ijn j in 01418 | UNIV I CO ™ op) Tp At ely 4 Corea a i a Digitized by the Internet Archive in 2009 with funding from University of Toronto http://www.archive.org/details/transactionsO7conn a te TRANSACTIONS CONNECTICUT ACADEMY ARTS AND SCIENCES. VOLUME VIL. NEW HAVEN: PUBLISHED BY THE ACADEMY. 1885 to 1888. OKHICERS OK WHE ACADERIY, 1887-88. President. WILLIAM H. BREWER. Vice-President. CHARLES 8S. HASTINGS. & l | mE: Corresponding Secretary. ADDISON VAN NAME. 4 4 / Recording Secretary. (1 (9 { A RUSSELL H. CHITTENDEN. “4! VW \ a4 ed Librarian. bead ADDISON VAN NAME. Treasurer. LEWIS E. OSBORN. Publishing Committee. HUBERT A. NEWTON, ELIAS LOOMIS, GEORGE J. BRUSH, ADDISON E. VERRILL, RUSSELL H. CHITTENDEN, EDWARD S. DANA, ADDISON VAN NAME. Auditing Committee. ADDISON E. VERRILL, HUBERT A. NEWTON, ADDISON VAN NAME, were IN ES. PAGE PiSteOr ON DDITIONS TO; TMH SUIBRARBY, -....2.-+--25----=-- Vv Art. I.—On tHe Law or Error 1n TARGET-SHOOTING. xt me MORNsR, 2-2. 20.24 kA kee I I].—ExtTENSIONS OF CERTAIN THEOREMS OF CLIFFORD AND oF CAYLEY IN THE GEOMETRY OF 7 Dt- MUONGCIONG - >yeta te MOORE, JR.,-2-=.2-.5-.. ° 9 Ill.—On Knots, wirnh a Census For Orpver TEN. Bye ee byrrameblates I=750 52.02. 2) .-252 IV.—Tue Amytoryric Action or DrastasE oF MAtt, AS MODIFIED BY VARIOUS CONDITIONS, STUDIED QUANTITATIVELY. By R. H. Cuirrenpen and (Gi, AIS GMC TOTES Se i ee a V.— INFLUENCE OF CERTAIN THERAPEUTIC AND Toxic AGENTS ON THE AMYLOLYTIC ACTION OF SALIVA. By R. H. Currrenpen and H. M. Painrer,.-- 60 VI.— INFLUENCE oF VARIOUS INORGANIC AND ALKALOID SALTS ON THE PROTEOLYTIC ACTION :OF PEPSIN- Hyprocutoric Acip. By R. H. Cuirrenpren SONS 3 ah rr {iLL Ra eee. 84 VIL—Inrivence or various THERAPEUTIC AND Toxic SUBSTANCES ON THE ProrEoLtytTic ACTION OF THE PANcREATIC FERMENT. By R. H. Carr- Pinang: Gr WW UMMINS, 22.22. 2-2 <2 108 VIIL—InFivencre oF TEMPERATURE ON THE RELATIVE AMYLOLYTIC ACTION OF SALIVA AND THE Dtas- TASE OF Marr. By R. H. Currrenpen and Deeb rmvemiinereeee. 2 oe eee E25 1X.—INr.vueEnce or Birr, Bite Satts anp Brine Acris on AMYLOLYTIC AND Prorreotytic Action. By bo ~T R. H. CairrenpEn and G. W. Cummins, ---.-- 134 X.—ABSORPTION OF ARSENIC BY THE Brain. By R. feCrrrnpen and H. EK. Smrrg;)._---..--- - 149 XI.—INFLUENCE oF Porasstum AND Ammonium Bro- MIDES ON Merapouism. By R. H. Currrenpen a VG ULBRRT “eae ots... 158 1V CONTENTS. XII.—InFLUENCE oF CINCHONIDINE SULPHATE ON METra- BOLISM. By R. H. Cuirrenpen and H. H. War ngOusm 225 foo eb a XII.—Tuer Post-mortem ForMATION OF SUGAR IN THE LivER IN THE PRESENCE OF PEPTONES. By R. H. CairrenpEen and A. LAMBERT, --_-- -_-. XITV.—GLosuLin AND GLoBULOSE Bopirs. By W. Ktn and JR, Hi (CaIrrENDEN, ..._.. --2 2:2 XV.—Peptonrts. By W. Kiune and R. H. Cairren- XVI.—On tHe DenuypRATION OF GLUCOSE IN THE SroMacH AND Inrestines. By R. H. Cuirren- 2 2a ee XVIL.—InF.vence or Uranium SALts ON THE AMYLOLY- Tic AcTION OF SALIVA AND THE PROTEOLYTIC Action oF Prrsin anp Trypsin. By R. H. CHITTENDEN and M. T. Hurcurnson, .---.--- XVUI.—Tuer Revative Disrrisution or ANTIMONY IN THE ORGANS AND TISSUES OF THE BODY, UNDER VARYING CONDITIONS. by R. H. CuirrenpEN and Josmpa A. BLAKn, _-.2.._.2 4 soeeee XIX.—InNFLuENcE or Antimonious OxtpE on Meta- BoLIsM. By R. H. Currrenpen and Josmps Ay BUAKE,. 20.2 62.2... .. 2a XX.—On some Meratiic Compounps or ALBUMIN AND Myosin. By R. H. Currrenpen and Henry H. Wair8Housn, 22..-.... <2. 2 XXI.—Eac—Arsumin anp ALBUMOSES. By R. H. Curr- TENDEN and Percy R. Boiron, _2_)seeeee XXII.—Casern anp rts Primary Creavace Propvucrs. By R. H. Currrenpen and H. M. Painter, .- XXII.—InNFiuENcE or some OrGantc AND INORGANIC SUBSTANCES ON. Gas METABOLISM. By R. H. CuITreNDEN and G. W. Cummins. Plate 8,-_- XXIV.—Nrw EnGianp Sprpers or tHE Famity Crxirton- ina. By J. H. Emerron. » Plates 9-11, ____-2 PAGE 166 274 293 ADDONS TO. THE LIBRARY OF THE Connecticut Academy of Arts and Sciences, By Girt AnD EXcHANGE, FROM JAN. 1, 1887, to Ava. 1, 1888. ALBANY.—Wew York Staite Library. Annual report. LXVII-LXIX, 1884-86. 8°. New York State Museum of Natural History. Report. XXXII, XXXVIII, XX XIX, 1878-85. 8°. American Association for the Advancement of Science. Proceedings. Meeting xxxviI, xxxvit, 1886-87. Salem, 1857-88. 8°. ANNAPOLIS.— United States Naval Institute. Proceedings. -Vol. XIII, XIV. 1, 2, 1887-88. 8°. BALTIMORE.—Johns Hopkins University. American chemical journal. Vol. VIII. 6, IX, X. 1-4, 1886-88. 8°. Studies from the biological laboratory. Vol. III. 9, TV. 1-4, 1887-88. 8°. University circulars. No. 63, 1888. 4°. . Observations on the embryology of Insects and Arachnids. By Adam T. Bruce. 1887. 4°. Boston. Scientific Society. Science observer. Vol. V. 1-3, 1886-87. 8°. —— American Academy of Arts and Sciences. Proceedings. Vol. XXII, 1886-87. 8°. Society of Natural History. Memoirs. Vol. IV. 1-6, 1886-88. +4°. Proceedings. Vol. XXIII pp. 289-528, 1886-88. 8°. BRooOKLYN.—Lntomological Society. Entomologica Americana. Vol. II. 9-12, III, IV. 1-4, 1886-88. 8°. CAMBRIDGE.— Harvard College. Annual reports of the president and treasurer. 1885-6, 1886-7. 8°. Record of the commemoration, Noy. 5-8, 1886, of the 250th anniversary of the founding of Harvard College. 1887. 8°. Astronomical Observatory of Harvard College. Annals. Vol. XIII 2, XVII, XVIII. 1-5, 1887-88. 4°. Annual report. XLI, XLII, 1886-87. 8°. Henry Draper Memorial Annual report. I, II, 1857-88. 4°. Boyden Fund. Circular, no. 2, 1887. 4°. Museum of Comparative Zoilogy at Harvard College. Memoirs. Vol. XV, XVI. 1, 2, 1887. 4°. Bulletin. Vol. XII. 2-9, XIV, XV, XVI. 1, XVII. 1, 1886-88. 8°. Annual report. 1886-87. 8°. Entomological Club. : Psyche. No. 135-137,'141-146, 1885-88. 8°. XX Additions to the Library. CHAPEL HiLu.—Hlisha Mitchell Scientific Society. Journal, 1885-86, 1887, 8°. CHARLESTON.—Lilliott Society of Science and Art. Proceedings. Vol. II pp. 81-160, 1875-87. 8°. Cuicaco.—The American antiquarian and oriental journal. Vol. IX, X. 1-3, 1886- Seige CINCINNATI.— Observatory. Publications. No. 9, 1887. 8°. Society of Natural History. Journal. Vol. IX. 4, X, XI. 1, 1886-88, 8°. DAVENPORY.— Academy of Natural Sciences. Elephant pipes and inscribed tablets in the museum of the Academy. By Charles EK. Putnam. 2ded. 1886. 8°. FRANKFORT.—Kentucky Geological Survey. Bulletin. No. 1, 1879. 8°. Chemical analyses. A. Vol. II, 1585. 89. On the fossil Brachiopods of the Ohio valley. By N.S. Shaler. 4°. On the prehistoric remains of Kentucky. By L. Carr and N.S. Shaler, 4°. Information for emigrants. By John R. Procter. 1888. 8°. Notes on the rocks of Central Kentucky, with list of fossils. By W. M, Linney. 1887. 8°. Report on the geology of Mercer county. By W. M. Linney. 1887. 8°. Report on the Pound Gap region. By A. R. Crandall. i885, 8°. Report. of a reconnoissance of apart of the Breckenridge coal district. By Charles J. Norwood. 8°. Report of the geology of a section near Compton, Wolfe county. By P. N. Moore. * 8°. Report on the progress of the survey, 1882-83, 1884-85, 1886-87. By John R. Procter. 8°. Geological maps. 14 sheets. GRANVILLE.—Dennison University. Bulletin of the scientific laboratories. Vol. I-III, 1875-78. 8°. HARRISBURG.—Second Geological Survey of Pennsylvania. > Annual report. 1886, pt. I-III, with atlas. 8°. Atlas of Western Middle Anthracite field. Pt. II (AA). 1887. 89. Atlas to report on Bucks and Montgomery counties. (C7). 1888. 89, Maptson.— Washburne Observatory. Publications. Vol. V, 1886. 8°. MERIDEN.— Scientific Association. Transactions. Vol. II, 1885-86. 8°. MIDDLETOWN.— Wesleyan University. Annual report of the curators of the museum. XVII, 1887-88. 8°. MINNEAPOLIS.— Geological and Natural History Survey of Minnesota. Annual report. XV, 1886. 8°. Bulletin. No. 2-4, 1887. 8°. — Minnesota Academy of Natural Sciences. Bulletin. 1875, 1876, 1878-79. 8°. Mr. HamiInron.—Lick Observatory. Publications. Vol. I, 1887. 4°. New ORLEANS.—Academy of Sciences. Papers. Vol, I. 1, 1886-87. 8°. New Yorxk.—Academy of Sciences. Annals. Vol. IIT, 11, 12, IV. 1-4, 1886-88, 80°. Transactions. Vol. IV, V. 7-8, VI, 1884-87. 89, American Geographical Society. Bulletin. Vol. XVII. 4,5, XVIII. 2-5, XIX, XX. 1, 2, 1885-88. 8°, Additions to the Library. XXi New York.—dAmerican Museum of Natural History. Bulletin. Vol. I. 8, IL. 1, 1886-87. 8°. Annual report. 1887-85. 8°. Astor Library. Annual report. XXXVIII, 1886. 8°. Microscopical Society. : Journal. Vol. III, IV. 1, 2, 1887-88. 8°. Torrey Botanical Club. Bulletin. Vol. XIII. 10-12, XIV, XV. 1-7, 1886-88. 89, PHILADELPHIA.—Academy of Natural Sciences. Journal. Series II. Vol. VIII. 4, IX. 1, 2, 1874-88, 40°, Pranklin Institute. Journal. Vol. CXXIII-CXXV, CXXVI. 1, 2, 1887-88. 80. Wagner Free Institute of Science. _ Transactions. Vol. I, 1887. 8°. PouGHKEEPSIE. Vassar Brothers Institute, Transactions. Vol. IV, 1885-87. 8°. RocHESTER.— Warner Observatory. History and work. Vol. I, 1883-86. 8°. SACRAMENTO.— California State Mining Bureau. Annual report of the state mineralogist. VII, 1887. 8°. SALEM.—Lssex Institute. Bulletin. Vol. XVIII. 4-12, XIX, 1886-87. 8°. San FrRANcISCcO.— California Academy of Sciences. Memoirs. Vol. II. 1, 1888. 4°. Bulletin. No. 6-8, 1887. 8°. Technical Society of the Pacifie Coast. Transactions and proceedings. Vol. IV, V. 1, 1887-88. 8°. SanTA BARBARA.—Sociely of Natural History. Bulletin. No.1, 1887. 8°. SAVANNAH.— Georgia Historical Society. The life and services of the Hon, Maj. Gen. Samuel Elbert, of Georgia. By Charles C. Jones, Jr. 1887. 8°. TopEeKA.— Washburn College Laboratory of Natural History. Bulletin. Vol. I. 8, 1887. 89. Trenton, N. J.—Natural History Society. Journal. No. 2, 3, 1887-88. 8°. WASHINGTON.— Bureau of Education. Report of the commissioner of education. 1884-85, 1885-86. 8°. Circulars of information. 1886 i, 1887 i-iii. 8°. Chief Signal Officer. Annual report. 1885, 1886, 1887 pt. 1. 89°. United States Geological Survey. Annual report. V, VI, 1885-84, 1884-85. 8°. Bulletin. No. 30-39, 1886-87. 8°. Monographs. Vol. X, XI, XII, 1885-86. 4°. Mineral resources of the United States. 1885, 1886. 8°. —— United States Naval Observatory. Astronomical and. meteorological observations. 1882, 1883. 4°. Smithsonian Institution. Annual report. 1884, 1885. 8°. Annual report of the Bureau of Ethnology. IV, 1882-83. 8°. Bibliography of the Eskimo language. By James C. Pilling. 1887. 8°. Bibliography of the Siouan languages. By James C. Pilling. 1887. 8°. The use of gold and other metals among the ancient inhabitants of Chiri- qui, Isthmus of Darien. By William H. Holmes. 1887. 8°. XXii Additions to the Library. WASHINGTON.—Smithsonian Institution. Work in mound exploration of the Bureau of Ethnology. By Cyrus Thomas. 1887. 8°. Perforated stones from California. By Henry W. Henshaw. 1887. 8°. WORCESTER.—American Antiquarian Society. Proceedings. New series. Vol. IV. 3,4, V. 1, 1886-87. 8°. AmiENS.—Socicélé Linnéenne du Nord dela France. Bulletin. No. 151-174, 1885-86. 8°. AMSTERDAM.—Kon. Akademie van Wetenschappen. Jaarboek. 1885. 8°. Verslagen en mededeelingen. Afdeel. natuurkunde. 3dereeks. Deel II. 1886. 8°. AvuasBpurG.—WNaturhistorischer Verein. Bericht. XXXIII-XXXVII, 1882-87. 8°. AUXERR®.—Socicté des Sciences Historiques et Naturelles de 0 Yonne. Bulletin. Tome XL. 2, XLI, 1886-87. 82. BAMBERG.—WNaturforschende Gesellschaft. Bericht. XIV, 1887. 8°. BAsEL.—WNaturforschende Gesellschaft. Verhandlungen. Theil VIII. 1, 2, 1886-87. 8°. BataviA.—Kon. Natuurkundige Vereeniging in Nederlandsch-Indié. Natuurkundige tijdschrift. Deel XLVI, XLVII, 1887. 9°. ——Magnetical and Meteorological Observatory. Observations. Vol. VI supplement, VII, IX, 1886-87. 4°. BERGEN.— Museum. Aarsberetning. 1886. 89° Beri .—Konigliche Sternwarte. Berliner astronomisches Jahrbuch. 1889, 1890. 8°. Botogna.—R. Accademia delle Scienze dell’ Istituto di Bologna. Rendiconto, Anno 1886-87. 8°. BompBay.—Bombay Branch of the Royal Asiatic Society. Journal. No. XLV, XLVI, 1887. 8°. Index to Transactions vol. I-III, and Journal vol. I-XVIJ. 1886. 8°. Government Observatory. Magnetical and meterological observations. 1885. 4°. Bonn.—Noaturhistorischer Verein der preussischen Rheinlande, Westfalens und des Reg.—Bezirks Osnabriick. Verhandlungen. Jahrg. XLIII. 2, XLIV, 1886-87. 8°. BorpEAuUx.—Académie Nationale des Sciences, Belles-Lettres et Arts. Actes. Année XLVII, 1885. 8°. Société Linnéenne. Actes. Tome XXXIX, 1885. 8°. Procés-verbaux. 1886, 8°. —— Société des Sciences Physiques et Naturelles. Mémoires. 3° sér. Tome II, 2, appendice iii, IfT. 1, 1886. 8°. BRrAuNsScHWEIG.— Verein fiir Naturwissenschaft. Jahresbericht. III, 1V, 1881-86. 8°. BREMEN.—WNaturwissenschaftlicher Verein. Abhandlungen. Bd. 1X. 4, X. 1, 2, 1887-88. 8°. BRESLAU.-—Schlesische Gesellschaft fiir vaterliindische Cultur. Jahres-Bericht. LXIII u. Erginz., LXIV u. Ergiinz., 1885-86. 8°. Brinn.—Natiirforscher Verein. Verhandlungen. Bd. XXIV, X XY, 1885-86. 8°. Bericht der meteorologischen Commission. V, 1885. 8°. . EE EEE a, Additions to the Library. XXili BRUXELLES.— Académie Royale des Sciences, des Lettres et des Beaux-Arts de Belgique Mémoires. Tome XLVI, 1886. 4°. Mémoires couronnés et mémoires des savants étrangers. Tome XLVII, XLVIII, 1886. 4°. Mémoires couronnés et autres mémoires. Tome XXXVII— XXXIX, 1886. 8°. Bulletins. 3° sér. Tome IX -XII, 1885-86. 8°. Annuaire. Année LII, LIII, 1886-87. 8°. Notices biographiques et bibliographiques. 1886. 5°. Catalogue des livres de la bibliothéque. Partie I, II. 1, 2. 1881-87. 6°. Musée Royal de ? Histoire Naturelle. Bulletin. Tome IV. 4, 1886. 8°. Socicté Entomologique de Belgique. Annales. Tome XXX, 1886. Table générale, tome IT-XXX. 1887. 8°. — Société Royale Belge de Géographie. Bulletin. Année X. 2-6, XI, 1886-87. 8°. Société Royale de Botanique. Bulletin. Tome XXV. 2, XXVI. 1, 1886-87. 8°. Société Royale Malacologique de Belgique. Annales. Tome XXI, 1886. 8°. Procés-verbaux. Tome XV pp. 97-143, XVI pp. 1-80, 1886-87. 8°. Statuts. 2° éd. 1886. 8°. Bucarest.—Jnustitut Météorologique de Roumanie. Annales. Tome I, II, 1885-86. 4°. Serviciultii meteorologicti in Europa. Note de calétoria de inginerti St- C. Hepites. 1884, 4°. Bupapvest.—Kon. ung. Central-Anstalt fiir Meteorologie und Hrdmagnetismus. Jahrbiicher, Jahrg. XV, 1885. 4°. BurEnos AtrEes.—Museo Publico. Anales. Entrega XIII, XIV, 1883-85, 4°, Atlas de la description physique de la République Argentine. Par H. Bur- meister. Section II. Mammiféres. Livr. 1-3, 1881-86. f°. ——Sociedad Cientifica Argentina. Anales. Tome XXII. 5,6, XXIII, XXIV, XXV. 1, 1886-88. 8°. Estudio critico y comparativo de las reglas de Descartes y de Newton re- specto al numero de raices de las ecuaciones numericas. Por Arturo Orzabel. 1886. 8°. Capn.—Société Linnéenne de Normandie. Bulletin. 4° sér. Vol. I, 1886-87. 8°. CatcuTra.—Asiatie Society of Bengal. Journal. Vol. LV, pt. i, no. 3, 4; pt. ii, no. 3-5; LVI, pt. i, no. 1-3; pt. ii; LV, pt. ii, no. 1; 1886-88, 8°. Proceedings. 1826, no. 8-10, 1887, 1888, no. 1-3. 8°. Descriptions of new Indian Lepidopterous insects from the collection of the late Mr. W.S. Atkinson. Pt. III, 1888. 4°. Geological Survey of India, Palzeontologia Indica. Ser. I, vol. i, pt. 1, (reprint); X, vol. iv, pt. 3, XII, vol. iv, pt. 2; XIII, vol. i, pt. 6; 1886-87. 4°. Memoirs. Vol. XXIV. 1, 1887. 8°. Records. Vol. XX, XXI. 1, 2, 1887-88. 8°. Manual of the geology of India. Pt. IV. Mineralogy. 1887. 8°. Catalogue of the remains of Siwalik Vertebrata in the Indian museum, Calcutta. Pt. 1, Mammalia. Pt. 2, Aves, Reptilia and Pisces. Pleisto- cene and pre-historic Vertebrata. 1885-86. 8°. Meteorological Department of the Government of India. Indian meteorological memoirs. Vol. LY. 2-4, 1884-86. f°. XXiV Additions to the Library. CaLcurra.—Meteorological Department of the Government of India. Report on the meteorology of India. 1885, 1886. f°. Report on the administration of the meteorological department. 1885-86, 1886-87. f°. Meteorological observations recorded at six stations in India. 1886 July- Dee., 1887. f°. Cyclone memoirs. Pt. I, 1887. 8°. Charts of the Bay of Bengal and adjacent sea north of the equator show- ing the mean pressure, winds and currents in each month of the year. 1886s Le. showing the specific gravity, temperature and currents of the sea surface. 1887. f°. Memoirs of the winds and monsoons of the Arabian Sea and North Indian Ocean. By W.L. Dallas. 1887. 4°. CAMBKIDGE.—Philosophical Society. Transactions. Vol. XIV. 1, 2, 1885-87. 4°. Proceedings. Vol. V. 6, VI. 1-3, 1886-87. 4°. Casspeu.— Verein fiir Naturkunde. Bericht. XX XII-X XXIII, 1884-86. 89°. CHEKBOURG.—Soci¢té Nationale des Sciences Naturelles. Mémoires. Tome XXV, 1887. &°. I CuRIstTIANta.—Kong. Norske Universitet. Norges vextrige. Af Dr. F. C. Schiibeler. Bd. I. 2, If. 1. 1886. 49°. Joannis Agricole Islebiensis apothegmata nonnulla nune primum edidit ‘ Dr. L. Daae. 1886. 4°. Norwegisches meteorologisches Institut. Jahrbuch. 1885. 4°. Norwegian North-Atlantic Expedition, 1876-78. Publication XVI-XVIII. 1886-87. 40°. . Videnskabs Selskabet. Forhandlingar. 1886. 8°. Cuur.—Natursorschende Gesellschaft Graubiindens. Jahres-Bericht. Neue Folge. Jahrg. XXIX, XXX, 1884-85, 1885-86. 8°. CorpoBa.—Academia Nacional de Ciencias. Actas. Tomo VY. 3, 1886. 4°. Boletin. Tomo IX, X. 1, 1886-87. 8°. Danzia.—WNaturforschende Gesellschaft. Schriften. Neue Folge. Bd. IV. 4, VI. 4, 1878-84. 8°. Dison.—Académie des Sciences, Arts et Belles-Lettres. Mémoires. 3° sér. Tome IX, 1885-86. 8°, Dorpat.—Gelehrte Estnische Gesellschaft. Sitzungsberichte. 1886. 8°. Naturforscher— Gesellschaft bei der Universitit Dorpat. Archiy fiir die Naturkunde Liy-Ehst-und Kurlands. Ser. I. Bd. IX. 4, 1887. 8°. Sitzungsberichte. Bd. VIII. 1, 2, 1886-87. 8°. Schriften, II-IV, 1887-88. 8°. Drespven.—Naturwissenschaftliche Gesellschaft Isis. Sitzungsberichte und Abhandlungen. 1885 Juli-Dee., 1886, i887. 8°, DuBLin.— Royal Lrish Academy. ; Transactions. Vol. XXVI. 2%, XXVII. 6-8, XXVIII. 21-25, 1879-86, 40. Proceedings. Ser. II. Science. Vol. IV. 5, 1886. 8°. Cunningham memoirs. No. I-IV, 1880-87. 4°, EDINBURGH.— Geological Society. Transactions. Vol. V. 2, 3, 1887. 8°. Catalogue of the library. 1887. 89. — a —s Additions to the Library. XXV Epinpureu.—Royal Physical Society. Proceedings. Vol. IX. 1, 1885-86. 8°. Royal Society. Proceedings. Vol. XII pp. 245-1008, XIII, XIV, 1883-87. 8°. List of members. Noy. 1887. 4°. Emprn.—Waturforschende Gesellschaft. Jahresbericht. LX XI, 1885-86. 8°. ErrFurt.—Kon. Akademie gemeinniitziger Wissenschaften. Jahrbiicher. Neue Folge. Heft XIV, 1886. 8°. Firenze.— Biblioteca Nazionale Centrale. Bollettino delle pubblicazioni Italiane ricevute per diritto di stampa. No. 25-61, 1887-88. 8°. FRANKFURT A. M.—Deutsche malakozoologische Gesellschaft. Nachrichtsblatt. Jahrg. XVIII. 11, 12, XIX, XX. 1-6, 1886-88. 8°. Senckenbergische naturforschende Gesellschaft. Abhandlungen. Bd. XIV. 2,3, XV. 1, 2, 1886-88. 4°. Bericht. 1886, 1887. 8°. FREIBURG IN B.—WNaturforschende Gesellschaft. Verhandlungen. Bd. VIII. 3, 1885. 8°. Berichte. Bd. I, 1886. 8°. GENEVE.—Jnstitut National Genevois. Bulletin. Tome XXVII, XXVIII, 1885-88, 8°. Mémoires. Tome XVI, 1883-86. 4°. -Société de Physique et @ Histoire Naturelle. Mémoires. Tome XXIX. 2, 1886-87. 4°. GIESSEN.— Oberhessische Gesellschaft fiir Natur-und Heilkunde, Bericht. XXV, 1887. 8°. Guascow.—Natural History Society. Proceedings and transactions. New series. Vol. I. 3, 1885-86. 8°. —— Philosophical Society. Proceedings. Vol. XVII, XVIII, 1885-86, 1856-87. 8°. GorLITzZ.—Naturforschende Gesellschaft. Abhandlungen. Bd. XIX, 1887. 8°. GOrrincgen.—Konigl. Gesellschaft der Wissenschaften. Nachrichten. 1886, 1887. 8°. Gutstrow.— Verein der Freunde der Naturgeschichte in Mecklenburg. Archiv. Jahrg. XL, XLI, 1886-87. 8°. Hapana.—feal Colegio de Belen. Observaciones magneticas y meteorologicas. 1885 iy, 1886 i-iii. 4°. Hanmax,— Department of Mines. Report. 1887. 8°. Hauie.—Kais. Leopoldinisch-Carolinische deutsche Akademie der Naturforscher. Leopoldina. Heft XXII, XXIII, 1886-87. 4°. Naturforschende Gesellschaft. Abhandlungen. Bd. XVI. 4, 1886. 4°. Bericht. 1885, 1886. 8°. Naturwissenschaftlicher Verein fiir Sachsen und Thiiringen. Zeitschrift fiir die gesammten Naturwissenschaften. Bd. LIX. 3-6, LX, 1886-87. 8°. Hampure.—Deutsche Seewarte. Archiy. Jahrg. VIII, IX, 1885-86, 4°. Monatliche Uebersicht der Witterung. 1886, 1887, 1888 Januar, Feb- ruar. 8°. XXV1 Additions to the Library. HampBure.—Naturwissenschaftlicher Verein.” Abhandlungen. Bd. IX, X, 1886-87. 4°. Wissenschaftliche Anstalten. Jahrbuch. Jahrg. IV, V, 1886-87. HANNOVER.—WNaturhistorische Gesellschaft. Jahresbericht. XXXIV-XXXVII, 1883-1887. 8°. Hartem.— Musée Teyler. Archives. Série II. Vol. III. 1, 1887. 8°. Catalogue de la bibliothéque. Livr. 5, 6. 1886, 8°. Liste alphébetique de la correspondence de Christian Huyghens. 4°, Société Hollandaise des Sciences. Archives néerlandaises des sciences exactes et naturelles. Tome XXI. 2-5, XXII, 1886-87. 8°. HELSINGFORS.—Societas Scientiarum Fennica. Ofversigt af forhandlingar. XXVII, 1884-85. Bidrag till kiinnedom af Finlands natur och folk. Haft. XLIII, XLIV, 1886-87. 8°. Exploration internationale des régions polaires, 1882-83 et 1884-85. Expé- dition polaire finlandaise. Tome I, II. 1886-S7. 4°. Institut Météorologique Central. Observations. Vol. I, IT. 1, 1882-83. 4°. HonekonG.— Observatory. Observations and researches. 1886. f°. JENA.— Medicinisch-naturwissenschaftliche Gesellschaft. Jenaische Zeitschrift fiir Naturwissenschaft, Bd. XX, XXI, XXII. 1, 2, 1887-88. 8°. A LBL.—LKonigl. Christian Albrechts- Universitat. Dissertationen, etc. (48). 1386-87. Naturwissenschaftlicher Verein fiir Schleswig-Holstein. Schriften. Bd. VII. 1, 1888. 8°. Kiny.—Kievskie Obshchestvo Iestestvoispytatelet. Zapiski. Tom. VII, VIII and suppt., 1883-87. 8° and 4°. Ky6BENHAVN.—Kon. Danske Videnskabernes Selskab. x Oversigt over forhandlinger. 1886 ii, iii, 1887 i, ii. 8°. KOnIGSBERG.—Kinigl. physikalisch-Gkonomische Gesellschaft. Schriften. Jahrg. XXVII, 1887. 4°. Krakow.—XK. k. Sternwarte. Materyaly do klimatografii Galicyi. Rok 1886. 8°. LAUSANNE.— Société Vaudoise des Sciences Naturelles. Bulletin. 38° sér. No. 94-96, 1886-87. 8°. Lreps.— Yorkshire Geological and Polytechnic Society. Proceedings. New series. Vol. IX. 2, 3, 1886-88. 8°. Lempen.—WNederlandsche Dierkundige Vereeniging. Tijdscrift. Ser. II. Deel I. 3, 4, Il. 1, 2, 1886-88. 8°. LrrpziG.—Astronomische Gesellschaft. Vierteljahrsschrift. Jahrg. XXII, 1887. 8°. Publication XVIII. 1886. 4°. Kon. sichsische Gesellschaft der Wissenschaften. Berichte. Math.-physische Classe. Bd. XX XVIII, XX XIX, i886-87. 8°. Verein fiir Erdkunde. Mittheilungen. 1884-1886. 8° and f°. Zoologischer Anzeiger. No. 241-282, 1887-88. 8°. Litan.—Société Géologique de Belgique. Annales. Tome XIII. 1, 1887. 8°. Procés-verbal de Vassemblée générale du 21 Noy. 1886, 8°. Additions to the Library. Xxvil LifieE.—Société Royale des Sciences. Mémoires. 2° sér. Tome XIII, XIV, 1886-88, 8°, Linz.—Museum Francisco-Carolinum. Bericht. XLIV, XLV, 1886. 8°. Lispoa,—Sociedade de Geographia. Boletim. Serie VI. 7-12, VII. 1-8, 1886-87, 8°. Elogio historico do Antonio Augusto (Aguiar. Por Gomez de Brito. LSS, (8o. ' LiverPoo..—Literary and Philosophical Society. Proceedings. No. XX XIX, XJ, 1884-86, 8°. Lonpon.—Geological Society. Quarterly journal. Vol. XLIII, XLIV. 1, 2, 1887-88. 89, List. 1887. 8°. Linnean Society. Journal. Zoology. No. 114-117, 126-29, 1886-87. 8°. Botany. No. 145-149, 151, 158, 1886-87. 8°. Proceedings. 1883-87. 8°. List. 1886-87. 8°, Mathematical Society. Proceedings. No. 273-320, 1886-85, 8°. List of members. 1887. 8°, Royal Meteorological Society. Quarterly journal. New series. No. 61-66, 1886-88, 8°, List of fellows, March 1, 1888. 8°. Royal Historical Society. Transactions, New series. Vol. III. 3, 4, 1886. 8°. England and Napoleon in 1803, being the despatches of Lord Wentworth and others. Edited by Oscar Browning. 1887. 8°. The teaching of history in schools. An address by Oscar Browning. 1887. 8°, Royal Microscopical Society. Journal. 1887, 1888 i-iii, 8°. — Royal Society. Philosophical transactions. Vol. CLXXYVII, 1886-87. 4°, Proceedings. No. 248-269, 1886-88. 8°. List of council and members. 1886. 4°. Zoological Society. List of vertebrated animals now or lately living in the gardens of the Zoological Society of London. 8th ed. 1883, 8°, Lounp.— Universitet. Acta. Tom. XXII, XXIII, 1885-87, 4°. Luxempoure.—Institut Royal Grand-Ducal. Publications. Section des sciences mathématiques et naturelles. Tome KX, 1886. 8°. Observations météorologiques. Vol. III, IV, 1887. 8°. Lyon.— Musée Guimet. Annales. Tome X-—XIT, 1886-87. 4°. Revue de Vhistoire des religions. Tome XIV, 2,3, XV, XVI. 1, 1886-87. Sécurité dans les théatres. Par M. Emile Guimet. 1887, 8°. Lyme Reeis.—Rousdon Observatory. Publications. Vol. IV. Meteorological observations for 1887, 4°, Mavpras.—Government Observatory. Observations of the fixed stars made with the meridian circle, 1862-67. 40, XXVill Additions to the Lnbrary. Manvrip.— Comision del Mapa Geologico de Espana. Boletin. Tomo XII. 2, XIII. 2, 1885-86. 8°. Memorias. Descripcion fisica y geologica de la provincia de Alaya. Por D. Ramon Adan de Yarza. 1885. 8°. Descripcion fisica y geologica de la provincia de Zamora, Por D. Gabriel Puig y Larraz. 1883. 8°. Observatorio. Observaciones meteorologicas. 1882-83, 1884-85. 8°. Resumen da las observacionés efectuadas en la peninsula, 1883. 8°. Sociedad Hspanola de Historia Natural. Anales. Tomo XV. 3, XVI, XVII. 1, 1886-88. 