. 0< ^O, ,or ^CHa -CHCOH) • CH • [CH(OH)]2 • CH I O 1 o CH • CH(OH)CH • CH • CH(OH)CH20H o< >o HOCHi • CH(OH)CH • CH • CH(OH)CH I O 1 and that they indicate that starch itself, and the non-reducing dex- trins which are not fermented by yeast, contain the ring which can be opened only by special ferments. In the light of all the observed properties of starch, we are obliged to accept the hypothesis that the molecule of this substance is large and very complex. Further experimental work, perhaps from the synthetical Standpoint, will doubtless throw much more light upon the limits of magnitude of the molecule. Laboratory of Biological Chemistry, Columbia University, College of Physicians and Surgeons, New York. 28 Pringsheim : Berichte, 1913, xlv, p. 2533 ; 191S, xlvii, p. 2565. Landw. Vers. Stat., 1914, Ixxxiv, p. 267. Naturwissenschaften, 1915, iii, p. 95- 29 Schardinger: Zentralbl. f. Bacteriologie (II Abt.), IQOS, xiv, p. 772; 1907, xix, p. 161 ; 1909, xxii, p. 198; 191 1, xxix, p. 188. THE BEHAVIOR OF TARTARIC ACID AND THE TARTRATES IN THE ANIMAL ORGANISM MAX KAHN Introduction. In a recent paper by Carles,^ attention was drawn to the important part played by tartaric acid in wine manu- facture, and a prophesy was made that within ten years the con- sumption of tartaric acid would be increased ten-fold. Tartaric acid and tartrates are used extensively, not only in wines but also in baking powders, in medicated waters and in candies. Com- paratively little experimental work has been done on the pharma- cology of tartaric acid and tartrates. The importance of exact knowledge in these connections is evident. Dextro-tartaric acid was isolated first by Scheele, in 1768, and obtained in crystalline form by Retzius, in 1770, who designated it Sal essentiale tartari. Its composition and salts were studied by Gay-Lussac, Berzelius, and others; its optical properties, espe- cially, by Biot (1815) and Pasteur (i84i).2 Four optical isomers are known: dextro-, levo-j para- (racemic), and m^.yo-tartaric acids. Tartaric acid is one of the most widely distributed acids in the plant kingdom. It is present in the free State, or in the form of salts, in grapes, mountain-ash berries, tamarinds,^ tomatoes,^ cu- cumbers, potatoes, black pepper, pineapple and in leguminous plants.^ It has also been found in grape leaves,® senna leaves,'^ liverwort,^ ferns, beet juice, and fungi.'^ 1 Carles : Repert pharm., 1913, xxiv, p. 387. 2 Hare, Caspari and Rusby : National Dispensatory, 1905, p. 90. 3 Adam : Zeit. d. östcrr. Apoth.-Vereins, 1905, xliii, p. 797. ^Albahary: Compt. rend., 1907, cxlv, p. 137. ß Müller: Arch. d. Pharmaz., 1883, ccxxi, p. 42; Hecke! and Schlagden- hauffen : Jour. d. pharm, et d. chim., 1889, xix, p. 11. ^ Piti : Ber. d. d. ehem. Ges., 1873, vi, p. 1313. ^ Wallis : Pharmaceut. Jour., 1912, xxxv, p. 644. 8 Zopf : Die Pilze, Breslau, 1890. ö Fritsch : Arch. d. Pharmas., 1889, ccxxvii, p. 193. 398 igis] Max Kahn 399 Behavior toward bacteria and fungi. Tartaric acid is pro duced, in the fermentation of fruit and grape juice, by the Apicii- latus yeast, in which process carbohydrates are oxidized to tartaric acid. Calcium tartrate may be fermented, by the Bacillus tartar- icus, to acetic acid, succinic acid, carbon dioxid and hydrogen.^* In the presence of ammonium nitrate, tartaric acid is fermented to propionic acid, acetic acid and carbon dioxid/^ Certain yeasts use tartaric acid as food, absorbing it in their growth.^^ Pasteur^^ found that Pcnicillium glaucum so affects /j-tartaric acid (racemic) that it is changed to /-tartaric acid. Yeast ferments c?-tartaric acid much more easily than the / form. The final carbonaceous product in the catabolism of tartaric acid hf yeast is carbon dioxid.^* The peculiar affinity of yeast for the f/-acid is quite significant, as will be seen below, where the metab- olism of the various tartaric acids in the animal body is considered. The mycoderms have no effect on tartaric acid.^^ According to Waterman,^^ /- and ^/-tartaric acids may be utilized by Aspergil- lus niger as a source of carbon. Racemic acid is scarcely attacked by this organism but, after a prolonged period, mutation occurs. Neuberg and Czapski^^* found that a concentration of 0.45 M of c?-tartaric acid retards the fermentation of glucose. General observations on toxicity. Certain of the earlier writers on the toxicology of tartaric acid considered this substance entirely non-poisonous. Christison^''' concluded, from the results of an experiment by him and Coindet, in which they administered per OS to a cat 3.75 gm. of tartaric acid dissolved in water, that this acid is wholly non-toxic. He also cited the experience of Dr. Sibbald, of Edinburgh, who accidentally ingested 22.5 gm. of the acid without suffering any ill effects. Wibmer stated that tartaric ^f*Pasteur: Compt. rend., 1858, xlvi, p. 615; 1863, Ivi, p. 416. Grimbert and Fiquet : Jour. de pharm, et d. chim., 1898, vii, p. 97. 11 König: Ber. d. d. ehem. Ges., 1881, xiv, p. 21 1. 12 Bail : Centr. f. Bakter. u. Parasitenk., 1902, viii, p. 567. isPasteur: Compt. rend., 1858, xlvi, p. 615. 1* Karezag: Biochem. Zeit., 1912, xxxviii, p. 516. 15 Meissner : Ber. d. königl. Wurttemb. Weinbau-Versuch., 1904, p. 72. 16 Watermann : Chem. Zentralbl., 1914, i, p. 485. 16a Neuberg and Czapski : Biochem. Zeit., 1914, Ixvii, p. 51. !'■ Christison: Abhandlung über die Gifte, 1831, p. 212. 400 Tartaric Acid in the Organism [June-September acid is injurious to the alimentary canal, hindering digestion, be- ing more toxic, in these relations, than citric acid; also more diu- retic than the latter acid.^^ Soon after the appearance of Wibmer's publication, certain cases of fatal poisoning were reported. In Watkins' case/^ a young man, 24 years of age, took 30 gm. of tartaric acid in mistake for bitter salts. He suffered from violent pains and debility, and died on the ninth day. Devergie^^ described another case of fatal poisoning with tartaric acid. Belloc^^ wrote of a case of poisoning by Rochelle salt. These cases caused a change in the attitude of toxicologists toward the tartaric acids. Orfila^^ regarded them as very irritating and therefore considered them toxic. He also treated a patient who, when intoxicated, took 120 gm. of potassium tartrate and died on the fourth day afterward. Van Hasselt^^ regarded tar- taric acid as toxic only in large doses. He considered it neither more nor less toxic than citric acid. He described its effects as resembling those of oxalic acid poisoning, differing only in a slower rate in the Initiation of the Symptoms. Hermann^* expressed the opinion that tartaric acid is toxic only in the free State; when in Union with a base, its toxicity is that of the base to which it is at- tached. Jaksch^^ stated that tartaric acid causes gastro-intestinal catarrh, with cramps and diarrhea. He did not mention the dose. Trevithick^® reported the case of a woman, 6y years old, who took 12 gm. of tartaric acid by mistake. She suffered pains all over the body, vomiting and diarrhea ensuing. On the fourth day delirium developed, temperature became subnormal, pulse very weak. Death occurred on the seventh day. 18 Wibmer : Wirkung der Arzneimittel und Gifte, Munich, 1842, v, p. 319. 19 Watkins : Jour. d. chim. med., 1845, i, p. 220. 20 Devergie : Ann. d'Hyg., 1845, xi, p. 432. 2iBelloc: Cours de med. leg., 139, cited by Taylor; Die Gifte in gericht- lichen Medizin, Cologne, 1863, ii, p. 131. 22 Orfila: Lehr. d. Toxikologie, Braunschweig, 1852, i, p. 154. 23 Van Hasselt: Allgemeine Giftlehre und die Gifte des Pflanzenreichs, Braun schweig, 1862, i, p. 532. 24 Hermann : Lehrb. d. exp. Toxikol., Berlin, 1874, p. 153. 25 Jaksch : Die Vergiftungen, Vienna, 1897, p. 43. 26 Trevithick : Brit. Med. Jour., June 24, 1903. ipis] Max Kahn 401 Experimental toxicology. Mitscherlich^'^ was one of the first to conduct a series of experiments to determine the toxicity of tartaric acid. In his work on rabbits and cats, he found that 10 gm. of the acid were necessary to kill a rabbit, when the substance was administered per os. In a cat, 5 gm. failed to induce Symp- toms. He also found that rabbits are more susceptible to citric acid than to tartaric, 5 gm. of the former being sufficient to cause death. After painting the skin of a frog with dilute citric or tartaric acid, Goltz and Bobrick^^ observed very marked slowing of the heart, with final stoppage. In 1893, Chabrie,^^ investigating the differences in toxicity of the various stereoisomeric tartaric acids, found that /-tartaric acid is the most toxic, whereas cf-tartaric acid is only half as toxic. He determined the lethal dose f or rabbits in the f ollowing way : Certain quantities of the acids were necessary to kill rabbits of the same weight in a certain time. There was a definite ratio between these doses, which he expressed by the function y. He suggested the f ollowing formula: I p ' 1000 where p is the dose used, P the weight of the animal in gm., and T the time in minutes. From his experiments he found, X( l ) =0.031; X{d) =0.014; X(f) =0.008; X(m) = 0.006. Attention has already been called to the difference in the behavior of these stereoisomeres in yeast fermentation. Chabrie also re- ported that /^-tartaric acid (racemic) is only one fourth as toxic as the £/-acid, and that the w-acid is wholly non-toxic. 27 Mitscherlich : De acidi acetici, oxalici, tartarici, formici et boracici effectu in animalibus observato, Berlin, 1845, p. 27. 28 Goltz and Bobrick : Königsberg med. Jahrb., 1863, iv, p. 95. 29 Chabrie : Conipt. rend., 1893, cxvi, p. 1410. 402 Tartaric Acid in the Organism [June-September Chio,^° accepting Chabrie's conclusions as correct, explained the different toxicologic behaviors as follows : The f our stereo- isomeric acids, which are different in their toxic behavior, modify in vitro the concentration of the H ions in the same manner; in the guinea pig they produce a very mild reaction. They fix cal- cium with a different activity in Solutions of calcium carbonate in water (saturated with carbon dioxid), in beef serum, or in dog serum. The degrees of their toxicity are not in accord with the slight Variation in concentration of H ions which is effected by them in the circulating blood, but depend on the extent to which they abstract calcium from the tissues of the organism}'^ Vietinghoff-ScheeP- found that after subcutaneous or intra- venous administration of tartrates into frogs, mice and rabbits, neutral sodium citrate is more toxic than neutral sodium tartrate, as is the case when the acids are administered in the free State. In Order to produce initial toxic Symptoms in a rabbit he was obliged to inject i gm. of the tartrate, whereas 0.2 gm. of the citrate was sufficient to induce poisonous effects. Steinfeld^^ made several experiments on frogs, and observed that the injection of 0.06 to 0.08 gm. of tartaric acid had no effect; 0.3 gm. induced paralysis of the nerves and muscles and death. A cat that received 2 gm. of the acid per os showed no toxic Symptoms. Salant and Smith^* tested the toxicity of the sodium salts of d- and /-tartaric acid on frogs and rabbits. These isomeres were found equally toxic in these animals, thus contradicting the earlier work of Chabrie on the subject. In the experiments on rabbits, the diet proved to be an important factor in the determinations of tolerance for this substance. Animals which were fed on oats, or oats and cabbage, succumbed to a dose of 0.4 gm. of the salt per k., when given by subcutaneous injection. Suppression of urine was usually observed on the first day and death usually occurred so Chio : Arch. intern, pharmacodyn., 1913, xxii, p. 473. 31 See Chemical Abstracts, 1913, vii, p. 2622. 32 Vietinghoff-Scheel : Arch. intern, de pharmacodyn. et de therapie, 1902, X, p. 145. 33 Steinfeld: cited by Vietinghoff-Scheel (footnote 32). 3* Salant and Smith : Proc. Soc. Exp. Biol. and Med., 1913, x, p. 170. igis] Max Kahn 403 in 6 to 7 days. During fasting, slightly smaller doses were fatal to some rabbits. Resistance was considerably increased when the diet was changed to carrots. Animals on a carrot diet stood i.o gm. per k. by subcutaneous injection, but 1.2 to 1.5 gm. per k. was toxic. A moderate degree of tolerance for tartrates was induced in animals fed on oats and cabbage. By gradually increasing the dose, a large proportion (6 out of 9) of the rabbits survived 0.8 gm. per k., which is twice the fatal dose. Rabbits which were receiving carrots did not acquire tolerance for tartrates. Sodium tartrate was much less toxic when given by mouth. The minimum fatal dose was 5 gm. per k. Cats are less susceptible to tartrates than rabbits, as has long been known. Subcutaneous injection of I gm. per k. into cats induced slight diarrhea in some individuals, and had no effect whatever in others. One and one-half gm. per k. proved fatal to one cat, but was without action in another. Of four cats that received 2 gm. per k., three died and one survived. When sodium tartrate was given by mouth, vomiting frequently occurred. In one case, however, when 10 gm. per k. were fed, diarrhea was the only effect observed. Upon infusoria and algae, tartaric acid acts very destructively. Bokorny^^ noticed that tartaric acid, at a concentration of 0.05 per- cent, kills Spirogyra and Spharoplea in 34 min; o.oi percent was fatal in several days. Neutral salts of this acid have no toxic effect on these organisms. Vietinghoff-Scheel came to the same conclusion. He found that concentrations less than i percent of the neutral salts were without effect on infusoria. Experimental pharmacology. . Several observers have in- vestigated the changes in the hlood due to the administration of tartaric acid or its salts. Freudberg^^ found that 5 to 10 gm. of the acid administered per os to dogs, caused reduction in the al- kalinity of the blood, amounting to 16 percent. Wallace and Cushny^'^ observed that sodium tartrate is absorbed at about the same rate as the sulfate. Vietinghoff-Scheel did not observe any effect of neutral sodium 35 Bokorny : Pflüger's Arch., 1896, Ixiv, p. 278. 36 Freudberg : Virchow's Arch., 1891, cxxv, p. 566. ^''Wallace and Cushny: Amer. Jour. Physiol., 1898, i, p. 411. 404 Tartaric Acid in the Organism [June-September tartrate on blood coagulation. Buglia and Karczag,^^ however, noted an inhibiting effect. Vietinghoff-Scheel found that small quantities of sodium tartrate inhibited the coagulation of casein by rennin, comparatively large amounts preventing it. These results were ascribed to abstraction of calcium from the blood or milk by the tartrate. Chiari^^ found that lo c.c. of 5 percent sol. of sodium tartrate, administered per os, caused perceptible reduction in the amount of soluble calcium in the intestines, the tartrate precipitat- ing much of the calcium, which passed into the feces. Karezag'*" injected, intravenously into dogs, 0.2 gm. each of the various tartaric acids. He found that c?-tartaric acid caused transitory Stimulation of the vagns centre, whereas the /-acid in- duced more prolonged Stimulation. The p- (racemic) and m-acids were intermediate in their effects between the d- and /-acids. On perfusing an isolated turtle-/z^ar^ with n/ioo sol., the acids caused decrease in the f orce of the Systole and loss of tone. The m-acid was less toxic than the other acids, on the ventricle. Both Sakai^^ and Gros^^ also reported depression of isolated frog hearts per- fused with n/100 sol. of tartrates. Salant and Hecht^^ found that perfusion of isolated hearts of dogs, cats, and frogs, with sodium tartrate, caused depression. "Thus, perfusion of the cat's heart with Solutions of w/io, n/20, n/40 and n/ioo sodium tartrate, in defibrinated blood diluted with Locke sol., or in Locke sol. alone, was followed by diminished activity of the heart, which became more marked as the concentra- tion of sodium tartrate was increased. It may be remarked, how- ever, that the action did not vary in the same ratio as the concen- tration. The effect of various dilutions was even better exempli- fied in experiments on frog hearts, for which very dilute sol. were employed. When w/300 sodium tartrate sol, was perfused for 30 to 60 seconds, a slight cardiac depression was observed, the Systole 38 Buglia and Karezag : Rendiconti d. r. Acc. dei Lincei Roma, 1909, xviii, P. 474- 39 Chiari : Arch. f. exp. Path. u. Pharmacol., 1910, Ixiii, p. 434. 4° Karezag: Zeit. f. BioL, 1910, liii, p. 218. *^ Sakai : Zeit. f. BioL, 1914, Ixiv, p. i. *2 Gros : Arch. f. exp. Path. u. Pharm., 1913, Ixxi, p. 395. *3 Salant and Hecht: Amer. Jour. Physiol., 1915, xxxvi, p. 132. iQis] Max Kahn 405 alone being decreased in some experiments, in others the decrease affected the diastole as well as the Systole. This may be regarded as the minimum concentration which can produce any effect, since a n/400 sol. proved to be without any action. . . . A very noticeable difference in the action of sodium tartrate was also obtained by varying the perfusion time." They also found that the citrate was more depressant than the tartrate. Brion^^ found that the various stereoisomeres of tartaric acid are differently cataholised in the animal organism. He observed, as Chabrie did before, that the four tartaric acids are differently oxidized in the body. The /- and w-acids are oxidized more rapidly and more completely than the c?