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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Kligler, Arthur Knudson, Mathilde Koch, Walter M. Kraus, Alfred H. Kropff, A. V. S. Lambert, Marguerite T. Lee, Victor E. Levine, Sidney Liebovitz, Charles C. Lieb. Mabel C. Little, B. E. Livingston, Leon Loewe, Alfred P. Lothrop. Daniel R. Lucas, Wm. H. McCastline, Mary G. McCormick, Louise McDanell, Jas. P. McKelvy, Alice H. McKinney, Grace MacLeod, C. A. Mathewson, H. A. Mattill. Clarence E. May, Gustave M. Meyer, Sergius Morgulis, Max Morse, Agnes F. Morgan, H. O. Mosenthal, J. Howard Mueller, Hermann J. Muller, Archibald E. Olpp, B. S. Oppenheimer, R. C. Osburn, Reuben Ottenberg, Charles Packard, A. M. Pappenheimer, Edwards A. Park, Olive G. Patterson, Almeda Perry, W. H. Peterson. Louis Pine, Helene M. Pope, E. R. Posner, P. W. Punnett, Jessie Moore Rahe, A. N. Richards, Anna E. Richardson, Winifred J. Robinson, David E. Roelkey, and the executive editorial sub-committee : Edgar G. Miller, Jr., Chairman, Benjamin Horowitz, Paul E. 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«. ,— I M '»" ' 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 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 + N tt P 1 '5 + N P + «1 n '5 1 N a P 1 'S 1 N P Percentage of positive findings m tfi 1 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 2 7 2 I 6 42 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 1.0975 o 0.784 o 1.095 0.5965 0.7234JO 0.65461O 1.0725 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 0.8649 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 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 = (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