8°. Real Academia de Ciencias Hxactas, Fisicas y Naturales. Memorias. Tomo XI, XII, XIII. 1, 1887. +°. Revista de los progresos de las ciencias exactas. Tomo XXI. 7-9, XXII. 1-4, 1886-87. 8°. Anuario. 1888. 8°. MAGDEBURG.— Naturwissenschaftlicher Verein. Jahresbericht und Abhandlungen. 1887. 8°. Das Innere der Erde. Vortrag yon Dr. phil. Ernst Heintzmann am 8. Mai, 1888, 8°. MANCHESTER.—Literary and Philosophical Society. Memoirs. Series (II. Vol. X, 1887. 89. Proceedings. Vol. XXV, XXVI, 1885-87. 8°. Marsure.— Gesellschaft zur Beforderung der gesammten Naturwissenschaften. Sitzungsberichte. Jahrg. 1886, 1887. 8°. MELBOURNE.—WNational Museum. Prodromus of the zoology of Victoria. Decade I-XV. 1878-87, 8°. Mprz.—Académie. Mémoires. 3° sér. Année XIII, 1883-84, 8°. MEXICcO.— Observatorio Meteorologico-Magnetico Central. Boletin mensuel. Tomo I. 1-5, 1888. 8°. Museo Nacional. Anales. Tomo III. 11, IV. 1, 2,.1886-88. 4°. Sociedad Cientifica ‘Antonio Alzate.” Memorias. Tomo T. 1-5, 8-10, 12, 1887-88. 8°. Sociedad de Geographia y Estadistica. Boletin. Epoca III. Tomo VI. 4-9, 1887. 8°. Sociedad Mexicana de Historia Natural. La naturaleza. Tomo VII. 19-24. Ser. II. Tomo I. 1-3. 1886-88. 4°. Minano.—Real Istituto Lombardo di Scienze e Lettere. Rendiconto. Serie II. Vol. XVIII, XIX, 1885-86. 8°. Real Osservatorio di Brera. Pubblicazioni. No. VII. 8, XXVII-X XXII, 1885-87. 4°. MopeEna.—Regia Accademia delle Scienze, Lettere ed Arti. Memorie. Tomo XX.3. Serie Il. Tomo IV. 1886. 4°. Societd det Naturalisti. ; Memorie. Ser. III. Vol. V. VI, 1886-87. 8°. Rendiconti.. Ser. ITI. Vol. III pp. 1-128, 1886. 8°. MONTPELLIER.—Académie des Sciences et Lettres. Mémoires. Section des lettres. Tome VIII. 1, 1886-87. 4°. Section des sciences. Tome XI, 1, 1885-86. 4°. MontREAL.—Natural History Society. The Canadian record of science. Vol. IT. 5, 6, 1887. 8°, Moscou.—Société Impériale des Natwralistes. Bulletin. Année 1886, 1887. 8°, Vv ee Additions to the Library. XXiX Moscou.—Société Impériale des Naturalistes. Meteorologische Beobachtungen am Observatorium der landwirth. Akad- emie bei Moskau. Jahr. 1886 ii, 1887i. 4°. MincuHen.—Koén. bayerische Akademie der Wissenschaften. Sitzungsberichte. Philosph.-philolog. und histor. Classe. 1886, 1887 Bd. I, Is -3.0 8°: Mathemat.-physikal. Classe. 1886, 1887 Heft 1-3. 8°. Inhaltsverzeichniss. Jahrg. 1876-1885. 8°. Gediichtnisrede auf Joseph v. Fraunhofer. Von Carl Max y. Bauern- feind. 1887. 4°. Gedichtnissrede auf Carl Theodor y. Siebold. Von Richard Hertwig. 1886. 4°. Gediichtnissrede auf Leopold v. Ranke. Von W.v. Giesebrecht. 1887. 4°. Ueber historisehe Dramen der Roemer. Festrede yon Dr. Karl Meiser. 1887. 4°, MUunster.— Westfiilischer Provincial- Verein fiir Wissenschaft und Kunst. Jahresbericht. XIV, 1885. 8°. Die Kunst- und Geschichts-Denkmiler der Provinz Westfalen. Stiick IT; Kreis Warendorf. 1586. 4°. Nancy.— Wirzsure.—Physikalisch-medicinische Gesellschaft. Sitzungsberichte. Jahrg. 1886, 1887. 8°. Ztricu.—Naturforschende Gesellschaft. Vierteljahrschrift. Jahrg: XXX, XXXI. 1, 2, 1885-86. 89. Review of the data for the study of the pre historic chronology of America. By Daniel G. Brinton, M.D. Salem, 1887. 8°, Prom the Author. Verities in Verses. 2d ed. London, 1887-88, 89. Copyright and patents for inventions. By R. A. Macfie. Edinburgh, 1879-1883. 2v. 8°. Prom the Author. Evolution of the faunas of the lower Lias.. By Alpheus Hyatt. Salem, 1888, 8°, Value in classification of the stages of growth and decline, with propositions for anew nomenclature. By Alpheus Hyatt. Salem, 1888. 8°. From the Author. Campagnes scientifique du yacht monégasque l Hirondelle. 3° année, 1887. Ex- cursions Zoologiques dans les iles de Fayal et de San Miguel. Par Jules de Guerne. Paris, 1888, 8°, Résultats des campagnes scientifiques accomplies sur son yacht, par 8. A. le Prince Albert de Monaco. Vol.I, Hydrographie et zoologie. (Programme). Monaco, 1888. 4°. From His Highness Prince Albert of Monaco. a Additions to the Library. XXxili Supplementa faunae Coleoptorum in Transsilvania scripsit Alexander Ormay. Nagy-Szeben, 1888. 8°. From the Author. Estudio critico y comparativo de las reglas de Descartes y de Newton respecto al numero de raices de las ecuaciones numericas. Por Arturo Orzabel. Buenos Aires, 1886. 89. From the Author. Heights of the White Mountains. By Edward C. Pickering. 8°. Observations on variable stars in 1886. By Edward C. Pickering. 8°. F From the Author. Osseryazioni astronomiche e fisiche sull’ asse di rotazione e sulla topografia del pianeta Marte, fatte nella Reale Specola di Brera in Milano coll’ equatoriale di Merz. Memoria terza di G. V. Schiaparelli. Roma, 1886. 4°. From the Author. SFr aN (On Os, 46555 os ; whit ye Ps Ly Pe + piney i math yeqe! ary gt Pon! 1a OLE Mat Be ath ‘ ~ EG" js wr rer ari Wes “hy nn A ‘ 7. Pt SS Reals al ss at teal HVAT te see : - ‘ t r 1 t at Oy Ga URS Sa kT Lee 7 ae toa Ho sal i a BR erie ien ors ; aan : - ‘ SiG: i teh cae hh ¢ f “ : , hho h aft 4 - CP PGP: Os Pee el cn ee iw it ; ‘ iat ives ae re) F ra Mit : rat ie rae | x4 a . ’ ss ; ve “ E | . a » r i ‘ ri to < a y 5 <= “ J . * = Ry. = ‘ - & ‘ ? a ‘ - x ae. - a t oat . ‘ x Pus be oe - a <—- ' x. = , : 4 y= ie -¢ ¢ ‘ S ADDITIONS TO: THE LIBRARY OF THE Connecticut Academy of Arts and Sciences, By Grrr Anp ExcuancE, From JuLy 1, 1885, ro Dec. 31, 1886. American Association for the Advancement of Science. Proceedings. Meeting xxxiv, xxxv, 1884-85. 8°. Salem, 1885-86. 8°. ANNAPOLIS.— United States Naval Institute. Proceedings. Vol. VIII, 1X, XI. 3, 4, XII, 1882-86. 8°. BALTIMORE.—Johns Hopkins University. American chemical journal. Vol. VII. 2-6, VIII. 1-5, 1885-86. 89, Studies from the biological laboratory. Vol. III. 4-8, 1885-86. 8°. Boston.—Amateur Scientific Society. Science observer. Vol. IV. 12, 1886. 8°. ——American Academy of Arts and Sciences. Proceedings. Vol. XX, XXJ, 1884-86. 8°. — Society of Natural History. Memoirs. Vol. III. 12,13, 1886. 4°. Proceedings. Vol. XX. 3, XXIII. 1, 2, 1880-86. 89. BrooxLyn.—Fntomological Society. Entomologica Americana. Vol. I. 3-12, II. 1-3, 1885-86. 8°. Papilio. Vol. IV. Philad., 1884. 8°. BROOKVILLE.—Society of Natural History. Bulletin. No. 2, 1886. 8°. BurraLo.—Society of Natural Sciences. Bulletin. Vol. V. 1, 2, 1886. 89. CAMBRIDGE.—Harvard College. Annual reports of the president and treasurer. 1884-85. 8°. Astronomical Observatory of Harvard College. Annals. Vol. XV. 1, XVI, 1886. 4°. Annual report. 1884-85, 1885-86. 8°. Museum of Comparative Zodlogy at Harvard College. Memoirs. Vol. X. 2,4, XIV. 1 pt. 1, 1885. 4°. ‘Bulletin. Vol. XI. 10, 11, XII, XIII. 1, 1885-86. 8°. Annual report. 1884-85, 1885-86. 8°. Entomological Club. Psyche. No. 129-134, 1885. 8°. CHARLESTON.—Zilliott Society of Science and Art. Proceedings. Vol. II pp. 1-40, 1859-60. 8°. Cuicaco.—Astronomical Society. Annual report. 1885. 8°. The American antiquarian and oriental journal. Vol. Vil. 5, 6, VIII, 1885-86. 8°. CINCINNATI.— Observatory. Publications. No. 8, 1885. 89. Society of Natural History. Journal. Vol. VIII. 2-4, TX. 1-3, 1855-86. 8°. vi Additions to the Library. Davenrort.—Academy of Natural Sciences. Proceedings. Vol. IV, 1882-84. 8°. HARRIsBuRG.—Second Geological Survey of Pennsylvania. Annual report for 1885, with atlas. 8°. Report of progress. AA and four atlases, C!-C’, Di-D2,. D3 v. 1, 2 and atlas, D5, E, F1, F?, G'-G’, H®-H’, I*, I’ and atlas, I4, K1-K4, L, M!-M3, N, 01, 02, Pv. 1-3 and atlas, P2, P’, Q'-Q*, R and atlas, R? and atlas, T and atlas, T?-T1, V, V2, X, Z, 1875-84. 8°. Grand atlas. Divisions I. 1, II. 1, 2, III.1,1V.1, V.1. fol. Mavpison.— Wisconsin Academy of Sciences, Arts and Letters. Transactions. Vol. VI, 1881-83. 8°. Washburne Observatory. Publications. Vol. III, IV, 1885-86. MINNEAPOLIS.— Geological and Natural History Survey of Minnesota. Annual report. XII-XIV, 1883-86, 8°. Geology of Minnesota. Final report. Vol. I, 1884, 4°. —— Minnesota Academy of Natural Sciences. Bulletin. Vol. II. 5, 1880-82. 8°. New Yorx.—Academy of Science. Annais. Vol. III. 9, 10, 1855-86. 8°. Transactions. Vol. III, V. 1-6, 1883-86. 8°. American Geographical Society. Bulletin. 1884 v, 1885 i-iii, 18861. 8°. —— American Museum of Natural Sciences. Bulletin. Vol. I. 6, 7, 1885-86. 8°. —— Microscopical Society. Journal, Vol. I, II. 1-7, 1885-86. 8°. Torrey Botanical Club. Bulletin. Vol. XII. 6-12, XIII. 1-9, 1885-86. 8°. PawtucKket.— The ornithologist and odlogist. Vol. X. 7-12, 1885. 8°. PHILADELPHIA.—American Entomological Society. List of the Coleoptera of America north of Mexico. By Samuel Henshaw. Philad. 1885. 8°. Re Franklin Institute. Journal. Vol. CXX. 2-6, CXXI, CXXIT, 1885-86. 8°. PouGHKEEPSI£.— Vassar Brothers Institute. Transactions. Vol. III. 1, 1884-85. 89°. Ricumonp.— Virginia State Library. Calendar of Virginia state papers. Vol. I~V, 1652-1792. 8°. Sr. Louis.—Academy of Science. Transactions. Vol. IV. 4, 1878-86. 8°. SaLem.—Lssex institute. Bulletin. Vol. XVII, XVIII. 1-5, 1885-86. 8°. San FRANcISCO.—California Academy of Sciences. Memoirs. Vol. I. 1, 1868. 4°. Bulletin. No. 4, 5, 1886. 8°. — — Technical Society of the Pacifie Coast. Transactions. and proceedings. Vol. I, IJ, III. 1-5, 1884-86. 8°. SepaLia.—Natural History Society. Bulletin. No.1, 1885. 8°. Toprka.—Kansas Academy of Science. Transactions. Vol. IX, 1883-84. 89. Washburn College Laboratory of Natural History. Bulletin. Vol. [. 1-7, 1884-86. 8°, Pe a Additions to the Library. vil UNIVERSITY OF VIRGINIA.—Leander McCormick Observatory. Publications. Vol. I. 1-3, 1855-86. 8°. Report‘of the director. 1885-86. 8°. WasuHineton.— Bureau of Education. Report of the Commissioner of Education. 1883-84. 8°, Circulars of information. 1885 i-v, 1886 i. 8°.: Chief Signal Officer. Annual report. 1884. 8°. Professional papers. No. 18, 1885. 4°. United States Geological Survey. Annual report. III, IV, 1882-83, 1883-84, 8°. Bulletin. No. 7-29, 1884-86, 8°. Monographs. Vol. VIII, 1884. 4°. —— United States Naval Observatory. Astronomical and meteorological observations for 1881. 4°. Report of the superintendent. 1885-86. 8°. ——Smithsonian Institution. Annual report. 1883. 8°. Annual report of the Bureau of Ethnology. II, 1881-82. 8°. WILKES-BAaRRE.— Wyoming Historical and Geological Society. Proceedings and collections. 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BaseL.—Naturforschende Gesellschaft. Verhandlungen. Theil VII. 3, 1835. 8°. Batavia.—Kon. Natuurkundige Vereeniging in Nederlandsch-Indic. Natuurkundige tijdsehrift. Deel XLIV, XLV, 1885-86. 8°. Catalogus der bibliotheek. 1884. 8°. — Magnetical and Meteorological Observatory. Observations. Vol. VI, 1885. 4°. BERGEN,— Museum. Bidrag til Myzostomernes anatomi og histologi. Af F. Nansen. 1885. 4°. BERwin.—Konigl. Sternwarte. Berliner astronomisches Jahrbuch. 1870-78, 1888. 8°. BompBay.—Bombay Branch of the Royal Asiatic Society. Journal. No. XLIII, 1885. 8°. viil Additions to the Library. BoMBAY.— Government Observatory. Magnetical and meteorological observations. 1884, 4°. Sassoon Mechanies’ Institute. Annual report. 1884-85, §&°, Bonn.—WNaturhistorischer Verein der preussischen Pivots Westfalens und des Reg.—Bezirks Osnabriick. Verhandlungen. Jahrg. XLII, XLII. 1, 1885. 8°. Autoren- und Sachregister zu Bd. I-XL. 8°. BorDEAUX.—Académie Nationale des Sciences, Belles-Lettres et Arts. Actes. Année XLIV-XLVI, 1882-84, 8°. Société Linnéenne. Actes. Tome XXXVII, XX XVIII, 1883-84. 8°. Société des Sciences Physiques et Naturelles. Mémoires. 3° sér. Tome I, II. 1, appendice i, ii, 1884-85. 8°. BREMEN.—WNaturwissenschaftlicher Verein. Abhandlungen. Bd. IX. 2, 3, 1885-86. 8°, BRESLAU.—Schlesische Gesellschaft fiir vaterlindische Cultur. Jahres-Bericht. LXII, 1884. 8°. Brtnn.—WNaturforscher Verein. Verhandlungen. Bd. XXIII, 1884. 8°. Bericht der meteorologischen Commission. 1883. 8°. BRUXELLES.— Académie Royale des Sciences, des Lettres et des Beaux-Arts de Belgique. Mémoires. Tome XLV, 1884. 4°. Mémoires couronnés et mémoires des savants étrangers. Tome XLV, XLVI, 1883-84. 4°. Mémoires couronnés et autres mémoires. Tome XXXVI, 1884, 8°. Bulletins. 3° sér, Tome VI-VIII, 1883-84, 8°, Annuaire. 1884, 1885. 8°, Société Royale Belge de Géographie. Bulletin. Année IX, 1885. 8°. —Société Entomologique de Belgique. Annales. Tome XXIX, 1885. 89. Societé Royale Malacologique de Belgique. Annales. Tome, XV, XIX, XX, 1880-85. 8°. Procés-verbaux. Tome XIV, XV pp. 1-96, 1885-86. 8°. Statuts. 2¢éd. 1886. 8°. ——Société Royale de Botanique. Bulletin. Tome XXIV, XXVI, 1885-86. 8°. Musée Royal de V Histoire Naturelle. Bulletin. Tome III. 3, 4, IV. 1, 2, 3, 1884-85, 1885-86. 8°. Bupaprst.—Kon. ung. Central-Anstalt fiir Metecorologie und Hrdmagnetismus. Jahrbiicher. Jahrg. X-XIV, 1880-84, 4°. Bugenos AirEs.—Sociedad Cientifico. Argentina. Anales. Tome XIX. 4, XX, XXI, XXII. 1-4, 1885-86. 8°. Examen de la propuesta y proyecto del puerto del Sr. D. Eduardo Madero. Por Luis A. Huergo. Parte i, ii, 1886. 8°. CaLcuTrTa.—Asiatic Society of Bengal. Journal. Vol, LIM. ii. 3, LIV. i, ii, LV. i. 1, 2, ii. 1, 2, 1884-86. 8°. Proceedings. 1885, 1886, No. 1-7. 8°, pengene Pye sortonr of the Asiatic Society of Bengal anne 1784 to 1883. 5 Geological Survey of India, ' ; ag are el Indica. Ser, II, vol. i. 2 fasc. 5; IV, vol. i. 5; X, vol. . 6-8, iv. 1, 2; XIII, vol. i. 4 fase. 5, 5; XIV, vol. i. 3 fase. 5, 6; pee as 4°, Memoirs. . Vol. XXI. 8, 4, 1885. 8°. Records. Vol. XVIII. 3, 4, XIX, 1884-85. 8°, 9 «* Additions to the Library. ix CaLcuTtTa.—WMeteorological Department of the Government of India. Indian meteorological memoirs. Vol. II. 3, 4, III. 1, IV. 1, 1884-86. fo. Report on the meteorology of India. 1883, 1884. f°. Report on the administration of the meteorological department. 1883-84. £o% Meteorological observations recorded at six stations in India, 1885, 1886, Jan.-June. f°. CAMBRIDGE.—Philosophical Society. Transactions. Vol. XIII. 3, 1885. 4°. Proceedings. Vol. V. 4, 5, 1885-86. 8°. CATANIA.—Accademia Gioenia di Scienze Naturali. Atti. Serie II]. Tomo XIX, 1886. 4°. CHERBOURG.—Société Nationale des Sciences Naturelles. Mémoires. - Tome XXIV, 1884. &9. Catalogue de la bibliothéque. II. 3, 1883. 8°. CuRISTIANIA.—Kong. Norske Universitet. Antinoos; eine kunstarchaologische Untersuchung. Von Dr. L. Dietrich- son. Universitatsprogramm. 1854. 8°. Om humanisten og satirikeren Johan Laurenberg. Af Dr. L. Daae. Universitets-program. 1884. 8°. ——Norwegische Commission der Huropiischen Gradmessung. Geodatische Arbeiten. Heft IV, 1885. 49. Vandstandsobservationer. Heft. III, 1885. 49. Norwegisches meteorologisches Institut. Jahrbuch. 1881-84. 4°. Norwegian North- Atlantic Expedition, 1876-78. Publication XIV, XV, 1885-86. 4°. — Videnskabs Selskabet. Forhandlingar, 1884, 1885. 8°. Cuur.—WNaturforschende Gesellschaft Graubiindens. Jahresbericht. Neue Folge. Jahrg. XXVIII, 1883-84. 8°. CorpoBa.—Academia Nacional de Ciencias. Actas. Tomo III. 2, IV. 1, V. 2, 1878-84. 4°. Boletin. Tomo II. 1, 3, 4, III. 5, VI. 2, 3, VIII. 2-4, 1875-86. 8°, Informe oficial de la comision cientifica de la expedicion al Rio Negro en 1879. Entrega I-III, 1881-82. 4°. Danzic.—WNaturforschende Gesellschaft. Schriften. Neue Folge. Bd. VI. 2, 3, 1885-86. 8°. Dison.—Académie des Sciences, Arts et Belles-Lettres. Mémoires. 3° sér. Tome VIII, 1883-84. 8°. ' Dorpat.—Gelehrte Estnische Gesellschaft. Sitzungsberichte. 1885. 8°. Natur forscher- Gesellschaft. Archiv fiir die Naturkunde Liy-Ehst- und Kurlands. Ser. I. Bd. IX. 3. etl Pb Cwpk el Soo. | 8°. 7 Sitzungsberichte. Bd. VII, 2, 1885.- 8°. DRESDEN.-—Waturwissenschaftliche Gesellschaft Isis. Sitzungsberichte und Abhandlungen. 1884, 1885 Jan.—June. 8°. Verein fiir Hrdkunde. Jahresbericht. XXI, 1885. 8°. Verzeichniss von Forschern in wissenschaftlicher Landes- und Volkskunde Mittel-Europas. Bearbeitet von Paul Emil Richter. 1856. 8°. Dusiin.—Royal Geological Society of Ireland. Journal. Vol. XVI. 3, XVII. 1, 1886, §&°. Xx Additions to the Library. Dusiin.—Royal Lrish Academy. Transactions. Vol. XXVIII. 17-20, 1884-85. 4°. Proceedings. Ser. If. Science. Vol. IV. 3, 4. Polite Literature and Antiquities. Vol. II. 6. 1885, 8°. EDINBURGH.— Geological Society. Transactions. Vol. IV. 3, VI, 1883-85. 8°. Royal Observatory. Astronomical observations. Vol XV, 1878-86. 4°. Micrometrical measures of gaseous spectra under high dispersion. By C. Piazzi Smyth. 1886. 4°. Royal Physical Society. Proceedings. Vol. VIII, 1883-85. 8°. Empen.—WNaturforschende Gesellschaft. Jabresbericht. LXX, 1884-85. 8°. FIRENZE.— Biblioteca Nazionale Centrale. Bollettino delle pubblicazioni Italiane ricevute per diritto di stampa. 1886, No. 1-24. 8°. ; —— -R. Istituto di Studi Superiori Pratici e di Perfezionamento. Pubblicazioni. Sezione di filosofia e di filologia : Della interpetrazione panteistica di Platone. Di Alessandro Chiappelli. USS ss Stato e chiesa negli scritti politici A. D. 1122-1347. Studio storico di Francesco Scaduto. 1882. &°. L’invito di Eudossia a Genserico. Studio critico del Prof. Guiseppe Morosi. 1882. 8°. fl primo sinologo C. Matteo Ricci. Per Lodovico Nocentini. 1882. 8°. —Sezione de scienze fisiche e naturali: Sulle convulsioni epilettiche per veleni. Ricerche dei Dottori A. Rovighi eG. Santini. 1882. 8°. ——Sezione di medicina e chirurgia: Archivio della scuola d’anatomia patologica. Vol. I, 1881. 8°. FRANKFURT A. M.—Deutsche malakozoologische Gesellschaft. Nachrichtsblatt. Jahrg. XVII. 7-11, XVIII. 1-10, 1885-86. 8°. Senckenbergische naturforschende Gesellschaft. Abhandlungen. Bd. XIV. 1, 1886. 4°. Bericht. 1885. 8°. Reiseerinnerungen aus Algerien. Von Dr. W. Kobelt. 1885. 8°. GENEVE.—Jnstitut National Genevois. Bulletin. Tome XXV, XXVI, 1884-85. 8°. Societé de Physique et @ Histoire Naturelle. Mémoires. Tome XXIX. 1, 1884-85. 4°. GENOVA,— Museo Civico di Storia Naturale. Annali. Vol. XVITI-XXII, 1882-85. 8°. G1ESSEN.— Oberhessische Gesellschaft fiir Natur- und Heilkunde. Bericht. XXIV, 1886. 8°. GLASGOW.— Geological Society. ; Transactions. Vol. I-VII, 1860-84. 8°. Natural History Society. Proceedings and transactions. New Series. Vol. I. 2, 1884-85, 8°. Index to proceedings, vol. I-V. 1885. 8°. Philosophical Society. Proceedings. Vol. XVI, 1884-85. 8°. G6TTINGEN.—K Onigl. Gesellschaft der Wissenschaften. Nachrichten. 1885. 8°. Gistrow.— Verein der Freunde der Naturgeschichte in Mecklenburg. Archiv. Jahrg, XX XTX, 1885. 8°. Additions to the Library. xi Hapana.—Real Colegio de Belen. Observaciones magneticas y meteorologicas. 1885 i-iii. 4°. Havie.—Kais. Leopoldinisch- Carolinische deutsche Akademie der Naturforscher. Leopoldina. Heft XXI, 1885. 4°. Naturforschende Gesellschaft. Abhandlungen. Bd. XVI. 3, 1585. 4°. Bericht. 1884. 4°. Naturwissenschaftlicher Verein fiir Sachsen und Thitringen. Zeitschrift fiir die gesammten Naturwissenschaften. Bd. LVIII. 2-6, LIX. 1, 2, 1885-86. 8°. HamBure.—Deutsche Seewarte. Arehiv. Jahrg. VI, VII, 1883-84. 40°. Monatliche Uebersicht der Witterung. 1884 Nov., Dez.; 1885. 8°. Wissenschaftliche Anstalten. Jahrbuch. Jahrg. II, III, 1885-86. 8°, HAnnover.—WNaturhistorische Gesellschaft. Jahresbericht. XX XIII, 1883-84. 8°. HarLeM,— Musée Teyler. Archives. Série II. Vol. II. 2-4, 1885-86. 8°. Catalogue de la bibliothéque. Livr. 1-4, 1885-86. 8°. Société Hollandaise des Sciences. Archives néerlandaises des sciences exactes et naturelles. Tome XX, XXI. 1, 1885-86. 8°. HELSINGFORS. tia Scientiarum Hennica. Acta. Tom. XIV, 1885. 4°. Ofversigt af forhandlingar. XXVI, 1883-84. 8°. Bidrag till kannedom af Finlands natur och folk. Haft. XXIXX-XLII, 1884-85. 8°. Societas pro Fauna et Flora Fennica. Acta. Vol. II, 1881-85. 8°. Meddelanden. Haft. XII, XIII, 1855-86. 8°. Beobachtungen tiber die periodischen Erscheinungen des Pflanzenlebens in Finland. 1883. 4°. Hopart.—fRoyal Society of Tasmania. Catalogue of the library. 1885. 8°. HoneKxone.— Observatory. Observations and researches. 1885. f°. JENA.— Medicinisch-naturwissenschaftliche Gesellschaft. Jenaische Zeitschrift fiir Naturwissenschaft. Bd. XVIII. 4, XIX. 1-4, Supplement 1, 2, 1885-86. 8°. KiEL.—Naturwissenschaftlicher Verein fiir Schleswig- Holstein. Schriften. Bd. VI. 2, 1886. 8°. — Christian: Albrechts- Universitit. Dissertationen, etc., 1884-85, (38); 1885-86, (80). KsbBennavn.—Kon. Diie Videnskabernes Selskab. Oversigt over forhandlinger. 1884 iii, 1885, 1886 i. 8°. KoniesBerc.—Kénigl. physikalisch-ikonomische Gesellschaft. Schriften. Jahrg. XXV, XXVI, 1884-85. 4°. ® Krakow.—K. k. Sternwarte. Materyaly do klimatografii Galicyi. Rok 1884-85. 8°. LAUSANNE.—Société Vaudoise des Sciences Naturelles. Bulletin. 2¢ sér. No. 92, 93, 1885-86. 8°. _ Leeps.— Yorkshire Geological and Polytechnic Society. Proceedings. New series. Vol. IX. 1, 1885, 8°. va xii - Additions to the Library. Leiprn.—WNederlandsche Dierkundige Vereeniging. Tijdschrift. Ser. II. Deel [. 1, 2, 1885. 8°. Lurrpzie.—Astronomische Gesellschaft. Vierteljahrsschrift. Jahrg. XX, XXI. 1, 2, 4, 1885-86. 8°. Publication. XVIII, 1886. 4°. Kon. stichsische Gesellschaft der Wissenschaften. Berichte. Math.-physische Classe. Bd. XXXVI, XXXVII, 1884-85. 8°. Naturforschende Gesellschaft. Sitzungsberichte. Jahrg. Xi, XII, 1884-85. 8°. Zoologischer Anzeiger. No. 198-240, 1885-86. 8°. Li&e@E.—Société Géologique de Belgique. Annales. Tome X, XII, 1882-83, 1884-85. 8°. Catalogue des ouvrages de géologie, de minéralogie et de paléontologie ainsi que des cartes géologiques qui se trouvent dans les pee bibliothéques de Belgique. Par G. Dewalque. 1884, 8°, Société Royale des Sciences. Mémoires. 2°sér. Tome XI, XII, 1885. 8°. LisBpoa.—Sociedade de Geographia. Boletin. Serie IV. 12, V. 1, 2, 4-12, VI. 1-6, 1884-86. 8°. Subsidios para a historia do jornalismo nas provincias ultramarinas Portu- guezas. Por Brito Aranha. 1885. 8°. LiIvERPOOL.—Literary and Philosophical Society. Proceedings. No. XX XVIII, 1883-84. 8°. Lonpon.— Geological Society. Quarterly jqurnal. Vol. XLI.3, 4, XLII, 1885-86. 8°. List. 1886. 8°. Linnean Society. Journal. Zoology, no. 103-113; Botany, no. 134-144, 150. 1884-86, 8°, List. 1884-85, 1885-86. 8°. ‘ Index perfectus ad Caroli Linnzi species plantanum nempe earum primam editionem, collatore F. de Mueller, Melbourne, 1880, 8°. Mathematical Society. Proceedings. No. 240-272, 1884-86. 8°. > Royal Meteorological Society. Quarterly journal. New series. No, 55-60, 1885-86. 8°. List of fellows. 1885. 8°. Royal Historical Society. Transactions. New series. Vol. III. 1, 2, 1885-86. 8°. ——Royal Microscopical Society. Journal. Ser. II. Vol. V. 3-6, VI, 1885-86. 8°. Royal Society. Philosophical transactions. Vol. CLXXV, CLXXYVI, 1884-85. 4°, Proceedings. No. 232-247, 1885-86. 8°. List of council and members. 1884, 1885. 4°. Lunp,- - Universitet, Acta. Tom. XIX, XXI, 1882-83.. 4°. Universitets-biblioteks acecssions-katalog. 1883, 1885. 8°. Lyon.—Académie des Sciences, Belles-Lettres et Arts. Mémoires. Classe des sciences. Tome XXVIII, 1885, 8°. Recherches historiques sur les mots plantes males et plantes femelles. Par le Dr. Saint-Lager. Paris, 1884, 8°. Musée Guimet. Annales. Tome VIII, IX, 1885-86. 4°. Revue de Vhistoire des religions. Tome XI. 2, 3, XII, XI, XLV. 15 1885-86. 8°, , - — «°°. . ae Additions to the Library. xii] MApDRAS.— Government Observatory. Magnetical observations, 1851-55. Madras, 1884, 4°. Telegraphic determination of difference of longitude. 1884. 4°. Administrative report of the meteorological reporter for 1884-85. 8°. Meteorological observations at Singapore, 1841-45. Madras, 1851. 4°. Manprip.— Comision del Mapa Geologico de Espana. Boletin. Tomo XII. 1, XIII. 1, 1885-86. 8°. Memorias. Descripcion fisica y geologica de la provincia de Guipuzcoa. Por D. Ramon Adan de Yarza, 1884. 5°. Sociedad Espanola de Historia Natural. Anales. Tomo XIV. 3, XV. 1, 2, 1885-86. 8°. Artropodos del viaje al Pacifico verificado de 1862 a 1865 por una comision de naturalistas enviada por el gobierno Espanol. Insectos neuropteros y ortopteros, por Ignacio Bolivar. 1884. 4°. MaGpEBurG.—WNaturwissenschaftlicher Verein. Jahresbericht und Abhandlungen. 1885. 8°. MARBuRG.— Gesellschaft zur Beforderung der gesammten Naturwissenschaften. Sitzungsberichte. Jahrg. 1884, 1885. 68°. Metz,—Académie. Mémoires. 3° sér. Année XII, 1882-83. 8°. Mexico.— Museo Nacional. Anales. Tomo III. 7-10, 1885-86, 4°. Ministerio de Fomento. Boletin. Seccion meteorologica. Tomo X 43-146, 1885-86. f°. Estudios de meteorologia comparada. Por Mariano Barcena y Miguel Perez. Tomo I, 1885. 8°. Sociedad Mexicana de Historia Natural. La naturaleza. Tomo VII. 5-18, 1885-86. 4°. MIDDELBURG.—Zeeuwsch Genootschap der Wetenschappen. Archief. Deel VI. 1, 2, 1885-86. 4°. Naamlijst van directeuren enleden. Verslag van het verhandelde in de algemeene vergadering, 1880-84, 8°. Mixiano —Real Istituto Lombardo di Scienze e Lettere. Rendiconto. Serie II. Vol. XVII, 1884. 8°. Mopena.— Regia Accademia delle Scienze, Lettere ed Arti. Memorie. Tomo XX. Serie II. Tomo II, 1885. 4°. Societa dei Naturalisti. Memorie. Ser. III. Vol. IJ]-IV, 1853-85. 8°. Rendiconti. Ser. II. Vol. I pp. 105-140, IT pp. 1-178, 1883-86. 8°. MONTPELLIER.—Académie des Sciences et Lettres. Mémoires. Section des lettres. Tome VII. 2, 3, 1884-86. 4°. Section des sciences. Tome X. 3, 1883-84. 4°, Section de médecine. Tome VI. 1, 1885-6, 4°, MontTrReAL.— British Association for the Advancement of Science. Canadian economics. 1885. 8°. Natural History Society. The Canadian record of science. Vol. I. 3, 4, II. 1-4, 1885-86. 8°. Moscovu.—Sociéte Impériale des Naturalistes. Nouveaux mémoires. Tome XY. 1-3, 1584-85. 4°. Bulletin. 1884 ii-iv, 1885. 8°. Meteorologische Beobachtungen am Obseryatorium der Jandwirth. Akad- emie zu Moskau, Jahrg. 1886i. 4°. Xiv Additions to the Library. Minonen.—Kén. bayerische Akademie der Wissenschaften. Sitzungsberichte. Philosoph.