-acid and still more so than the in- active />-acid (racemic). However, Brion's results have not been corroborated. Neuberg and Saneyoschi^^ found that, in the same dog, there is a difference in the amount of oxidation of a given tartaric acid at various times. They did not notice any difference in the oxidation of the various tartaric acids. They also found that administration of p-tartaric (racemic) acid resulted in the ex- cretion of this inactive acid in the urine. Underhill, Wells and Goldschmidt/^ found that sodium tartrate administered subcutane- ously to rabbits (dose 0.5 to 0.765 gm.) could not be recovered in the urine, and concluded that the disintegrative influence of the salt upon the convoluted tubules is sufficient to account for the failure of the salt to appear in the urine. The amount of tartaric acid that may be oxidized in the body has been variously estimated. Freudberg found that the greater portion is oxidized. Vietinghoff-Scheel stated that the acid " is very easily burnt " in the animal organism. Both Piotrowsky^^ and Magawly^^ found that tartaric acid is almost completely con- sumed when given per os, only slight traces of alkali or calcium salt being excreted in the urine. Pohl^^ administered sodium tar- trate per OS to dogs and rabbits, and found that only about 33 per- *4 Brion : Zeit f. physiol. Chem., 1898, xxxv, p. 283. *5 Neuberg and Saneyoschi: Biochem. Zeit., 191 1, xxxvi, p. 32. 46 Underhill, Wells and Goldschmidt : Jour. Exp. Med., 1913, xviii, p. 317. •^^ Piotrowsky : Dissertation, Dorpat, 1856. ■*s Magawly : Dissertation, Dorpat, 1856 ; cited by Vietinghoflf-Scheel. 49 Pohl : Arch. exp. Path., 1896, xxxvii, p. 424. 4o6 Tartaric Acid in the Organism [June-September Cent was oxidized in the organism, the rest having been excreted unchanged in the urine. Baumgarten^° observed that tartaric acid is oxidized as well by diabetic animals as by normal ones. He fed normal and pancre- atectomized dogs with tartaric acid and observed equal amounts of oxidation. It was reported by Baer and Blum^^ that subcutaneous admin- istration of 8.8 gm. of sodium tartrate, to phlorhkiniaed dogs weighing about 12 k., caused great diminution in the urinary Out- put of nitrogen and glucose. UnderhilP^ substantiated these find- ings. His experience shows that sodium tartrate, subcutaneously administered to phlorhizinized rabbits and dogs, induces disinte- grative changes in the kidney tubuli sufficient to account for the diminished elimination of urinary nitrogen and glucose, as observed by Baer and Blum. Under strictly comparable experimental con- ditions similar results were obtained by Underhill in normal ani- mals, "thus demonstrating that sodium tartrate acts specifically in this direction and that phlorhizin contributes little or nothing to the detrimental influence under discussion." In a paper on the experimental nephritis induced by salts of tartaric acid, Pearce and Ringer^^ found that the administration of IG to 15 gm. of Rochelle salt to dogs {per os, intraperitoneally, or subcutaneously) caused severe renal lesions, with the excretion in the urine of casts and albumin. Anuria occurred in some cases. The histological changes in the kidneys were necrosis of the con- voluted tubules, with fatty changes in the loops of Henle and some- times also in the collecting tubules. The mode of administration did not influence the character of the renal lesions. When tartrates were given by mouth, diarrhea was induced, which tended to cause rapid removal of the salt from the intestine, and thus probably prevented the severer types of renal lesions by reducing the amount of tartrate absorbed. In 1903, MacCallum^* stated that the tar- ßo Baumgarten : Zeit. f. exp. Path. u. Ther., 1905, ii, p. 53- ßißaer and Blum: Hofmeister' s Beitr., 1907, x, p. 80; ibid., 1908, xi, p. 102; Arch. f. exp. Path. u. Pharm., 191 1, Ixv, p. i. 52 Underhill: Jour. Biol. Chem., 1912, xii, p. 115. 53 Pearce and Ringer : Jour. Med. Res., 1913, xxix, p. 57. 5* MacCallum : Amer. Jour. PhysioL, 1903, x, p. loi. igis] Max Kahn 407 trates may be administered subcutaneously, with no bad results. He did not State the doses to which he referred, but he classed the tartrates with the sulfates and the citrates. Post^^ found that Rochelle salt, administered in ordinary doses by mouth to human beings, did not cause albuminuria or cylinduria, or aggravate an existing nephritis. Connio,^^ in his experiments on dogs and rabbits, showed that the intravenous administration of tartaric acid induced marked al- buminuria, nephritis and death. The nephritis produced was essen- tially glomerular, though the tubules were also involved. Given per OS it induced vomiting and finally nephritis and death.^''' Underhill and his coworkers^^ have recently added much to our knowledge of the pathological effects of tartrates. In their ex- periments on rabbits and dogs they found that there is no strict relation between the dose of tartrate and the extent of damage inflicted. Histological study of the affected kidney tissue revealed that it was the epithelium of the convoluted tubules that was mostly involved, and to a less extent the loops of Henle. The glomerulus and the interstitial tissue remained intact. Neither the liver nor the adrenals showed any ill effects of the tartrate administration. The most effective way of administering the tartrates was found to be subcutaneously (in doses of 1.5 to 2.5 gm., to rabbits). Under- nutrition increased susceptibility to the influences that caused renal lesions. It was also observed that the kidneys in tartrate nephritis lost the power of excreting urea injected intravenously, but the power of excreting chlorids so administered remained unimpaired. Dakin^^ investigated the coefficient of intestinal ahsorption of the various tartaric acids. He found that all of the acids were absorbed equally, no selective absorption having been noticed. In liver perfusion experiments, Ohta^^ found that, upon addi- tion of 6 gm. of either d- or /^-tartaric (racemic) acid to the perfus- ing blood, acetone was formed in the liver. The perfusion of succinic acid was not followed by this result. 55 Post: Jour. Amer. Med. Assoc, 1913, Ixii, p. 592. 56 Connio: Arch. di antropol. crimin., 1911, xxxii, p. 438, 67 Connio : Path. riv. quindicin. Geneva, 1910, iii, p. 428. 68 Underhill, Wells and Goldschmidt : Jour. Exp. Med., 1913, xviii, p. 322. 69 Dakin : Jour. Biol. Chem., 1908, iv, p. 437. 60 Ohta : Biochem. Zeit., 1912, xlv, p. 167. 4o8 Tartaric Acid in the Organism [June-September Dr. William Salant^^ and his coworkers have made a special and interesting study of the influenae of sodium tartrate on the circulation: "Experiments with various concentrations of sodium tartrate were made on dogs. When the tartrate Solution was in- jected into an animal under chloretone anesthesia the following results were obtained : The amplitude of cardiac pulsation, as shown by the Cushny myocardiograph, was decreased even when dilute Solutions were employed, the Systole being more affected than the diastole. There was also moderate slowing of the heart. Blood- pressure was not affected to any appreciable extent. The volume of the kidney, as shown by the oncometer, was only slightly in- creased. When ether or morphine anesthesia was used, the cardiac effects were not quite so uniform. In deep ether anesthesia cardiac amplitude and rate, after the injection of sodium tartrate, were the same as under chloretone anesthesia. When anesthesia was lighter, sodium tartrate was without effect on the rate or amplitude in some experiments; in others, the rate was increased by 15-25 percent, amplitude also being distinctly increased. A rise of blood-pressure accompanied injection of sodium tartrate; o.i to 0.4 gm. per k. producing a rise of 10 to 25 percent. In some experiments blood- pressure rose 40 to 50 percent. Recovery followed invariably. In curarized animals the action of sodium tartrate was more marked ; 3-4 c.c. per k. of 2.5 percent sodium tartrate causing a rise of blood-pressure varying between 45 and 200 percent, which was also the case after the injection of 10 percent sodium tartrate sol. Suc- cessive injections made at short intervals produced the same effect, thus showing absence of accumulation. When the concentration was increased 20 percent, the action was reversed, and a distinct fall in blood-pressure was observed. The volume of the kidney was markedly increased. This was out of any proportion to the rise of blood-pressure and was very constant. The increase was simul- taneous with the rise in blood-pressure, but recovery was much slower. Microscopic examination of the kidneys of rabbits that died as a result of the administration of sodium tartrate showed very marked congestion." 81 Private communication to the author. See also Salant and Hecht: Amer. Jour. of PhysioL, 1915, xxxvi, p. 126. iQis] Max Kahn 409 Dr. Salant has also observed, in some of his tartrate experi- ments, that " when both vagi were cut the blood-pressure rose slightly, while the cardiac action was hardly affected." Biochemical Lahoratory of the Western Pennsylvania Hospital, Pittsburgh, Pa, DOCTORATES IN BIOLOGICAL CHEMISTRY Conferred by American Universities, 1914-15 The names of recent recipients of the Ph.D. degree in blochem- ical science, with the subjects of the dissertations, are arranged below in university groups. Brown University. — Ralph Gibney Huriin: Histogenesis and distribution of the connective-tissue pigmentation of the silky fowl. — Benjamin Samuel Levine: Rem oval of natural impurities of cot- ton cloth by action of bacteria. — Courtland Sawin Mitdge: Effect of sterilization on sugars in culture media. — George Hathorn Smith: Parenteral digestion of bacterial protein.— ^/&^r^ Whitman Szveet: Sanitary surv^ey of the Seekonk River. Columbia University. — Arthur Donaldson Emmett: Metabo- lism studies of fatigue, rest and recuperation. — Frederick Grosvenor Goodridge: Biochemical studies of mercaptan. — Edward Gray Grif- fin: Inosite and pinite, and some of their derivatives. — Mildred Alhro Hoge: Influence of temperature on the development of a Mendelian character. — Israel Jacob Kligler: Biochemical studies and differen- tiation of oral bacteria, with special reference to dental caries. — Dora Estelle Neun: Examination of certain methods for the study of proteolytic action. — Percy Withers Pnnnett: Study of the prod- ucts of the action of different amylases. — Arthur Percival Tanberg: Experiments on the amylase of Aspergillus oryzcE. — Arthur Wal- dorf Spittell Thomas: Influence of certain acids and salts upon the activity of malt amylase. Cornell University. — Miliard Alschuler Klein: Studies in the drying of soils. — Leonard Amby Maynard: Fixation of nitrogen by sweet clover. — James Kemp Plummer: Effect of oxygen and carbon dioxid on nitrification and ammonification in soils. — William Jacob Robbins: Digestion of starch by Penicillium (Camembertii). — James Kennith Wilson: Physiological studies of Bacillus radi- cola of soy bean {Sojus max Piper) and of factors influencing nodule production. 410 I9I5] P.H.D. 411 Harvard University. — Thorne Martin Carpenter: Comparison o£ methods for determining the respiratory exchange in man. — Frederick Simonds Hammett: Uric acid in tissues. — Guilford Bevil Reed: Studies in plant oxidases. Johns Hopkins University. — Walter Hatheral Coolidge: Os- motic-pressure measurements of glucose solutions at 10° and 20°. — James Eugene Levering Holmes: Difference in chemical behavior of free and combined water, as illustrated by the saponification of esters. — Forman Taylor McLean: Preliminary study of climatic conditions in Maryland, as related to the growth of soy-bean seed- lings. — Arnos Sentman Musselman: Osmotic-pressure measure- ments of glucose Solutions at 30°, 40°, 50° and 60°. — Lyde Stuart Pratt: Esterification of benzoic acid by mercaptans. — John Wesley Shive: Study of physiological balance in nutrient media resulting in a simplified culture-solution for plants. Northwestern University. — Siegel Buckborough: Structure of maitose and its oxidation products with alkaline peroxid of hydrogen. University of California. — Oscar Leo Brauer: Rate of conver- sion of cinchonin into cinchotoxin. — Richard Morris Holman: Ori- entation of terrestrial roots, with particular reference to the medium in which they are grown. — Charles Walter Porter: Temperature coefficients and the effects of acids, bases and neutral salts in reac- tion velocities of the triphenylmethane dyes. University of Chicago. — Joseph Stuart Caldwell: Study of the effects of certain antagonistic solutions upon the growth of Zea mays. — Walter Lee Gaines: Contribution to the physiology of lac- tation. — Edwin Frederick Hirsch: Experimental study of the influ- ence of iodin and iodides on the absorption of granulation tissue and fat-free tubercle bacilli. — Charles Edwin King: Origin of the dia- stases of blood and lymph. — Julian Herman Lewis: Absorption of substances injected subcutaneously, and the inhibitory action of heterologous protein-mixtures on anaphylaxis. — Agnes Fay Morgan: (I) Viscosities of various methyl and ethyl imido-benzoates, and of the sodium salts of para- and meta-nitrobenzoylchloroamides in moderately concentrated aqueous solutions; (II) Molecular rear- rangement of some triaryl methylchloroamines. — George Burton Rigg: Decay and soll toxins. — Cläre Christman Todd: Action of alkaline hydrogen peroxid on cf-galactose. 412 Doctorates in Biological Chemistry [June-September University of Illinois. — William Leonidas Budison: Availabil- ity of mineral phosphates for plant nutrition. — Harry Peach Cor- son: Manganese in water supplies. — Wallace Macfarlane: Solubility of lime carbonates in relation to their endurance in soils. — Harald Hansen Mitchell: Feeding experiments on the Substitution of pro- teins by definite mixtures of isolated amino-acids. — Fred Weaver Muncie: Effect of large applications of commercial fertilizers upon carnations. — Morris Miller Wells: Relations of fishes to ions in their natural environment : (I) Reactions and resistance to acidity, alkalinity, and neutrality; (II) Reaction and resistance to salts. — Frank Archihald Wyatt: Influence of calcium and magnesium Com- pounds on plant growth. University of Michigan. — George Herbert Coons: Study of the factors involved in the growth and pycnidia formation of Plenodro- mus fuscomaculaits. University of Minnesota. — Morris Joslin Blish: Chemical Con- stitution of wheat proteins and their relation to baking " strength " in flour. University of Wisconsin. — John Nicholas Lowe: Action of chemical Stimuli on the chromatophores of brook trout, Salvelinus fontinalis mitchill. — Charles August Mann: Chemistry of San Pal- metto berries. — Howard Edward Pidling: Movement of water in serotid soils. Washington University. — Alva Raymond Davis: Enzyme ac- tion in the marine algse. — Joseph Charles Gilman: Cabbage yellows and the relation of temperature to its occurrence. — Melvin Clarence Merrill: Electrolytic determination of exosmosis from the roots of plants subjected to the action of various agents. Yale University. — Joseph Suniner Bat es: Synthesis of dipeptid- hydantoins, together with a short study of Michigan hard-wood tar. — Emil Jacob Baumann: Question of fat absorption from the stomach. — Isaac Faust Harris: Chemical and physiological studies of the castor bean and soy bean. — Byron Murray Hendrix: Studies in the physiological action of some protein derivatives. — Edward Frederick Kohmann: Constitution of mono- and di-nitrotyrosin, and the xanthoproteic and Millon reactions. — Walter Moody Scott: Hydroxyl derivatives of Phenylalanin, and their biochemical inter- IQIS] P.H.D. 413 est. — Raymond Louis Stehle: Röle o£ the digestive glands in the excretion of endogenous uric acid. — Richard Wrenshall: Synthesis of a-amino-8-phenylvalerianic acid. Universities that conferred Ph.D. degrees in the natural and exact sciences, but at which there were no biochemical candidates are named below : Catholic University Clark University George Washington Univ. Indiana University Mass. Inst, of Technology New York University Ohio State University Princeton University Stanford University Tulane University University of Iowa University of Missouri University of Nebraska Univ. of North Carolina Univ. of Pennsylvania University of Pittsburgh Number of awards of the Ph.D. degree, by American universities, to biochemical candidates: 191 2, igi3, 19 14 and 191 5 1912 Brown University i Clark University o Columbia University 11 Cornell University 5 Harvard University l Indiana University o Johns Hopkins University . . i New York University o Northwestern University . . . o University of California ... S University of Chicago 8 University of Illinois 5 University of Michigan 2 University of Minnesota . . . o University of Missouri 0 University of Pennsylvania., o University of Wisconsin ... 