-philolog. und histor. Classe. 1885, 8°, — Mathemat.-physikal. Classe. 1885. 8°. J. A. Schmeller. Eine Denkrede von Konrad Hofmann. 1885, 4°, Sage und Forschung. Festrede von F. Ohlenschlager. 1885. 4°. Zum. Begriff und Wesen der romischen Provinz. Festrede von Alois von Brinz. 1885. 4°. Konigliche Sternwarte. Annalen. Supplbd. X, XIV, 1871-84. 8°. Minsrer.— Westfiilischer Provincial- Verein fiir Wissenschatt und Kunst. Jahresbericht. XIII, (884. 8°. Nanoy.—Académie de Stanislas. Mémoires. 5° sér. Tome II, III, 1884-86. 8°. Napoit.—R. Accademia delle Scienze Fisiche e Matematiche. , Rediconto. Anno XXII-XXIV, XXYV. 1-8, 1883-86. 4°. ZLoologische Station. a Mittheilungen. Bd. VI. 2-4, 1885-86. 8°. NEWCASTLE-UPON-TYNE.— North of England Institute of Mining and Mechanical Engineers. . Transactions. Vol. XXXIV. 46, XX XV. 1-4, 1885-86. 8°. Ntrnpere.—WNaturhistorische Gesellschaft. Jahresbericht. 1885. 8°. OpEssa.—Socicté des Natwralistes dela Nouvelle Russie. Zapiski. Tom. IX. 2, X, XI. 1, 1885-86. 8°. Supplément, 1886. 4°. —Matematicheskoe otdielenie. Tom. I-VI, 1878-85. 8°. Orrawa.— Geoloyical and Natural History Survey of Canada. Report of progress, 1862-83-84, with maps. Montreal, 1885. 8°. Summary report, 1885. 8°. Contributions to Canadian paleontology. Vol. I. 1. Montreal, 1885. 8°. Royal Society of Canada. Proceedings and transactions. Vol. I, III, i884-85. 4°. OxrorvD.— Radcliffe Library. Catalogue of books added, 1884, 1885. 8°. mi PALERMO.—R. Accademia di Scienze, Lettere e Belle Arti. Bollettino. Anno II, JIT. 1=3, 1885-86. 49. Paris.— Ecole Polytechnique. Journal. Cahier LV, 1885. 4°. Catalogue de la bibliothéque. Société Nationale d Acclimatation. . Bulletin. 4° sér. Tome II. 6-12, III. 1, 3-8, 10, 1885-86. 8°. Société Géologique de France. Bulletin. 38° sér. Tome XII. 9, XIII, XIV. 1-7, 1884-86. 8°. Société Mathématique de France. Bulletin. Tome IX. 5, X. 3, XI. 3,5, XIII. 5, 6, XTV.1-4, 1881-86. 8°. Pisa.—Societd Toscana di Scienze Natwrali. Memorie. Vol. VI. 2, VII, 1885-86." 89. Processi verbali. Vol. IV pp. 231-262, V pp. 1-118, 1885-86. 89. PoTtsDAM.—Astrophysikalisches Observatorium. Publicationen. Bd. IV. 1, V, 1885-86. 4°. Prag.—K. k. Sternwarte. : Magnetische und meteorologische Beobachtungen. Jahrg. XLV, XLVI, 1884-85, 4°, Astronomische Beobachtungen, 1884, 4°. EE Additions to the Library. XV PuLKova.—WNicolai-Hauptsternwarte. Jahresbericht. 1882-83, 1883-84, 1884-85. 8°. Tabule quantitatum Besselianarum pro annis 1885 ad 1889. Ed. Otto Struve. Petrop., 1885. 8°. Die Beschliisse der Washingtoner Meridianconferenz. Von Otto Struve. St. Petersb., 1885. &°. REGENSBURG.—Zoologisch-mineralogischer Verein. ‘ Correspondenz-Blatt. Jahrg. XX XIX, 1885. 8°. Historischer Verein von Oberpfalz und Regensburg. Verhandlungen. Bd. XX XVIII, XXXIX, 1884-85. 8°. Riga.— Naturforscher Verein. Correspondenzblatt. Jahrg. XX VII-X XIX, 1884-86. 8°, Rio DE JANEIRO. — Instituto Historico, Geographico e Ethnographico do Brasil. Revista trimensal. Tomo XLVI-XLVII, 1883-84. 8°. Catalogo dos manuscriptos, 1884, 8°. Catalogo das cartas geographicas, hydrographicas, atlas, planos e vistas existentes no bibliotheca. 1885. 8°. Museu Nacional. Archivos. Vol. VI, 1885. 4°. Lettre a M. Ernest Renan a propos de l’inscription phénicienne apocryphe. Par Ladislau Netto. 1885. 8°. Conférence faite au muséum national le 4 Noy. 1884. Par Ladislau Netto. 1885. 8°. Roma.—Biblioteca Nazionale Centrale Vittorio Hmanuele. Bollettino delle opere moderne Sstraniere acquistate dalle biblioteche pubbliche governative del regno d'Italia. 1886, No. 1-4. 8°. Reale Accademia dei Lincei. Memorie della classe di scienze morali, storiche e filologiche. Ser. III. Vol. VIII, X, XI, XIII, 1883-84. 40°. Memorie della classe di scienze fisiche, matematiche e naturali. Ser. III. Vol. XIV-XIX. Ser. 4. Vol. II, 1883-85. 4°. -Rendiconti. Vol. I. 13-25, 27, 28, II. i. 1, 4-14; ii. 1-11; 1885-86. 4°, Annuario. 1856. 16°. Reale Comitato Geologico @ Italia. Bollettino. Vol. XV—XVI, 1884-85. 8°. Relazione sul servizio minerario nel 1882. 8°. —— Ufficio Centrale di Meteorologia Italiana. Annali. Ser. II. Vol. V, 1888. 4° RorrerRDAM.—Satauvsch Genootschap der Proefondervindelijke Wijsbegeerte. Nieuwe verhandelingen. 2de reeks. Deel III. 2, 1885. 4°. Sr. GALLEN.—Naturwissenschaftliche Gesellschaft. Bericht. 1883-84. 8°. St. PerersBurG.— Comité Géologique. Mémoires. Tome I. 1-4; IT. 1-3, ITI. 1, 1883-85. 4°. Bulletins. Tome I-III, IV. 1-7, V. 1-6, 1882-86. 8°. Bibliotheque géologique de la Russie. I, 1885. 8°. Hortus Petropolitanus. Acta. Tom. IX. 2, 1886. 8°. ——Imp. Russ. Geograf, Obshtchestvo. Otchet. God 1885. 8°. Kais. Akademie der Wissenschaften. Repertorium der Meteorologie. Bd. IX, 1885. 4°. ——Physikalisches Centralobservatorium.. Annalen. Jahrg. 1884. 4°. Schweizerische naturforschende Gesellschaft. Verhandlungen. Jahresversammlung LXVII, LX VIII, 1884-85. 8°. « Xvi Additions to the Library. SrockHoLm.—Zntomologisk Porening. Entomologisk tidskrift. Arg. VI, 1885. 8°. Kong. Svenska Vetenskaps Akademien. Handlingar. Ny foljd. Bd. XVIII, XTX, 1880-81. 4°. Bihang. Bd. VI-VIII, 1881-83. 8°. Ofversigt. Arg. XXXVIII-XL, 1878-79. 8°. Meteorologiska iagttagelser. Bd. XX, XXI, 1878-79. 4°. Lefnadsteckningar. Bd. II. 2, 1883. 8°. SrurreaRT.— Verein fiir vaterliindische Naturkunde in Wirttemberg. Jahreshefte. Jahrg. XLI, XLII, 1885-86. 8°» SypneEY.— Observatory. Results of rain and river observations, 1885. 8°. Royal Society of New South Wales. Journal and proceedings. Vol. XVIII, 1884. 8°. Annual report of the department of mines, New South Wales. 1885. f°. TacuBAYa.— Observatorio Astronomico Nacional. Anuario. Ano XI, 1886. 89°. Coordenadas geograficas, determinadas por Angel Anguiano. Mexico, 1886. 8°. THRONDHJEM.—Kon. Norske Videnskabers Selskab. Skrifter. 1884. 8°. TiFiis.—Physicalisches Observatorium. Magnetische Beobachtungen. 1879-1883. 8°. Meteorologische Beobachtungen. 1878-1884. 8°. : Beobachtungen der Temperatur des Erdbodens. 1880-83. 8°. Tox1o.—Imperial University of Japan. Abhandlungen. No. XI, 1885. 4°. Calendar. 1886-87. 8°. Seismological Society of Japan. Transactions. Vol. VII. 1, IX, 1885-86. 8°. Torino.—Musei di Zoologia ed Anatomia Comparata, Bollettino. Vol. I. 1-15, 1886. 8°. Toronro.— Canadian Institute. Proceedings. Ser. III. Vol. III. 2, IV. 1, 1885-86. Meteorological Service of the Dominion of Canada. Report. 1881. Ottawa, 1883. 8°. TouLousn.— Académie des Sciences, Inscriptions et Belles-Lettres. Mémoires. 8 sér. Tome VI, VII, 1884-85. 8°. Upsata.—Regia Societas Scientiarum. Nova acta. Ser. III. Vol. XII. 2, XIII. 1, 1885-86. 4°. Urrecur.—Kon. Nederlandsch Meteorologisch Instituut. Nederlandsch meteorologisch jaarboek. 1885, 4°. Provinciaal Utrechtsch Genootschap van Kunsten en Wetenschappen. Verslag van het verhandelde in de algemeene vergadering. 1885, 8°. Aanteekeningen van het verhandelde in de sectie-vergaderingen. 1884-85. tofer ‘ Vennzia.—IJstituto Veneto di Scienze, Lettere ed Arti. Atti. Ser. VI. Vol. II. 3-10, III. 1-9, 1883-85. 8°. Notarisia. Anno I. 1, 2, 1886. 8°. VicENZA.—Accademia Olimpica. Atti. Vol. XVIII, 1883. 38°. Wien.—Kais. Akademie der Wissenschaften. Sitzungsberichte. Mathemat.-naturwiss. Classe. Abth. I. Bd. XC, XCI. 1-4, 1884-85. 8°. ——~K. k. Central-Anstalt fiir Meteorologie und Erdmagnetismus. Jahrbiicher. Neue Folge. XX, X XI, 1883-84. 4°. Additions to the Library. Xvii Wien.—K&K. k. geologische Reichsanstalt. Jahrbuch. Bd. XXXV, XXXVI. 1, 1885-86. 8°. Verhandlungen. Jahrg. 1885, 1886 i-iv. 8°. Wien.—K. k. Naturhistorisches Hofmuseum. Annalen. Bd. I. 1, 2,4, 1886. 8°. — K. k. zoologisch-botanische Gesellschaft. Verhandlungen. Bd. XX XIV-XXXVI,.1884-86. 8°. Oesterreichische Gesellschaft fiir Meteorologie. Zeitschrift. Bd. XX. 7-12, 1885. 8°. WIESBADEN.—WNassauischer Verein fiir Naturkunde. Jahrbiicher. Jahrg. XX XIX, 1886. 8°. WtrzpurG.—Physikalisch-medicinische Gesellschaft. Sitzungsberichte. Jahrg. 1885. 8°. ZtvRicH.—Naturforschende Gesellschaft. Vierteljahrsschrift. Jahrg. XX VI-X XIX, 1881-84. 8°. Barus, (Carl) and Strouhal, (Vincent). The electrical and magnetic properties of the iron carburets. Washington, 1885. 8°. From the Authors. Harden, William. A suggestion as to the origin of the, plan of Savannah. Savannah. 1885. 8°. From, the Author. Hirn, G. A. Notice sur les lois du frottement. Paris, 1884. 4°. From the Author. Klossovsky, A. Les orages au sud de la Russie. Odessa, 1886. 8°. Les orages en Russie. Odessa, 1886. 8°. From the Author. Lewis, H. Carvill. Marginal kames. Philadelphia, 1886. 8°. A great trap dyke across Southeastern Pennsylvania. Philadelphia, 1885. 8°, From the Author. MacLeod, Jules. La structure des trachées et la circulation péritrachéenne. Bruxelles, 1880. 8°. Prom the Author. Pickering, Edward C. An investigation in stellar photography conducted at the Harvard College Observatory. Camb. 1886. 4°. From the Author. Russell, H. C. Local variations and vibrations of the earth’s surface. Sydney, 1885. 8°, Annual address, Royal Society of New South Wales. Sydney, 1885. 8°. From the Author. Searle, Arthur, The apparent position of the zodiacal light. Boston. 4°. From the Author. I.—On tHe Law or Error in Tarcer-Snootine. By E. L. De Forxst, Watertown, Conn. THE complete expression for the symmetrical law of error in the position of points in a plane is h h lxd. 2 9,,9 g—- — I o— (hia +ho y’), (1) where z denotes the probability that an error committed will fall within any small rectangle dzdy whose codrdinates, at its middle point, are z and y. The axes should be taken to coincide with the free axes of the group of shot-marks, when these last are regarded as the masses of material points all equal to each other. The origin is at their centre of gravity, and is the point for which the proba- bility zis a maximum. (Compare my article in The Analyst, Des Moines, Iowa, vol. viii, p. 73.) Though dz and dy are in strictness infinitesimals, the formula is evidently approximately true when they are regarded as any small finite distances. Points for which z is a given quantity will lie in an ellipse, and all such ellipses are similar and concentric as long as the constants 4, and h, remain the same. These are determined by the relations 1 1 h,= p,/2” h,= ee where p, and , are the quadratic mean errors in the « and y direc- tions. If the probability of deviation from the maximum is the same in all directions, then P=, + p,=2p,'= 2p," (3) is the squared q. m. error measured directly from the origin, and (2) ii : Az=h=h.=— 4 Caer (4) is the constant to be introduced in (1). Denoting «*+-y’ by 2”, (1) is reduced to ) . Iida dy —hir? => ———“e 5 se (5) where the ellipses of equal probability have become circles, and the axes may be taken in any convenient direction. As this formula is TRANS. CoNN. ACAD., Vou. VII. 1 SEPT., 1885, 2 EF. L. De Forest—Law of Error in Target-Shooting. the simplest, it is often adopted in discussing. the errors of target- shooting. Even when the form (1) is retained, it is robbed of a portion of its generality by assuming that the axes of X and Y are respectively horizontal and vertical, instead of being coincident with the free axes. Although this assumption seems to have been uni- versally made, it has appeared to me to be of doubtful propriety. The only reasons I have seen stated for its adoption are, that errors caused by the wind are horizontal, while those which depend on the range and the force of gravity are vertical. This takes no account of errors produced by other causes, such as defects or peculiarities in the weapon, imperfect sighting, and fatigue or nervousness in the marksman. ‘These may act obliquely, and so far as we know, are as likely to occur in one direction as another. As a result of accident, it will happen in general, that the centre of gravity of the shot-marks does not exactly coincide with the true | point aimed at, namely, the centre of the target. Accidental devia- tions from the centre of gravity, or from the free axes drawn through it, are thus of the nature of residual errors, while such deviations from the centre of the target, or from axes drawn through it parallel to the former ones, are of the nature of true errors. In any given case, we can compute the amount of probable deviation of the centre of gravity from the centre of the target. If the actual deviation falls within this amount, or does not much exceed it, we may pre- sume that it is purely accidental, and shifting the position of the computed probability surface (1) so as to make its origin coincide with the centre of the target while its codrdinate axes remain par- allel to their former positions, we shall have the law of probability of error for future shots. But if the actual deviation is far beyond the probable amount, it indicates the probable existence of some constant causes of error, likely to affect future shots in the same way, and the probability surface must not be shifted, unless we also correct the aim of future shots to correspond with it. It will in general happen also, as the result of accident, that an actual group of shot-marks will be more elongated in one direction than in the direction at right angles to it, so that one of the squared q. m. errors ,° p,’ will be greater than the other, even when the probability of error is really the same in all directions. The con- stants A, and h, computed by (2) will thus appear to be different, and the law of error will seem to be as in (1), when it is really of, the simpler form (5). But here too, in any given case, we can com- 2 pute the amount of probable difference between p,’ and p,’*, sappos- =< oN phe oe - = y ? E. L, De Forest—Law of Error in Target-Shooting. 2 ing it to be accidental. Then if the actual difference falls within this amount, or does not much exceed it, we may presume that the probability of error is really the same in all directions, and that formula (5) may be properly used, the axes being taken horizontal and vertical, simply because those directions are most convenient. On the other hand, if the actual difference between p,? and ,’ is much in excess of its probable value, we must presume that the ob- served elongation of the group of shot-marks is due to constant causes, likely to have a similar effect on future shots, so that (1) is the most suitable formula to express the law of error, and the axes assumed should be the free axes of the group. It has seemed to me that the question whether formula (1) should be used, and if so, whether the coérdinate axes should be made coincident with the free axes, is not a mere matter of opinion, but should be decided by some definite test. like the above, applied to an extended set of observa- tions. The most suitable observations for this purpose within my reach are those given by Didion at the close of his Caleul des Proba- bilités appliqué au Tir des Projectiles. Paris, 1858. His first table gives the positions of 125 shot-marks made by spherical bullets, fired from a rifled pistol at 50 metres, under a charge of one gramme of powder. The weapon was placed on a rest, and aimed ata point 0°430 metres above the centre of the target. The positions of the shot-marks were referred to axes taken hori- zontally and vertically through that centre. The arith. mean of their ordinates is the ordinate of their centre of gravity, and the arith. mean of their abscissas is its abscissa. We easily find the abscissa «w and ordinate v of each shot-mark, referred to axes taken horizontally and vertically through the centre of gravity, and ex- pressed in centimeters. The sums of their squares and of their _ products are pe [we] = 44211, [v7] = 55766, [wv] = — 6655. The angle m which a free axis makes with the U axis is given by tan 2Q9 = at? tual (6) [u"]—[o*y Hence log. tan 2—~=:06140, and Ga 240 31 or Gis iy, (7) These two values, differing by 90°, represent the inclinations of the two free axes of X and Y to the U axis. Denoting them by gy’ and gp’ +90°, the codrdinates of a shot-mark referred to the free axes will be x= ucos p'+ vsin g’, y=VvCOS g — usin g’, (8) 4 E. L. De Forest—Law of Error in Target-Shooting. and the sums of their squares are [a*] = [v’] cos’ p+ [v7] sin’ g' + [w] sin 29", (9) [y?] = [v’] cos’ gp! + [u’] sin’ p’— [wv] sin 29’, from which we readily find [a] = 41172 [y?] = 58806. The squared q. m. errors are therefore a ; p= sea = 332°03, ie Bue = 474-24, (10) where 7 is the number of shots. Since the codrdinates of the centre of gravity are the arith. means of those of the shot-marks, the semi-axes of the ellipse of probable error in the position of the centre of gravity are a= 11774. = 1°92, b= 11774 = =9-29. (11) n Vn : (Analyst, viii, p. 77). In the case we are considering, the horizontal and vertical codrdinates of the centre of the target referred to the centre of gravity are u =)-02; v = 8'25, and when referred to the X and Y axes they are by (8) a = 3°44, y = 7°50. (12) These values compared with a and 6 in (11) show that the actual dis- tance of the centre of gravity from the centre of the target is much greater than it probably would be if it were purely accidental. Hence, to represent the probabilities of error in future shots, the sur- face (1) should in this instance remain with its vertex at the centre of gravity, and not be shifted to the centre of the target, unless the aim is corrected at the same time. Having thus determined the most probable position of the origin, we wish next to know whether the actual difference between p,* and p, is much in excess of what might be expected if it were acci- dental. Its probable amount may be found approximately as fol- lows. When the q. m. error ¢ is computed from n observations, the probable error of this determination is known to be 2H AE é 6 aa (13) and consequently the probable error of «* will be 674587 4/ (14) n E.. L. De Forest—Law of Error in Target-Shooting. 5 Therefore, when ,* and p,* are computed as in (10), if the proba- bility of error in the « and y directions is really the same, the proba- ble difference between p,’ and p,*, occurring from accidental causes, will be Qe A*= 6745 —_, (15) n or, if we take approximately &=4(p,°+~,,°), 6745(p,” ; jak fires (p,"+ Ps ) (16) Vn To apply this to the case in hand, we substitute for p,* and 9,” their numerical values as in (10), and so get for the probable difference A? = 48°64. (17) The actual value 474°24 — 332°03 = 142-21, is so much larger that we are obliged to conclude that in this case it is probably not an accidental but a real difference, likely to affect the distribution of future shots in the same way. Hence (1) is the proper formula to use, and the codrdinate axes ought to be taken not horizontally and vertically, but coincident with the free axes of the group of shot-marks. Didion also gives a table of the positions of the shot-marks made by firing a pistol, apparently similar and with equal charge, 250 times at 100 metres distance, aiming at a point 1°47 m. above the centre of the target. By the same procedure as before, we find [w?] = 1392560, [»v7]=1742890, [uv] = — 301930, where uv and v are expressed in centimetres. The inclinations of the free axes to the U axis then are by (6) p= 29° 56’, g' + 90° = 119° 56’, (18) and with codrdinates referring to these axes*we have by (9) [a] = 1218600, [y?] = 1916800, so that the squared q. m. errors are by (10) p= 4894-0, p,’= 7698°0. (19) The semi-axes of the ellipse of probable error in the position of the centre of gravity are by (11) O== 5°21, ' 6 = 6°53. (20) But the horizontal and vertical coérdinates of the centre of the tar- get are Ua leT2, Oil Be 6 E.. L. De Forest—Law of Error in Target-Shooting. which referred to the free axes become a= 9°38, y = 12°85. (21) Comparing these with (20), we see that the actual distance of the centre of gravity from the centre of the target is too great to be considered accidental, and we infer as in the former case, that to rep- . resent the probabilities of future shots, the vertex of the probability surface should remain at the centre of gravity, and not be changed to the centre of the target, unless the aim is also changed. The probable difference in this case between p,* and p,* is by (16) A*= 537°2. (22) The actual difference. however is 7698°0 — 4894°0 = 2804°0, a value so much greater that we are obliged to conclude that it is probably not accidental. This is what might be expected from the results already obtained with the same kind of weapon at shorter range. If there is really a greater liability to error in one direction than in another, it will nat- urally show itself at all ranges, with only such differences as might occur by accident. The angles which the direction of greatest error makes with the X axis at the two ranges here considered g +90°= 114°31' and g@’+ 90°=119°56’, are not very different from each other. Didion finally gives the codrdinates of the trajectories of 100 can- non balls fired, under constant charges and angles of elevation, at distances of 200, 400 and 600 metres. The heights are reckoned, not from the centre of a target, but from the plane of the platform on which the gun-carriage stands. By an easy reduction, we get the codrdinates « and v referred to axes taken horizontally and vertically through the centre of gravity, and expressed in metres. For 200 metres range, we find Peet 11°62, [v7] = 16°39, [uv] = — 0°18, and (6) gives Geo las gp’ + 90°= 92° 15’. (23) Then by (9), . fet] == "11°81, [y*] = 16:40; and by (10), p= 1193, pi, ="1657, The probable difference between p,’ and p,? is by (16) A= "0102: (24) E.. L. De Forest—Law of Error in Target-Shooting. 2 The actual difference is greater, being "1657 — 1193 = *0464. We omit the discussion for the 400 and 600 metre ranges, since the shots here are apparently the same as those used at 200 metres, and the differences in relative position for the same shot at different ranges seem to be largely due to accidental variations in the projec- jectile or the powder. From the three series of trials retained, namely, two of pistol shots and one of cannon shots, it appears that the test requires us to use formula (1), and to make the codrdinate axes coincide with the free axes. This therefore, it seems to me, had better be generally done when accuracy is desired, unless indeed our results should hereafter be invalidated by those of other and more extended experiments. We might also inquire what are the probable values, arising from accidental causes, of the cubes of the c. m. inequalities in ~ and y, when the law of error in either direction is suspected to be unsym- metrical. This question finds an answer in my article on the Unsym- metrical Probability Curve, Analyst, vol. x, p. 74. See also Trans. of the Conn. Academy, vol. vi, part 1. For determining a, and a, the easiest way will perhaps be to compute the values of [w*], [v*], [wv] and [wv*]. Then from (8) we have [2*j=[:4 ]cos* p' +[v*]sin’g' } + 8sing’cosq’ ([u’v |cosg’ +[uv*|sing’) | 2 3 3 ! eh dl (25) [yJ=[o"]eos' gy —[u"]sin' p | —3ssing’cosp'([uv" |cosp’—[u'v]sing’) | and @, and a, are obtained like a in Analyst, ix, p. 161. For ex- ample, from the first table here tried we get [u*] = 322100, [u*v | = — 9600, [v*] = 264700, [ wv" | = 37400, and consequently by (7) and (29) [a] = 269207, [y*] = 133301, and the cubed inequalities are c= mle) == 21 DS78, f= aly) = 1066'5. (26) From accidental causes alone, they would probably be something like (6°) = er45p,4/ 1° = + 14137, | We t (15. 4 9413-1, | (f)') = ae 6745 ,'4/ — J 8 EF. L. De Forest—Law of Error in Target-Shooting. The actual value of ¢,° falls far within the probable amount, and that of ¢,° does not so much exceed the probable one as to make us confi- dent that it is anything more than accidental. But if both the ine- qualities were taken into account, we should have a, = 29,°> 6,°='30832 6,=/,°= 332°03 f (28) a, = 2p,’ 6,°= "88934 b,=,;=474:24 and the equation of the surface will be of the same form as in formula (34) of my article in the Zransactions. 'The sub-index of the first 6, should fall inside the bracket, instead of outside as there printed. a t « é | | . IT.—Extensions OF CERTAIN THEOREMS OF CLIFFORD AND OF CAYLEY IN THE GEOMETRY OF n DimeENsions. By ELIAKImM Hastines Moors, sr., Denver, Cotorapo. J. GENERAL THEOREMS. CiirForD, at the beginning of his “ Classification of Loci” (Mathe- matical Papers, p. 305-331), proves the following theorems : A. Every proper curve of the n” order is in a flat space of 2 dimensions or less. B. A curve of order 7 in flat space of & dimensions (and no less) may be represented, point for point, on a curve of order »—A+42 in a plane, whence C. A curve of order 7 in flat space of 2 dimensions (and no less) is always wnicursal. These theorems may be extended. Clifford’s nomenclature* and methods of proof are adhered to throughout. * For the convenience of the reader who may not have at hand a copy of Clifford’s Mathematical Papers, the definitions (p. 305-6) are given here. “By a curve we mean a continuous one-dimensional aggregate of any sort of ele- ments, and therefore not merely a curve in the ordinary geometrical sense, but also a singly infinite system of curves, surfaces, complexes, &c., such that one condition is sufficient to determine a finite number of them. The elements may be regarded as determined by / coordinates; and then, if these be connected by k—1 equations of any order, the curve is either the whole aggregate of common solutions of these equations, or, when this breaks up into algebraically distinct parts, the curve is one of these parts. It is thus convenient to employ still further the language of geometry, and to speak of such a curve as the complete or partial intersection of kK—1 loci in flat space of & dimensions, or, as we shall sometimes say, in a k-flat. Ifa certain number, say h, of the equations are linear, it is evidently possible by a linear transformation to make these equations equate hk of the coordinates to zero; it is then convenient to leave these coordinates out of consideration altogether, and only to regard the remain- ing k—h—1 equations between k—h/ coordinates. In this case the curve will, therefore, be regarded as a curve in a flat space of /—A dimensions. And, in general, when we speak of a curve as in flat space of & dimensions, we mean that it cannot exist in flat space of /—1 dimensions. x x* By a surface we shall mean, in general, a continuous two-dimensional k aggregate (which may also be called a two-spread or two-way locus) of any elements whatever, curves, surfaces, complexes, &c., defined by the whole or a portion of the system of solutions of kK—2 equations among k coordinates. We shall assume that none of these equations are linear, and then shall speak of the surface as in a flat Trans. Conn. Acap., Vou. VII. 2 Sept., 1885. 