4 Washington University o Yale University _6 Total number of awards of degrees 49 Number of universities awarding the degree 11 Men and Women 1913 1914 1915 Total 1912 1913 Women 1914 1915 Total 0 I 5 7 0 0 0 G 0 0 I 0 I 0 G I G I 7 7 9 34 I 0 I 2 4 2 4 5 16 0 I I 0 2 I 4 3 9 G 0 0 0 0 0 I 0 I 0 G G G 0 I 2 6 IG 0 0 I 0 I 0 I 0 I 0 G 0 0 0 0 0 I I 0 0 0 0 0 0 2 3 IG I G I 0 2 4 3 8 23 I G G I 2 0 3 7 15 0 0 0 0 G 0 I 1 4 0 G G 0 0 0 I I 2 0 0 G 0 0 I 0 0 I 0 0 G 0 0 0 I 0 I G G 0 0 0 3 4 3 14 0 I 0 0 I 2 0 3 5 0 0 0 G G A _6 _8 24 I I 0 0 2 25 42 63 179 4 3 5 3 15 9 16 14 — 4 3 5 2 . P. H. D. THE BIOCHEMICAL SOCIETY, ENGLAND. Proceedings Reported by R. H. A. Flimmer^ Secretary I. HONORARY SECRETARY'S ANNOUNCEMENT OF THE SCIEN- TIFIC PURPOSE AND SCORE OF THE MEETING ON MAY 5, 1915^ The Meeting is to be devoted to a discussion of " Methods adopted in the estimation of the nitrogenous constituents of extracts derived f rom albuminous substances such as meat extracts and simi- lar products, with special reference to the interpretation of the results." The following scheme, which represents the chemical methods commonly made use of in the examination of nitrogenous extracts, is intended to serve as the basis of discussion : Meat Fibre and Coagulable Albumenoids. Ten (10) grammes of the extract are dissolved in 100 c.c. of cold water. Five (5) drops of acetic acid are added and the Solution brought to the boil and allowed to boil gently for 5 minutes. It is then filtered and the precipitate is washed with warm water, and the nitrogen de- termined by Kjeldahl's method in the ordinary way. N X 6.25 = Meat fibre and coagulable albumenoids. Albumoses and Peptones. The filtrate from the meat fibre and coagulable albumenoids is made up to a definite volume and an amount corresponding with 5 grammes of the original extract pipetted out. To this is added a few drops of hydrochloric acid and then bromine water in large excess. The precipitate is then collected in tubes by means of a centrifugal machine, washed with bromine water in the same way, dissolved in hot water, and Kjeldahled. N X 6.25 = Albumoses and peptones. Some analysts are in the habit of saturating the filtrate from the coagulable albumenoids with zinc sulphate for the purpose of pre- cipitating the albumoses. In that case the nitrogen so obtained is iThe last previous meeting was held March 11. See Biochemical Bul- letin, 1915, iv, p. 174. 414 I9I5] R' H. A. Flimmer 41 5 deducted from the nitrogen contained in the bromine precipitate and the residue is calculated as peptone. It will be interesting to have the views of the Meeting as to whether this particular Separation has any practical Utility. When gelatine is present this is also precipitated by the bromine and the amount as determined below will obviously have to be sub- tracted from the total precipitate in order to arrive at the percentage of albumoses and peptones. Gelatine. Five to twenty grammes are dissolved in hot water and evaporated to dryness with sand. The dried mass is then ground finely in a mortar, placed in a beaker, and washed four times with ice-cold alcohol, about 50 c.c. of alcohol being used for each washing. The alcohol is pumped through an asbestos filter surrounded with ice. The sand is then extracted several times with ice-cold alcohol and water, gradually decreasing the strength of the alcohol with each extraction until the final washing-solution contains only 10 percent of alcohol by weight. In all about 5-6 washings of about 50 c.c- each are required. The gelatine in the beaker and on the asbestos filter is then dissolved in boiling water and the nitrogen determined in the usual manner. N X 5.44 = Gelatine. Creatine and Creatinine. A 10 percent Solution of the extract in distilled water is first prepared. Several 10 c.c. quantities of this Solution, representing i gramme of extract, are pipetted into small beakers and to each 10 c.c. of normal hydrochloric acid are added; the beakers are placed in an autoclave and heated for half an hour at a temperature of 120° C. ; the whole of the creatine present is thus converted into Creatinine. The conversion may also be carried out by dissolving 10 grammes of the extract in a 100 c.c. flask in about 90 c.c. of one-third normal hydrochloric acid, heating in a boiling water bath for 4 hours, allowing to cool and making up to 100 c.c. Ten c.c. of this converted Solution, representing i gramme of extract, are used for each colour experiment. Fifty milligrammes of pure crystallised creatine (which contains one molecule of water of crystallization), are similarly converted with hydrochloric acid and the Solution made up to 100 c.c. To the Contents of one of the beakers, or to the Solution of one gramme of 41 6 The Biochemical Society^ England [June-September extract otherwise converted, cooled to 20° C, 30 c.c. of a saturated picric acid Solution and 15 c.c. of a 10 percent sodium hydroxid Solu- tion are added. After standing for 5 minutes the coloured liquid is made up to 500 c.c. The colour is matched in any suitable colourim- eter against that given by the Standard Creatinine Solution. It can also be matched against 8 millimetres of a Solution of potassium bichromate containing 24.54 grammes of bichromate per litre. the colour of which corresponds with 10 milligrammes of creatine in 500 c.c. of liquid. Residual Nitrogen usually returned as " Meat Bases." The sum of the percentages of nitrogen existing in the form of coagulable proteins, and albumoses and peptones (and of course gelatine, when present) is subtracted from the total nitrogen: the residual nitrogen multiplied by 6.25 is usually returned as " meat bases." It is clear that when creatine and Creatinine are estimated, the nitrogen present in these substances may be deducted from the total nitrogen, in which case the expression " meat bases " must be qualified by the additional words " other than creatine and Creatinine." Perhaps, however, the best mode of expression is to State the percentage of "meat bases" as above, and to point out in an independent Statement that this con- tains such and such a percentage of combined creatine and Creatinine expressed as Creatinine. It is suggested that no useful purpose is to be served by making separate estimations of these two bases, It will be interesting to have the views of the Members on the employment of the arbitrary factor 6.25 for the conversion of re- sidual nitrogen into the equivalent of so-called "meat bases" and also on the question of the food value of the various groups of con- stituents mentioned in the above Classification. In Order to make the discussion as practical as possible, it is par- ticularly requested that proposals in reference to new or alternative processes of analysis should not be introduced except on the basis of experimental data. IL PROCEEDINGS OF THE SOCIETY OF PUBLIC ANALYSTS AND OTHER ANALYTICAL CHEMISTS, AND THE BIOCHEMICAL SOCIETY, IN JOINT SESSION Reported by E. Richards Bolton, Joint Hon. Secretary May 5. Society of Public Analysts and Other Analyti- I9I5] R' H. A. Flimmer 4^7 CAL Chemists. I. Ordinary meeting: held at the Chemical So- ciety's rooms, Burlington House, London. Mr. A. Chaston Chap- man, President, in the chair. The following were elected members of the Society: Honorary Members — Sir William Crookes and Prof. Meldola. Ordinary Members — Messrs. Paul Seidelin Arup, Francis How- ard Carr, Alexander Scott Dodd and Harri Heap. Certificates were read for the first time in favour of Prof. Arthur William Crossley, Mr. Daniel James Davies, Dr. Martin Onslow Forster, Prof. Herbert Jackson, Mr. Frederic Ion Richardson, Prof. William Jackson Pope, Prof. James Charles Philip and Mr. George Henry Warburton. 2. The ordinary meeting was followed by a Joint meeting of The SOCIETY of public analysts and other analytical chem- ists^ AND THE BiocHEMiCAL SOCIETY. Mr. A. Chaston Chapman, representing both Societies, in the Chair. The Meeting was devoted to a discussion on " Methods adopted in the estimation of the nitrogenous constituents of extracts derived from'albuminous substances, such as meat extracts and similar prod- ucts, with special reference to the interpretation of the results." The discussion was opened by the Chairman {Mr. Chapman), who drew attention to the necessity of dividing the products of pro- tein hydrolysis into certain groups or categories for analytical pur- poses. From the technical point of view the purposes to be served by such analyses were, firstly, to indicate the general character of the process by which any particular extract had been prepared ; secondly, to throw some light on the source of the extract and its genuineness or otherwise; and lastly to furnish Information as to the physio- logical properties or dietetic value. The bromine method, properly applied, precipitated gelatine, gelatine-peptone, syntonin, albumoses and peptones, but not Creatinine or other ' meat-bases.' Saturation of the Solution with zinc sulphate in presence of a little acid might be resorted to for the purpose of separating the albumoses from the pep- tones ; and this process, although its results were not very definite, might be useful in so far as it threw some light on the extent to which the original protein matter had been hydrolysed. Bigelow and Cook, in the United States, precipitated the albumoses and peptones by 41 8 The Biochemical Society, England [June-September means of tannln in the presence of salt; and, the albumoses being precipitated by zinc sulphate, the peptones were obtained by differ- ence. For the determination of gelatine, the ice-water-alcohol method of Stutzer was perhaps the most used and most generally considered to give the best results, though some inaccuracy might be caused by the fact that substances rieh in albumoses yielded an appreciable proportion of their nitrogen by this method. As to the determination of creatine and Creatinine there seemed little to be said from the chemical side, Folin's method having been thoroughly worked out. The use of ammonium salts had been occa- sionally alleged, but no case of this very crude form of aduheration had come under his notice, though he had met with a sample of extract which, on distillation with magnesium oxide, yielded a con- siderable quantity of ammonia, due, apparently, to some putrefactive change. The residual nitrogen was of ten returned as * meat-bases,' some analysts using for conversion the factor 3.12 originally sug- gested by Stutzer on the basis of the nitrogen percentage of crea- tine. Hehner, however, had suggested the use of the ordinary pro- tein factor of 6.25, which at least had the merit of not involving any assumption or giving to the results an apparent accuracy they did not in fact possess; though on the other band, to those who did not understand the matter, the quantity of 'meat-bases' might be made to appear much larger than it really was, while a considerable error was thrown on the proportion of non-nitrogenous extractives, which was arrived at by difference. The best plan was to retum the actual nitrogen percentages. Prof. F. Gowland Hopkins said that the animal body dealt, not with the intact proteins, or even with the albumoses and peptones, but with the free amino-acids, which were the individual constituents of the protein molecule. The actual way in which the different amino- acids were grouped in the protein molecule was not of much conse- quence, but the efifects produced by the individual amino-acids were of extreme importance. He described physiological experiments which he had made showing that when rats were given a diet includ- ing a complete amino-acid mixture corresponding to the proteins of an ordinary diet, their growth was almost exactly normal, while, when arginine and histidine were removed from the amino-acid mix- iQis] R. H. A. Plimmer 4^9 ture, the growth ceased immediately, being resumed when arginine and histidine were again added. The removal of tryptophane from the amino-acid mixture also produced similar results ; and Osborne and Mendel in America had shown cystine to be similarly essential. It did not follow that this was the case with every amino-acid, and the question as to which of the amino-acids were essential in that way offered a large field for investigation. Recent work at Cambridge indicated that certain acids could be removed from the amino-acid mixture without affecting the rate of growth. With regard to the minimum quantity of any amino-acid required for nutrition, experi- ment had shown that, in the case of rats, at any rate, the critical minimum for arginine lay somewhere between 2.5 and i percent. It appeared that the functions of the individual amino-acids were not confined merely to flesh formation. The effect of feeding ani- mals on zein, which was deficient in both tryptophane and lysine, was not only to restrict growth but also to shorten the survival, and the same was observed with zein plus lysine; but with zein pliis tryptophane the animal was able to maintain its weight for a long period, although it did not grow. It was clear from this that the tryptophane exercised some other function than the mere supply of material for growth. Further work on this subject is proceeding at Cambridge. Dr. E. P. Cathcart, referring to creatine and Creatinine, said that the observations at present available were so scanty that it could not be stated with certainty that creatine and Creatinine had a special niche in the organism. Creatinine, of course, was a constant ex- cretory product, but creatine was not, except in the case of infants, though it might be made to appear if an animal were starved or treated with certain drugs. Folin had stated that creatine was a fairly valuable food-stuff, but he (Dr. Cathcart) did not think, on the evidence available, that it was. He did not think that any end would be gained by the separate estimation of creatine and Creatinine in meat extract, since a large part of the original creatine would prob- ably be converted into Creatinine during the process of manufacture. Mr. A. R. Tankard thought that it was important in some cases to separately estimate the meat fibre and coagulable albuminoids, with a view to the detection of extraneous matters which were some- 420 The Biochemical Society^ England [June-September times added. For some time past he had adopted the method of centrifugalising the bromine precipitate, mixing the residue with water, and centrifugahsing again. He made a practice of separating the albumoses and peptones, but agreed that in most ordinary cases this was of Httle value. There were, however, on the market cer- tain so-called ' f ortified ' meat extracts which contained albumoses and peptones in very much larger proportion than occurred in ordir nary meat extracts. With regard to the factor used for Converting the residual nitrogen into ' meat-bases ' he remarked that this was the only instance which he knew of in chemistry in which a factor known to be wrong was used merely for convenience. Mr. E. Hinks remarked that the use of a factor of approximately 3 for calculating the * meat-bases ' f rom the residual nitrogen seemed to be justified by the fact that the proportions of nitrogenous and non-nitrogenous extractives thus obtained were about equal, this being what one would expect in the case of such a product as meat extract. Dr. Percival Hartley described his experience of Van Slyke's method of determining amino-nitrogen, which was based on the fact that when protein and protein degradation products were treated with nitrous acid, nitrogen gas was given off. The results of com- plete analyses of various proteins showed that the Constitution of serum albumen was quite different to that of globulins, the former containing a much larger proportion of lysine and of cystine than the latter. Another interesting result was that the globulins exhib- ited no difference in chemical composition — euglobulin and Pseudo- globulin apparently being closely related. Another point brought out was that the free amino-nitrogen of native proteins appeared to be approximately one-half of the lysine content, which if substantiated, would appear to afford a simple method of estimating lysine without hydrolysis of the protein. Furthermore, the proportion of free amino-nitrogen indicated to what extent the protein had been di- gested or peptonised. Further remarks were made by Dr. Rideal, Prof. Barger, Prof. Harden, Dr. G. S. Walpole, Dr. Cathcart, Prof. Gowland Hopkim and the Chairman. igis] R' H. A. Plimmer 421 III. SCIENTIFIC PROGRAM OF THE BIOCHEMICAL SOCIETY, JUNE 12 June 12. ROTHAMSTED EXPERIMENT STATION, HaRPENDEN, Herts. W. E. Brenchley: The effect of the concentration of nutrient Solutions upon the growth of plants in water culture. IV. A. Davis: The periodic Variation of the sugars in the foliage leaves of plants during the day and night. W. A. Davis and G. C. Sawyer: The Variation of the starch content of the potato leaf during day and night and its relation to the sugars present. E. H. Richards: The loss of nitrogen during the bacterial de- composition of the nitrogen Compounds of animal excretions. CO A. Appleyard: The -77-^ ratio in soll oxidations. W. Weir: The effect of soluble humus on plant growth. E. Horton: Methods for the extraction of organic Compounds from soil. /. Prescott and E. J. Russell: The reaction between soil phos- phates and dilute acids. IV. PROVISIONAL SCHEDULE OF MEETINGS FOR 191S-16 May 5. Joint Meeting with Society of Public Analysts, Chem- ical Society, Burlington House, London, W. June 12. Rothamsted Experimental Station, Harpenden, Herts. Nov. IG. Physiological Laboratory, King's College, London. Dec. 14. Lister Institute, London. Feb. 12. University of Leeds. March 9. Institute of Physiology, University College, London. V. OFFICERS, igi5-i6 Committee:^ Hon. Treasurer, J. A. Gardner; Hon. Secretary, R. H. A. Plimmer; Editors of the Biochemical Journal, W. M. Bayliss and A. Harden; Ordinary Members, G. Barger, V. H. 2 The business of the Society is conducted by a committee consisting of a treasurer, secretary, the editors of the Biochemical Journal, and twelve ordinary members. 422 The Biochemical Society, England [June-September Blackman, A. Chaston Chapman, W. A. Davis, A. E. Garrod, W. D. Halliburton, T. A. Henry, W. H. Hurtley, G. W. Monier- Wil- liams, J. Lorrain Smith, H. M. Vernon, T. B. Wood. VI LIST OF THE MEMBERS ELECTED SINCE THE PUBLICATION, IN THE BIOCHEMICAL BULLETIN, OF THE ORIGINAL LIST OF MEMBERS3 Anrep, Dr. G. von, Univ. College, London, W. C. Atkin, Dr. E. E., Lister Institute, Chelsea Gardens, London, S. W. AuLD, Prof. S. M. J., University College, Reading. BoTTOMLEY, Prof. W. B., King's College, Strand, London, W.C. Brah MACHART, Dr. U. N., 19 Grey Street, Calcutta, India. BuRN, J. H., Esq., Brockwell Hall, Herne Hill, S.E. Carr, f. H., Esq., F.I.C., Edgmont, Derby Road, Nottingham. CoMPTON, Dr. Arthur, Imperial Cancer Research Fund, 8-11 Queen Square, Bloomsbury, W.C. Cornish, Miss Elfreda, C.V., M.Sc, 50 Morgan Road, Reading. Cruickshank, Dr. E. W. H., Univ. College, Gower Street, Lon- don, W.C. CuLLis, Miss Winifred, D.Sc, London Seh. of Med. for Women, 8 Hunter St., London, W.C. Daish, A. J., Esq., Rosemount, Tennyson Road, Harpenden, Herts. Drummond, J. C, Esq., B.Sc, Cancer Hosp. Research Inst, Bromp- ton, London, S.W. DuDLEY, Dr. H. W., The University, Leeds. Edie, E. S., Esq., B.Sc, The University, Aberdeen. EuLER, Prof. H. von, Stockholms Högskola, Stockholm, Sweden. FiNLOW, R. S., Esq., B.Sc, Dacca, Bengal, India. Fräser, Miss Mary T., B.Sc, 18 The Park, Ealing, W. Ginsberg, Dr. W., Dahlmannstrasse i, Kiel, Germany. Graham, Dr. G., 17 Bentinck Street, Alanchester Square, Lon- don, W. Grüner, Dr. O. C, 29 Park Square, Leeds. Harman, H. W., Esq., 24 Holborn Viaduct, London, E.C. Harris, Prof. D. Fräser, Dalhousie University, Halifax, N.S. HiNKS, E., Esq., F.I.C, 16 Southwark Street, London, S.E. 8 Biochemical Bulletin, 1913, ii, p. 447. I9I5] R- H. 'A. Flimmer 423 Horton, E., Esq., B.Sc, Rothamsted Experimental Station, Har- penden, Herts. HosEASON, J. H., Esq., 2 Bridge Street, Manchester. Laidlaw, C. G. P., Esq., 3 More's Garden, Chelsea, London, S.W. Latham, P. W., Esq., 263a St. James's Court, Buckingham Gate, London, S.W. Leathes, Prof. J. B., F.R.S., University, Sheffield. Leetham, Miss Constance, B.Sc., London Seh. of Med. for Women, 8 Hunter St., London, W.C. Lefebure, V., Esq., B.Sc, 25 Belitha Villas, Bransbury, London, N. LoGiE, W. J., Esq., 14 Duckers Road, Edgbaston, Birmingham. Macallum, Dr. A. Bruce, University, Toronto, Canada. Marrack, John R., Esq., Research Hospital, Cambridge. Mockeridge, Miss Florence A., B.Sc, King's College, Strand, W.C. Newton, L. O., Esq., 41 Bennett Park, Blackheath, Kent. Oliver, G. Y., Esq., 6 Ridgmount Gardens, London, W.C. Orr, Dr. J. B., Marischal College, Aberdeen. Poulton, Dr. E. P., 24 St. Thomas's Street, London Bridge, S.E. Raper, Dr. H. S., The University, Leeds. Roberts, F. F., Esq., M.A., Cläre College, Cambridge. Robinson, Fred^k., Esq., M.Sc, The HolHes, Mile End, Stockport. RoBisoN, Dr. R., 78 Primrose Mansions, Battersea Park, London, S.W. Scott, Prof. F. H., University of Minnesota, Minneapolis, U. S. A. Stead, A., Esq., Grootfontein Seh. of Agric, Middleburg, Cape Province, S. Afr. Steele, V., Esq., 55 Amersham Road, New Gross, London, S.E. Stephenson, Miss Marjory, Univ. College, Gower Street, London. W.C. SuRVEYOR, Dr. N. M., 145 Queen's Road, Bombay, India. Trevan, J. W., Esq., M.B., St. Bartholomew's Hospital, London, E.C.' Tribe, Mrs. Enid M., B.Sc, London Seh. of Med. for Women, 8 Hunter St., London, W. C. Wager, Harold, Esq., F.R.S., Hendre, West Park, Leeds. 424 The Biochemical Society, England [June-September Warner, C. H., Esq., F.I.C., 24 Gordon Street, Gordon Square, London, W.C. WiDDOWS, Miss Sibyl T., B.Sc, Braeside, Beaconsfield Road, Lon- don, S.E. Williams, Dr. R. Stenhouse, University College, Reading. WiNFiELD, G., Esq., 31 Panton Street, Cambridge. University College, London. AMERICAN CHEMICAL SOCIETY, Proceedings of the Division of Biological Chemistry, New Orleans, La., March 31-April 3, 1915.^ REPORTED BY PAUL E. HOWE The American Chemical Society held its fiftieth meeting in New Orleans, La., March 31-April 3, 191 5. The Division of Biological Chemistry held its meeting at the Hotel Grunewald, on April 2nd. The f ollowing papers were presented : C. L. Aisberg: Control of cotton-seed products. — C. C. Boss: Emetine, the specific remedy for pyorrhcea alveolaris. — Charles Mann: Saw palmetto, a biochemical study. — E. R. Miller and /. M. Moseley: Volatile oils of some species of Solidago. — E. R. Miller: Volatile oils of several species of Eupatorium; Volatile oil of Achil- lea millefolium L; Some volatile oils from the genus Pycnanthe- mum. — W. J. V. Osterhout: Artificial photosynthesis by chlorophyl. — W. E. Tottingham: Röle of chlorin in plant nutrition. — G. S. Fraps: Nitrification studies. — C. B. Lipman: The form of nitrogen in nitrogenous materials as an index to nitrifiability. — M. X. Sulli- van: Formation of Creatinin by bacteria; Amount of Creatinin in plants. — Edward Gudeman: Toilet papers, a source of infection. — Adolph Bernard: Simple colorimetric method for the determination of free reducing sugars and total carbohydrate in miscellaneous food products. — /. P. Atkinson: Reducing action of certain carbohydrates on distillation. — Sara S. Graves: Precipitant for ammonia. — W. Denis: Phenols and phenol derivatives in urine. — /. H. Long: Physi- ological activity of combined hydrochloric acid; Combinations of proteins with halogen acids. — Charles Baskerville: Rate of evapora- tion of ether from oils and its application in oil-ether colonic anes- thesia. — H. S. Grindley and E. C. Eckstein: Free amid nitrogen and 1 For accounts of the Organization of the Biochemical Division and suc- cessive meetings see Biochem. Bull., 191 i, i, p. 94; 1913, iii, p. 76; 1914, iii, p. 444. 425 426 American Chemical Society [June-September the free amino-acld nitrogen of feedingstuffs. — H. S. Grindley, W. E. Joseph and M. E. Slater: Quantitative determination of the amino-acids of the mixed proteins of feedingstuffs. — R. S. Potter and R. S. Snyder: Nitrogen distribution according to the Van Slyke method in soils and their " humic acids " ; Amino-acid nitrogen in soils variously treated. — Max Kahn: Urinary mucin. — M. Kahn and F. G. Goodridge: Cystin. — F. G. Goodridge: Biochemical studies of mercaptan. — M. Kahn and Francis Huber: Metabohsm studies of multiple myeloma with Bence-Jones albumose. — M. Kahn and 5. Schneider: Study of the mineral metabolism of diabetics. — A. F. Hess and M. Kahn: Mineral metabolism of two cases of hemophilia. — Jacob Rosenbloom: Ethereal sulfates of the urine in various dis- eases; Modification of Gerhardt's test for diacetic acid; Influence of low and high protein intake on the excretion of acetone, diacetic acid, and i^-hydroxybutyric acid in diabetes. — W. M. Clark: Ad- justment of the reaction of bacteria culture media; Final hydrogen- ion concentration of cultures oi B. coli. — E. H. Walters and W. M. Clark: Relation of propionic fermentation to the development of " eyes " in Ementhaler cheese. — 6*. L. Jodidi: Factor to be used for the calculation of the phosphoric acid in Neumann's method. — S. L. Jodidi and E. H. Kelhogg: Factor to be used for the calculation of phosphoric acid in Neumann's method. I. Factor as influenced by the water used for washing the yellow precipitate. — R. E. Swain and E. R. Harding: Quantitative estimation of alantoin. — Lewis Knndson: Influence of certain sugars on the growth and respiration of vetch. — W. J. Robbins: Influence of certain inorganic substances on the digestion of starch by Penicillium camemberti. — W. M. Clark and H. A. Lubs: Differentiation of organisms of the colon group by means of indicators. — C C Johns and Arno Viehoever: Studies on the saponins of Chlorogalum pomeridianum and of Agave lechu- guilla; Alkaloids of Amianthium miisca:torium. Biochemical Lahoratory of Columbia University, College of Physicians and Surgeons, New York. AMERICAN PHILOSOPHICAL SOCIETY General meeting — April 22 to 24, 1915 REPORTED BY JOSEPH S. HEPBURN New members. — The following, among others, were elected members: John J. Abel (Johns Hopkins Univ.), John M. Coulter (Univ. of Chicago), William J. Gies (Columbia Univ.), Philip B. Hawk (Jefferson Med. Coli, Phila.), Thomas H. Morgan (Colum- bia Univ.), Raymond Pearl (Maine Agric. Exp. Station), Theobald Smith (Harvard Univ.). PaPERS of BIOLOGICAL and CHEMICAL INTEREST. M. H. Jacobs: Heredity in protozoa. — T. H. Morgan: Constitution of hereditary material. — G. H. Parker: Problem of adaptation as illus- trated by the für seals of the Pribilof Islands. — Edward M. East: Interpretation of sterility in hybrids. — G. H. Shidl: Heterosis and the effects of inbreeding. — B. M. Davis: Significance of sterility in Oenothera. — G. F. Atkinson: Morphology and development of 'Agaricus rodmani. — William Trelease: Large-fruited American oaks. — M. V. Cobb: Relationships of the white oaks of Eastern North America. — L. H. Bailey: Present need in systematic botany. — M. T. Bogert: Convenient form of receiver for fractional distil- lations under diminished pressure. — /. R. Tuttle and M. T. Bogert: Cymene carboxylic acids. — E. Plant and M. T. Bogert: Syringic acid and its derivatives. — W. J. Gies: Relation- of ductless glands to dentition and ossification. — P. B. Hawk: Gastro-intestinal studies. — Charles Baskerville: Rate of evaporation of ether from oils and its application in oil-ether colonic anesthesia. — A. J. Smith: Oral endamebiosis. — /. T. W. Marshall: New form of nephelometer. — W. M. Davis: New evidence for Darwin's theory of coral reefs. — Stewart Paton: Certain factors eonditioning nervous responses. Philadelphia, Pa. 427 BIOCHEMICAL NEWS, NOTES AND COMMENT EDITORI AL SUB-COMMITTEE: Benjamin Horowitz, William J. Gies, Hattie L. Heft, Joseph S. Hepburn, Paul E. Howe, Edgar G. Miller, Jr., William A. Perlzweig Contents. — (I). General: Necrology, 428; appointments, 429; honors, 431; lectures and addresses, 432; medals, 433; prizes, 434; associations, societies, etc., 434; miscellaneous items, 435. (II). War Notes: Necrology, 443; university items, 444; booze bombs, 444; honors, 445; notes on the sanitation of military quarters and battlefields, 446; food notes, 447; medical notes, 450; miscellaneous items, 453. (III). Col. Univ. Biochem. Assoc: (i) General notes— honors, 453; appoint- ments, 454; dental graduates, 454; associations and societies, 454; miscellaneous items, 455; (2) Proc. of the Assoc— 23"^ (sixth annual) meeting, 456; (3) Co- lumbia Biochem. Dep't — appointments, 456; associations and societies, 457; ad- dresses and reports, 457; awards of higher degrees, 458; summer session, 459; miscellaneous items, 459. I. GENERAL Necrology.^ — A. Arnaud, who occupied the chair of ehem. at the Museum of Nat Hist., Paris; distinguished for work in ehem. and pharmacol. Paul Ehrlich, director, Royal Inst, for Exp. Therapeutics, Frank fort. Erich Harnack, director, Inst, of Pharmacol., Halle. A. Sheridan Lea, formerly Univ. lecturer in physiol., Cam- bridge; well known for his researches in physiol. ehem., and his " Chemical basis of the animal body." Friedrich Löffler, director, Robert Koch Inst, for Infec. Dis- eases, Berlin; discoverer of the diphtheria bacillus. John U. Nef, head of the dep't of ehem., Univ. of Chicago; eminent for his contributions to organie ehem. Albert Plaut, vice-p., N. Y. Coli, of Pharmaey. Dr. Proskauer, director, Berlin Bureau of Food and Trades 1 War necrology, page 443. 428 1915] General 429 Inspection; an authority in the field of water-supplies and sewage- disposal. Ludwig Tohler, chief, Children's Hosp., Breslau; investigator of the pathol. of nutrition. Body of Prof. Hildebrandt recovered. In 1912, Dr. Hilde- brandt, instructor in pharmacoL, Univ. of Halle, lost his life during a mountain-climbing trip. His body was recently found in a crevice, by soldiers crossing the Bozen district. Appointments.^ Digestive Ferments Co. (Detroit, Mich.) : /. W. M. Bunker (Harvard Univ.), direc, bacteriol. research dep't Fordham Univ., Med. Seh. (N. Y .City) : Lewis W. Fetzer (U. S. Dep't of Agric), prof., physiol. and biochem., vice Alwyn Knaiier, resigned; Carl P. Sherwin, assis. prof., biochem.; John A. Killian, instr., physiol. and biochem. McGill Univ., Med. Seh. : F. W. Skirrow, assis. prof., ehem. : /. C. Meakins and F. B. Gurd, lect's, immunology. Mass. Agric. Coli. : Paid Serex, instr., ehem. Montana Agric. Coli.: R. H. Bogue (Mass. Agric. Coli.), assis. prof., agric. ehem. N. Y. City Dep't of Health : L. P. Brown (State Food and Drug Commiss., Tenn.), direc, Bur. of Food Inspec. ; Matthias Nicoll, Jr., assis. direc. of lab's. N. Y. Post-Grad. Med. Seh. and Hosp. : Morris Fine, adj. prof., path. ehem. (prom.). Okla. Agric. and Mech. Coli: L. C. Raiford (Univ., Chicago), prof., ehem. Parke, Davis and Co. (Detroit) : S. P. Miller (Mass. Agric. Coli.), chemist. Queen's Univ., Med. Seh. (Kingston, Ont.) : /. 0. Halverson (Jeff. Med. Coli.), lect., biol. ehem. Rockef eller Inst. Med. Research. Associates (promotions) : C. J. West, ehem. ; Michael Heidelberger, ehem. ; Angelia M. Court- ney, ehem. — Assistants (promotions) : C. H. Allen, ehem.; /. K. Senior, ehem. ; G. E. Cullen, ehem. ; Marian Vinograd, ehem. — New appoint's: E. A. Wildman, feil., ehem.; A. L. Meyer, assis., physiol. and pharmacol. 2 In this summary institutions from which appointments were made are named in parenthesis. See also pages 454 and 456. 430 Biochemical News, Notes and Comment [June-September Sta'n for Exp. Evol, Carnegie Inst'n: A. F. Blakeslee (Conn. Agric. Coli.), plant geneticist. U. S. Dep't of Agric, Bur. of Chem. : F. B. La Forge (Rocke- f eller Inst.), expert, org. chem. Food Research Lab. (Phila.) ; T. E. Harper, Jr., chemist's aid, fish-handling investigations. U. S. Pub. Health Serv., Pellagra Hosp. (Spartanburg, S. C.) : /. R. Murlittj temp. biochem. ; M. X. Sullivan (Div. of Soil Fer-' tility, U. S. Dep't of Agric), permanent biochem. Univ., Cal. : H. T. Chickering (Rockefeiler Inst), "research associate in pathol." He will be associated with Prof. F. P. Gay in investigations on the treatment of typhoid by the use of sensitized Vaccine. This research associateship, established with a gift by Mr. J. K. Moffit, one of the Univ's regents, is additional to a research associateship in pathol., for which other donors recently agreed to provide an annual gift of $1,200, and an eventual endowment of $25,000. Hooper Foundation for Med. Research: ^. H. Hurwitz (Harvard Med. Seh.) : instr., research med. Univ., Halle, Pharmacol. Inst.: R. Magnus (Utrecht), prof., pharmacol. and direc, in succession to Prof. Hamack, deceased. Univ., 111. : H. B. Lewis (Univ. of Penn.), assoc, physiol. chem. Agric. Exp. Sta. : Robert Stewart (Utah Agric Coli.), assoc. prof., soil fertility; assis. chief, soil fertility work. Univ., Minn. : Jean MacKinnon, assis. prof., nutrition. Univ., Oregon: H. B. Myers (N. Y. Univ. and Bell. Hosp. Med. Coli.), prof., mat. med. and pharmacol. Univ., Penn. : A. L Ringer, assis. prof., physiol. chem. {re- signed) ; R. L. Stehle and B. M. Hendrix, instrs., physiol. chem. Univ., So. Cal., Seh. of Med. : C. G. MacArthur, instr., biochem. and toxicol. Univ., W. Va., Coli, of Agric, Exp. Sta'n: /. L. Coiilter (Geo. Peabody Coli.), dean. Coli, of Agric; direc Exp. Sta'n, vice E. D. Sanderson, resigned. Va. State Dep't of Agric. : /. B. Weems, chief chemist, to suc- ceed Dr. Magruder, resigned. West. Resc've Univ., Med. Seh. : P. J. Hanslik, assoc, phar- macol. (prom.); G. E. Simpson, instr., org. and biol. chem. (prom.) ; H. H. McGregor, instr., biochem.; R. J. Collins, de- monstr., pharmacol. 191 5] General 431 Yale Univ., Med. Seh. : C.-E. A. Winslow, Anna M. L. Lauder prof. of public health. He will resign his positions in the N, Y. State Dep't o£ Health and Teachers' Coli., to take up this work in the fall, but will continue to act as curator of public health at the Amer. Mus. of Nat. Hist., N. Y. City. Honors. Posthumous. A monument to the late Prof, /. H. van't Hoff was unveiled at Rotterdam, Apr. 17. It consists of a bronze statue, double life-size, in sitting position, placed in front of the school at which van't Hoff was educated. The monument is about 30 ft. high, and the statue itself is flanked by female figures representing Imagination and Reason. On the front of the base is the f ollowing inscription : Van't Hoff 1852-1911 Physiciam chemiae adiunxit The centenary of the birth of David Waldie, who suggested, to Sir James Simpson, a trial of Chloroform as an anesthetic, has been commemorated with a bronze tablet on the house in Linlithgow where he lived for some time. The tablet shows a portrait of Waldie, with an inscription in which he is described as a pioneer in anesthetic research. HoNORARY DEGREEs. Washington Univ. (Med. Seh.), Apr. 30: ScD., Otto Folin; LL.D., R. H. Chittenden, S. J. Meltzer, W. H. Howell. V. C. Vaughan, LL.D., Jeff. Med. Coli. John A. Kolmer, M.S., Villanova Coli. F. E. Stewart, Phar.D., Medico-Chi. Coli., Phila. Dr. Stewart is prof. of mat. med. in the Medico-Chi. Coli, and Organizer of the Nat'l Bur. of Med. and Foods. Phila. Coli, of Pharm., Pharm. M. : E. G. Eherle, ed., Jour. Amer. Pharmaceut. Assoc; C. A. Mayo, pres., Amer. Pharmaceut. Assoc, ed. of Amer. Druggist; W. M. Mittlebach, pres., Nat'l Assoc. Boards of Pharmacy ; H. M. Whelpley, treas., Amer. Pharmaceut. Assoc. and Board of Trustees of U. S. Pharmacop. Conven., prof. of micros- copy and dean of St. Louis Coli, of Pharm., prof. of mat. med. and pharm, of Washington Univ.; W. L. Cliffe, trustee, Phila. Coli, of Pharm. 