10 FE. H. Moore, jr.—Theorems of Clifford and Cayley. Tueorrem A, Hvery proper r-spread of the n” order is in a flat space of n+r—1 dimensions or less. For through n+1 points of the 7-spread we can draw an #-flat, R,; this meets the r-spread 8, in a number of points greater than its order, and, therefore, contains a curve, or l-spread §, of the r-spread §,. An (n+1)flat R,,, drawn through this -flat R, and an external point of the 7-spread S,, for a similar reason, contains a 2-spread §, of the r-spread §$,. Thus, finally, there is reached an (n+r—1)flat R,,., which completely contains the 7-spread 5,. An r-spread of order », say S,,,,, may lie in a flat space of & dimensions, where Kl n+r—1; when k=n+r—1, the §,,,,,,4,. may be called a full skew r-spread of order n. 5 TurorEM B. An r-spread of order nin a flat space of k dimen- sions (and no less), say 8,,,,., may be represented, point for point, on an y-spread of order n—k-+-r-++-1 in an (r+1)flat, 8, pacts, ep Join P, an arbitrary fixed point, to Q, a variable point, both being on the r-spread; the resulting (7-+1)spread 8,,, is of the order »—1; for a (k—r)flat R,_, through P meets the 7-spread 8, elsewhere in nm—I1 points Q, and, therefore, meets the §,,, in the n—1 lines PQ, i. e., in a curve of ordern—1. Each line PQ meets a fixed (A—1)flat R,. in a point Q’ corresponding to Q; the (7+1)spread S,,,; meets the fixed flat R,_, in an r spread of order n—1. The 7-spread of the n order in k-flat S,,., is thus projected into one of order n—1 in a (k—1)flat, S,,.,..1. A second projection from an arbitrary point upon a fixed (k—2)flat R,_, gives an r-spread of order n—2 in a (k—2)flat, S, , » 0: Thus, finally, after A—r+1 successive projections the original S,2, 18 represented, point for point, on an 7-spread of order n—k-+7-+-1 in an (7+ 1)flat, 8, ge a But the result may be reached at once. Through any k-r-1 fixed points P of the r-spread and a variable point Q pass a (A—r+1)flat R,-;, cutting a fixed (r+1)flat R,,,, in a point Q’ corresponding to Q. ‘Thus the S,,,,, is represented, point for point, space of k dimensions. We shall in certain cases go further, and speak of an h-spread or h-way locus, viz: a locus determined by the whole or an algebraically separate portion of the system of solutions of kK—h equations among & coordinates; if none of these equations are linear, the h-way locus will be said to be in & dimensions.” A proper curve or spread is one which does not break up into two or more alge- braically distinct parts. a FE. H. Moore, jr.— Theorems of Clifford and Cayley. 1] on an r-spread in the (7+-1)flat, 8. (24-41, 41, the order of which is n—k+r-+1, since a (A—r)flat R,_, through the /-r+1 fixed points P meets the 7-spread 8, ,,,, in »—k+7r-+1 additional points Q which correspond to the n—k+7r-+1 points Q’ in which the line of inter- section of the R,_, with the fixed R,,, meets the projected 7-spread S,, n—k+r-+1, r+1° Treorem C. A quadric r-spread isin an (7+-1)flat, and is wniewrsal, its points having (e. g., by projection from a point on it) a one-one cor- respondence with those of an 7-flat R,. Hence, a full skew r-spread of order 2, S,,n,n4,1, 18 always unicursal, since, by theorem B, it may be represented on an 7-spread of order n—-+-r + 1=2 in an (7+ 1)flat. Not only so, but every flat section of a full skew r-spread is étself a full skew spread, and, therefore, wnieursal. For an s-flat R, cuts a full skew S,,..% @tior+n) In an S 42k, 2,2 =r, uu, Which is full skew, since k’4+1=7" +n, ie., s+1=—(r+s—/s)+n, using the given relation, 4-+-1=r-+n7. Il. Furt Skew Two-Spreaps. 1. The abbildung-system. The full skew two-spread of order m in Ryiy, So,m,mp1y 18 Unicur- sal; unicursal (I, C); an R,,_,, the intersection of two R,,, and so the axis of a pencil of R,,, meets the two-spread in m points (m being the order of the spread); further, in the R,,,,, the all-including flat, there are 0”! m-flats R,,, i. e., m+2 asyzygetic R,. Hence, there is a representation or Adbdildung of the Sem, mys point for point, on a plane y, y.¥;, A; to an KR,,-section corresponds a wni- cursal curve, say of order n, "; to the m points of intersection of an R,,_, correspond m points of intersection of a pencil of curves &”; the Abbildung of the (m-+1)ply infinite system of R,,-inter- sections S,, m,, is the system of curves ©”, (m-1)ply infinite or lin- early derivable from m+2 asyzygetic curves of the system, all of the curve of intersection with any m-flat R,, Sinn 18s also which are unicursal and have the equivalent of x’—m common points of intersection, say base-points of the system. A Cremona transformation may, be found which will change an abbildung-system G” of the spread 8S, », myi into any other abbildung-system @”’ of the same spread, since there is a one-one correspondence between the points of the two coincident planes 2, containing the two abnild- 12 E. H. Moore, jr.— Theorems of Clifford and Cayley. ung-systems. There is, therefore, no loss of generality in assuming as the abbildung-system, the system of curves @” having as base- points an (m—1)ple pt. @ and m—1 other points, 9,,--+ Pm: the conditions given above are satisfied by this system ; for the curves are unicursal; the asyzygetic number is 2m+1—m—l=m-+2; the base-points are equivalent to (m—1)?+(m—1)=m(m—1)=0'—m=n*—m common points of intersection. This abbildung-system may be simplified. Transform the plane *, by a quadric transformation, having ©, Pn and mo, as fun- damental points. A curve &”, of order m, passing through the two fundamental points Dn, Pm—2» and having an (m—1)ple pt. at @, the third fun- damental point, and passing through m—3s fixed points 9, .. . Bn—39 is transformed into a curve @’ of order m—1=2. m—(m—1 +1+1), having an (m—2)ple pt. at @, (since the line Dn—1 Bm—» Meets the &” in m—2 additional pts.), passing through the m—3 fixed points M1. - B'n—s ae hee to the pts. $i. . - Pn, and not pass- ing through 9,4 [®,,-2], (since the line @©%,, [O#n.] does not meet the @” except in @ and Bn— [@ and 9),,.]). Thus such a quadric transformation reduces by unity the order of the curves of the abbildung-system, and deletes two of the base- points. By 7 such quadric transformations, the abbildung-system of curves &”, with @, an (#—1)ple point, and m—1 other points 9, as base-points, is changed into a system of curves @””" with @, an (m—r—1)ple point, and m—1—2r other points 9, as base-points. The simplest abbildung-systems are, evidently, (a) m even=2m',r=m'—1. &”""* vith @ as m’ple point and through one pt. A. (0) m odd=2m" +1, r=m". G""*' with © as m"ple point. 2. The canonical form of the equations of the So, m, m4 Let the X, (s=1,...m-+3) be the homogeneous coordinates in R,,4, and the y, (s=1, 2, 3) be the homogeneous coordinates in R,. (a) meven=2m'. @is y,, Yo, Ys=0, 0,15 A i8 Yi, Yo, Ys=1, O, O. By the simplified abbildung-system set Yr r r ON Ae ene 2K, ae GaN D. Cane ial ot et hey. eter” «ide Me: | ail et Aaiee hes D. Caray Me m+1 m-+2 Yi myer OF mti—1 ye LY mi—2 pie eu te ae 4," YE mt “ie Y; Ys mt, Ue mT a 0a 1 s— m—2 ae mi— Pei Sie, at Ui Ys o5 . porate ee wives eee PN megs AK 8 ha WME ete oe E. H: Moore, jr.— Theorems of Clifford and Cayley. 13 Setting y.=ty, and y,=wy., this becomes Ne tr Ny ONG fey t easi.os Nm gee 7 Ney) Mme He Pinte ruces ana 5 (ie cee hae ROARS Tigatw on on as Pune = ee sad whence the plexus of equations _ ap. Care De hd os EOE SR eee. Corer. Gann ae Xetall > er Re eX ys ws: 5 AS Fe | Se (6) m odd=2m" +1. @'is ¥,, Yes Ys=O, 0, 1. In accordance with the simplified abbildung-system, set DEE INES DSR OE) TP a CME NA gre: ees ee eae momen e i ty.) os 2. 3h. Maninyes Momige ts m+1 m+-2 PU Yat, Ye: yi Ys PS cosa e. Gus a Ua ae i Pa ee Us te Poe a Uy Ya Ya Ys” |, - or, setting Yo=ty, and ¥,=uUy,, 2 OE se ES Ee, COD. Can tre peed: SRR (VAR att ide ee te et arael Lebstie se acl, ate | whence the plexus of equations X, ? X,, X;, corals sc Xing ? Xnngs ’ Xue 5) Xs ema et » ORE —0 rr Ne es) nerd Amys yes = Rts hats In each case the canonical form exhibits the S,,»,,,, a8 the locus of the line of intersection of corresponding m-flats R,, of m projective pencils of R,,; ~X,—éX,=0, X,—¢X,=0, &e. In fact, these right lines on and ruling the S,,,,, 4, correspond to the right lines on the plane %, through the multiple point ©; y.—ty,=0 is met by a C™*' or C”"*! (which corresponds to an R,,-section of So m,m+i) in only one point. A full skew curve O of order Nn ide “, corresponds to the point @. @ is (0, 0,1); or w= »,t¢indeterminate ; and, therefore, @ corresponds ey | Rete Aasyes-<-- Xn |]9 Rd Wa) ee 5 Gene aa (0) to 2. GaSe. es PSS es ==). Cedi) Xiang ’ bois 5 cumeciee Xn |- Dw 9/19) sh (0) one OO ’ is I or the (unicursal) curve (2) min an 7; flat. Cf. Clifford, p. 310. ” E (6) m m’- (a) A line A corresponds to the point A. For q is (1, 0, 0); or _ wu indeterminate, ¢==0; and, therefore, A corresponds to Se Hen en OM =). Carte. ONT pH tr seer = Os m m-flats R,, in R,,,, intersect in R,, a right line. 14 E. H. Moore, jr.—Theorems of Clifford and Cayley. A point corresponds to the dine OA, viz: the intersection of the full-skew curve O and the line A. y,=0; or w= », t=0;5 RS sede dee = Ne 0, g. Ch pea. CRA > SRoieae = Kee 3. Tangent and osculating flats. On the two-spread let Q be a point and C a curve passing through Q; there are at Q a tangent line R,, and osculating 2-flat R,, osculating R,... R, to the curve C, determined by 2, 3... (#+1) consecutive points (Q included) of the curve; the oscu- lating ¢flat R, meets the curve C in ¢+1 points at P. The tangent — plane R, at Q to the two-spread is the locus of tangent lines R, to all curves C through Q; every m-flat R,, through this tangent plane R, meets the spread in a curve having a double-pt. at Q. Suppose there were an s-flat R, such that every R,, through it met the two-spread in a curve having a (¢+1)ple pt. at Q. Take any curve C through the point P; every R,, through the R, meets the curve C in ¢+1 points at Q, and, therefore, contains the osculating R, to C at Q; hence the R, contains the osculating ¢-flats R to all curves C through Q, or say it meets the two-spread at ¢ consecutive points in every direction from Q. In the case of this full skew two-spread it will be shown that at every pt. Q there is an osculating R,,_, containing the osculating R, to every curve through Q; but probably s=2¢ for the general two-spread in any number of dimensions, R,; (2¢2%, of course). . Considerations from the abbildung-system. The general abbildung-system (cf. §1) of curves ¢” has as base-pts. the (m—1)ple pt. ©, and the m —1 pts. 9; there are m+2 asyzyge- tic curves of the system. Consider those curves of the system which. have a (¢+1)ple pt. at @, say &” (@‘*t’); these correspond to the R,,-sections of the two-spread which have a (¢+1)ple pt. at Q. Such a curve @” (@)‘t') includes the line @@ ¢ times, and, besides, a sup- plementary curve ¢”~*‘ (@), passing through @ and the m—1 pts. 9 and having an (#—t—1)ple pt. at @. For say the curve &” (@t') includes the line ®@ « times, and, therefore, also a supplementary curve @”* (Q‘***) with (¢+1—2)ple pt. at @ and (m—a#—1)ple point at ®; the line ©) meets the supplementary curves in (m—a—1)+(¢+1—x)=m+t—2e pts. and will be again thrown off, if m—w# TX qu — Xo i=, TX —Xnis =0. 2m'(=m) R,, meeting in R,, the line rz. The additional R,,, vX,—X,,,,.=0, determines the point tv on the line 7. 16 FE. H. Moore, jr.—Theorems of Clifford and Cayley. t=1.. R,, the osculating R,,,., along z‘t'-?, the locus of tan- gent planes of points v along the line rT. { TR Or X a KV =0, TE Rong — 2 at ae TE ie ket 90; TO Kg — oT a a es Zi fa — BT) Ae east =O, T Xn —2TX,45+ hee 2(m'’—1)=m—2 R,, meeting in R,. The additional R,, v(7X,—X,) —(t7Xn2—Xm4s)=0, determines the tangent plane R, at the point rv. And so, in general, the law of formation is clear. The osculating R,,,, along z’t', the locus of the osculating R,, of points v along the line 7. The equations may be written X'(z—X')#1=0, Xe = eee X?(r—X’)*7=0, KM (7 — XO er (a Xt =0; AMAT — Oe where, after the binomial expansion and multiplication by the exter- nal factor, the exponents of the X are to be exchanged for corres- ponding subscripts ; X” is to be changed to X,. 2(m'—t)=m—2t R,, meeting in Ry, ,. The additional R,,, (vX’'—X™'*) (r—X’)'=0 (the exponents be- coming subscripts, as above), determines the osculating 2¢-flat R., at the point tv. elie The osculating R,, , along rt‘ lies in the osculating R,,,, along T'*'; the two R,,, X'(r—X’)'=0, X™'*(7—X’)'=0, with the m—2¢ equas, of the R.,,, written above are easily seen to be equivalent to the m—2(¢—1) equations of the Ry_,, X"(r—X’)'=0 X”"'**(r—X")'=0 Xn (7 —X?)'=0 X"+#9(7_ X=, Thus the equations show that the singly infinite system of osculat- — ing 2¢-flats R, at points v along the generator 7 lie in the oscu- lating (2¢+1)flat R,,, along 7‘t'; and, at the same time, form a pencil of R., in the R,,,, having as an aaés the osculating (2¢—1) — flat R,, along 7‘; and the osculating flats of the pencil are homo- graphic* with their points of osculation along the generator T. Compare the well known theorem: Salmon; Geometry of Three * The only equation introducing v is linear in v. E.. H. Moore, jr.— Theorems of Clifford and Cayley. 17 Dimensions, $459; “the anharmonic ratio of four tangent planes passing through a generator of a ruled surface is equal to that of their four points of contact.” (6) m odd = 2m" +1. There is one more equation in each case from the first group of coordinates, X, . . Xnwi2, than from the second group, Xiyw 3... X42; the v equation is of exactly the same nature; the conclusions stated above hold equally for m odd or even. As the line z generates the two-spread S,,,,,,.4: the osculating R,,, along zt’ generates a (2¢+2)spread of order (¢+1) (m—2zt) Berp2, HpI\m—29, m-b1 + Let the m—2t asyzygetic R,, determining R,,,, in terms of 7 be A,, Anr,---- Agii3 the A involve r to the power ¢+1; let An,2» Am—y,2) > + - Axti,e, be what the A become when the coordi- nates of a point P, in the (m-+1)flat are substituted for the current coordinates. The (2é+2)spread is met by an (m—2¢—1)flat in say x points P,; any point in the (m—2t—1)flat may be given in terms of m—2t asyzygetic points P,,.. ~~. Poi; Say PA pPntAnaP met 00 ¢ + Ne Peits ier. 2G), A). CE 2) ee. Cai soa Ag Xs, ott y etc. Substituting the coordinates of P, in the m—2¢ A,, and eliminating from the m—2¢ A,, the m—2¢ A which enter homogeneously in the first degree, we have the determinant of the order m—2¢ Ag m9 1 m—1 9 O08 ive 2t-+1 =), AO m9 ebdarg m=—139 ° ° js Uy 2t-+-1 DOO Et e's Poti 19 o Avcorts, +1 an equation of degree (¢+-1)(m—2¢) in 7; for each value of 7, there is one set of values of the A, one point P,. Hence the order of the - (2¢+2)spread, the locus of osculating Ry,, is (¢+1)(m—2z), as stated. Through a point P, an osculating R,,, along 7‘t’ may be _ drawn; this contains the osculating R.,., along 7‘; the R,, joining the point P, with this R,,, is the osculating R,, at some point v of the line tz. This may be expressed thus; through an Re, may be passed (¢+1)(m—2t) m-flats R,, which meet the spread in a _ (¢4+1)ple line z; and (¢+1)(m—2t) (m—1)flats R,,_,; which meet _ the spread in a (¢+-1)ple point rv. TRANS. ConN. ACAD., VoL. VII. 3 SepT., 1885. 18 EE. H. Moore, jr.—Theorems of Clifford and Cayley. (a) meven=2m'. The simplified abbildung-system; § 1. @”'*! may degenerate into the line 7 taken m’ times, and a line through 4; for this the limiting case t=m'—1. = The (2¢+-2)spread, locus of osculating R,,,, along tt! is of order | (t+1)(m—2t). But the (m-—2¢)spread, locus of osculating R,, «4 along 7”’~ is also of order (m! —t)(m— 2m! —t—1)=(t+1)(m— 20). For instance, t=0; the two-spread locus of lines R, (i. e., the orig- inal two-spread) is of order m; and, also, the m-spread locus of oscu- lating R,,_, along 7” is of order m. Thus, when m is even, the orders of the (2¢+-2)spread for t=t,, tty, are equal, if ¢,+-¢,=m'—1. (b) m odd = 2m"-+-1. t=m; since the curve ¢”"*' in the limiting case degenerates into the line 7 taken ¢--+-]=m”-++1 times. For t=m" the order of the (2¢4+2) = 2m" +2 = (m-+1)spread is (m"+1)(m—2m")=m'+1; i e., through every point Ry xs» of Ry: may be passed m”+1 R,, meeting the 2-spread in an (m" + 1)ple line rT. ¢ There is no symmetry analogous to that for m even. 4. Ourves on the two-spread. (b) m odd = 2m"-+-1. The abbildung-system, @"t' having © as m’-ple pt. Let us denote the unique curve of order m” in an m’-flat corre- sponding to ® by O; and the right lines of the spread by tr. A curve on the spread of order p, meeting the unique curve O in ¢ points and every line 7 in 7 points may be written C? (O/7’). A curve on the abbildung plane @°(@') of the order s with a ¢-ple point at © is met by a curve of the system @”*! in s(m"+1j)—tm" points and by any line 7 in s—¢ points; therefore, it corresponds to a curve on the spread of order s(m”+-1)—tm", which meets the nnique curve O in ¢ points, and every line 7 in (s —f) points, say Cher Ora): So a curve G’ (@‘t*“) transforms into Ct)" (O'r™), A curve ©’ (O’), of order p, meeting the curve O in q pts., must have prog and p=q (mod. m” +1); say p=s(m" +1) —tin", G==t. : s(m’+1)—tn"=p. (s20). s1,t=1,p=1. A linet on X, corresponds to a line 7 on the spread. ss, t=s, ps. 8 lines 7 correspond to s lines 7. xsi die Tet bok aPIRrte ym pein ite E.. H. Moore, jr.— Theorems of Clifford and Cayley. 19 If s>t, p=m"+1; which shows that the curve O of order m” is unique, since it is the only curve of so low an order on the two-spread (the lines 7 excepted). Curves on the two-spread and in flats of less than m+-1=2i”-+-2 dimensions are full skew curves. Such a curve is an R,,-intersection or a part of an KR,,-intersection ; therefore, its abbildung is a curve @'(@°'); any m"+1--s lines 7 belong to the supplementary system, of which the asyzygetic number is m”+2-—s; therefore, m”+2—s asyzygetic R,, meet in an R,,,,,, in which the curve O@"4)—G—Dmomit lies. A curve C”"’** in R,,,,,, is a full skew curve. (I; theorem C.) The general plane curve @’ (¢=0; not through ©) corresponds to a C+) which does not meet the unique curve. The plane is a full skew 2-spread 8,5 (m=2m"+1=1, m’=0). All the geometry of plane curves depending upon intersections and tangencies and the order of curves is immediately applicable to the general full skew two-spread of odd order m=2m"-+1, the curves Ct” on the two- spread corresponding completely to the curves @’ of the plane. A curve Cm"'t)" (O’) meeting the unique curve O ¢ times is a par- ticular case of C%""t», and in fact plays the same réle as a C%™"'*) having a ¢-ple point. A few examples are given. There is a double infinity of curves Ct’; two meet in one point; one is determined by two points; they correspond to the lines @' of the plane. A line 7 together with the unique curve O is a special case of a curve C”"*, Five points determine a curve C%"’t); which corresponds to a conic @? of the plane. Pascal’s theorem becomes : If six points P'.. P® lie on a curve Ct” the three points of intersection of the two curves C””t' joining P'P*®, P*P®; P?P®, P®P*®; P§P', P®P*, respectively, lie on another curve C”"*’. To a curve Cty im (O'r*) there are s(s —1) —¢(¢+1)= (s+2)(s—t—1) tangent lines 7, and s(s - 1)—¢(¢—1)=(s—t)(s+¢—1) tangent curves C”’*’ in a pencil through a point P.* An m-spread of order P meets the 2-spread in a curve C”” meeting the unique curve O m’P times, and every line 7 in P points; ee = COMIN" (OR 7”). sa=(m" +-1) P, tm" P., To an m-spread of order P in Roniio-mi, there are mP(P—1) tangent lines lying entirely on the Sy mm, mit Two curves C (O'7r* *), C (O”r""), meet in ss’ —¢#t’ points; in particu- lar, two curves C (O'r*“) meet in s*—¢ points; one is determined by * These formulz are similar to some given by Chasles, Comptes Rendus, 1861; cf. _ the following (a), 20 EF. H. Moore, jr.— Theorems of Clifford and Cayley. $}s(s4+3)—t(t+1)} points. Hence two curves C (O'7r*“) through ${s(s+3)—¢(¢+1)}—1 points determine a pencil of such curves through these and ${s(s—3)—¢(¢~1)}-+1 additional points. (a) meven=2m'. The simplified abbildung-system, iets having ® as m'-ple point and 4 as an ordinary point. Curves @?*' (@,4’) of order p+1 having © as p-ple pt., through A, correspond to full skew curves of order m'+-p; these are the only curves on the spread in a flat of less than m-+1 dimensions. The proof is like that of (b) for m odd. In particular:—A line y,—Ty,=0 through © corresponds to a line 7 on the spread (§ 2); two lines 7 do not meet. A line v through A corresponds to a full skew curve v of order m’ on the spread; two curves v do not meet. Through every point on the spread pass one line 7 and one curve C”, v; a line 7 and a curve v meet in one point. @ corresponds to a curve O of order m’, meeting every line rT. A corresponds to a line A of order m’, meeting every curve v. The curve O meets the line A in a point OA. (§ 2.) In fact, the curve O, the line A, the point OA in no way differ from an ordinary curve v, line 7, point tu of the spread. Observe that a curve @' (@'A") of order s (having O a ¢ple and A an u-ple point), corresponds to a curve C®%™+¢™ (z7*“v'") of order (s—t)m'+-(s—w) meeting every line 7 in s—¢ points and every curve v in s—w points: it passes s—¢—wu times through the point OA and meets the line A elsewhere in x points, the curve O elsewhere in ¢ points; (.*. in all, it meets the line A in s—¢ points, the curve O in s—wu points). ANCUIVe Go (Qo —gro) with an (s—t—z)ple point at say @ corresponds to a curve C®%"' 6 (ry) with an (s—t—vu)- ple point at Q. Thus the order and character of intersection with the lines 7 and curves v and the (s—t—w)ple point are exactly the same. The asyzygetic numbers are equal; as shown by the follow- ing equality (where v=s— ¢—w, s’=s++v=2s —t—u, =s—u,w'=s—t), (s+1)(s +2)—¢ ‘ +1) —u . +1) =(s'+-1)(s' +2) —¢ c +1)—w o +1)—0(v+1) Qe The statement above is cee and it is therefore proper to con- sider only curves which have no especial relation to the point OA; i, e., in the abbildung-plane, only curves @* (@'4") where s=¢+2. The spread is ruled with the lines 7 (§2); and also with the curves , C™, The curves v correspond to y;—vy,=0; the (m'+2)spread E.. H. Moore, jr.— Theorems of Clifford and Cayley. 21 = Dae ’ D.C > xy, oth ST 1S he 8. ve we, 1 Daddy Bae 5) 0. ore fa Te et LO Xn, +19 ®, +-2 is cut in the curves v by m’-flats, the intersection of corresponding R,, in the m'+1 projective pencils of m-flats R,, , re MeO XK ig UNO 27) bs OX) RL), OO, A curve 6" (@'A") corresponds to a curve C+ (r"v") of order wm! +t meeting each line 7 in w points, each curve v in ¢ points. Since the number of intersections of curves on the two-spread with the lines 7, the curves v and with each other, and all intersection- and tangency-properties, depend only on the abbildung-curves, it is clear that there is a complete correspondence between the curves on a two- spread of even order m=2m’, and those on an ordinary hyperboloid or quadric (m'=1); the two systems of ruling curves, the lines 7 and the curves v of order m’, answer to the two systems of generators on the quadric (only, in the latter case, the two systems being of the same order are indistinguishable). Hence many of Chasles’ results (Comptes Rendus, liii, 1861) concerning “ Propriétés générales des courbes gauches tracées sur l’hyperboloide” apply in this more == () general case; for example: A curve C'™'“(r"v’) is determined by tu-+(¢-++w) points. Two curves Cv™"'* (rv), GC" (rv), meet in é’ + ut’ points. All curves C’'"‘(r"v') going through tw+(t+wu)—1 fixed points form a pencil passing through tua—(¢+w)+1 other fixed points; since any two meet in 2¢w points. To a curve C’’**(r"v') 2t(w—1) lines 7 are tangent. 2u(t—1) curves v are tangent. 2tu curves C”’*! (r'v"') of a pencil through a pt. P are tangents. These numbers are easily derived by considering the correspond- ing abbildung-curves. The curve C'"'*'(r"v') corresponds to a curve C"*'(r"v') on an ordinary quadric ; on the quadric there is no distinction between the curves C"t’(r"v'), Cv‘ (z'v"); i. e., two curves of the same order ¢ +, which meet the generators of one system 7 in / points, and those of the other system v in ‘, points. If, then, there is a theorem about curves of order u,m’++t, (r=1, 2,.. 