432 Biochemical News, Notes and Comment [June-September Dinners and celebrations. A testimonial dinner was ten- dered to Dr. Leo Loeh at the Univ. Club, St. Louis, May 25, by mem- bers of the med. profession of St. Louis, the scientific faculties of Washington and St. Louis Univ's, and members of the Biol. So- ciety of St. Louis. Dr. /. P. Lobenhojfer, on June 15, completed 15 years of con- tinuous Service as chemist at the Touro Infirmary, New Orleans.. He was presented by his assistants with a gold smoking set and the Board of Direc. adopted a resolution expressing their keen apprecia- tion of his Services. Prof. /. M. Bartlett, on April 30, completed 30 years of con- tinuous Service as chemist at the Maine Agric. Exp. Sta'n. This period includes the entire history of the Station. In recognition of this unusual length of Service in one institution, a reception in Prof. Bartlett's honor was held in the Station building on the evening of Apr. 30, and he was presented with a commemorative volume. This volume was composed of a series of congratulatory letters from nearly all of the 109 different persons, now living, who have, at one time or another, been associated with Prof. Bartlett in the work of the Station. A celebration which, owing to present circumstances, has taken on a personal character, was recently held at the Pasteur Inst. ; the occasion was the 70th birthday anniv. of Prof. Elie Metchnikoff. Prof. Gaston Darboux and Dr. Roux, on behalf of the Acad. des Sciences and Pasteur Inst., resp., reviewed the career and the works of the Russian scientist. Prof. Metchnikoff gave the audience an interesting talk on the Prolongation of life. Paris letter: Jour. Amer. Med. Assoc, 191 5, Ixiv, p. 2082. MiscELLANEOus. The name of Curie, in honor of the dis- coverer of radium, has been given to a small park (Place de Curie) formed by the demolition of the old rue Dauphine in Paris. — Royal Roumanian crosses of the first class for Sanitätsverdienst (merit in sanitary service) have been conferred on von Behring and Ehrlich. Lectures and addresses. Cutter Lectures. The Cutter Lectures in preventive medicine, for 19 15, were given at the Har- vard Med. Seh. by V. C. Vaughan and Joseph Goldherger. Dr. Vaughan discussed The phenomena of infection, April 14, 15 and 16; Dr. Goldberger, Diet and pellagra, in one lecture, April 12. I9I5] General 433 MiscELLANEOus ITEMS. Clcveland Acad. of Med., May 21 : R. M. Pearce (Univ. of Penn.), Relation of the spieen to blood de- struction and regeneration, and to hemolytic jaundice. Coli, of Phys. and Surg. (Phila.), April 12: V. C. Vaughan (Univ., Mich.), Phases of modern military Hygiene and camp sani- tation, particularly in reference to war mortality. Columbia Univ. ; Phi Lamhda Upsilon Lect., May 6 : David T. Day (U. S. Bur. of Mines), Petroleum, illustrated with motion pictures. Jeff. Med. Coli., June 15: V. C. Vaughan (Univ., Mich.), A doctor's Ideals. King's Coli. (London), Alay 31, June 2, 7 and 9: T. B. Brodie (Univ., Toronto), Gases of the blood. N. Y. Bot. Card. Summer series : Wm. Mansfield (N. Y. Coli, of Pharm. ) , Poisonous plants of eastern U. S. Late-summer series : W. A. Murrill, The use of mushrooms for food. Ohio State Univ., Apr. g: A. J. Carlson (Univ., Chicago), Re- cent contributions to the physiology of the stomach. Univ., Chicago; P/ii Lamhda Upsilon Lect., May 21 : /. U. Nef, Chemistry of enzyme action. Vassar Coli, May 10: F. G. Benedict (Carnegie Nutr. Lab.), (i) Investigations in the Nutr. Lab. of the Carnegie Inst'n of Washington; (2) Women as research assistants. Washington Univ., Med. Seh. (dedication week, April) : Otto Polin (Harvard Med. Seh.), (i) The utiUzation of food protein; (2) Tissue metaboHsm, with special reference to Creatinin; (3) Pro- tein metabolism, with special reference to uric acid; (4) The occur- rence and significance of phenols and phenol derivatives in the urine. Medals. Baly medal, Royal Coli, of Phys. (London) : To F. G. Hopkins.— Dany medal (Royal Soc.) : To W. J. Pope, for his researches on stereochem. and on the relations between crystal- line form and ehem. structure.— Franklin medal (the highest recognition in the gift of the Franklin Inst., Pa.) : To H. K. Onnes, for his low-temp. research, and io T.A. Edison, for his "numerous basic inventions and discoveries forming the foundation of world- wide Industries, signally contributing to the well-being, comfort and pleasure of the human race." 434 Biochemical News, Notes and Comment [June-September The bronze thesis-medal of the Science Club, Univ. of Wis., was awarded, at commencement, to Walter Pitz for a thesis on The effect of elemental sulfur and of calcium sulfate on certain of the higher and lower forms of plant life. This medal is awarded annu- ally to a senior in the Univ. of Wis. for quality and quantity of research in the preparation of a thesis in physical or natural science, or pure mathematics, or their useful applications. Prizes. The Royal Soc, Edinburgh, has awarded the Mak- dougall-Brisbane prize, for 1912-14, to C. R. Marshall (Dundee and St. Andrews), for his studies on the pharmacol. action of tetra- alkyl ammonium Compounds. The OsiRis PRizE, $20,000, which the Inst, of France gives every three years as a reward for the most remarkable work or discovery by Frenchmen in science, art, letters or industry, was awarded jointly to Prof 's Widal (^) and Chantemesse (ji), and Dr. Vin- cent (^), whose names are connected with the development of anti- typhoid inoculation. As the Osiris prize can be given only to French- men, the Inst, decided to award, in this connection, a special prize to Sir Almroth Wright, who first prepared anti-typhoid serum. Associations, societies, etc.: Officers-elect. Amer. Assoc. Immunologists. Pres., /. IV. Johling; vice-p., G. P. Sanhorn, W. T. Councüman, J. A. Kolmer; treas., W. J. Stone; sec, M. J. Synnott. Amer. Med. Assoc. Pres., Surg. Gen., R. P. Bliie. Amer. Neurolog. Assoc. Pres., L. F. Barker. Amer. Pharmaceut. Assoc. Pres., F. L. Wulling, dean. Coli, of Pharm., Univ. Minn. Amer. Soc. Trop. Med. Pres., M. J. Rosenau. Assoc. Amer. Physicians. Pres., Henry Sewall. German Assoc. Scientific Men and Physicians. Pres., F. von Müller. Medical Brotherhood. See page 294. New Orleans Acad. Sciences. Pres., Gustav Mann. SiGMA Xi, DisT. Columbia Chapt. Vice-p., Isaac K. Phelps. Soc. Exp. Biol. and Med. Pres., Graham Lusk (re-elec); vice-p., G. N. Calkins; sec-treas., H. C. Jackson; addit. members of the Council, Wm. J. Gies and John Auer. 191 5] General 435 Members elect. Amer. Philosoph. Soc. : J. J. Abel, J. M. Coulter, Wm. J. Gies, P. B. Hawk, Theobald Smith. Nat'l Acad Sciences. F. R. Lillie, Graham Lusk, Alexander Smith, V. C. Vaughan. Soc. Exp. BiOL. AND Med. G. M. Baehr, Olaf Bergeim, War- ren Coleman, D. J. Edwards, P. A. Kober, J. A. Kolmer, H. B. Lewis, W. G. Lyle, S. S. Maxwell, W. F. Petersen, G. H. Whipple. Miscellaneous items. Personal. Dr. Rokiiro Nakaseko (M.S., Yale: Ph.D., Johns Hopkins) has lately been a visitor in this country. He is now in charge of the A. C. James Lab., Muro- machi-Demizu, Kyoto, Japan. Meritorious exhibit. The commit. on awards for scientific exhibits, at the San Francisco meeting of the Amer. Med. Assoc, granted a gold medal to Dr. M. H. Fischer, for his exhibit on Newer experiments in the physiology and pathology of kidney functions. Lab. kinks. Method of paraffining labeis. Instead of going to the trouble of melting paraffin whenever you wish to apply this as a protective coating, the paraffin can be dissolved in carbon tetra- chlorid, making a saturated sol. This may be applied as a varnish. The carbon tetrachlorid evaporates, leaving a coating of paraffin behind. Gasoline or benzine might be used but carbon tetrachlorid is preferred, as its vapor is not inflammable. D. L. Randall : Chem- ist- Analyst, 1915, no. 13, p. 25. Cleaning Solution for glass and porcelain. The following sol. for removing org. material from glass- or porcelain-ware has been found by the author much superior to the well-known potassium- bichromate-sulfuric-acid mixture. The glass- or porcelain-appar. to be cleaned is placed in a large evap. dish containing sulfuric acid and a little nitric acid. If the appar. is not completely immersed, it is turned after a few minutes so that the cleansing sol. comes in con- tact with every part. The acid should be kept very warm but not hot enough to evolve sulfur trioxid or to distill the nitric acid. Thick glassware, e. g., suction flasks, should be placed in the cold mixture, which is then heated. The sol. is permanent except that enough nitric acid must be added, from time to time, to keep it white or at least yellow in color rather than black. This mixture has been found especially useful in cleaning dyes, gums and waxes from glassware. 43^ Biochemical News, Notes and Comment [June-September A thin film of dye will be removed almost instantly, but the re- moval of masses of org. material requires longer treatment, F. C. Mathers: Chemist-Analyst, 191 5, no. 13, p. 10. Food notes. Bork acid proscribed. Constitutionality of the 111. pure-food law, prohibiting, in effect, sale of food preservatives containing boric acid, was upheld, June 21, by the U. S. Supreme Court. Justice Hughes stated, for the court, that the law must be upheld as valid unless the defendant shows there is no doubt that boric acid is wholesome, which, the court held, he had failed to do. Diet treatment of pellagra. An arrangement has been entered into between the U. S. Pub.-Health Serv. and the Epworth Orphan- age, Columbia, S. C, for the application of the diet treatment of Pellagra among the children, in the orphanage, afflicted with the dis- ease. The Service will prescribe the diet and furnish the protein portions of it. All necessary facilities will be given the gov't officials in this work. John Barleycorn on the run. Physicians favor prohihition. The Acad. of Med., Edmonton, Alberta, has adopted the following temperance resol. : That the Acad. of Med., City of Edmonton, favors Prohibition in the Province of Alberta and endorses the pro- posed liquor act for the suppression of the liquor traffic in Alberta, Can. " Fine old whiskey" as danger ous as the cheap raw product. The Weekly Bulletin of the N. Y. State Health Dep't quotes from the summary of the Investigation concerning the physiological as- pects of the liquor problem, by Dr. J. S. Billings, which was pre- pared for the Commit. of Fifty, showing that the common idea, that a large degree of the injury to health from the use of alcoholic drinks is caused by injurious substances in the liquor, such as fusel oil and furfurol, which have not been properly removed, is errone- ous, as is also the notion that cheap liquors contain larger quantities of such ingredients than others. The injurious effects of the fusel oil are trifling in comparison with those of the ethyl alcohol. The general conclusion is that " fine old brandies and whiskies " are nearly as likely to produce ill effects as the cheaper varieties of the present time, if taken in the same quantities; and that the injurious effect is in proportion to the ethyl alcohol contained. Jour. Anier. Med. Assoc., 1915, Ixv, p. 885. 191 5] General 437 N. Y. Health Dep't temperance Crusade. The N. Y. City Dep't of Health recently pubHshed the results obtained from a study of the effects of alcohol on human Hfe. Forty-three leading Hfe insurance companies have furnished their records on about 2,000,000 lives, f or a period of 25 years. The report states that nothing has been more conclusively proved than that the steady free use of alcohohc bever- ages, or occasional excess, is detrimental to the individual. Among men who admitted that they had taken alcohol occasionally to excess in the past, but whose habits were considered satis facto ry when they were insured, the extra-mortality was equivalent to a reduction of more than 4 years in the average expectation of Hfe for these men. The report further states that available statistics justify the State- ment that total abstainers have a mortality during the working years of life of about one-half that for those who take two glasses of whiskey a day. ToxicoLOGiCAL NOTES, Wood alcohol in toilet preparations. Investigation by inspectors of the N. Y. City Health Dep't last year showed that more than one-third of the toilet preparations sold in the city contained wood alcohol, which is forbidden by the Sanitary Code. Of more than 300 preparations taken from barber shops, manicuring establishments and supply-houses, during the past few months, however, only a small proportion contained this deadly poison. Systematic inspections will be continued. Retail druggists endorse patent-medicine campaign. At a meet- ing of the pharmacists of N. Y. City, July 2, under the auspices of the Bronx Co. Pharmaceut. Assoc, resolutions were adopted endors- ing the Health Dep't's campaign against f raudulent patent medicines. This change in the attitude of the pharmacists of the city is very gratifying. It may now be expected that N. Y. City pharmacists will do all in their power to uphold the Dep't of Health in its endeavor to stop the local sale of the many f raudulent cure-alls now on the market. Drug victims fill city prisons. At a recent Conference on crime and environment, Dr. Katherine B. Davis, Commiss. of Corrections, N. Y. City, stated that on Mar. i, 1914, there were 4,647 persons in the correctional inst's of that city, but on Mar. i, 191 5, there were approx. 7,500, an increase of almost 50 percent. Dr. Davis attrib- 43 S Biochemical News, Notes and Comment [June-September Utes this increase to the drug Crusades of the past 15 months, which have increased the number of commitments of users of habit-form- ing drugs as well as of persons convicted of selling narcotics illegally. Fall in the production-cost of radium. Sec'y of the In- terior Lane authorizes the statement that the production of radium from Colorado carnotite-ores by the Bur. of Mines, in connection with the Nat'l Radium Inst., has passed the exper, stage in its new process and is now on a successful manufacturing basis. He says: The cost of I gram of radium bromid during Mar., Apr. and May of the present year was $36,050, This includes the cost of ore, In- surance, repairs, amortization, allowance for plant and equipment, cost of Bur.-of-Mines Cooperation, and all expenses incident to the production of high-grade radium bromid. When it is considered that radium (bromid) has been selling for $120,000 to $160,000 a gram, it will be seen just what the Bur. of Mines has accomplished along these lines. BoTANicAL NOTES. N. Y. Botüfi. Garden anniv. The 20th anniv. of the opening of the N. Y. Botan. Garden was celebrated by Amer. botanists at the Garden, Sept. 6-1 1. At the initial meeting, the delegates and visitors were welcomed, on behalf of the B'd of Managers, by Dr. W. Gilman Thompson, pres., and on behalf of the Scientific Directors, by Dr. H. H. Rusby, chairman. Relation of botany to medicine. " The most important develop- ment of modern biology came when the great principle of the exist- ence of cells was transferred from botany to zoology by Theodor Schwann, at the beginning of the iQth Century. When Virchow took the further step of applying the cell-doctrine to pathol., he made per- haps the greatest advance in modern medicine. He used to declare in later life that when these two far-reaching developments were made by himself and Schwann, the rising generation of scientific investigators in Germany were quite as much interested in botanic Problems as in microscopic anatomy. It was this breadth of interest, he declared, that gave them the larger outlook which enabled them to see beyond the bounds of what had been hitherto known, to newer phases of knowledge." Jour. Amer. Med. Assoc, 1915, Ixiv, p. 2142. I9I5] General 439 Obesity of THE HAND, Intevesting case of fat-metaholism. A defect in the back of the band of a girl of 12 was remedied with a Aap taken from her abdomen. It healed in place and answered its purpose perfectly until lata in life. After 30 she became obese. Then the patch on her hand increased in size proportional to the increasing thickness of the abdominal wall. Jour. Amer. Med. Assoc, 191 5, Ixiv, p. 2106. HoMEOPATHic PROGRESS. The Hahncmann Med. Coli., San Francisco, conveyed all its property to the Univ. of Cal, and has discontinued separate Instruction. Instead, two professors will be maintained in the Univ. of Cal. Med. Seh. in homeopathic mat. med. and in homeopathic therap. Instruction in these subjects will be offered as elective courses. Students wishing eventually to become homeopathic practitioners will be given the same instruction, in the Univ. of Cal. Med. Seh., as all the other students will receive. Anti-vivisection BILL UNCONSTITUTIONAL. Gov. Johnson has declined to approve the anfi-vivisection bill passed by the Cal. Legis- lature at its last Session. The commit. on med. instr. of the regents of the Univ. of Cal., the deans of the Cal. and Stanford Med. Sch's, the biolog. and agric. investigators, the med. practitioners, and many other Citizens, had protested against the measure as an unwarrant- able interference with science. In declining to approve the bill the Gov. announced that its provision, that any humane officer should be permitted to invade any scientific lab. without a searph Warrant, was an unconstitutional interference with personal liberty and the rights of privacy. Keen Fellowship. Prof. W. W. Keen has established the Corinna Borden Keen Research Fellowship in Jeff. Med. Coli., the income from which now amounts to $1,000. The gift provides that the recipient of the fellowship shall spend at least one year wherever he can obtain the best facilities for research in the line of work he shall select, after consultation with the faculty, and that he shall pub- lish at least one paper, embodying the results of his work, as the "Corinna Borden Keen Research Fellow of the Jefferson Medical College." Applications, stating the line of investigation which the candidate desires to follow, should be forwarded to Dr. R. V. Patter- son, Sub-Dean, Jeff. Med. Coli., Phila. 440 Biochemical News, Notes and Comment [June-September BlOCHEM. WORK IN THE BUREAU OF SciENCE, PhILIPPINE Islands. In response to our request for a statement of the nature of the biochem. work done, or in progress, under the auspices of the Philippine Bur. of Science, we have received the foUowing from Acting-Direc, Dr. J. A. Johnston, dated. Mar. 3, 1915 : The Bureau of Science has no regulär staff devoted to biochem. work and has carried on no work dealing strictly with the ehem. of life processes. A few practical problems have involved studies more or less border- ing on the biochemical. Of such might be mentioned the investiga- tion of the enzymes of the nipa palm, the constituents of certain foods, active principles of medicinal plants, the etiology of beriberi, the treatment of leprosy, and the relation of soil moisture and en- vironmental conditions to plant growth. All of these are of inci- dental interest to biochemistry. Many problems still to be attacked seem to offer attractive opportunities to physiol. and pathol. chemists. A PHARMACEUTic EXPER. STATION. " During reccut years the number of med. schools which have been provided with facilities for research, through state grants or by private endowments, has rapidly increased. While much is still to be hoped for, the outlook for med. advance is far brighter in this respect than it is in the related brauch, pharmacy. Pharm, schools, whether privately owned or controlled by the State, are almost without exception devoted to the routine Instruction of students and are, as a rule, doing nothing toward the advance of pharmacy as a science. A notable exception to this back- ward condition of pharmac. education was the establishment of a pharmac. exper. sta'n by the Wis. Legislature, in 19 13. The Statute creating the exper. sta'n provides that it further the cultivation and investigation of medic. plants. In the first annual report of the direc. of the sta'n,^ Edward Kremers, a well-known authority and author on plant chemistry, gives an indication of what may be expected from this pioneer work. The sta'n not only is engaged in the exper. cultivation of medic. plants, but also cooperates with the gov't in this field, and offers its help and advice to those who wish to engage in this relatively new Amer. industry. While drug-plant cultiv, is to be a prominent feature of the sta'n's scope, the work planned by the 3 Report of the Direc. of the Pharmac. Exper. Sta'n, Bull. 542, Univ. Wis., Dec, 1914. iQis] General 44 1 director has a wider sphere. The present report shows that plant analysis, a most neglected subject, as well as the so-called synthetics, is to receive attention." Jour. Amer. Med. Assoc, 191 5, Ixv, p. 259. A HALF CENTURY OF ANTisEPTic SURGERY. " In a review o£ the scientific features in the development of modern surgery, Lee* has written: *With the discovery of practicable anesthetics, the battle was only half won. The Operation itself had lost much of its horror, but the tragedy of the subsequent days was unchanged. There were the almost inevitable suppuration of the wound, the putrefaction and sloughing off of tissue, the sickening odor, the high fever, the danger of hemorrhage, the slow healing, the complications of blood poison- ing, erysipelas, gangrene and tetanus, the physical and mental an- guish, and the uncertainty of the final outcome. The mortality from major Operations was from 50 to 100 per cent.' " Today, on the contrary, the opening of the abdomen, the ehest or the skull no longer is equivalent to signing the death Warrant of the patient. Pasteur proved that fermentation and putrefaction were neither spontaneous, on the one hand, nor due to occult causes, on the other, but are in reality the result of the activity of minute living organisms. Among the fruits of Pasteur's labors was the work of Joseph Lister. " It is sometimes stated that antiseptic surgery had its birth in 1867, when Lister reported, in the Lancet, his eleven cases of Com- pound fracture, with a 'prelim. note' on the antiseptic method of opening abscesses. He had furnished the first Solution of the prob- lem of how to prevent putrefaction in open wounds. As Paget^ remarks, ' Pasteur could prevent putrefaction in broth, by his aseptic method: but patients cannot be boiled, nor kept in filtered air, in flasks.' To kill the germs in the wound, Lister at first chose phenol (carbolic acid). To prevent any more germs from getting in, he left untouched the scab or crust formed by the antiseptic and blood together on the wound. His first case, in Mar., 1865, failed ; his next case, in Aug., was successful. " Half a Century has elapsed since those memorable experiences. * Lee : Scientific features of modern medicine, Columbia Univ. Press, N. Y., 1911. 6 Paget : Pasteur and after Pasteur, 1914, p. z^- 442 Biochemical News, Notes and Comment [June-September The wonderful strides which surgery has made during these years are fresh in our minds. The work of the surgeon is not confined to the repair of wounds, the correction of deformities, or the removal of tissues. Deficiencies may be supplied by transplantation ; trans- f usion is readily carried out ; a new era in reconstructive surgery has been inaugurated. There is no reason to believe that the end of these progressive advances in surgery in recent years is in sight As a recent writer has remarked, surgery is no longer merely an art of skilful cutting and sewing; it has risen to the higher level of a science. "The incalculable benefit of Lister's studies, and of that which has grown out of them, can best be appreciated in the contempla- tion of the surgical infections which, in the memory of physicians now hving, were once the dread of all operating surgeons. Park^ has thus recorded his impressions of the earlier days : ' I deem myself fortunate in this — that I have been a living witness of the benefit of the change f rom the old to the new, since when I began my work, in 1876 (over twenty years ago), as a hosp. intern, in one of the largest hosp. in this country, it happened that during my first winter's expe- rience — with but one or two exceptions — every patient operated upon in that hosp., and that by men who were esteemed the peers of any one in their day, died of blood poisoning, while I myself nearly per- ished from the same disease. This was in an absolutely new build- ing, where expenditure had been lavish; one whose walls were not reeking with germs, as is the case yet in many of the old and well- established institutions. With the introduction of the antiseptic method, during the two years following, this frightful mortality was reduced to the average of the day, and in the same Institution today is done as good work as that seen anywhere. The same was true without exception in the great hospitals of the Old World; and in Paris, where, 30 years ago, famous surgeons would go from one end of the building to the other, handling one patient after another with- out ever washing their hands, and where erysipelas and contagion of various kinds were thoroughly distributed, as it were, impartially, now the successors of these very same men, employing modern methods, get results which challenge comparison.' 6 Park: An epitome of the history of medicine, 1898, p. 326. 1915] War Notes 443 "Those who fail to recall the historical development of the modern surgical technic are sometimes wont to assume that because certain current aseptic procedures have entirely superseded the anti- septic devices introduced by Lister, the advantages of his precautions are no longer essential. It must be remembered, however, that anti- septic and aseptic are nothing other than two ways of arriving at one result. In the words of Sir William Osler: *It is the difference be- tween tweedledum and tweedledee. They are both applications of the same principle.' We cannot summarize the Situation better than by again quoting Paget : ' It is true,' he writes, ' that Lister was more afraid of the powers of the air, half a Century ago, than sur- geons are now; it is true that his use of a phenol spray, to sterilize the air around the wound, has been given up; but the law of all Operations remains today that law which was revealed to Lister in 1865, in the hght of Pasteur's work on putrefaction. From the very first, antiseptic surgery had in itself the making of aseptic surgery.' "The principles defended and applied by Lister find applications in everyday lif e at the hands of the layman ; their value is f urther attested in the prominence which they play in the modern military regime, The soldier on the European battlefield has been taught to apply an antiseptic dressing promptly to the wound he receives in action, and the enlightened teachings of Lister follow him through all the subsequent stages of his treatment to recovery. After a lapse of 50 years we may well pause to recall that Lister was instrumental in saving more lives than the armies of the greatest general or poten- tate have destroyed. Let us mark the anniversary on our medical calendar and pay a tribute to the masterful genius of Lister, * a man serene through controversy, a spirit pf invincible patience and of radiant purity.'" Editorial: Jour. Amer. Med. Assoc, 1915, Ixv, p. 171. II. WAR NOTES Necrology. — Dr. Chaillou, head of the anti-rabies dep't of the Pasteur Inst. G. C. M. Mathison, known for his work on the physiol. of respiration. Max Rappart, assis, in ehem. to Fischer, Univ. of Berlin. E. Rhode, instr. in the physiol. and pharmacol. of the heart, 444 Biochemical News, Notes and Comment [June-September Univ. of Heidelberg; died of pneumonia while engaged in Red-Cross Service. Hugo Lüthje, chief of the Med. Clinic, Kiel Univ., author of exhaustive studies of nutritional diseases; died of typhus contracted during a visit of inspection to a camp of Russian prisoners. University items. Dr. V. E. Henderson, pro f. of pharm, and pharmacol., Univ. of Toronto, commands the detention camp at Kapuskasing, Ontario. Among the British scientific men in mihtary service are Dr. /. A. Gunn (reader in pharmacol., Oxford), Lieut. R.A.M.C, and Sir Wm. Osler (regius prof. of med. Oxford), Hon. Col. S. Midland Div. R.A.M.C. Dr. /. George Adami (prof. of pathol., McGill Univ.), is now serving as a member of the British War Office, having charge of the preparation of a medical history of the war. Booze bombs. Prohibition of alcoholic drinks in the French army. The example set months ago by Russia, on the " liquor question," has been f ollowed by other nations. The f ollow- ing note relates to conditions in France. " An order of Gen. Gallieni, mil. gov. of Paris, forbidding the sale of alcoholic drinks to soldiers garrisoning the defences of Paris, was published in July. Now, when the physical and moral energy of soldiers ought to be carried to their highest degree of intensity, the order explains, it is important that the campaign against alcohol, which destroys both, should be carried on without faltering. Hence it is forbidden to seil alcohol and alcoholic drinks (absinthe, vermuth, bitters, aperitifs, liqueurs, etc.) to soldiers of any grades. Liquor sellers will have their estab- lishments closed temporarily for the first offense and permanently for the second." Paris letter: Joiir. Amer. Med. Assoc, 1915, Ixv, p. 636. On beer and brandy in the German army. " In the Journal {Amer. Med. Assoc), Mdij 15, 191 5, p. 1663, appears editorial com- ment entitled ' Alcohol in the European Armies,' which is based on figures obtained from the British Med. Jour. discussing the daily consumption of alcohol by the armies in the field. So far as these figures are intended to apply to the Gennan troops, they are without basis and fact. It is said, in the edit. comment, that the German 1915] War Notes 445 soldier is allowed 1,793 S^- of beer and 20 gm. of brandy daily, amounting to a total of 70.7 gm. of alcohol a day. I do not know how you or the British Med. Jour. happened to take the figure 1,793 gm. of beer, but that this could not possibly be true should have occurred to you. Let us assume, for the sake of argument, that there are 3,000,000 soldiers at the front, although there are probably more than that. They would receive 5,379,000 kg. of beer daily. A liter of beer, including packing, weighs, according to the estimation of the breweries, 1.65 kg., so that the daily allowance for the army would total 8,075,350 kg. The ordinary freight car carrying beer will hold 10,000 kg. Therefore, 887 such cars filled with beer would have to be shipped to the front daily and as many returned. Such freight traffic, under present conditions, is an impossibility because so many other things must be transported to the front at this time. In reality there is no regulär alcohol allowance or consumption. The soldiers are given coffee and tea as stimulants, and not alcohol; and if, by any chance, a keg of beer should happen to fall into the hands of the soldiers, it would be cause for celebration. You must be pre- pared to find unsolved in the final analysis the problem as to how much military efficiency among the German troops may be increased by means of so-called ' Dutch courage.' " Very respectfully yours, Walther Straub, M.D., Professor of pharmacology, University of Freiburg in Breisgau, July 10, 191 5 {Jour. Amer. Med. Assoc, 1915, Ixv, p. 732). Honors. The Paris Acad. of Sciences, in secret Session, passed a resolution removing from its membership four German scientific men, including Emil Fischer. Prof. Roentgen was 70 years old, Mar. 27. In honor of the occasion the Kaiser presented him with an Iron Gross. In the ac- companying message of congratulation, the Kaiser said : " The Ger- man nation cannot be grateful enough to the discoverer of the rays for whom they are named. The many advantages from the use of the rays are being rendered apparent by the war more than ever before." 44^ Biochemical News, Notes and Comment [June-September Sir Wm. Ramsay, recently writing on German " Kultur," shows that o£ the 58 awards made during the past 12 years by the Swedish Nobel Commit., only 17, or merely 30 percent, were received by Germans and Austrians. The ratio of German and Austrian foreign members and associates of the principal academies of the world is only 28 percent. " Our foreign exchanges now contain an occasional article with the subheading, ' Dedicated to Elias Metchnikoff on the occasion of his 70th birthday, May 16, 191 5.' These articles had been prepared for an international Festschrift, which was to have been presented to him on that date, but the war broke up the plans for the volume, and the contributions are being published separately in various neutral Journals. He is a native of southern Russia but has had a lab. at the Paris Pasteur Inst, since its erection, over 27 years ago. His discoveries in phagocytosis were made previously in Italy. Although he studied under German physicians, he never took a degree in medicine." Jour. Amer. Med. Assoc, 191 5, Ixiv, p. 2080. (See page 246.) Notes on the sanitation of military quarters and battlefields. Dr. F. Bordas, Substitute prof., Coli, de France, has suggested to the Acad. des sciences that the disinfection of cantonments, trenches occupied by troops, and battlefields, be effected by copious spraying with petroleum emulsified in water by means of a suitable amount of rosin soap. " In the present war, in which the battle f ronts extend in France and Belgium alone over more than 375 miles, the question of the sanitation of battlefields has a special importance. ... In general, the use of chemical agents and spray applications (coal tar, phenol, ferrous sulfate, zinc sulfate, chlorinated lime, etc.), is recommended. These chemical agents, however, are rather expensive and it is not always possible to obtain them. It is therefore of interest to recall that, in 1871, the Conseil d'hygiene publique recommended, and ap- plied with success, an altogether different method, based on the power possessed by vegetable growths of absorbing and transforming decaying animal substances. By this method bodies are left where they fall, but covered with a layer of earth no thicker than about 15 to 20 inches. It is necessary, however, to take care to bring the I9I5] War Notes 447 earth for the mound from a distance of about i to 2 yards, In order not to disturb the soil impregnated with liquids of decomposition and to avoid forming trenches at the side which would dry the mound too rapidly. The mound is then planted with grains and plants that grow rapidly and which are especially avid o£ nitrogen or the products of decomposition. Among the best for this use are the Helianthus annuus or common sunflower and the Balsamita suaveolens, otherwise known as the Chrysanthemum balsamita or baisam herb, whose assimilative powers are remarkable. Other plants that are very greedy for nitrogen may be employed, such as the Galega officinalis or goat's rue, the Helianthus tuberosus or Jerusalem artichoke, the Sinapis arvensis or wild mustard, and some forage plants." Paris letter: Jour. Amer. Med. Assoc, 1915, Ixv, p. 813. Food notes. Conditions in Mexico. Distressing economic conditions prevail in Mexico. Mr. C. J. O'Connor, the repr. of the Amer. Red Gross in Mexico City, reports that the amount of food necessary for an individual per day costs at present nearly $10 Mexican, while the laborer's wage is $0.75 Mexican. Civilians in Saltillo are reported to be eating donkey flesh and cactus. No corn or beans can be purchased at any price. The food supply in Germany. "The restrictions that were imposed on the consumption of bread are f ortunately being reduced more and more. When it was learned officially that there is a large reserve of grains to carry us to the harvest (7 millionen