8’) meeting the lines 7 in w, pts. and the curves v in ¢, pts., the same theorem will be true about cor- responding curves of order ¢,m'+-u, meeting the lines 7 in ¢, pts. and the curves v in w, pts. A curve OC’ (rv) must have P=um' +t. 22 kh. H. Moore, jr.— Theorems of Clifford and Cayley. An m-spread of order P intersects the 2-spread in a curve of order mP=2m' P, meeting the lines 7 in uw= P points, and the curves v in (=n _P points: way C7 (rut?) = Cores ry). 2t(u—1)=2m' P(P—-1)=mP(P—1) lines 7 are tangent to this curve. There are mP(P—1) lines 7 lying entirely on the two-spread Som, mii and tangent to an m-spread S,, pm of order P.* (m odd or even ; cf. § 4, b). The curves on a full skew two-spread of even order, m=2m', then, have an exact correspondence with those on an ordinary quadric, m=2; those on a full skew two-spread of odd order, m=2m" +1, have almost as exact a correspondence with the curves on a plane, m=1; the unique curve O is a singularity, but curves meeting it in ¢ points play very much the same réle on the two-spread as curves having a ¢-ple pt. at an ordinary point of the spread. These close correspondences with the hyperboloid and plane curves are the marked features of the theory of curves on full skew two-spreads. III. Spreaps oF Opp OrpdERS ON QUADRICS. The known theorems, that a curve 8, of odd order on an ordinary quadricone, cone-Q, ,, passes an odd number of times through the vertex V, and that a general quadric 3-spread Q; , contains no 2-spreads 8, of odd order (ef. Clifford, Mathematical Papers, p. 644), may be extended. Z Q,,.4: Will denote a general quadric r-spread in R,.,, and cone-Q, ,,, a quadric 7-spread in R,,, formed by joining a (general) Q,_1,, to a vertex-point V in R,,,. The section of Q,,,, made by a tangent r-flat R, is a cone-Q, ,,. The section of a cone-Q,,,, by an R, through the vertex V is a cone-Q,_,,.; but that by an arbitrary R, is a (general) Q,_,,,. S, will denote an 7-spread. A,. The general Q,,,.,,,; has on it no 7-spread of odd order unless r, which contains the projection through P of §, ; if B, holds, the preceding consideration shows that A., must also. Hence, if the propositions hold for R,, (i. e., the B), they do for R,,,,, (i. e. the A). If the A (m,=n) hold for R,,,,, the B (m,;=n+1) will hold for Pies is B,. The Qoris,ony2 Contains an 7-spread S, of odd order; the tan- gent R,,,, at a pt. V (not on the 7-spread 8,), cuts the quad- ric in a Cone-Qyn on11, Containing an (7—1)spread of odd order not passing through the vertex V ; hence, by A.,,, r—1lm; (III, Aj). + If an R,, passes through V, it lies completely in the fangent R’om at V. ¢ Thus the intersection of an 7, (the projection of an R,,) with the fixed 7/1 lies completely on the fixed q’; and likewise two 7m and the fixed 7’om_—; intersect on the fixed q’. § A quadric m-spread passing through V would project into an m-flat 7» having a quadric (m—2)spread on q’. E.. H. Moore, jr.— Theorems of Clifford and Cayley. 25 (2) m even. Two R,, of opposite systems in general do not intersect, but may motersect in R,, or R,, or .: . or R,,_,. Two R,, of the same system in general intersect in a point, but may me intersect in an R,, or R,, or... or R,». This may be expressed (R,= a point, 0=no intersection), begin- ning with the most infrequent cases : Two R,, of "oue} system intersect in m in general nf m even m odd runes 9 or R,,-4 > or oo a ONE R, , 0 > > . a»: eercOnmen Or Mrs sors). RR, (1). A few considerations to be used in the proof are given here. In 7,,, two 7,, meet in a pt. 7); if in another pt., then in a line 7, ; if two 7,, have an 7, common, and also another pt., then they have an | 7,,, 1 common. Two 7, intersecting on g’ in 7, may intersect in another point ; and thus in 7,,,; but they do not intersect in #0 other asyzygetic points, for then the intersection with the fixed 7’,,,., would not lie entirely on the qg’. (Cf. foot-note f, $1.) If two r,, intersect only in an 7, lying on the q’, the two correspond- ing R,, meet the R,,, joining this common r, to the fixed point V in two R, (which were both projected into the common r,) which in the R,,, intersect in an R,,. ‘As a special case, if two 7, intersect in a point r, on gq’, the two corresponding R,, [intersect the line R, , join- ing 7, to V, in two points R, and] do not intersect. (2) meven. The7,,, on q’ intersect according to (4), m—1 being odd. On @ fra. and 7,_,,, in general do not intersect, but may in- tersect In 7,7; .... Or 7,3. Twor, through them must intersect in a point, at least. Hence, in general, m being even, two R,, of the _ same system intersect in a point. In the particular cases, if the two Tm, intersect entirely on g’, the two R,, intersect in R,, R, ... or R,,_.; but if the two r,, intersect also in a point not on q’, the two R,,, intersect in R,, R,... or R On q’ 7,-1,, and 7,,_;,, intersect in a point 7,; but may intersect In 7%, 7%... OF 7%, ». Two 7, through them would in general in- __tersect in no external point; hence, in general, m being even, two R,,, of opposite systems do not intersect. In the particular cases, if the two 7,, intersect entirely on g’, the two R,, intersect in R,, R, j ...or R,,;; but if the 7,, intersect also in a point not on gq’, the metwo ht, intersect in R,, R; .. . or R,_,. m—3 * m - Trans. Conn. AoapD., Vou. VII. 4 SEpt., 1885, 26 E.. H. Moore, jr.—Theorems of Clifford and Cayley. Thus, the (4) holding for 7—1 odd, the (7) hold for m even, (3) m odd. By similar considerations it is shown that, the (¢) holding for m even, the (/) hold for m-+1 edd. But the (4) hold for m=1 on the quadrie Q,., in R, (as might be shown immediately by the projection from V on a plane); and, therefore, (4) and (/) are true in general. 3. One m-flat of each system passes through every R,,, of the Q.,: The R,_; projects into anr”” having an 7,_, on g’, through which (suppose) there pass an 7, ,,, and an7,,,; the original r,, — and the 7,,,,, intersecting in 7,,,, determine an 7,, the projection of an R,,,, through the R,,_,. This is true on the Q,,; therefore, in general, Every pt. in R,,,,, has a polar R,,, with reference to the Q,,,; the polar R,,, of every pt. in an R, passes through a certain R,,,_, which is called the polar of the R,. (Clifford.) If the s-flat R, lie on the Q,,,, the polar R,,, of every point in it, i. e., the tangent R,,, at that point, passes through the s-flat itself; hence the polar P,,,_, must include the original s-flat R,. The m-flats R,,, are self-polar. The R,,, and R,,,, through an R,,_, taken together lie in and determine the polar R,,,,, of the R,,_,. More generally, two R,,, intersecting in an R, lie in and determine the polar R,,,_, of the R,. EM 8: wey PP iste > = fi IIT.—On Kwors, with a Census ror Orper Tern, By C..N. Lirrite, Linconn, N ex. 1. Gauss in 1833* called attention to the importance of the study of the ways in which cords might be linked. Nothing, however, appears to have been written upon the subject until in 1847 Listing published his Vorstudien zur Topologie.t In this he briefly but in a masterly way touched upon the subject of knots, established some of the fundamental propositions, and proposed a notation which, as slightly modified by Prof. Tait, furnishes the point of. view for the present paper. In a communication{ to Prof. Tait in 1877, Listing points out the fragmentary character of his own contribution to the subject, and says that the type-symbol used by him is “ nichts weiter als ein derartiger Fingerzeig.” It is to Professor Tait, however, that the greater part of our pres- ent knowledge of the subject is due. He, independently of Listing, obtained the fundamental propositions and found the knots and their forms for orders from three to seven inclusive.§ In 1884 Kirkman || published the forms of knots of orders eight and nine, and immediately Tait, making use of Kirkman’s work, extended his census of knots to these orders.4 2. Professor Tait has shown that any closed plane curve of n cross- ings divides its plane into n+2 compartments; that these compart- ments are in two groups; that, at the crossings, like compartments are vertically opposite. We shall call these compartments of the plane parts. A part is represented by the number equal to the num- ber of double points on its perimeter. The sum of the numbers representing the parts of either group is 2n, that is, these numbers together constitute a partition of 2n. The partitions for the two groups together make up Listing’s type-symbol. As it can lead to * ‘Kine Hauptaufgabe aus dem Grenzgebiet der Geometria Situs und der Geo- metria Magnitudinis wird die sein, die Umschlingungen zweier geschlossener oder unendlicher Linien zu zaihlen.”—Werke. GOttingen. 1867, vol. v, p. 605. 2 . + Gottingen Studien, 1847. I have been-able to see only Tait’s apparently full Jc ertaceiagniey( hi abstract in Proc. Roy. Soc. Edin., vol. ix, pp. 306-309. ¢ Proc. Roy. Soc. Edin., vol. ix, p. 316. § On Knots, Trans. Roy. Soc. Edin., xxviii, 145-191, 1876-77 | Trans. Roy. Soc. Edin., xxxii, 281-309. { Trans. Roy. Soc. Edin., xxxii, 327-342 28 C. N. Littleh— Knots, with a Census for Order Ten. no ambiguity we shall also call the number representing a compart- ment a part, and either group of compartments a partition. Since every closed plane curve of 7 crossings, having double points only, may be read alternately over and under at the crossings, every such curve which gives parts, none greater than m or less than 2, may be taken as a projection of a reduced knot of » crossings. We call such curves knot-forms or briefly, forms, and regard two forms as distinct if they do not have the same parts similarly arranged. The first part of the problem is to find all the different knot-forms of any: order. Since the same knot may be transformed so as to be projected into more than one knot-form, the second part of the problem is, from the complete series of knot-forms of 7 crossings to find all the different n-fold knots. Knots exist for which the law of over and under does not hold; these are not considered in the present paper. 3. It is unnecessary to do more than allude to two very distinct and very ingenious methods devised and used, the one by Tait and the other by Kirkman, for the solution of the first part of this problem. We may perhaps infer from Professor Tait’s opinion* that “ a full study of 10-fold and 11-fold knottiness seems to be relegated to the somewhat distant future,” that they were more laborious than proves to be necessary. 4. A third method, based on Listing’s type-symbol, is thus de- scribed by Professor Tait at page 168 of his first memoir. “ Write all the partitions of 2n, in which no one shall ke greater than » and no one less than 2. Join each of these sets of numbers into a group, so that each number has as many lines terminating in it as it contains units. Then join the middle points of these lines (which must not intersect one another), by a continuous line which intersects itself at these middle points and there only. When this can be done we have the projection of a Anot. When more continuous lines than one are required we have the projection of a linkage.” On page 160 of the same memoir, he says, speaking of this method: ‘“‘ But we can never be quite sure that we get all possible results by a semi-tentative process of this kind. And we have to try an immensely greater number of partitions than there are knots, as the great majority give links of greater or less complexity.” It seems possible however, with the help of some simple theorems to make the “ Partition Method ” exhaustive, and wholly to do away with the drawing of links. *TIn 1884. Trans. R. S. E., xxxii, 328 seeudieen ica eee set Abs = as Py por “ - OE OE A ah LE Ne MLD ET St a Ne PEO NS a eeihtederl uicaeda Mipguinairt & § C. N. Littl—Knots, with a Census for Order Ten. 29 5. An inspection of form Aa of Plate I will make clear some-yerms already introduced and others that we shall now require. Regarding the curve Aq as alone in a plane, it divides it into twelve parts, two 9-gons, two 3-gons and eight 2-gons. The external 3-gon or am- plecum differs in no way from the other parts. Of these twelve parts, two 9-gons and one 2-gon form one group—the leading par- tition; the two 3-gons and seven 2-gons form the other group—the subordinate partition. The terms leading and subordinate are rela- tive merely, but that partition will be taken as leading which has the smaller number of parts. The type-symbol for Aa is sae 6. The double points common to the perimeters of two parts of the same partition will be called bonds of those parts, and the parts are said to be bound by these bonds. It is well known that a type- symbol does not determine a form. For this, it is necessary to know the numbers of bonds between the several parts of either par- tition, together with the arrangement of these parts. In general the parts of a given partition may be bound in more than one way giving forms that may be projections of either links or knots. Each set of numbers of bonds of the several parts of the given partition is a clutch of that partition. The class p of a partition is the number of parts in it. The class of a form is the class of its leading partition. The order of any par- tition is equal to and is the same as the order of all knot-forms derivable from it. The deficiency % of a partition is its order minus its greatest part. 7. Let the parts of 2n be A, B,C, ... P arranged in order of magnitude, and the numbers of bonds of each part be respectively a, P, y,... 2. Let the number of bonds common to any two parts as A and B be (AB). Then PAU POANGN fo ic° sie": (AP)=a | MENA GBO) bei. \. f des (BP)=6 | RM eHO NS on, 85, (CP)=y ae eee). OR 2! or m equations with $7(2—1) unknown quantities which can have only positive integral values. The possible solutions of (@) will evidently give all the clutches for this partition. 30 C. N. Littlh— Knots, with a Census for Order Ten. 8. I, Toeorem.—If a part be solely bound to a second part, or if any g parts (¢ *p—2) be bound mutually in any way and all free bonds of these parts go to a single part, then this portion of the form constitutes a separate knot (unless there be linkage) and the string concerned in it may be drawn tight without affecting the remainder of the knot-form. Such knots are not considered as belonging to — order . In particular a 2-gon so bound throws out from consideration a clutch. 9. Il. Tororem.—No knot-form of the nth order has as leading par- tition one whose class exceeds %+ 2. Adding equations (a) above, dividing by two, and subtracting the first and any other, say the second, we find —(AB)+(CD)+(CE)+ ...(CP)+ ... (OP)=n—a—f, =xu— fi. Therefore, (AB) x 6—. In a similar way (AC) xy— (AD) xd—x (AP) xa— Adding (AB)+(AC)+ ... (AP)xf#+y+ ... #—(p—1)z Kn+ u—(p—1)% xKn— (p—2)x, we have then the two conditions (AB)+(AC)+ ... (AP) xn—(p—2)x (b) =N— Ht. Now suppose, if possible, p=%+3 (AB) + (AC) + MORRIS 2.0) yas —n—nx Kn—(H+1)" Kn—u— xn’. To the minimum values of (AB), (AC), etc., (that is, to 6—x, y—x,...) must be added 1° in all, and to no one more than x—1, by I. The «+2 smallest parts of % square are evidently (%—1)* (x—2)**. By adding these «+2 parts in any way to the mini- mum values, a clutch will be given in which each of four parts will have a single bond not going to A, and each of ~—2 parts will have two. A part of the latter kind cannot have its two free bonds ear- ried to a second part of the same kind, by I. If two parts be joined by a single bond there will be left two free bonds. Ultimately it will be necessary to join two parts of the first kind to a combina- tion of parts having but two free bonds, and I will appiy. Tf any of the +2 parts of %° be diminished by s then will s parts of 2n be ~ - O. N. Little—Knots, with a Census for Order Ten. 31 added to those of the first kind, and however the free bonds may be arranged, ultimately the same result as before will be reached. Therefore p cannot equal %+3 and still give knot-forms. Much less can p be greater than %+°3. 10. A given clutch of a leading partition does not uniquely deter- mine a form. The following proposition however holds. III. Tseorem.—All or none of the forms determined by any given clutch of a partition are knot-forms of the order considered. For, all forms to be had from any clutch of a given partition may be obtained by taking all the possible different changes (consistent with the given clutch) of relative position of the various parts. But these can all be effected by successive interchanges of the con- nections of two parts, whether such connections are direct (by a single bond), by a 2-gon, by a 3-gon, or are more complicated. We may therefore confine the attention to a definite portion of the knot and keep the remainder fixed. Let A and B, (Fig. 1, Plate I), be two parts connected as shown. Two strings, or two parts of a single string, are involved. If there were more all but two would be closed. Let the ends of these strings, or parts of a single string, leave the portion of the form under consideration at @ and ¢ on the perimeter of A, and 6 and d on that of B. Cut at these points and call the ends @ and a’, band 0’, cand ¢’, d and d'; a, b,c and d remain fixed. Now revolve through 180° about the axis AB, and join the free ends. Before the change there may be three cases. The strings may be of | we or) 24 After the change a@’ is joined to c, and ¢’ to ab ae ad a,; 6 tod, and d' to b. tee become ee as bd a b Ub ae ae ac a'e be bd bdae ad acbud Therefore coming up to this part of the knot on any string, we must leave on the same string before and after the change. If ines _ the form was a knot before the change it will be one after, and if a link before it will be a link after. _ 11. C@oils.—A succession of n 2-gons constitutes an n-cozl, which may be open or closed. Since at the 3rd or 2n+ 1st crossing of a coil the strings have the relative position of the first crossing, if the coil be closed by carrying around the ends to the beginning and 82 C. N. Little— Knots, with a Census for Order Ten. joining them so as to preserve the law of over and under one string will be formed. While if from the 27th crossing the strings are car- ried around, that string over at the Ist is under at the 2vth and, on joining, there will be two strings. Hence, as is well known, ree is always a knot, while ye always a link. ; 12. For the purpose of distinguishing between clutches giving knots and those giving links, it follows from Theorem III that we may take the direct bonds between any two parts together, and these form open coils of the subordinate partition ; and further it is evident from § 11 that any odd open coil (even number of bonds), may be dropped,-and any even coil (odd number of bonds) may be replaced by a single bond. If the resulting clutch gives link, so would the original. If the resulting clutch be still too complex for easy recog- nition of its character, the clutch of the subordinate partition of the | resulting form may perhaps be still farther reduced in the same way. If the clutches of lower orders were at hand they also could be used for settling the question. 13. We have the following theorems for throwing out clutches unproductive of knot forms. , IV. Turorem.—If a part be joined to other parts in every case by an even number of bonds, there is linkage. For, the string about this part is closed by Section 12. V. TurorremM.—If two parts are connected by two 2-gons (of the same partition with the parts) there is linking. For they may be put in succession by Theorem III. When this is done there is a 4-gon of the negative partition bound to two other parts in each case by two bonds, and IV applies. VI. Turorem.—An odd part joined to one part by an odd number of bonds and to other parts in every cage by an even number of bonds may be dropped; for, by'Theorem III it becomes a loop with a single crossing, and this can have no effect on the question of linking. In particular a 3-gon joined by one bond to one part and by two bonds to a second part may be dropped. If two odd parts are joined by an odd number of bonds, and are joined to other parts in every case by an even number of bonds there is linkage. In particular two 3-gons so joined throw out the clutch. VIL. Turorrem.—If two 3-gons, C and D, are themselves joined directly and are joined to A and B in each case by a single bond there is linkage. C. N. Little—Knots, with a Census for Order Ten. 33 For, HCM (fig. 2, Plate I) under we will say at C is over at H and M. LCN is then over at C and under at L and N. LHN is over at L, under at H, and over at N. Its continuation is therefore NML which is over at N, under at M, and over at L. 14. Crass III, OrpER ».—In this class we have . (AB) + (AC) + (BC)=n and an unique solution of equations (a), § 9. Therefore (BC)=x, (AB)=f6—x and (AC)=y—x. If two or three of these quantities be even, the clutch to which they belong will give a linkage, by Theorem IV. In other cases by § 12 there is a knot. Suppose 7 to be odd and a@ even; then x is odd, and f and y are both odd, or both even. In the first case the clutch gives a link, in the second a knot. Suppose 7 to be odd, and a odd; then ~ is even, and of (@ and y one must be odd and the other even. The clutch gives a link. Suppose 7 to be even and a even; then x is even and /, y are both odd or both even. In the first case a knot, in the second a link. Suppose 2 to be even and @ odd; then x is odd, and of f and y one must be odd and one even, and there is a knot. This proves the following : VIII. Turorem.—In odd orders only partitions of 2 into three even parts, give knots, while in even orders, only these partitions give links. 15. Crass 1V, OrpER n. Here — (AB) + (CD)=x—£f —(AC) + (BD)=x—y —(AD)+(BC)=x—6 (AB) + (AC)+(AD) oe The minimum values of (AB), (AC), (AD), (BC), (BD), (CD), are respectively S—x, y—u,d—x,0, 0,0. To get every clutch we must add in every possible way to the minimum values of (AB), (AC), (AD), all the partitions of into not more than three parts, none greater than x—1. But evidently we must increase the minimum values of any quantity of the second set (BC), (BD), (CD), by the same number that we increase the corresponding quantity of the first “set. The following scheme which considers in detail every possible case, expresses clearly the propositions for determining whether clutches of partitions of this class furnish knots or links. Let e or o indicate whether a number be even or odd. Trans. Conn. Acap., Vou. VII. 5 Sept., 1885, Ey a OC. N. Littleh—Knots, with a Census for Order Ten. 2wee epee aupee, n, Kk | Partition. | Add k= tA Boe ale Form Ore 6 e)] © evere eee eee eee eee Link. IV. e 00 e000 ooe rt §12, V. eeoo0 eee e0oo eoo eee . Ve eoo eee ooe % Ves o0eo ooe o0eo Knot. |§12, VI §11. ®0| 0000] 000 eee 000 000 Link. §12, VIL. oee oee ee 0 a §12, VI. ooee 000 e000 oee 000 Knot. |§12, VI, 11. oee 000 eeo ie §12, VI, 11. eoe eeo eoe Link. 1\e © €]/ Ooe eee oee oee eee e IV. e0oo 000 ooe ‘“ §12, V o0eo eeo 0e0 Knot. $12 e|0000| eee 000 000 ee6 rs §12, e0o oe ooe ee $12 Oo] eecee 000 000 eee 000 Link. IVE oee eoo eeo . VE ee00] 000 oee € 00 000 & §12, V oee eee eeo ut VE eoe ooe eoe Knot. §12. The first line of this scheme says that when 7 is even and x even, and 2n divided into four even parts, the minimum values of (AB), (AC), (AD), will be even, and that if a partition of x into three even parts be added to 6—x, y—x, d—x the numbers constituting the clutch will be even, and the form a link by Prop. IV. One of the propositions proved in this scheme is worthy of separate enunciation. IX. Turorem.—In even orders partitions of 2m into four even or four odd parts give link-forms only. In odd orders partitions into four even parts give link-forms only. 16. X. TaHrorem.—In all orders partitions of six even parts give link-forms only. For, an even number may be divided into five parts, of which four, two or none shall be odd. After the application of §12 the only parts to be found in the leading partition will be 4-gons and 2-gons. Every form under consideration will be reduced so as to have as lead- ing partition one of the following, with a clutch of which every number is 1. . Or, 42 ADS AAR, AAD ere In the lower orders 4°2°, 4°2*, 42°, and 2° have been found to give no knots. If the form given by 4° be drawn it is found in any particular case to consist of four closed curves, and therefore by Theorem III is always a link-form, On drawing 4*2’ it also proves to be a linkage, PEELS LINE SORE AES : C. N. Litile—Knots, with a Census for Order Ten. 35 The partition 4°2 with the given clutch can not exist in a plane. This happens in two cases in order 10. On drawing the forms in such cases additional crossings will be found to be necessary. It is therefore true that in all orders, partitions of six even parts give link- forms only. A synthetic proof of this theorem is possible. 17. Instead of continuing the consideration of the general subject we shall now illustrate the method of determining the knot-forms of any order by a particular consideration of order 10. The number of partitions of 20 into parts none greater than 10 or less than 2 is 107.