Doppel- zentnern Getreide) , the bread ration per capita was increased. The authorities regarded it as especially important that the bread supply for working people should be made more ample. However, every one soon became accustomed to the restrictions on bread, and no one complains any more. This is the more readily understood as the consumption of meat and of bread used to be above what was actually required — a Luxuskonsumtion. With regard to other foods, conditions have righted themselves so that the menus show scarcely any difTerence from those of peace times. The only thing in this line that reminds us of the war is the increased cost of meat and other articles of food except certain vegetables. It is remark- able, moreover, that other countries, even the neutral ones, are hav- 44^ Biochemical News, Notes and Comment [June-September ing a similar experience, suffering from higher prices to almost the same degree as we are. "Some official figures have been pubhshed recently comparing the prices now and a year ago : I kg. wheat flour, May, 19 14, 0.37 mark; May, 1915,0.55 mark ikg.ryeflour, " 0.29 " " 0.55 " . I kg. potatoes, " 0.07 " " 0.15 " ikg.butter, " 2.61 " " 3.54 " I kg. milk, " 0.20 " " 0.24 " i-egg, " 0.07 " " o.ii " ikg.sugar, " 0.50 " " 0.58 " ikg.coffee, " 3-o8 " " 3-35 " ikg.rice, " 0.48 " " 1.22 " " It should be added that the prices in May were in part lower than in the preceding months. In London, according to the London corresp. of the Swiss Zürich Post, flour is 71 percent higher than last year; butter, 24 percent; meat, 30 percent; tea, 25 percent; sugar, 88 percent; fish, 50 or 75 percent; refrigerated meat, 100 percent. " Naturally, the increased cost of living would bear particularly hard on the poorer classes, especially those families whose bread- winner is serving in the army, if it were not for the assistance ren- dered by the State, local organizations and private philanthropy. Without exaggeration I can affirm that of my own knowledge I am not aware of any distress from the high cost of living at present more than in times of peace, and the lay press has not published any- thing suggesting this in any part of the empire. My assertion will receive more credence when the amount of the sums that have been contributed for assistance is realized. . . . The social insurance companies had spent for relief purposes, in connection with the war, up to June I, $3,250,000 of their reserve funds. Besides this, $14,000,000 have been loaned to communities for relief purposes, and they took up $72,500,000 of the war loans." Berlin letter: Jour. Amer. Med. Assoc, 19 15, Ixv, p. 441. The feeding of prisoners of war in Germany. " The War Dep't recently organized a course of Instruction for the ofiicials in iQisl War Notes 449 Charge of the concentr. camps of war prisoners. The chief aim of the course was to instruct the officials in the essential principles of the physiol. of nutrition and the art of cooking. The officials came from 129 different concentr. camps, from all over Germany, and leading specialists delivered lectures on the scientific bases for, and the practical features of, the feeding of the prisoners, Dr. Neu- mann, direc. of the inst., at Berlin, for research on the utilization of grains, spoke on the essentials of bread as food. This is a particu- larly difficult subject, as the bread-ration has had to be reduced for the prisoners as well as for the civilian populace. As the French and Russians are accustomed to make much u.se of bread, the Minister of War had large amounts of second-grade rye and wheat, which were not suitable alone for making bread, mixed with other kinds of flour. This mixed flour was used to bake a supplementary bread-supply for the concentr. camps, where it was sold to the prisoners through the canteens for 50 pfennigs per kg. or, as rolls or cakes, by weight, 75 gm. for 5 pfennigs. It can also be supplied gratuitously, on a physician's order, to certain undernourished per- sons and certain others engaged in manual labor. The Standard dietary suggested by the "Minister of War is, for every prisoner of war, military or civilian, 85 gm. protein, 40 gm. fat and 475 gm. carbohydrate, a total of 2,700 calories. Those who have to do manual labor, particularly the inmates of the work-camps, are to receive 10 percent more. This ration is to be given in three nourish- ing and palatable meals: in the moming, coffee with 30 gm. of sugar, or a soup containing 100 gm. of solids. A soup has proved particularly satisfactory that is made of 30 gm. soy beans, 60 gm. flour (Stärkemehl) and 10 gm. fat. The dinner is to consist of 750 gm. potatoes, and 300 gm. fresh or canned vegetables or 40 gm. dried vegetables. Every third day meat is to be given with the above. It was recommended to give fresh meat twice to corned meat once. The amount is prescribed as 120 gm. meat with bones or 100 gm. without bones. On the two intervening days, 200 gm. fish is recommended or 150 gm. pod vegetables, with the addition of bacon, fat or pickled meat. Soy beans in the form of flour were particularly recommended. Salt, spices and fat are not to be spared. For supper, baked potatoes with herring or sausage or cheese have 450 Biochemical News, Notes and Comment [June-September proved satisfactory; the potatoes can be glven in the form of a salad. Thick soups made f rom beans or flour have also proved use- ful for supper. Rice with baked or stewed fruit may also be used. Opportunity for tea drinking is given the Russians. Where skim- milk is obtainable, it shall be supplied abundantly to the prisoners. It was emphasized that the tastes of the prisoners should be con- sidered as much as possible in the selection and preparation of the dishes." Berlin letter: Jour. Amer. Med. Assoc, 1915, Ixv, p. 544- Medical notes. "Medical-supply Exports doubled. The U. S. Bur. of Foreign and Domestic Commerce has estimated that during the year ended June 30, 191 5, the exports of medicines and surgical Instruments amounted to $35,744,000 as compared with $19,916,000 for the preceding year. War helps medical women in England. A statement signed by Premier Asquith, Lord Curzon, and Hon. A. J. Balfour calls the attention of the British public to the work in London of the School of Med. for Women, which has now doubled its plant in an endeavor to cope with the war-time increase of opportunities for women physicians. The statement begins as follows: "The war constitutes the turning point in the Position of medical women, for whom there are new openings and new opportunities in many directions." Medicinal plants in Germany. The Münch. med. Woch. States that the German Minister of the Interior has appealed to the apothecaries to stimulate the collection and drying of medicinal plants and parts of plants in their districts. They can then prepare them for med. use, each in his own laboratory or by exchanging them with others. He explains that large amounts of the plant- drugs used in making medicines have always hitherto been imported from other countries for this purpose. The war has rendered it very difficult to Import them now or has shut off the supply com- pletely. A list of plants, useful for the purpose, is given, including the flowers of arnica, chamomile, linden, eider and mallow, and the leaves of digitalis, walnut, belladonna, colt's-foot, henbane, stramony, buck-bean, and various herbs and berries. Children, it is urged, can be taught to collect them, and also the elderly and the otherwise iQis] War Notes 45 1 incapacitated, so that there need be no difficulty in collecting an adequate supply. Rapid whitening of the hair after explosion of a mine. "At one of the recent sessions of the Societe med. des höpitaux, Paris, Dr. Lebar reported the case of a soldier, aged 33, who, hav- ing been blown into the air by the explosion of a mine, next day had locks of white hair on the left side of his head. The decoloration of the hairs was complete from base to extremity. The longest as well as the shortest were white and there was not a brown one among them. All the hairs that became suddenly white are still firmly implanted. It was suggested that the general nervous shock caused by the explosion of the mine set in motion the medullary cells of the hair, the pigmentophagic röle of which has been shown by Metchnikoff." Paris letter: Jour. Amer. Med. Assoc, 1915, Ixv, p. 183. The so-called new antiseptic. "Recently the newspapers have contained announcements of a new antiseptic or germicide that has proved, or is to prove, of great value in the treatment of the wounded in the present war. Credit for its discovery is given to Drs. Carrel and Dakin. " The antiseptic referred to is that which Dr. Dakin,''' of the Herter Laboratory, N. Y. City — now serving as bacteriologist in a war hosp. at Compiegne, France — announced in a paper read before the Acad. des sciences, Paris. It is made by the well-known process O'f adding sodium carbonate to a sol. of chlorinated lime. The mixture is thoroughly shaken, and after half an hour the liquid is siphoned off from the precipitate of calcium carbonate and filtered through cotton. To this clear liquid,- sufficient boric acid is added to make the preparation neutral or acid, the amount required being determined by titration with phenolthalein. Such a sol. was found to kill staphylococci in two hours. " According to the British Med. Jour.^ about a year ago Prof. Cohen, of the Univ. of Leeds, entered into communication with Dr. Dakin, a former Student, regarding research on antiseptics for surgical use. The arrangement was that the substances elaborated ''Dakin: Presse med. (society proceedings), Aug. 5, 1915. 8 Research in Antiseptics, Brit. Med. Jour., Aug. 14, 1915, p. 261. 452 Biochemical News, Notes and Comment [June-September by Prof. Cohen should be tested bacteriologically by Dr. Dakin, and that the most promising should be tried chnically by Dr. Carrel. " At about the same time, undertheauspicesof the EngUsh med. research commit., a similar research by Prof. Lorrain Smith, with the assistance of Prof. Drennan of the Univ. of Otago, N. Z., Dr. Rettie, a chemical expert, and Lieut. W. Campbell of the British army med. corps, was undertaken in the Univ. of Edinburgh. Their results were reported in the British Med. Jour.^ The sub- stance which they prepared was made by rubbing chlorinated lime to a fine powder and mixing it with an equal weight of powdered boric acid. The ideal antiseptic for the field, they concluded, was a dry powder to be applied direct, which, it was believed, has advan- tage over a sol. because it is more portable, and water is often not procurable. " Chlorinated lime, the basis of the so-called new antiseptic prepa- ration, is well known as a powerful disinfectant. Its alkalinity, however, makes it destructive to living tissues except in dilute sol. The same may be said of sol. of chlorinated potash (Javelle water), which has been largely used by French surgeons in the present war, and of sol. of chlorinated soda (Labarraque's sol.). The advan- tage claimed for the new mixture is that the preparation, being practically neutral and unirritating to the tissues, may be applied in greater strength than that in which it is possible to use chlorinated lime, Javelle water or Labarraque's sol. Experiments indicate also that the germicidal activity of chlorinated lime is increased by such treatment of the calcium hypochlorite as has been described. Such increase in germicidal activity is generally attributed to the libera- tion of hypochlorous acid. It has been found that the activity of ordinary bleaching powder is greatly increased by passing through it carbonic acid gas. Any other acid, as boric acid, will do as well. " From the ehem. point of view, therefore, there is nothing new in this method. That the practical application of such a mixture is not wholly new is proved by an earlier article published by Vincent. ^° He suggested the application to ulcerating and gangrenous wounds ^ Brit. Med. Jour., July 24, 1915, p. 129; abstr., Jour, Amer. Med. Assoc, Aug. 21, 191S, p. 744- 10 Vincent: Presse med., 1914, xxii, No. 70; abstr., Jour. Amer. Med. Assoc, Nov. 28, 1914, p. 1986. 1915] Columbia University Biochemical Association 453 o£ a mixture of chlorinated lime and boric acld. Editorial: Jour. Amer. Med. Assoc, 191 5, Ixv, p. 880. Miscellaneous items. "Chemical Abstracts will be the only complete record of chemical research reported during the war period." E. J. Crane: /. Ind. and Eng. CJiem., 1915, vii, p. 465. A deputation from the Royal Soc. and the Chem. Soc. was received by the pres't of the Boards of Trade and Educ, in London, May 6. The dep. was introduced by Sir Wm. Crookes, pres't of the Royal Soc. Prof. W. H. Perkins, Sir Wm. Tilden, Prof. P. Frankland, Prof. W. J. Pope and Dr. M. O. Forster spoke in Support of memorials from the two societies, indicating the steps which might be taken immediately to improve the Status and effici- ency of the chemical Industries and those engaged in them in the United Kingdom. III. COLUMBIA UNIVERSITY BIOCHEMICAL ASSOCIATION I. General notes Honors. Hon. degrees. Drs. R. H. Chittenden and S. J. Meltzer were among the distinguished biologists who received the hon. degree of LL.D. on the occasion (Apr. 30) of the dedication of the new buildings of Washington Univ. Med. Seh., St. Louis. Medals. At the last annual commencement of the N. Y. Coli, of Dental and Oral Surgery (June 8), Dr. Louise C. Ball was the recipient of three medals and one "honorable mention," as foUows: Clarkson Cowl Gold Medal (highest award), for the best average stand in the final exam's in the füll course for three successive years in the Coli. ; Operative Dentistry Medal, for the highest mark in the final exam. in operative dentistry; Chemistry Medal, for the best thesis in dental chemistry; Sanger Medal, first hon. mention (second in rank), for the best work in theoretical and practical prosthetic dentistry, during three successive years in the Coli. At the com- mencement in 1914, Dr. Ball received the only medals offered to Juniors : Faculty Medal, for the highest marks in the final exam's of the jr. year; Oral Surgery Medal, for the best thesis on suppura- tion. Dr. Ball is vice-pres. of the class of 191 5, which has 77 members. SiLLMAN Biochem. Soc. Mcmbcrs of the soph. class. Coli, of 454 Biochemical News, Notes and Commenf [June-September Med., Baylor Univ., have organized the Sillman Biochemical So- ciety, which is named in honor of Prof. Maxwell Sillman. The society's object is the study of the relationships between the med. Sciences and chemistry, and to stimulate biochem. investigation in general. The officers for the next academic year are : Pres., W. W. Looney; vice-p., H. L. Farmer; sec-treas., W. N. Bunkley. Appointments.^^ Albany Med. Coli. : Arthur Knudson, assis. prof., biol. ehem. (prom.). Coli. City New York : L. J. Curtman, assis. prof., ehem. (prom.). Fordham Univ., Med. Seh. : D. R. Lucas, lab. assis., physiol. Iowa State Coli. (Ames) : Helen Monsch, head, Dep't of Foods. N. Y. State Dep't of Health (Albany), Div. of Lab's and Research: Tula L. Harkey (Nat'l Path. Lab., N. Y. City), lab. assis., bacteriol. Rockefeller Inst. Med. Research: M. Heidelberger, assoc, ehem. (prom.). Univ., Cal. : H. A. Mattill (Univ., Utah), assis. prof., nutrition. Univ., Neb., Coli, of Med. (Omaha) : Max Morse (Univ., Wis.), assis. prof., biochem. Vassar Coli.: Cora J. Beckwith, assoc. prof., zoology (prom.). Va. Agric. Exp. Sta'n (Blacksburg) : F. D. Fromme (Purdue Univ., Agric. Exp. Sta'n), plant pathologist and bacteriologist. West. Maryland Coli. (Westminster) : P. W. Punnett, prof., ehem. and biol. Dental graduates. At the recent commencement of the N. Y. Coli, of Dental and Oral Surgery, the degree of D.D.S. was con- ferred on Louise C. Ball and Siegfried J. Nilson. Associations and societies.^^ Officers-elect. Medical Brotherhood: Pres., vS. /. Meltser; councilor, N. B. Foster; hon. pres., R. H. Chittenden; members of advis. commit., C. L. Aisberg, John Howland, L. B. Mendel. Sigma Xi (Yale Chapt.) : L. L. Woodruff, pres. Members-elect. Amer. Philosoph. Soc. : P. B. Hawk. Nat'l Acad. Sciences: Alexander Smith. Sigma Xi (Columbia Chapt.) : Louise H. Gregory, E. G. Griffin, 11 See also page 456. 12 See also page 457. iQisl Columbia University Biochemical Association 455 /. H. Northrup, H. H. Plough, George Scatchard, A. P. Tanberg, J. R. Tuttle. Soc. Exp. Biol. and Med. : George M. Baehr, D. J. Edwards. Miscellaneous items. In his capacity as sec'y of the Assoc. of Off. Agric. ehem., Dr. C. L. Aisberg is a member of the ed. commit. of the newly founded Journal of that Assoc. Dr. Geo. D. Beal is one of the members of the ed. board of The Register of Phi Lambda Upsilon; also of The Illinois Chemist. Dr. G. Delgado Palacios, prof. of pathol. ehem., Univ. of Caracas and member of the Venezuelan Acad. of Med., is a visitor in this country and temporarily engaged here in chemical work. Address : ii8 East ii6th St., N. Y. City. See page 278. The Texas Public Health Assoc. has appointed Dr. L. B. Bibb, of Austin, to secure the Cooperation of other agencies in the State to undertake a thorough experimental investigation of the value of cottonseed meal as human food. Miss Anna E. Richardson, of the Domestic Economy Dep't, Univ. of Texas, has been appointed a member of this Commit., and, with the Cooperation of Miss Helen S. Green, has undertaken to investigate the nutritive value of cot- tonseed meal. The work is being carried on by metabolism experi- ments and extensive feeding of small animals. Dr. F. C. Phillips, emer. prof. of ehem., Univ. of Pittsburgh, was recently the guest of his pupils at a testimonial dinner, in Pittsburgh, when he was presented by them with a gift of $1,000. The move- ment that culminated in this cordial expression of affection for Prof. Phillips was inaugurated by Dr. Jacob Rosenbloom during his term of ofifice in the Columbia Biochem. Dep't. The directors of the Retail Drygoöds Assoc. of N. Y. City have voted to establish a Commit. on Health and Sanitation, to coöperate with the N. Y. Dep't of Health in protecting the health of the 50,000 employees of the retail drygoöds Stores in N. Y. City. The owners of large department Stores are planning to secure and main- tain the best working conditions for those in their employ. The Bur. of Public-Health Educ, under the direction of Dr. C. F. Bolduan, will establish a course of lectures and a publicity campaign for the purpose of teaching employees how to be sanitary and healthy. 