* These, arranged in dictionary order, are given in full in Table I. Of these the single partition of Class II gives a link, §11; the thirty-seven marked II are cut out by Theorem II. In Class III, Theorem VIII throws out 8°4 and 86’; in Class IV, Theorem IX the eight partitions marked IX ; and in Class X, Theorem X the three marked X. The partitions remaining in Classes III to VI inclusive are alone taken as leading partitions; for, all remaining partitions appear in every possible way as subordinate partitions (§ 2), and can, therefore, furnish no additional knot-forms. We have then to tabulate the clutches of those partitions still remaining in Classes III to VI. We at once write down Tables II and III in which are omitted all clutches that are thrown out by Sections 14 and 15. In Class V, a table with headings as shown in Table IV is used. We take for illustration the first partition, 72°. Here x=3, and f—x, y—u, O—u, e—xu are 4, —1, —1, —1. In this class we must add in every possible way to the minimum values of (AB), (AC), (AD), (AE) the partitions of 2% into four parts, or fewer, none greater than x—1. The only partitions of 6 meeting these conditions are 2° and 2°1°, which may be added in seven different ways to 4, —1, —1, —1, giving the different clutches of the table. Thus adding 1, 2, 2,1, to 6—x, y—x, 0O—x, e~u we have (AB), (AC), (AD), (AE), equal to 5, 1, 1, 0, respectively. Sub- tracting (AB) from f, ete. 2B =(BC) + (BD) + (BE) =2 2C =(BC) + (CD) + (CE) =1 2D =(BD) + (CD)+(DE)=1 2EK=(BE) + (CE) + (DE)=2. These quantities are put in the columns headed 2B, >C, SD, SE, *See Tait, Trans. Roy. Soc. Edin., vol. xxxii, p. 342. 36 C. N. Little—Knots, with a Census for Order Ten. and all the possible solutions of this set of equations here as throughout the work are written down by inspection. The rest of _ this table is made out in the same way. In Table V we must, in every possible way, add the partitions of 3% into four parts or fewer, none greater than x—1. In the general case we add the partitions of (p—3)x into p—1 parts none greater than x—1. In the com- plete Table IV there are 400 clutches, and in Table V 1000. It has not been thought necessary to publish more than a sample of either table. 18. Having completed the tables of clutches we next cast out in Tables IV and V all clutches unproductive of knot-forms. The theorems already established are sufficient for this purpose. The routine followed will be readily understood from eo its use in the samples given of Tables IV and V. Partition 7°2°: in clutch (1) A and B are connected by two . 2-gons, and V applies ; (4) becomes (3) by interchanging D and E; (7) becomes (6) by interchanging C and D; and in (2) and (5) we have a 2-gon solely bound to a single part, and I applies. | Partition 6° 42°: clutch (1) is thrown out by IV since a 6-gon B is © joined to A by four bonds and to C by two; Theorem IV casts out (4) because of C, (14) because of C, (17) because of B, and (20) be- cause of A; in (2), (3), (8), (9), (11), (12), (15) and (18) a 2-gon is solely joined toa single part, and I applies; in (5) B and C form a combi- nation solely bound to A, and I applies; I throws out also (10) where D and E are bound together and to C, and (13) where the same parts are bound to B; interchanging D and E (7) becomes (6); interchanging A and B (16) becomes (7). Partition 5°32: I throws out (2), (3), (6), (8), (14), (16), (19), (20), (26), (27), (30), (31), (83), (37) and (38) ; by interchanges of parts (5=(4, CN=(5), @H=(9), — (62)=(10), (=(), (18)=( 8), = (25)=( 4), (84) = (02), (13)=(10), (21)=(0), (28)=(23), (35)=( 1), (15)=(12), (22)=(21), (29)=(23), and (36)=( 9). In (4) D is dropped Py VI, and B thus is changed to a 4-gon; then the application of $12 leaves the two 3-gons A and C joined by two 2-gons, E aah the still farther reduced B; V, there- fore, shows that the clutch gives links only. In (9), drop C by VI and B becomes a 2-gon and A a 3-gon; the two 3-gons A and D are connected by the two 2-gons E and the reduced B; therefore the clutch gives links, by V. In (11) drop C by VI and the two 3-gons A and B are connected by the two 2-gons E and the reduced D; V applies. C.N. Little—Knots, with a Census for Order Ten. 37 In Table V, clutch No. (1) of 53° is thrown out by I. In (2) the two 3-gons E and F are joined together by a single bond and to A -in each case by two bonds, and VI applies; or we might use 1 Theorem VI throws out (4), since the two 3-gons B and F are joined to each other by a single bond and to other parts in each case by two bonds. In (5), drop F, by VI; B becomes a 2-gon joining D and E, and they are 3-gons connecting A and C. An obvious exten- sion of Theorem VII throws out the clutch. Since in (7) B and C are joined together and to A, I applies. This process is continued until all clutches which do not give knot-forms have been thrown out, as well as those clutches which are repetitions of clutches previously given. The samples given of Tables IV and V constitute about one-twentieth of the complete tables. 19. We are now ready to draw the forms which the remaining clutches furnish. Derryition.—The number of Circular arrangements of n things is the number of distinct ways in which x things can be arranged in a circle, the order whether direct or retrograde being of no con- sequence. . For illustration of the general method we consider in detail the productive clutches of partition 6°42*. In clutch (6) the two 6-gons are connected, by three bonds, by a 2 gon, and by a 4-gon which is connected with one 6-gon by two bonds and with the other by a 2-gon and one bond. There will be as many forms from this clutch as there are circular arrangements of aaa b (cd), where c and d may not be separated. These are aaa b (ed) aaa b (de) aab a (ed) aab a (de). The four forms C’c,, C’c,, O%c,, O’c,, can at once be drawn. See Plate V, Knot LXIV. In (19), the only other clutch of this partition that affords forms, a _ 6-gon is connected with a 4-gon directly, by a 2-gon, and by a 6-gon which is connected with the 4-gon by a single bond and by a 2-gon. _ There are two circular arrangements of a b (cd), namely ab (cd) and b a (cd); these furnish the two forms C*d,, C*d,. Knot XXX, Plate III. In practice the operations described in this section and “in the preceding are performed simultaneously. 20. In Class VI every productive partition is used as a leading ‘Saw 38 C. N. Little—Knots, with a Census for Order Ten. partition. But since the subordinate partitions here belong to the same class, every form, with certain exceptions, will be found twice ; this affords a check on the completeness of this part of the. work. The exceptions are the amphicheiral knot-forms. These have the same partitions similarly connected both as leading and as sub- ordinate partitions. These therefore appear but once. In the partition 53°, which was given as the shortest possible illustration of Table V, clutch (3) furnishes a single form D*s,, which already had been obtained under 54°32°; clutch (6) gives two which had been obtained, Du, under 643°2* and D’x, under 54°32”, Clutch (8) gives the only new knot-form from this partition, viz: the amphicheiral D*o. From the clutches of Classes III, IV, V and VI 364 ten-fold knot forms are obtained. 21. The Derivation of Knots from Knot-Forms.—Prof. Tait has _ not described the methods which he used in his derivation of the knots of lower orders from the knot-forms. In 1884 he says :* “the treatment to which I have subjected Kirkman’s collection of forms, in order to group together mere varieties or transformations of one special form, is undoubtedly still more tentative in its nature; and thus, though I have grouped together many widely different forms, I cannot be absolutely certain that all those groups are essentially dif- ferent from one another.” If a ten-fold knot be placed upon a plane in such a way as to have but ten crossings the eye will project it upon the plane in a form which will be found among the 364 above obtained. If the knot gives more than one form it will be possible to obtain any other of its forms by one or more turnings over of restricted portions of the knot while the remainder is held fixed. Now the string cannot issue from the portion of the knot that is turned at more than four points, for in that case the turning would introduce consecutive overs, and one or more additional crossings; the portion of the knot that is turned must therefore be wholly between two parts of the given knot form and in turning it we untwist two of the strings at one point and twist two at another, the result being simply to change the position of a single bond from one end of the connection to the other. The class of the form is therefore not changed, and all the forms of any knot belong to the same class. 22. Moreover in order 10 and lower orders all the forms coming from any clutch are obtained by changing the position of single * Trans. Roy. Soc. Edin., vol. xxxii, p. 327. a A a eons ‘ caneiaal -_— > i AGS SR IN a ily on, C. N. Littlh—Knots, with a Census for Order Ten. 39 bonds in the connections of pairs of parts. Therefore in these orders all forms from any clutch are forms of the same knot. The sub- ordinate partitions of these forms are then to be examined and all forms added which are obtained from them by changing the positions of connections of their parts, retaining the given clutch of the subor- dinate partition. These forms in turn are treated in the same way, but it will usually happen that no new form of the knot is obtained and the complete determination of the knot in all of its forms is finished. 23. We take for illustration Knot I of Class IV, shewn on Plate I. Ba, becomes Ba, by twisting about a vertical axis the 2-gon connect- ing the 8-gon and 7-gon. The first crossing below is opened and the strings above are crossed, the rest of the knot remaining fixed. Twisting the 2-gon again Ba, is obtained and nothing new is gotten by further changes of the forms. Since the negative partition in _ every case consists of two parts joined by three symmetrical comnec- tions, which have only one circular arrangement, there are no other forms of Knot L 24. The knots of Class III, order n, are unique; since three things have but one circular arrangement. 25. The knots of Classes III, [V, V will be found with their forms grouped together in Plates I-V. On Plates V—VII are figured the forms of Class VI grouped as they come from the clutches, except that no form is repeated. The knots of this class will be found in Table VI. Every knot-form is the projection of two knots, one of which is the perverted image of the other, and consequently each group of knot-forms belongs to two knots which are in general different. If a series of knot-forms contains any amphicheiral form then it will also contain the perversion of every form of the series not amphicheiral. The series consists of the forms of one knot and not of two. é 26. In order 10 I find, counting a knot and its irreconcilable perver- sion as two: Class. Forms. Knots. Knots. III, 6 12 6 IV, 25 30 15 ve 200 128 64 VI, 133 64 39 40 O. N. Littleh—Knots, with a Census for Order Ten. In lower orders Professor Tait has found: Orders. Forms. Knots. Knots. 3 1 2 1 4 iL u 1 5 2 4 2 6 3 5 3 7 10 14 7 8 27 31 18 9 100 82 4] The fourth column contains the numbers as they are given by Tait, the perversions of knots not being counted. In so long a labor as is involved in making such a census the opportunities for error are many. Any errors or omissions that may be found in the census are to be attributed to the writer rather than to the method, which is simple and direct. TaBLE I.—Partitions of 20. Gpass II. |Cu. 1V.—Contin’d Cu. V.--Contin’d.| Crass VI. |¢p. VII.-Conti’d 10° Bx) 9292 ' (8628 Tl. 1025 6495 Crass TIL 8732 | 8532? , | Il, 932% II | 63294 ( 1082 IX. 8642 II. 4 84222 It 8494 "5295 II } 1073 863° : 84372 | 83728 54394 : ) 1064 8522 334 ( 7524 53393 (105? 8543 7293 TI. ~ 74322 4394 922 IX. 843 76322 73522 423298 983 724.2 45422 BXe. 6224 43492 974 IDig ABE 75329 65323 369 965 7652 74239 XGA : VII. a4 7643 4498) 643222 Ctass VIL 875 EX 573 624.22 6342 VIII. 86? 754? : 62329 524.93 Li S628 F 7°26 EXE 682 65222 523292 IGE bite Crass IV. 6°53 65432 54732? 43995 10622 IX. oe 6533 54332 . gig ry, J 10532 ee 6432 53° -y 0492 | TX. 5 64232 x 4% Cuass XI. 1043? Cuass V. 5332 43372 ( 972? tr, J 1042* 52422 4°34 IT, 428 9632 ™ 1103222 52432 3727 IL. 4 9542 952" 5493 Crass VII. cee 9532 Ir. 2 94322 45 I. 82 aces (9423 {93% | IL 7325 910 TABLE II.—Clutches for p=3. Se a +4 HOS Partition. LOS ti r ROS Partition. 438 artition 428 Partition. az 3 929 91 1 An 974 631 Ac 815 532 Ad 983 y (iy ee: ) 965 5 41 Ae 7°6 43 3 Af 4] TABLE III.—Clutches for p=4. C. N. Littleh—Knots, with a Census for Order Ten. — bel oS ees _ A 2 (ez) rt HST rsG SECs 0) 4 es me = ea oY . In CiaMntionis MAAMtNMMRMNO - om & (ax) 7 SN Mies bsilsstGo : a 9 nN 29344 |-2-2-2-9-2 3 3211/0012 2/3 000/00 0/11 O)L. K=5 | | | 2-100} 1 0 0/0 0 1)(2) ae P10 010 ae | | 542322, 23334 | ,82221/01112/2001/000,2 0 0j@) VE | | 0111/1 10/00 0\(5) VIL | 0210/01 1/0°0 0\(6) Knot Du, of 645°2?, knot | | | | D2x, of 542322” 33333 | /2222 2/111 1 1)2'0.0 0) On Oem | | 1100/01 0/01 1((8) Kuot D'o | | Amph. * * | x * * * Sa en EO a ae Re Rae eee eek ee ee ae cae oi — ee - oT et BES: a * ule =< won ’ _ = 5 noe — : bz wrge . as 3 — . 5 = " M en wet CO A OC. N. Littlh—Knots, with a Census for Order Ten. 43 TABLE VI.—Knots of Class VI. Knot. No. Forms. Eel od te Date Day, Daz, Das. Das, Das. II 1 Db. Ill 4 Dei, Des, Des, Dey. IV | 4 De,, Deg, De;, Dey. YN De al) DNB ies Wal 4 Dk, Amph, Dkg||,* D?j Amph. WAU al DI Amph. VET oe) Om. 27. D2ts. IX 16 | Dn, Amph, Dngl, Dngl, Dnyl, D%i:, Dig Amph, D*h,|, Dhol), : D*h; Amph, D*’s Amph. axe 12 | Du, Due, Dus, Duy, Dus, D?x,, D?x2, D’xs, D?x,, D?x;, Doi, Dos. i XI eee 4 Dp,, Dpz, Dps, Dps, Dp;, Dpe, D*e,, D’e., Des, Dey, D’e;, D°e,. XII | 9 | Dai, Dao, Dqs. D*fi, D?f., D°fs, D’c, D2u, D2b. TUT mG DreeDr Drs D2q7,, D2q5, D*q3- XIV 3 Ds, D?e,;, D?eo. XV 2 DYE, eG XVI 9 Dy, Dv:, Dy,, Dyz, Dys, D’g:, D’ge, D?y, Dj. XVII 9 | Dwil, Dw. Amph, Dwsl, D7], Amph, D*l.l, D*l Amph. PEM) 6416.) Dxy, Dx9,-Dxs, D?a,,, D’aq, D?a. XIX 4 | Dz, Amph, Dz,||, D*b Amph. xXx l D*b Amph.. Mele eee S| Nt, it, DA: XXII 1 Dk. XXIII |) OF XXIV 1 D?n. Be. 3 | D?p,|, D’p. Amph. XXXVI 3 | D*r, Amph, D®ro. XX VII 3 D*s;, D?s,, D?s3. XXVIII 2 |-Dv;, Dv XXIX 2 | D?w., D’we XXX 2 | D?z;, Dz. XXXI 1 Dd Amph XXXII 1 | D’e Amph. XXXIII 4 oe Amph, D#f.|, D®q Amph. XXXIV 1 Dig. XXXV ee Deke XXXVI 1 | D§m Amph. XXXVII 1 | Do Amph. XXXVIII 1 | D*’p Amph. KXXIX 1." || abe * The symbol || indicates that a form and its perversion are both included. + The subordinate partition of Doz, Plate V. should be 643°2°. 1TV.—Tue Amyvoryric action oF Diasrase or Matt, as MODIFIED BY VARIOUS CONDITIONS; STUDIED QUANTITATIVELY. By R. H. CHITTENDEN AND Gro. W. Cummins, Pu.B. Tue close relationship existing between the diastase of malt and the amylolytic ferment of saliva has led us to make a careful study of the conditions favorable to the action of the former, in the hope of obtaining confirmation of previous results obtained with the sali- vary ferment.* The widespread use, moreover, of malt extracts as therapeutic agents lends to the work in question a practical interest, which in no wise detracts from its value. Falk+ has recorded that the diastase of malt loses its amylolytic power under the influence of dilute acid, similar to the ferment of. saliva; that it is made inactive by gastric juice and that the retard- ing influence of a dilute acid (say 0°0135 per cent.) on its amylolytic power is diminished by the presence of peptone, owing to the proba- ble formation of a peptone-acid compound. Falk, moreover, states that the retarding action of hydrochloric acid is due to destruction of the ferment, since on neutralization of the acid, amylolytic power is not restored. Kjeldahl{ has recorded that dilute acids in very small quantity retard the amylolytic action of diastase; if, however, smaller, minimum quantities of acid are added the amylolytic power of diastase is increased. The same inyestigator§ has also noticed a like accelerating action of very small quantities of acid on invertin. Basnitz|| has found that the presence of carbonic acid invariably in- creases the amylolytic power of diastase. Detmer{] has recorded the same fact and in addition, that small quantities of citric acid as well as of phosphoric and hydrochloric acid increase the diastatic power of malt. Larger quantities of these acids render the malt extract in- active. Detmer has also found that the presence of a very slight alkaline reaction diminishes the amylolytic power of the ferment. Brown and Heron** state that a malt extract neutralized with barium hydroxide has its amylolytic power somewhat weakened; thus im- * Chittenden and Smith, Trans. Conn. Acad., vol. vi, p. 343. + Virchow’s Archiv, vol. Ixxxiv, p. 119. { Jahresbericht fiir Thierchemie, 1879, p. 382. § Jahresbericht fiir Thierchemie, 1881, p. 449. || Berichte der deutsch chem. Gesell., vol. xi, p. 1443. §| Zeitschrift fiir physiol. chemie, vol. vii, p. 2. ** TLiebig’s Annalen der Chernie, vol. excix, pp. 236-238. Chittenden and Cummins—Action of Diastase of Malt. 45 plying that the ferment acts more vigorously in the naturally acid extract than in a neutral fluid. The same investigators found that making the extract faintly alkaline with sodium carbonate also diminished somewhat the activity of the ferment, while sodium hydroxide completely stopped the action of the ferment. A like re- sult was also obtained on the addition of 0-05 per cent. salicylic acid. Such are the recorded statements bearing on this question. Few quantitative results are given, and the influence of proteid matter, aside from its connection with dilute acid, has not been considered. Method employed. A fresh malt extract was prepared for each series of experiments, since the fluid tends rapidly to become acid, owing to the develop- ment of schizomycetes. The extract was prepared from coarsely ground malted barley, by simply extracting it with water at 40° C. for two to three hours (5 grams barley to 100 c. c. water), then filter- ing, neutralizing and diluting to 500 ¢. ¢. Owing to the great difficulty of obtaining perfectly neutral starch, that used in the present work was prepared from potatoes, thoroughly washed and dried, making a starch perfectly neutral to the most delicate test papers. The volume of each digestive mixture in the various experiments was 100 ¢.¢., containing 1 gram of starch pre- viously boiled with a portion of the water, a definite quantity of the malt extract and a given amount of acid, alkali, or proteid matter, except in the control, which was naturally free from the latter. In determining amylolytic power the digestive mixtures were warmed at 40° C. for thirty minutes, after which further ferment action was stopped by boiling the fluid. The extent of amylolytic action was then ascertained by determining in one-fourth of the fluid, made up to 100 c.¢., the amount of reducing bodies by means of Allihn’s* gravimetric method; the reducing bodies being then calculated, for the sake of convenience, to dextrose, from which in turn, was calcu- lated the percentage of starch converted. Influence of sodium carbonate on the amylolytic action of diastase. Previous experiments} with saliva have shown that the percentage of alkaline carbonate which absolutely or to a certain extent hinders its amylolytic action can be designated only for a definite mixture and not in a general sense, owing to variations in the amount of pro- * Zeitschrift fiir Analytische Chemie, vol. xxii, p. 448. + Chittenden and Smith. 46 Chittenden and Cummins—Amylolytic Action teid matter present and doubtless also in part to increase or decrease in the amount of ferment. It became necessary, therefore, at first, to ascertain something re- garding the relative amylolytic action of the malt extract, which contains some proteid matter. Three quantities of malt extract were employed, which by thirty minutes warming at 40° C. with 1 gram of starch in the manner already described, gave the follow- ing results : Total amount Malt extract. Wt. Cuin 4. reducing bodies. Starch converted. L0Jer(c; 071192 gram. , 0°2428 gram. 21°85 per cent. 15 01489 * 0°3038 27°34 2D 0°1543 0°3150 28°35 i It is interesting to note here that, as in the case of the amylolytic ferment of saliva, there is no quantitative relation between the amount of ferment and the extent of amylolytic action ; it is only when the ferment solution is greatly diluted that amylolytic action can be taken as a definite measure of the amount of ferment present. Kjeldahl* has likewise studied the influence of the quantity of diastase upon the amount of sugar formed under given conditions and he came to the conclusion that the formation of sugar was pro- portional to the amount of ferment only up to a certain point ; be- yond which, increase in the amount of ferment was not accompanied by proportional increase in the formation of sugar. Preliminary experiments showed us that the ferment of malt is very susceptible to the action of sodium carbonate; the addition of even 0°025 gram of the alkaline carbonate to 15 ¢. c. of ‘perfectly neutral malt extract, with subsequent dilution to 100 ¢. c. allowed no diastatic action whatever. Following are two series of experiments illustrating the action of different percentages of sodium carbonatet on the ferment under different degrees of dilution. a. with 15 c. e. of the standard malt extract. Total amount Na2Cos. Wt. Cuin 4. reducing bodies. Starch converted. 0 01371 gram. 0°2794 gram. 25.14 per cent. 00005 per cent. 0°1318 0°2684 24°15 0°0010 0°1274 0°2596 23.36 0-0020 0-0544 01124 10°11 j 0°0050 0:0197 0°0434 3°90 0:0080 0°0134 0:0312 2°80 00100 0°0135 0:0314 2°82 0°0125 0°0065 0:0148 . 1:33 0°0250 0 * Jahresbericht fiir Thierchemie, 1879, 381. + The standard solutions of sodium carbonate employed in these experiments were made from the chemically pure anhydrous salt. = of Diastase of Malt, as modified by various conditions, 47 b. with 30 c. c. of the standard malt extract. Total amount Na.Cos. Wt. Cu in 4. reducing bodies. Starch converted. 0 0°1650 gram. 0°3372 gram. 30°34 per cent. 0-001 per cent. 0°1578 0°3224 29°01 0°003 0°1465 0:2986 26°87 0°005 0°1147 0°2334 21-00 07010 00380 0-0796 716 0°025 00238 0°0516 4°64 0°050 0 It is evident from these results that the amylolytic power of dias- tase, like that of ptyaline, is diminished in proportion as the percent- age of alkaline carbonate is increased. Moreover, it would appear by comparison with results previously obtained* that diastase is far more susceptible to the action of sodium carbonate than ptyaline, and also that dilution of the malt extract does not so materially affect the retarding action of the different percentages of alkaline car- bonate as in the case of saliva. Both of these results, however, may be due either to the presence of a larger amount of proteid matter in the saliva or to the presence of a larger proportion of ferment, or in fact, to both. It is noticeable in both series of experiments that the amylolytic power of the ferment after gradually diminishing appears to receive a sudden check, which in the larger amount of malt ex- tract is produced by just double the percentage of carbonate requisite with the smaller amount of malt. In this way the effect of dilution is apparent and shows moreover that the exact influence of a given percentage of the alkaline carbonate can be designated only for a definite mixture. Destructive action of sodium carbonate on diastase. In order to ascertain how far the retarding action of sodium car- bonate is due to destruction of the ferment, the following experiment was tried. Six mixtures were made as follows: 1 2 3 4 5 6 Malt extract -._ 30 c.c. BOR EAC: 30) ice: 30 ee. 30 ce. 30 @.e. Na,Co; sol. .-.. 0 “ 1-25 “ 0-1% 2°5 “ O-1Z 2°5 “ 0°54 5 0-5¢10 “ 05% ae 20." Weis We 1-50 15 1g) 50 50 50 50 50 50 Percent. Na,Co, 0 0-0025 0-005 0-025 0-05 071 These were warmed at 40° C. for 1 hour, then neutralizing and b) > _ equalizing mixtures were added as follows: * Chittenden and Smith. 48 Chittenden und Cummins—Amylolytic Action 1 2 3 4 5 6 0-1 per cent. HCl 0 0°42 c.c. O86¢c¢. 4:25 c¢.c. 8:5 c.c. 16°96 cre: 0°5 i Na.Co; ees 10 ae. 9°75 9°5 7:5 50 0 0-1 a HCl J 16°95 16°55 161 12-7 8°45 0 The six solutions were now exactly alike; neutral to test papers and contained the same amounts of diastase and sodium chloride. - They had, however, been exposed to the action of the above percent- ages of sodium carbonate for 1 hour at 40° C. Their amylolytic power was now determined in the usual manner (action on 1 gram of starch in a total dilution of 100-¢. ¢.) with the following results : * Total amount No. Wt. Cuin 4. reducing bodies. Starch converted. ] i 01739 gram. 0°3558 gram. 32°02 per cent. 2 01737 073554 Seon 3 01745 0°3570 32°13 4 00341 00722 6°49 5 0°0319 0°0678 6°10 6 00281 0°0602 5°41 No destructive action is apparent until 0°025 per cent. sodium car- bonate is reached; warming the malt extract with 0°005 per cent. sodium carbonate causes no destruction whatever, while with 0°025 per cent. destruction is very great. The amount of malt extract (30 c. ¢.) experimented with, being the same as was used in deter- mining the influence of alkaline carbonate on the amylolytic power of the ferment, the two series of results are directly comparable and show plainly that the retarding action of small percentages, in the present case up to 0°005 per cent., is due to simple retardation witb- out destruction of the ferment. Beyond this point, however, as in the presence of 0°025 per cent. the greatly diminished amylolytic action is due to destruction of the ferment. Hence it would appear that in the case of the diastase of malt the destructive action of sodium carbonate is out of all proportion to its retarding action. This apparent difference, however, between diastase and the ptyaline of saliva is due, as we shall show later on, to the comparatively small amount of .proteid matter in the malt extract. Saliva very greatly diluted, so that the percentage of proteid matter is reduced to a minimum, shows similar results. Influence of neutral peptone on the amylolytic action of diastase. It was demonstrated some time since,t that the presence of neutral peptone tends to increase the amylolytic action of neutral saliva. * The two equalizing mixtures were united before being added to the main solutions, + Chittenden and Ely, Amer, Chem, Jour., yol, iv, 107, of Diastase of Malt, as modified by various conditions. 