45^ Biochemical News, Notes and Comment [June-September 2. Proceedings of the Association Edgar G. Miller, Jr., Secretary Twenty-third (sixth annual) meeting. The concluding quar- terly meeting of the Assoc, for 1914-15, was held in the Library of the Columbia Med. Seh., May 28, at 8 p. m. This meeting fol- lowed an informal dinner at 6.30 in the same room, which was greatly enjoyed by all in attendance. The feature of the program was Dr. C. F. Bolduan's interesting discussion of the " Educational lunch room," conducted by the Bur. of Public-Health Educ. of the Dep't of Health, N. Y. City, of which Dr. Bolduan is director. Abstracts of the papers comprising the biochem. proc. will be published in the next number of the Biochem. Bull. The matters of public interest in the exec. proc. are noted below. The commit. " to endeavor to quicken the interest of Amer. med. men" in the proposed "Medical Brotherhood," Organization of which was suggested by Dr. Meltzer at the fourth annual dinner (pages 263, 267 and 292), was appointed as follows: S. J. Meltzer, chairman; Carl L. Aisberg, Nellis B. Foster, Wm. J. Gies, A. J. Goldfarb, Alfred F. Hess, Lafayette B. Mendel. The time for the recurrent annual meeting was changed from that of the last regulär meeting of the academic year to that of the last regulär meeting of the calendar year. It was voted to reelect the present officers, to serve until the elec- tion of their successors at the regulär Dec. meeting. (The list of officers was printed on page 228.) The register of members of the Assoc, as published by the sec'y in No. 13 of the Biochem. Bull., was made the official role of membership to date. Miss Hattie L. Heft was unanimously elected assis. sec'y for the term ending Dec, 1915- The next regulär meeting of the Assoc will be held on Dec 3. 3. Columbia Biochemical Department Appointments. From the staEf. Dr. Arthur D. Emmett, instr. in this dep't during the past year, has been reappointed assis. Chief, animal nutr., Agric Exp. Station, Univ. of III, Urbana. 1915] Columbia University Biochemical Association AS7 FrOM THE BODY OF ADVANCED STUDENTS. Amer. MuS. Nat. Hist., Dep't Public Health (N. Y. City) : /. /. Kligler, scientific assis., in charge of the lab. of bacteriol. (part time). Columbia Univ., Seh. of Med. : /. Howard Mueller, Alonzo Clark Scholar. Cornell Univ. (Ithaca, N, Y.) : Mary F. Henry, instr., home economics. Lederle Lab. (N. Y. City) : /. /. Kligler, assis., bacteriol. and biochem. (part time). Montana Agric. Coli: Lilla A. Harkins, prof., domes. science; head of the dep't. Mt. Holyoke Coli. (So. Hadley, Mass.) : Anna B. Yates, instr., physiol. Ottawa High Seh. (111.) : Isabel Clegg, head, dep't of househ. arts. Penn. Hosp. (Phila.) : Mrs. Jennie D. Wood, head dietician. Rockefeiler Inst. Med. Research: G. E. Cullen, assis., ehem. (prom.). Trenton Public Seh. (N, J.) : Jennie P. Gase, Supervisor, domes. art and science. Univ., Minn. : Lucile Wheeler, assis. prof., foods and cookery. Appointments to the staff. L. H. Almy (U. S. Food-Re- search Lab., Phila.), univ. scholar, biol. ehem. Arnold K. Balls (U. S. Bur. of Chem.), assis., biochem. Adolph Bernard (N. Y, Post Grad. Med. Seh.), univ. scholar, biol. chem. Frederick G. Goodridge, assoc, biochem. (prom.). Sergius Morgulis (U. S. Bur. ofFisheries; resident in this lab., I9i4-'i5), reappointed to the instructorship held 1913-14. Associations and societies. Amer. Pharmaceut. Assoc. : V. E. Levine, member. Amer. Philosoph. Soc. : Wni. J. Gies, member. Medieal Brotherhood: Wm. J. Gies, first sec'y and member of the Exec. Com. Soc. Exp. Biol. and Med. : Wm. J. Gies, member of the Council. Addresses, lecture and reports. Prof. Gies was one of the Speakers at the i6th ann. dinner of the Alumni Assoc. of the Coli. 45 S Biochemical News, Notes and Comment [June-September of Dental and Oral Surgery of N. Y., at the Hotel Manhattan, April 17. He delivered the lecture at the annual Joint session of Sigma Xi and Phi Beta Kappa, at Columbia Univ., May 19, on Diseases of the teeth and bones, their causes and prevention, with some demonstrations. Prof. Gies attended the 46th annual meeting of the Dental So- ciety of the State of N. Y., in Albany, May 14, and there presented a report, in collaboration with E. G. Miller and W. A. Perlzweig, on the results of research on the relation of internal secretions and diet to dentition. He presented to the I4th Internat. Lord's Day Congr., Oakland, Cal., July 28, a report, in collaboration with A. D. Emmett and Katherine R. Coleman, on the results of research on the physio- logical influence of a recurrent weekly day of rest, as measured in terms of effects on general nutrition. Dr. Gies's duties during the summer session made it impossible for him to present the report in person. Awards of higher degrees at Columbia to students of biolog. ehem. DocTORS of Philosophy. Of the 28 recipients of the degree of Ph.D. under the Fac. of Pure Science, at Columbia's last commencement, 10 had taken "majors" or "minors," or both (or "extra" advanced courses) in the Biochem. Dep't. The names of the candidates, and the subjects of their major and minor courses, are given below. Name of candidate Arthur D. Emmett Frederiek G. Goodridge Edward G. Griffin Mildred A. Hoge Major biol. ehem. biol. ehem. ehem., org. zoology M inor ehem., food biol. ehem. ehem., phys. zoology Minor bacteriology pharmacology biol. ehem. r physiology 1 biol. ehem. Roscoe R. Hyde Israel J. Kligler zoology biol. ehem. bacteriology bacteriology physiology f bacteriology 1 pathology Alexander Lowy ehem., elec. ehem., org. f biol. ehem. \ edueation Percy W. Punnett Arthur P. Tanberg Arthur W. S. Thomas chemistry ehemistry chemistry chemistry chemistry ehem., inorg. biol. ehem. biol. ehem. biol. ehem. Masters of Arts. The A.M. degree was recently conferred upon the f ollowing advanced students in the Biochem. Dep't : A. K. Apisdorf, J. C. Baker, O. C. Bowes, H. B. Clough, Helen C. Coombs, Hasel Dotiham, W. J. Donvan, Jessie V. Farr, Helen G. Gates, Lucy 1915] Columbia University Biochemical Association 459 H. Gillett, Helen S. Green, C. P. Harris, F. W. Hartwell, Hattie L. Heft, Jacob Hoff mann, Lottie M. Hüll, Mabel M. Lutes, Marguerite L. McLean, Jeannette C. Mullikin, Alma M. Oswald, Almeda Perry, H. H. Plough, Helene M. Pope, W. H. Schliffer, Jr., J. J. Tanzola, M. K. Thornton, Jr., Lucile Wheeler, Anna B. Yates. DocTORS OF Pharmacy. The following students of biol. ehem. at the N. Y. Coli, of Pharm, received the degree of Phar.D. : Jose E. Argüello, S. E. Posin. Summer Session. Courses. The Dep't conducted five courses in nutrition, biochem. methods, and research, during the recent sum- mer session (July 6-Aug. 14). Two of these courses were given at Teachers Coli, by Prof. Gies, Dr. Emily C. Seaman and Miss Helen C. Coombs; three were given at the Coli, of Phys. and Surg. by Prof. Gies and Mr. W. A. Perkweig. The biochem. lab. at the Med. Seh. was open daily for research throughout the summer. Investigators. The workers named below were engaged in research, in the biochem. lab. at the Med. Seh., at various times dur- ing the summer vacation : B. Aronowitch, Robert Bersohn, O. C. Bowes, Katherine R. Coleman, A. D. Emmett, Wm. J. Gies, B. Horowitz, C. H. Jordan, I. J. Kligler, Arthur Knudson, S. Kubushiro, V. E. Levine, F. Low- enfels, E. G. Miller, Jr., Sergius Morgulis, A. Mutscheller, Wm. A. Perlzweig, Louis Pine, G. J. Rosenthal, Maxwell Sillman, J. R. Tuttle, Wm. Weinberger. Miscellaneous items. Dr. A. D. Emmett is one of the three members of the ed. board of The Register of Phi Lambda Upsilon. Dr. Benjamin Horowitz was one of the delegates from the Col- legiate Zionist League to the Zionisf Convention in Boston, June 27-July I. Dr. Gies recently served as chairman of a sub-committee of the Commit. on Food Inspection of the Advisory Council of the N. Y. City Dep't of Health, to " deal with the problem of the use of copper tanks by candy manufacturers." EDITORIAL WILLIAM J. GIES The opening pages of this issue of the Biochemical Bulletin. publish the notable address by Dr. Aleltzer, in which he proposed the Organization of a Medical Brotherhood for the Furtherance of International Morality. At page 292 of this issue B th h d ^^ present, also, a general Statement regarding the " origin, Organization and proceedings " of the Medical Brotherhood (prior to Oct. i). Although the Medical Brotherhood is not a biochemical Organi- zation, we give its afTairs a prominent place in this issue because the Organization of the Medical Brotherhood was proposed by a bio- chemist at a biochemical dinner, was endorsed at a subsequent bio- chemical meeting, includes in the membership of its Executive and Advisory Committees four past presidents and two past secretaries of the American Society of Biological Chemists, numbers among its members many biological chemists, invites all biochemists to rally to its Standard, and deserves universal support and encouragement. The official invitations to membership require (as the only con- dition of membership for those who are eligible to election) endorse- ment, by signature, of the following avowal: " I am in füll sympathy with the sentiments expressed in the Appeal (p. 300), and desire to be enrolled as a member of the Medi- cal Brotherhood (Fraternitas medicorum=^'F M..) ." The official announcements pertaining to membership also in- clude the following statement : " There is no membership fee. It is expected that the necessary expenses of Organization, distribution of literature, etc., will be paid from voluntary contributions." Dr. Meltzer's noble address (p. 279) and his stirring appeal (p. 300), leave nothing to be said that would add materially to the reasons why all who may be engaged in the practise of medicine anü in the advancement of medical sciences — biochemistry among them — should endorse the Brotherhood movement and enroll as members. 460 1915] William J. des 461 Because the significance of the f ollowing statements in the official " Appeal," in behalf of the Brotherhood, may not be fully noted by the casual reader, we single them out f or special attention here : "It is obvious that such a Brotherhood could not exercise an important infiuence at once. But our modest expectation f or prompt results should not prevent us from attempting now to take the first Step in the right direction. Many important results have often had small beginnings." " It should be expressly understood that it is not the object of the proposed Brotherhood to infiuence the feelings and views of any- one regarding the prohlems involved in the present war." Perhaps the most effective comment we can add to the foregoing is the f ollowing, from Lancet, on the " link of medicine " : We announce in another column the arrival in England from the United States of a complete medico-military unit, known as the " Chicago Unit," comprising the füll medical and nursing Organiza- tion for a general hospital of 1,040 beds. The establishment con- sists of 32 medical men (physicians, surgeons, specialists, a radiog- rapher and a pathologist) and a nursing staff of 75 women, includ- ing the matron, The unit has been recognized by the War Office. Every one of our readers will admit with gratitude the practical sympathy of a splendid sort which is thus displayed by the United States, while we understand that similar units may be expected to arrive from America, taking their departure from other great cities, and animated with the same quick and deep desire to minimize as far as possible the horrors of war. From a private communication which we have received from one of the staff of the Chicago Unit, it is easy to guess that there will be no dearth of applicants in other American centers for what will necessarily be very hard and per- haps dangerous work. The medical men and nurses of the Chicago Unit were selected from several hundreds of applicants, every man's post could have been filled at least six or seven times, and a brisk competition prevailed among candidates for the nursing staff. The same rivalry will hold good elsewhere. And here we may add that a generous Citizen of Chicago has personally offered to meet the large difference in pay between the salaries of military nurses in the British army and the salaries of nurses in the United States, where. 462 Editoriais [June-September as is well known, skilled assistance of every kind is rated at a higher figure than prevails with us in this country. We are certain that the American nurses did not need money as an incentive, but it is equally certain that some of the most experienced of them could not have undertaken to volunteer unless the pecuniary footing of their em- ployment had been made sure. Mediane is here proving itself a real link between nations. When we get down to the simple fact of a man in pain, in sickness, and perchance in peril of his life, the differences that exist between one country or State and another, arising out of political Conventions, trading regulations, unhealed quarreis or injudicious speeches, may, we see, disappear altogether, and the action of the United States displays this medical spirit splendidly. We must not forget that the war is practically a world war, and that its results now give rise to very serious mental and material trouble to the United States, the only first-class power still remaining neutral. While the greater part of the inhabitants of the United States, though maintaining political neutrality, are known to see eye to eye with the Allies in the rights of the quarrel, there is still a section among them who hold a dififerent view, and who make the satisfaction that comes from com- plete unanimity very hard to obtain. Again, the trade of America is embarrassed and her financial position is complicated in many directions by a struggle in whose origin she at any rate had no part. Because of all this the European war is a subject of painful anxiety on the part of the inhabitants of the United States, and their action in helping our armies in the field by providing hospitals in accord- ance with the recognized military pattern, to be placed under the control of the War Office, is one for which we must be very grateful. Nor must we forget to express appreciation of the fact that among American surgeons and physicians who have come to the help of our sick and wounded are some who have left lucrative practices and important positions on hospital stafTs, or in connection with universi- ties, in order to lend their aid in the hour of need. This alone would show that the medical inipulse is at the bottom of their action; the Citizens of the United States are not taking sides so much with the Allies against the German as with the sufferers against the triple alliance of disease, privation and injury. Lancet, London, July 3, 1915- CONTENTS PAGE The deplorable contrast between intranational and international ETHICS, and THE MISSION OF MEDICAL SCIENCE AND MEDICAL MEN. S. /. Meltser. 79 MeDICAL BROTHERHOOD FOR THE FURTHERANCE OF INTERNATIONAL MORAUTY. William J. Gies, Secretary. 292 ReSULTS OF STUDIES ON VITAMINES AND DEFICIENCY DISEASES, DURING THE YEARs 1913-1915. Casimir Funk 304 BiocHEMiSTRY OF coD-uvER oiL. Casimir Funk 365 The PROBLEM OF REjuv'ENESCENCE IN PROTOZOA. Lorande Loss Woodruff ... 37 » The CHEMICAL CONSTITUTION OF STARCH. Arthur W. Thomas 379 The behavior of tartaric acid and the tartrates in the animal organism. Max Kahn. 398 DOCTORATES IN BIOLOGICAL CHEMISTRY. P. H. D 4>0 The Biochemical Society, England. R. H. A. Flimmer, Secretary 4'4 American Chemical Society. Paul E. liowe 425 American Philosophical Society. Joseph S. Hepburn 427 Biochemical news, notes and comment. Benjamin Horowits and coUaborators. 428 Editorial. William J. Gies * 460 The Biochemical Bulletin is a monthly biochemical review. It publishes results of original investigations in biological chemistry, pre- liminary reports of researches, addresses, lectures, criticism, reviews, abstracts of papers, practical suggestions, biographical notes, historical summaries, bibliographies, quotations, questions, news items, proceed- ings of societies, personalia, views on current events in chemical biol- ogy, descriptions of new substances, methods and apparatus — any and all suitable items of personal and professional interest to students, investigators and practitioners of biochemistry. Subscrtption prices: Vol. I, $6.00; vol. II, $5.00; vol. III, $4.50; vol. IV, $3.50; vol. V, $2.75 ($3.00, foreign). Address remittances to the Biochemical Bulletin, 41 North Queen St., Lancaster, Pa., or 437 West sgth St, New York; manu- scripts and miscellaneous correspondence to the Managing Editor, William J. Gies, 437 West sgth St., New York. New York Botanical Garden übrai 3 5185 00259 9635