49 Langley and Eves* have confirmed this statement, although they do not believe in the theory of a direct stimulation of the ferment, advanced by one of us. We find now that neutral peptone added to a neutral solution of malt diastase, similarly increases its amylolytic action; the increase being even greater than noticed in the case of neutral saliva. Two series of experiments were tried with the fol- lowing results ; the peptone used being made perfectly neutral with a dilute solution of sodium carbonate. a, with 15 c.c. of the standard malt extract. Total amount Peptone. Wt. Cu in 44. reducing bodies. Starch conyerted. 0 ~0°1140 gram. 0:2320 gram. 20°88 per cent. 01 per cent. 071545 0°3154 28°38 02 071512 0°3084 27°15 0°3 071494 0°3048 27°43 0°5 01457 0°2970 26°73 10 071427 0°2910 : 26°19 b. with 30 ¢. ec. of the standard malt extract. 0 0°1785 gram. 0°3654 gram. 32°88 per cent. 0-1 per cent. 0°1847 03772 33°94 0°3 071912 0°3916 35°24 Peptone causes increased amylolytic action throughout; with 15 c. c, of malt extract, the smallest amount of peptone gives the greatest acceleration, which slowly diminishes as the percentage of peptone is increased; with 30 c. c. of malt extract, however, accel- eration, which is much less than in the preceding series, increases with the increase in peptone. It is hard to find any reason for this acceleration in amylolytic action, other than a direct stimulation of the ferment. . Influence of sodium carbonate on the amylolytic action of diastase in the presence of proteid matter. Proteid matter tends to prevent the retarding action of sodium carbonate on this ferment, as in the case of the salivary ferment. Thus, the addition of neutral peptone to a malt extract allows vigor- ous amylolytic action to take place in the presence of percentages of sodium carbonate, which alone would completely destroy the ferment. The following experiments, using 15 c. c. of the standard malt extract in each instance, illustrate the influence of peptone on the action of sodium carbonate. * Journal of Physiology, vol. iv, No. 1. TRANS. Conn. AcaD., Vou. VII. 7 Oct., 1885, 50 Chittenden and Cummins—Amylolytic Action With 0°5 per cent. neutral peptone. Total amount NayCO3. Wt. Cuin 4%. reducing bodies. Starch converted. 0 01388 gram. 0°2828 gram. 25°45 per cent. 0-001 per cent. 0°1443 0°2942 26°47 0-002 071431 0°2918 26°27 0-003 0°1485 0°3030 27°27 0°004 0°1404 0°2860 25°74 0°005 071406 0°2864 25°77 0-010 01317 0°2682 24°13 It is thus seen that the presence of 0°5 per cent. of neutral peptone entirely prevents the retarding action of the several percentages of sodium carbonate, except in the last experiment of the series where slight retardation is apparent. It is to be remembered here that even 0:005 per cent. of sodium carbonate alone, almost completely stops the action of the ferment. What at first sight appears to be strange in this last series of experiments, is that the first three per- centages of sodium carbonate cause a gradual increase in amylolytic action over that of the neutral fluid plus like percentage of peptone. The explanation of this, however, is quite simple. In studying the influence of neutral peptone on the action of the ferment (15 ¢. ¢. malt) it was found that the greatest acceleration of amylolytic action was obtained with the smallest percentage of peptone, and moreover that ferment action diminished in proportion as the peptone was increased. Now peptone undoubtedly prevents the action of sodium carbonate on the ferment by combining with it, forming an alkaline carbonate-proteid compound, possessed of but little retarding action ; hence in the above experiment the first action of the smallest percen- tages of sodium carbonate is to diminish the amount of free peptone, thus causing slight acceleration ; further on, however, the increased amount of alkaline-proteid body formed, counteracts the accelerating influence of the free peptone, when gradual retardation commences ; finally, increase in the percentage of sodium carbonate leads to the presence of free sodium carbonate, when amylolytic action comes to a sudden standstill. This point being reached, increasing the per- centage of peptone prevents the stoppage of ferment action. This is well illustrated by the following series of experiments, using larger percentages of both peptone and sodium carbonate, but 15 ¢. c. of — the malt extract, as before. 2 — - OMAK EPS wpa of Diastase of Malt, as modified by various conditions. 51 Total amount Starch Neutral peptone. Na ,CO3. Wt. Cu in 4. reaucing bodies. converted. 1‘0 per cent. 0 0°2078 gram. 0°4268 gram. 38°41 per cent. 10 0°010 per cent. 0°1583 0°3234 29°10 1:0 0°025 0°1529 073122 27:09 1:0 0-050 01351 0°2754 24°78 1-0 0°100 00086 0-0209 1°88 2°0 0-100 01341 0°2730 24°57 Here, as before, the retarding action of sodium carbonate is held in check by the peptone, although there is slight retardation due to the alkaline-proteid body formed. Finally, the percentage of sodium carbonate being increased beyond the necessary proportion of pep- tone, there is a sudden cessation of ferment action. Increasing the amount of peptone, however, prevents this retarding action; evi- dently the alkaline-proteid body is without much effect-on the fer- ment, only slowly diminishing its amylolytic power. Influence of acid-proteids on the amylolytic action of diastase. Falk has noticed that peptone prevents to a certain extent, the retarding action of dilute acid on this ‘ferment; no quantitative results, however, have been recorded, nor has any attempt been made to ascertain whether said action is due to simple retardation, or destruction of the ferment, or both. The action of acids, whether free or combined with proteid matter, on the diastase of malt is particularly important, in view of the rapid passage of the ferment into the stomach when taken in therapeutical preparations. Its ultimate fate must depend in great part upon the action of free and combined (proteid) hydrochloric acid upon it. It is, moreover, im- portant to compare the behavior of the ferment in this respect, with the amylolytic ferment of saliva. An aqueous extract of malt prepared as described, contains but little proteid matter; as a ruie 2°0-2°5 c.¢. of 071 per cent. hydro- chloric acid are required to completely saturate the proteid matter contained in 30c¢.c. of the neutral malt extract. This point was ascertained by use of the tropaeolin test for free acid as recommended _ by Danilewsky.* Thus, by way of illustration, in one instance 30 ¢. ¢. of carefully neutralized malt extract required the addition of 3:3 - ¢. ¢. 01 per cent. HCl to give the tropaeolin reaction for free acid, and since nothing smaller than 0:003 per cent. free HCl can be detected by this method, it follows, making the proper deduction, that 2°3 ¢. ¢. of 0-1 per cent. HCl are required to completely saturate * Centralbl. Med. Wiss., 1880; also description in Trans. Conn. Acad., vol. vi, p. 360. 52 Chittenden and Cummins—Amylolytic Action the proteid matter in 30 c. ¢. of the extract, which would then contain 0°0023 per cent. combined HCl in the form of acid-proteids.* Our first experiments were made to ascertain the influence of small percentages of combined acid on the action of the ferment, viz: the influence of such additions of dilute acid to the neutral malt extract. as would completely saturate the proteid matter present, without giving any free acid whatever. The results plainly show that the addition of very small quantities of dilute hydrochloric acid to a neutral solution of diastase increases the amylolytic action of the ferment. Following are a few of the results obtained. A. Malt extract, neutral to test papers, required 274 ¢. c. 071 per cent. HCl to completely saturate the proteid matter in 30 ¢. ¢c.; the fluid was then acid to test papers but contained no free acid. Per cent. combined HCl. Wt. Cu in 4. a. with 30-¢.c. of the malt extract. Total amount reducing bodies. Starch converted. 1 0 071630 gram. 0°3332 gram. 29°98 per cent. 0°0024 0°1749 0°3578 32°19 Ul 6. with 15 c. c. of the malt extract. 0 0°1285 gram. 0°2516 gram. 23°54 per cent. 0-0012 0°1460 0°2976 26°78 B. Malt extract, neutral to test papers, required 3:1 ¢. c. 0°1 per cent. _ HC] to saturate the proteid matter in 30 ¢. ¢. of the extract. . a. with 30 ¢. c. of the malt extract. Per cent. Total amount combined HCl. Wt. Cu in 4. reducing bodies. Starch converted. 0 0°1595 gram. 0°3258 gram. 29°32 per cent. 0-0031 0-176] 0°3602 32°41 b. with 15 ec.ec. of the malt extract. 0 0°1319 gram. 0°2686 gram. 24°17 per cent. 0°00155 071599 0°3266 AS oy) C. With 30 ¢. ¢. of malt extract; two distinct preparations. Per cent. Total amount combined HC}, Wt. Cu in 4, reducing bodies. Starch converted. . 0 071575 gram. 0°3214 gram. 28°92 per cent. 0:0023 01766 0°3612 32°50 0 0°168] 0°3438 30°94 - ¢ 0°0023 O-1LT70 0°3620 32°58 s * Doubtless, however, it is not wholly as HCl-proteid, since the extract frequently : contains a trace of sodium lactate, rat of Diastase of Malt, as modified by various conditions. 53 Such a degree of uniformity in the results, makes it evident that the slight. accelerating action of the acid-proteid is a constant one under the above conditions, and indeed all of our results in this direction clearly indicate such to be the case. Although acid-proteid thus accelerates amylolytic action, it is still able under slightly different conditions to retard the action of the ferment and even cause destruction ; thus by warming the amount of ferment (30 ¢. c. malt extract), used in the preceding experiment, at 40° C. for 1 hour with the acid necessary to saturate the proteids, in a total volume of 50 ¢.¢., thus doubling the percentage of acid, amylolytic action was found on subsequent neutralization and testing with starch paste, to be very much diminished. The following results obtained with the two malt extracts used in C illustrate this point : a. db, Cr d. Per cent. HCl __ 0 0:0046 0 0:0046 = SS —\ ‘ia a aa Malt extract_... 30c c¢. 30. che 30 ¢c. ec. 30) (eke: 0°1 percent. HCl 0 nie) te 0 2e3 mes al 40) setae ® seen DA De ce ig(erhs Ao" =) ies lige te 50°0 ce. 50°0 ec. ¢. 50:0 c. ¢. 50:0 ¢. ¢. These solutions were warmed at 40° C. for 1 hour, then neutraliz- ing and equalizing mixtures were added, after which amylolytic action was determined by adding starch paste, diluting to 100 ¢.c¢., warming at 40° C. for 30 minutes, ete., with the following results, expressed in the percentage of starch converted into sugar. a. b. C. d. a -_A_-— — 36-24 21-14. 29-25 22-96 Hence, it is apparent that under these conditions, acid-proteids may exert a destructive action on the ferment. This destructive action takes place with considerable degree of rapidity, as is shown by the following experiment, which at the same time illustrates the acceler- ating action of smaller percentages of combined acid and confirms the preceding experiments. The neutral malt extract required 2°4 c. c. 0-1 per cent. HCl to combine with the proteid matter in 30 ¢. ¢. — — —— Destructive action. Accelerating action. oo ———S SS = 1 Z oe 4 5 Malt extract_-_--- 30 '¢. c: 30} ere: 30° @e. 30) cc. 30) esc: 0-1 per cent. HCl 0 DA 24 ‘* 0 Dr t 10) BES eS ae 20) % Dione iGo TO* * T0* “* 50 a D0 “ 50 i 100 100 Per cent. HCl... 0 0°0048 0°0048 0 00024 * Containing one gram of starch. 54 Chittenden and Cummins—Amylolytic Action The amylolytic action of 4 and 5 was tested directly by warming the mixtures at 40° C. for 30 minutes and then determining the amount of reducing bodies. No. 1 (the control) was warmed at 40° C. for 30 minutes, while No. 2 was warmed at the same temperature for 15 minutes and No. 3 for 30 minutes. Neutralizing and equaliz- ing mixtures were added as follows: 1. a. 3. 0°1 per cent. Na2COs3-.-- 0 aHLayrey (2 3°5 ©. ¢. : 0-1 per cent. HCl_.---_- 2°4¢.¢. 0 0 0-1 per cent. NazCO3--- By 0 0 All three were then mixed with one gram of starch and made up to 100 ¢.¢. in order to determine amylolytic action; each solution was neutral and contained the same quantity of sodium chloride as well as the same amount of ferment and starch. Following are the results of all five: Total amount No. Wt. Cuin 4. reducing bodies. Starch converted. 1 0:1630 gram. 0°3382 gram. 29°98 per cent. + 2 071253 0°2554 22°98 3 071195 0°2434 21°90 44 0°1628 0°3322 * 29°89 5 0°1749 0°3578 32°19 It is thus seen that with this amount of ferment, 0-0024 per cent. combined acid causes an acceleration in amylolytic action (Nos. 4 and 5) amounting to over 2 per cent. in the quantity of starch con- verted, while warming the same amount of ferment with the same amount of acid, but under a less degree of dilution, causes a destruc- tion of the ferment amounting to 7 per cent. in the conversion of the starch; 15 minutes longer at 40° C. causes only a slightly increased destruction. Influence of acid-peptone. By increasing the amount of proteid matter, larger percentages of hydrochloric acid can be added to a malt extract without retarding the action of the ferment or even interfering with the accelerating action of the smaller percentages. As previously stated, Falk has shown that peptone prevents to a certain extent the retarding action of hydrochloric acid, but it does more than this, it causes accelera- tion in ferment action not only in neutral solution, as already shown, but in an acid solution likewise, provided there is an excess of pep- tone present, in which case the acid-peptone compound formed, causes greater acceleration than the same percentage of peptone alone would do if added to a neutral solution of the ferment. The follow- Py of Diastase of Malt, as modified by various conditions. 5A ing experiments, which we have repeated many times, testify to the accuracy of this statement: peptone. Gombinga HCL. Wt. Cuin 4. Hd ea beiee cid: 0°2 0 0°2118 gram. 0°4356 gram. 39°20 per cent. 0-2 0°0003 0°2168 04460 40°14 0°2 0°0005 O:211'5 0°4348 Boas 0°2 0-0030 0°2165 0°4454 40°18 0°2 0-0050 0°2175 0°4478 40°30 0°2 0 0-1730 0°3540 31°86 0-2 0°0003 071726 0°3532 31°78 0°2 0°0005 0°1802 0°3688 33°17 0:2 0:0010 0-1L794 0°3672 33°04 0°2 0°0030 071765 0°3610 32°49 0°2 0-0050° 01775 0°3632 32°68 Acceleration in amylolytic action is here quite noticeable ; the pep- tone, however, is in considerable excess. As the peptone approaches saturation, retardation commences as is shown by the following ex- periments ; such a point being reached, however, retardation can be completely prevented by increasing the amount of peptone. Per cent. Per cent. Total amount Starch Peptone. combined HCl, Wt. Cuin ¥. reducing bodies. converted. 0-2 0 0°1716 gram, 0°3508 gram. 31-57 per cent. 0°2 0-008 071732 0°3424 30°81 0-2 0°010 071631 0°333 30°00 0-2 0-012 0°1442 0°2940 26°46 0-5 0 0°1633 0°3338 30°04. 0°5 0-012 071652 0°3376 30°38 0°5 0°025 01170 0°2384 21°45 The peptone employed in this last experiment required 11°8 c. c. of 0-1 per cent. hydrochloric acid to completely saturate 0°2 gram, con- sequently in the fourth experiment the peptone was more than com- pletely saturated with acid, but the amount of free acid could have been only 0:0002 per cent. In the last experiment, however, retard- ing action is due wholly to acid-peptone, no free acid at all being present. Increasing the percentage of combined acid still further, the proteid matter at the same time being just saturated, causes far greater retardation in the action of the ferment. Per cent. Per cent. Total amount Starch Peptone. combined HCl. Wt. Cuin 4. reducing bodies. converted. 0°5 0 0°1607 gram. 0°3282 gram. 29°53 per cent. 0°5 0°012 0°1661 0°3394 30°54 0°5 0°015 071670 0*3412 28°70 0°5 0°025 0°1202 0°2448 22°03 0°65 0:035* 0°0317 00674 6°06 * With this percentage the peptone is exactly saturated with acid, 56 Chittenden and Cummins—Amylolytic Action All of these results show acceleration under the influence of small percentages of combined acid, followed, as the percentages are in- creased, by decided retardation, thus agreeing with the results pre- viously obtained with the salivary ferment. The destructive action of small percentages of acid-peptone on the _ ferment is not great in the presence of an excess of peptone, but as the peptone approaches saturation its destructive power is increased, and when completely saturated with acid, a moderate amount of peptone so combined will quickly and completely destroy the fer- ment. This is plainly shown in the following series of experiments : , if. 2. 3. 4. Malt extract.._..... 15¢ ¢. Lbvexes lbtenc lbiene Peptone sol.*______- 20 20 20ccem, 20 HCl 0:1 per cent. ---- 0 1 2 4° Ep Oe Se eee 3 Ib 14 13 11 50 50 50 50 Peptone) 22 eee ae-oe: 0-1 per cent. 0-l percent. 071 percent. 0:1 percent. Combined HCl_--- -- 0 0-002 0004 0-008 These mixtures were warmed at 40° C. for 30 minutes. 20 ¢. ¢. of the peptone solution together with 15 c.c. of the malt extract re- quired 4:0 c.c. of 0°1 per cent. hydrochloric acid to saturate the proteid matter, consequently in the above mixtures, 2 and 3 contained a large excess of uncombined peptone, while in 4 the proteid matter was just saturated. Neutralizing and equalizing mixtures were added to,each as follows : we 2. 3. 4. 0-1 per cent. Na,CO; | --- 0 LAC: SEeue 5:9icie: 01 Me Na.CO; _..-- Os) @, @ 4-4 279 0 ae a JEG) hE Sete eae I) 3°0 3°0 0 The solutions were now, on being diluted to 100 ¢. ¢., in every re- spect equal. Their amylolytic power on being tested with starch paste was as follows : Total amount Starch No. Wt. Cuin 4. reducing bodies. converted. ] 0°1595 gram. 0°3258 gram, 29°32 per cent. 2 01527 0°3118 28°06 3 0°1239 0°2522 22°69 4 trace. Hence, when the proteid matter present is only one-quarter satu- rated with acid, the acid-peptone so formed may exert some destruc- * The peptone solution contained 0°250 gram peptone in 100 c. c. water and was made neutral to test papers; 20 c. c. therefore contained 0:050 gram peptone. - — of Diastase of Malt, as modified by various conditions. 57 tive action on the ferment; when half saturated, destructive action is more pronounced ; when wholly saturated it is, under the above con- ditions, complete. Frequently, such small percentages of combined acid as the above will have no retarding effect whatever on amylolytic action, though if the ferment be warmed for half an hour with the same percentage of peptone and combined acid, its subsequent amylolytic power will be much reduced, owing to a partial destruction of the ferment. This is well illustrated by the following series of experiments : A. B. a (Fa aS a | tip 2. 3. 4. 5. 6. Malt extract_----- i5rene! 15 c. ¢. 15 cc. ili) © Gy (CC. Worenc: 0-1 per cent. HC]__ 0 l 2 0 2 4 Peptone sol. 0°5¢%_. 10 10 10 20 20 20 Ee Oe aa ck 2: WB 24 23 65* 63* 61* 50 50 50 100 100 100 Combined HCl__-. 0 0:002% 00044 0 00024 0-004% Peptone; -. = =-- 01% O01 01 01% 0-1 0-1 Nos. 4, 5 and 6 were warmed directly, with the starch, for 30 min- utes at 40° C. and the reducing bodies determined. Nos. 1, 2 and 3 -were warmed at 40° C. also for 30 minutes, then neutralizing and equalizing mixtures were added, the solutions diluted to 100 ¢. ¢. and warmed with 1 gram of starch for 30 minutes at 40° C. The follow- ing results show the amylolytic power of the six solutions: Total amount Starch Wt. Cuin 4. reducing bodies. converted. oh 0°1482 gram. 0°3024 gram. 27.21 per cent. A. 071412 0°2876 25°88 3 0°1351 072754 24°78 ( 4 0°1594 0°3256 29°30 B.+ 5 071628 0°3324 29°91 ie 071605 0°3282 29°52 In series A, we see a gradual decrease in the amylolytic power of the solutions ; this can be due to nothing but the destructive action of the acid-peptone compound, for the solutions are in every respect alike, being in the same degree of dilution and containing the same amount of sodium chloride, etc. The destruction is not great, as the peptones are in considerable excess. In series B, where the fer- ment is exposed to the direct action, in the presence of the starch, of the same percentage of peptone and acid there is no retardation of * Containing 1 gram of starch, TRANS. Conn. Acap., Vou. VII. 8 Oct., 1885, 58 Chittenden and Cummins—-Amylolytic Action yloty amylolytic action whatever; presumably because of the rapid action of the ferment and the slow retarding action of the acid-peptone. Influence of free acid on the amylolytic action of diastase. As might be expected, free hydrochloric acid, even in very small. quantity at once stops the amylolytic action of this ferment, quickly destroying it. It is interesting, however, to compare the action of very small percentages of free acid with results obtained in like manner with the salivary ferment. The first experiment gave the following results: the malt extract used, required per 30 ¢. ¢., 2°4 ¢. ¢. 0°1 per cent. hydrochloric acid to saturate the proteid matter. Per cent. Per cent. Total amount Starch combined HCl. free HCl. Wt. Cuinl. reducing bodies. converted. 0 0 0°1378 gram. 0°2808 gram. 25°27 per cent. 0:°0024 0 071584 0°3236 29°12 0°0024 0°00] 071486 0°3032 N29 0°0024 0°003 0°0805 0°1642 14-77 With another malt extract not so active, but containing the same percentage of acid-proteids, the following results were obtained : Total amount Free HCl. Wt. Cu in %. reducing bodies. Starch converted. 0 00780 gram. 0°1592 gram. 14°32 per cent. 0°0003 per cent. 00719 01470 13°23 0°0005 0°0631 01294 11°64 0:0020 0:0380 00796 7-16 0°0030 00091 0°0222 Ssh) From these two series of results it is quite evident that a very small percentage of free hydrochloric acid will stop the amylolytic action of this ferment. The main action of the acid is that of destruction, killing the ferment very quickly. The following is a sample of several experiments tried, to ascertain how far retardation is due to destruction of the ferment. it 2. 3. 4. Nentral malt \extract: ..2. 22-2 oseeec 30 cee. (30 cle: ~ 30) \cicisOmeaas 01 per cent. HCl to saturate proteids 2°2 22 te 7 PAT fe 22-8 0-1 per cent. HCl for free acid --.... 0 05 “ On 1:5 le ORS a eps See eee ee , 1938/2 Wiese 16:8 * 16s a HOt 50 500 “ 50°0 “ Pen.cent; tee EO a2. susnjso oes 0 0-001 0°002 0°003 These solutions were warmed at 40° C, for 30 minutes, then neu- tralizing and equalizing mixtures were added and the amylolytic power determined. No. 1 converted the usual amount of starch into sugar, but No. 2 showed only a trace of amylolytic power and Nos. 3 and 4 none at all. Evidently then 0-001 per cent. of free acid had of Diastase of Malt, as modified by various conditions. 5Y all but completely destroyed the ferment by 30 minutes warming at 40° C. In conclusion then, we have to notice a greater susceptibility on the part of this ferment to the action of acid-proteids and free acid than the salivary ferment. Whether this latter point constitutes any real difference, it is hard to say, since the apparent increase in amylolytic action noted in the presence of traces of free acid in the case of saliva (0°0001—0-0006 per cent. free HCl) involve such small quantities as to make the results somewhat questionable,* since such very small additions of acid might perhaps be used up by the phos- phates or other salts present. But taking the evidence of the results and comparing them with results obtained in like manner with the diastase of mait, it would certainly appear that the latter is more susceptible to the action of free acid than the salivary ferment, though both are very readily destroyed by a few thousandths of one per cent. of free HCl. ‘In other respects, the ferment of malt behaves similarly to the fer- ment of saliva; both act better in a neutral than in an alkaline solution; proteid matter too, prevents the retarding action of alka- line carbonate and thus, as in the case of saliva, the action of a given percentage of sodium carbonate on diastase is dependent in part, upon the concentration of the fluid and the consequent amount of proteid matter present. Neutral peptone, moreover, exerts a direct stimulating effect on the amylolytic action of neutral diastase. Greatest amylolytic action, as in the case of saliva, is, however, ob- served in the presence of proteid matter partially saturated with acid, but larger percentages of acid-proteids may cause complete destruc- tion of the ferment. The accelerating action of proteid matter is in great part due to its power of combining with both acid and alkaline carbonate, but in addition we cannot but recognize a direct stimula- tion of the ferment, as in the action of neutral peptone on a neutral solution of diastase. Lastly, it is evident from these results, that diastase taken into the stomach must sooner or later be completely destroyed, by either the free acid or the large percentage of acid-proteids; but in the first stage of digestion, in the absence of free acid and under the protect- ing influence of proteid matter the conversion of starch into sugar may still go on, though soon destined to feel the effects of the gradually increasing percentage of combined acid. ' * See Chittenden and Smith, Trans. Conn. Acad., yol. vi, p. 370. V.— INFLUENCE OF CERTAIN THERAPEUTIC AND Toxic. AGENTS ON THE AMYLOLYTIC ACTION OF Sativa. By R. H. CuirrenDEN AND H. M. Painter, B.A., Pu.B. Frew attempts have been made to ascertain, experimentally, the influence of therapeutic and toxic substances on amylolytic action. Yet in view of the important part which the ferment of saliva plays in the digestive processes of the body and in view likewise of the great susceptibility of the ferment, it would seem especially desirable to obtain accurate data regarding the effects of many substances on its amylolytic power. While many laborious investigations have, from time to time, been undertaken to ascertain the influence of some one or more substances on the metabolism of the body, the influence of the same sub- stances on the digestive processes has apparently been very little considered, with the exception, however, of the more common alkali and alkali-earth salts. Likewise too, the possible action of many toxic substances on the digestive processes, as in chronic cases of poisoning, has with a few exceptions been almost entire- ly ignored; yet in both of these instances it is possible that much light might be obtained by a knowledge of the influence of individual substances upon proteolytic and amylolytic action.* : With these thoughts in mind, the present investigation was under- taken, and the results which we present here plainly show the import- ance of the work, In selecting substances for study, we have chosen not only those noted for therapeutic or toxic power, but also those possessed of antiseptic or germicidal properties ; our object being to see how far the unformed ferment of the saliva corresponds, in its behavior towards these bodies, with the formed or organized ferments.t More- * An interesting table of comparisons by Wernitz shows the relative action of several therapeutic agents, on the various enzymes of physiological interest.—Brun- ton’s Pharmacology, p. 86. } A difference in action by the same substance upon formed and unformed ferments is, as stated by Brunton, a fact of great importance, for upon it may depend a useful application of the substance in medicine ; thus creosote, which has but a slight action upon pepsine and ptyaline, will kill bacteria in a dilution of 1 to 1000, and thus this agent can be used to arrest fermentation in the stomach depending on the presence of low organisms, while the proteolytic action of the digestive ferment is but little interfered with.—Brunton, p. 87. Chittenden and Painter—Action of Saliva. 61 over, only neutral bodies could be experimented with, since the smallest quantity of either free acid or alkali would exert its own peculiar destructive action on the ferment.* In view of this fact also, we have invariably used chemically pure salts and those frequently recrystallized to be sure of the absence of deleterious impurities. Method employed. A few preliminary experiments clearly indicated that the presence of very small percentages of foreign substances exercise a decided effect on the amylolytic action of saliva, and thus the investigation resolved itself into a study, not of the percentages requisite to com- pletely hinder the power of the ferment, under given conditions, but of the relative action of small percentages on the amylolytic power of the ferment. ‘This seemed to us the more important, since we soon found that substances which, present in comparatively large amount tended to hinder amylolytic action, would when present in small quantities actually increase the activity of the ferment. Hence, we deemed it best to use accurate quantitative methods for determina- tion of amylolytic action; such as would indicate small variations with certainty. The experiments were made in series, in which one digestion of each series served as a control for comparison. The volume of each digestive mixture was 100 ¢. ¢., in which was present 1 gram of per- fectly neutral potato starch, previously boiled with a portion of the water, 10 ¢. c. of a diluted neutral salivat and a given quantity of the substance to be experimented with. The mixtures were warmed at 40° C. for 30 minutes, after which further action of the ferment was stopped by heating the solution to boiling. The extent of amylo- lytic action was then ascertained by determining in one-fourth of the solution, the amount of reducing substances by Allihn’s{ gravimetric method. From the amount of reduced copper thus obtained, the total amount of reducing bodies was calculated (as dextrose), from which in turn was calculated the percentage of starch converted. * Ghittenden and Smith, Transactions Conn. Acad. Arts and Sciences, vol. vi, p. 343, + The saliva was human, mixed saliva, freshly collected. It was prepared for use by being filtered, made exactly neutral, then diluted in the proportion of 1:5, Thus in each digestion there were present 2 ¢. c. of undiluted saliva, { Zeitschrift fiir Analytische Chemie, Jahrgang xxii, p. 448, 62 Chittenden and Painter—Influence of Therapeutic Mercurie chloride. Sternberg* places mercuric chloride first in the list of germicides ; its presence to the extent of 0:003 per cent. being sufficient to pre- vent the development of the micrococcus of pus, while 0-005 per cent. destroys the vitality of the same bacterial organism. To our surprise the salt acts even more energetically on the unoy- ganized ferment of the saliva, as the following results show: Total amount Starch HxCle Wt. Cuin 4. reducing bodies. converted. 0 0°2385 gram. 04920 gram. 44°28 per cent. 0°0005 percent. 0°1277 0°2500 22°50 0-0010 0°0925 071880 16°92 0:0020 0°0395 00824 741 ; 00030 0°0060 ’ 0°0040 0 It is evident that the ferment of saliva is very susceptible to the action of this poison, and we have repeated the experiment, using still smaller percentages, with the following results: Total amount Starch HgCle, Wt. Cuin 4. reducing bodies. converted. 0 0°1635 gram. 0°3340 gram. 30°06 per cent. 0°0001 per cent. 01610 0°3288 29°59 00002 071570 0°3204 28°83 00003 0°1545 0°3152 28°36 The smallest possible addition, therefore, of mercuric chloride dimin- ishes the amylolytic power of saliva, in proportion to the amount of mercury salt added. Mercurie bromide, mercuric iodide and mercuric cyanide. These salts of mercury, vigorous in their action as poisons, and the two former as germicides likewise, would be expected from analogy to act similarly to the chloride. Such we find to be the case with the bromide and iodide, but with the cyanide there is to be noticed, to a slight extent, an action which we find common to many substances, viz: increasing the amylolytic power of the ferment * Amer. Jour. Med. Sciences, April, 1883, p. 321. For the action of the various salts studied in this work, on the organized ferments, see also Marcus and Pinet in Compt. Rend. Soc. de Biolog., 1882, pp. 718-724, or ab- stract in Jahresbericht fiir Thierchemie, 1882, p. 515; also Ch. Richet in Compt. Rend., vol. xevii, pp. 1004-1006, or in Jahresbericht fiir Thierchemie, 1883, p. 418, and Robert Koch, Jahresbericht fiir Thierchemie, 1881, p. 471, N. Jalan de la Croix, Jahresbericht fiir Thierchemie, 1881, p. 476. Brunton’s Pharmacology, p. 96, las “* - and Toxie Agents on the Amylolytic Action of Saliva. 62 when present in one percentage and diminishing it when the percent- age is increased. On account of the insolubility of mercuric iodide and bromide, these salts were dissolved in water containing potassium iodide and sodium chloride respectively, in such proportion that the various digestive mixtures contained the same percentages of these salts as they did of the mercury salts.* Following are the results obtained: Total amount Starch Mercury salt. Wt. Cu in 44. reducing bodies. converted. 0 0°1295 gram. 0°2636 gram. 28°72 per cent. HgBr. 0°0005 per cent. 01150 0°2344 21:09 0°0010 : 0°0770 071572 14°14 0:0020 0°0340 0°0720 6°48 Hgl, 0°0010 071257 0°2560 23°04 0°0020 071180 0°2404 21°63 Hg(CN). 00005 01375 0°2800 25°20 0°0010 071445 0°2944 26°49 0:0020 071242 0°2528 22°75 0°0030 0°1252 0°2552 22°96 0 0°1319 0°2684 24°15 Hg(CN), 0°0500 0°1025 0°2084 18°75 In this series of results, it is to be noticed that mercuric bromide is the most energetic in its hindering action; 0°0005 per cent. being even more effective than 0°002 per cent. of the iodide. With the cyanide, however, the first two percentages stimulate or in some way give rise to an increased amylolytic action and even 0°050 per cent. of the salt does not retard the action of the ferment as much as 0°001 per cent. of mercuric bromide. Cupric sulphate. With this salt the following results were obtained: Total amount Starch CuSO 4+5H 20. Wt. Cu in 4. reducing bodies. conyerted. 0 071712 gram. 0°3500 gram. 31°50 per cent. 0°0005 per cent. 0°1445 0°2944 26°49 0°0020 0°0530 0°1096 9°86 070100 0°0250 0°0540 4°86 0°0250 0 * Apparently, the double salts so formed act as vigorously as the mercury salt alone could do. 64 Chittenden and Painter—Influence of Therapeutic The hindering action of the copper salt is nearly as pronounced as that of mercuric chloride and even more so than the bromide and iodide of mercury. Lead acetate. With this salt, the smaller percentages experimented with show a - slight stimulating action; but the larger percentages fail to retard the amylolytic action of the ferment as the preceding salts. Total amount Starch Pb(CgH309)2+8Hy0. Wt. Cuin 4. reducing bodies. converted. 0 0°1630 gram. 03332 gram. 29°98 per cent. 0°0003 per cent. 071642 0°3356 30°20 0-0005 : 0°1635 0°3340 30°06 0°0010 0°1635 0°3340 30°06 0°0020 0°1595 0°3256 29°30 0°0050 071395 0°2840 25°56 © 0°0100 071402 0°2856 25°70 A second series of experiments, with still larger percentages of the lead salt, gave the following results: Total amount Starch Pb(C2H302)9+3H,0. Wt. Cu in 4. reducing bodies. converted. 0 0°1742 gram. 0°3564 gram. 32°07 per cent. 0°05 per cent. 0°1735 0°3548 31°93 0°10 071720 0°3516 31°64 0°30 0°1657 0°3384 30°45 0°50 0°1555 0°3172 28°54 1-00 01375 0°2800 25°20 3°00 0:0785 0°1600 14°40 5°00 00490 071016 9°14 » Thus, the presence of even five per cent. of lead acetate fails to completely prevent amylolytic action. Arsenious oxide. Owing to the comparative insolubility of this substance in neutral fluids, small percentages only could be experimented with. With these, the following results were obtained : Total amount Starch A803, Wt. Cu in 4. reducing bodies. converted. 0 0°1475 gram. 0°3004 gram. 27:03 per cent. 00003 per cent. 01507 03072 27°64 0:0005 01537 0°3136 28°22 0°0010 071475 : 0°3004 27°03 0°0020 071579 0°3204 28°83 00050 0°1390 0°2832 25°48 0:0900 0°1605 0°3276 29°48 Although the results obtained do not wholly accord with each other they still plainly show that arsenious acid, to the extent pres- rs Pree and Toxic Agents on the Amylolytic Action of Saliva. 65 ent in these experiments, stimulates the amylolytic action of the ferment ; a fact which might be expected, assuming that the acid combines with the proteids of the saliva, for as has been elsewhere* shown, acid-proteids when present in not too large an amount increase the amylolytic action of the salivary ferment. Schafer and Béhmf state that arsenious acid has no influence whatever on the conversion of starch into sugar by a glycerine ex- tract of the pancreas. Possibly they sought only for retarding action, or it may be that the pancreatic ferment differs in this respect from the ferment of saliva. Arsenic acid. This substance being still more acid than the preceding, might naturally be expected to diminish amylolytic action, when present in quantities which in the preceding would increase the activity of the ferment ; and indeed there is to be seen in the results, a slight in- crease, followed by a rapid decrease of amylolytic action, Total amount Starch H3AsOq. Wt. Cu in 4. reducing bodies. converted. 0 01755 gram. 0°3588 gram. 32°29 per cent. 0°0005 per cent. 0°1765 0°3608 32°47 0:0010 071635 0°3340 30°06 0:0030 0°0310 0°0660 5°94 0°0050 0 With 0°005 per cent.. of arsenic acid present in the fluid, no reduc- ing bodies were formed in the thirty minutes of the experiment, but the solution did become clear, showing the formation of soluble products. The same fact was observed in the presence of larger percentages of the acid; the starch solution becoming clear, after the addition of saliva, even in the presence of one per cent. of the acid, although, as before, no reducing bodies were formed. Ammonium ursenate. With this salt the following results were obtained: Total amount Starch (NH4)3 A804. Wt. Cu in 4%. reducing bodies. converted. 0 0°1527 gram. 0°3112 gram. 28°08 per cent. 0:0005 per cent 0°1620 0°3308 2971 00010 0°1630 0°3340 30°06 00050 0°1675 0°3420 30°78 0‘0150 071745 0°3568 32°11 0:0250 =. 071700 0°3476 31:28 * Chittenden and Smith, Trans. Conn. Acad., vol. vi, p. 343. + Abstract in Jahresbericht fir Thierchemie, 1872, p. 365. Trans. Conn. Acap., Vou. VII. 9 Oct., 1885. 66 Chittenden and Painter—Influence of Therapeutic In a second series, larger percentages were used with the following results : Total amount Starch (NH4)3 A804. Wt. Cu in 4. reducing bodies. converted. 0 0°1712 gram. 0°3500 gram. 31°50 per cent. 0°05 per cent. 071475 0°3004 27°03 0°10 01147 0°2332 20°98 0°50 0°0165 0°0368 3°31 1:00 The solution became clear but no reducing bodies were formed. With this salt, a very decided stimulation of the ferment is to be observed in the presence of small percentages, while increased amounts of the salt ultimately stop diastatic action. Potassium antimony tartrate. Two series of experiments were tried with this salt, with the fol- lowing results : Total amount Starch K(SbO)C4H4Og. Wt. Cu in 4. reducing bodies. converted. . 0 0:1540 gram. 0°3144 gram. 28°29 per cent. 0:001 per cent. 0°1610 0°3288 29°59 0°005 0°1660 0°3392 30°52 0°010— 0°1760 0°3600 32°40 0°050 01760 0°3600 32°40 0-100 071745 0°3568 Sitti! 0°200 0°1750 0°3580 32°22 0 071545 0°3152 , 28°36 0°10 0°1850 0°3788 34-09 0°30 01640 0°3352 OF Gps 0°50 0°2565 0°5304 47°73 1:00 0°1570 ; 0°3204 28°83 2°00 071232 0°2504 22°53 5:00 0°0470 0°0976 8-78 Here we have an illustration, more forcible than with any other salt, of the power possessed by many substances of both increasing and diminishing the action of the ferment. One* of us has for some time held that the addition of very small quantities of hydrochloric acid to neutral saliva tends to increase the amylolytic power of the. ferment; that this takes place even when the proteids present are completely saturated with the acid, or in other words when there is present a very small amount of free acid, provided the acid-proteids are not present in too large an amount. It is well known that free hydrochloric acid, when present to the extent of a few thousandths of one per cent. completely stops the action of the ferment. Langley * Chittenden and Smith, Trans. Conn. Acad., vol. vi, p. 360. 7 Gt and Toxie Agents on the Amylolytic Action of Saliva. 67 and Eves* make this divergence of action of one and the same sub- stance a ground for questioning the accuracy of such a view, for, say they, “since 0°0015 per cent. HCl decreases amylolytic action it seems very unlikely that 00005 per cent. should increase it.” The action of many neutral salts here experimented with, where both stimulation and retardation are obtained, plainly show that such a double action, dependent simply on quantity is not an impossible one, Stannous chloride. With this salt very marked results were obtained as follows: Total amount Starch SnCl» - Wt. Cu in 4%. ‘reducing bodies. converted. 0 01475 gram. 0°3004 gram. 27°03 per cent. 0°0003 per cent, 0°1582 0°3232 29°08 0:0010 The solution became clear, but no reduction. 0°0050 The starch was not at all altered in appearance. Here there is stimulation, followed by rapid and complete stopping of amylolytic action. Zine sulphate. Total amount Starch ZnSO4+7H;0. Wt. Cu in ¥. reducing bodies. converted. 0 071495 gram. 0°3048 gram. 27°43 per cert. 0-0003 per cent. 071490 0°3040 27°36 0°0005 071510 0°3088 27°79 0°0010 01475 0°3004 27°03 0-0020 071440 0°2936 26°42 0°0050 071360 0°2772 24-94 0°0100 .0°1260 0°2576 23°18 0 0°1375 02800 25°20 0-05 per cent. 00775 0°1480 13°32 0°10 0-0650 0°1332 11°98 0°30 0°0450 0°0936 8°42 0°44 0 These two series of experiments plainly show a gradually dimin- ished amylolytic action, as the percentage of the zinc salt is in- creased, until with 0-4 per cent. a complete stoppage is effected. Kjeldahl+ found a like retarding action on the addition of zine sulphate to a malt extract. In this connection it is interesting to note that Sternbergt finds zinc sulphate devoid of germicide value, even when used in the proportion of 20 per cent. * Journal of Physiology, vol. iv, No. I. + Jahresbericht fiir Thierchemie, 1879, p. 382. ¢ Amer. Jour. Med. Sciences, April, 1883, p, 330. 68 Chittenden and Painter—Influence of Therapeutic Ferric chloride. i With this salt we obtained the following results: - Total amount Starch FegCl¢- Wt. Cu in 4. reducing bodies. converted. 0 0-1740 gram, 0°3560 gram. 32°04 per cent. 0:0005 percent. 0°1597 03260 29°34 0°0020 0:0437 0:0908 817 0°0100 00095 0°0236 2°12 4 0:0250 0 Sternberg states that tincture of ferric chloride is effective as a germicide (upon micrococcus) when present to the extent of 4 per cent. On the unformed ferment of the saliva, it is, as the results show, much more active, its hindering action being directly propor- tional to the percentage of iron salt present. ferrous sulphate. With this salt of iron quite different results were obtained; and as we wished simply to compare its action with that of the ferric salt, only very small percentages were experimented with. Total amount Starch FeSO4+7H20O. Wt. Cu in 4. reducing bodies. converted. 0 071245 gram. 0°2532 gram. 22-78 per cent. 0°0005 per cent. 071037 0°2108 18°97 0:0020 01323 0°2692 24°22 0°0100 071365 0°2780 25°02 Here there is decided stimulation with the two larger pertentages, while the smallest per cent. shows an apparent decrease of amylolytic action. | Kjeldahl* found that this salt exercised a strong hindering action on the amylolytic ferment of malt. Potassium permangunate. Sternberg places this salt next to mercuric chloride in germicide value, it being efficacious in 0°12 per cent. With the unformed fer- ment of the saliva it is likewise active, although no more so than many other salts experimented with. Following are the results: Total amount Starch Ky MngQg. Wt. Cu in 4. reducing bodies, converted, 0 0°1475 gram. 0°3004 gram. 27°03 per cent. 0005 per cent. 0°1012 0°2060 18°54 0°025 0 * Jahresbericht fiir Thierchemie, 1879, p. 382, and Toxic Agents on the Amylolytic Action of Saliva. 69 Magnesium sulphate. With this salt we obtained the following results: Mgs0,+7H20. Wt. Cu in 4. ota ie Eonverted: 0 071475 gram. 0°3004 gram. 27-03 per cent. 0-025 per cent. 0°1597 0°3260 29°34 0°500 0°0510 01056 9°50 Here, there is a slight increase of diastatic action with the smallest percentage, while 0°5 per cent. of the salt greatly retards the action of the ferment. Pfeiffer* has likewise noticed the retarding effect of this salt on salivary digestion. | Potassium cyanide. This salt, so powerful as a poison, was found to have a decided effect also on the salivary ferment, causing a rapid decrease in amylolytic action. Total amount Starch KCN. Wt. Cu in 4. reducing bodies. converted. 0 0°1245 gram. 0°2532 gram. 22°78 per cent. 0°0005 per cent. 0°1080 0°2200 19°80 0-0010 0°0896 0°1828 16°45 0°0030 0°0330 0°0700 6°30 With 1:0 per cent. and even with 5:0 per cent. of potassium cyanide, the starch solutions became clear on the addition of saliva, showing that the ferment was able to effect some change, although in neither case were any reducing bodies formed. It is our intention at some future time, to study the exact nature of the products formed under such conditions. The ferment appears to be peculiarly affected; for while a very small percentage of a substance like potassium cyanide or borax will completely prevent the formation of reducing bodies, increasing the amount of substance added a hundred-fold, has no effect on the clearing up of the starch solution by the ferment. Some light may be thrown upon the nature of the ferment or its mode of action. Potassium ferrocyanide. A preliminary experiment showed that this salt was less active than the cyanide and therefore larger percentages were used, with the following results: Total amount Starch K4Fe(CN) ¢+3H,0. Wt. Cu in 4. reducing bodies. converted. 0 071417 gram. 0°2884 gram. 25°96 per cent. 0-025 per cent. 0°1497 0°3052 27°46 0-100 071375 0°2800 25°20 0°250 071025 0°2084 18°80 * Centralbl, Med. Wiss., 1885, p. 328, abstract. 70 Chittenden and Painter—Influence of Therapeutic Here, unlike the cyanide, there is stimulation of the ferment. Retardation of amylolytic action requires much larger percentages ; thus 1:0 per cent. of ferrocyanide completely prevented the formation of reducing bodies, although soluble starch was apparently formed, as also in the presence of 5:0 per cent. of the salt. Potassium ferricyanide. The action of this salt is almost identical with that of the ferro- cyanide. Total amount Starch KgFee(CN))2- Wt. Cu in 44. reducing bodies. converted. 0 01417 gram. 0°2884 gram. 25°96 per cent. 0°025 per eent. Onl5S 0°3088 27°79 0°100 0°1295 0°2636 23°%2 0°250 0°0975 0°1984 17°85 Like the ferrocyanide, this salt in 1:0 and 5:0 per cent. solutions allows the partial conversion of starch into soluble products, but — no reducing bodies are formed. Potassium nitrate and potassium chlorate. Potassium Total amount Starch salt. Wt. Cuin ¥%. reducing bodies. converted. 0 0°1513 gram. 03080 gram. 27-72 per cent. KNQ3. 0°20 percent. 0°1550 0°3164 28°47 0°50 0°1528 0°3108 27-97 1:00 0°1462 0°2976 26°78 KC1O3. 0°20 071581 0°3228 29:05aes 0°50 0°1580 0°3228 29°05 1:00 0°1600 0°3268 29°41 With 5:0 per cent. of the salts, the following results were obtained: Potassium : Total amount Starch Salt. Wt. Cuin 4. reducing bodies. converted. 0 0°1672 gram. 0°3416 gram. 30°74 per cent. KNO; (5:0 pr. ct.) «0°15 59 0°3176 28°58 KCI1O; (5:0 pr. ct.) 071351 02752 24°76 With these two salts it is very obvious that small fractions of one per cent. decidedly increase amylolytic action and that potassium chlorate is the more energetic of the two in this respect. With one per cent. of the salts, potassium chlorate still shows increased action, while the nitrate causes a decrease of amylolytic activity; in the presence of 5 per cent. of the salts, on the other hand, potassium chlorate causes the greatest decrease in ferment action. Of these two oxidizing agents, potassium chlorate does not appear to have been hitherto experimented with, but with potassium nitrate and Toxic Agents on the Amylolytic Action of Saliva. 71 O. Nasse* found increased amylolytic action with human saliva in the presence of 4°0 per cent. of the salt. Possibly this difference in our results is dependent in part upon difference in the relative amount of salt and ferment. Sodium tetraborate [Na,B,O,+10H,O}]. With this salt experiments were tried with quantities varying from 0-050 to 3:0 per cent. and in each instance the starch was dissolved, but no reducing bodies whatever were formed. Dumast has pre- viously noted a like retarding effect on the diastatic action of emul- sin, diastase and other like ferments. Sternberg states that this salt is without germicide value, even though used in a saturated solution ; its antiseptic power, 1. e. its capacity for preventing the multiplica- tion of bacterial organisms, is, however, considerable. Potassium bromide and potassium iodide. These two common therapeutic agents gave the following results: Total amount Starch Salt used. Wt. Cuin 4%. reducing bodies. converted. 0 01483 gram. 0°3020 gram. 27°18 per cent. KBr. 0°5 per cent. 0°1566 0°3192 28°72 3°0 0°1450 0°2956 26°60 5°0 071314 0°2668 23°51 KI, 0°5 0°1550 0°3164 28°47 3°0 071557 0°3172 28°54 50 0°1467 0°2984 26°85 Both of these salts show a stimulating action which is more per- sistent in the case of the iodide than with the bromide; 5:0 per cent. of the bromide causes a marked diminution of amylolytic action. Sodium chloride. Previous experiments have been tried with this salt by several investigators, notably by O. Nasse{ and E. Pfeiffer.§ The former found that the presence of 4°0 per cent. of the salt [the only percent- age experimented with] caused an increase in the ferment action of saliva [128:100]; the latter experimenter likewise found that the * Pfliiger’s Archiv. fiir Physiologie, vol. xi, p. 150. + Berichte der deutsch. Chem. Gesell., vol. v, p. 826. t Pfliiger’s Archiv fir Physiologie, vol. xi, p. 155. § Centralbl. med. Wiss., 1885, p. 329, 72 Chittenden and Painter—Influence of Therapeutic presence of the salt, in concentrations up to 2 per cent., greatly in- creased the amylolytic action of saliva. Our results with different percentages are as follows : NaCl. Wt. Cuin 4. cadieine holies, conercett 0 0°1660 gram. 0°3392 gram. 30°52 per cent. 0°3 per cent. 0°1765 0°3608 32°47 0-5 0°1750 * 0°3580 32°22 10 01715 0°3504 31°53 2°0 O-1715 0°3504 31°53 3-0 01770 0°3620 32°58 5:0 071630 0°3332 29°98 These accord with the results mentioned above and show, more- over, that with 5:0 per cent. of the salt, hindering action just com- mences. Increasing the amount of salt beyond this point, however, only slowly diminishes the action of the ferment; thus, in the pres- ence of 10°0 per cent. of the salt, 22°78 per cent. of starch was con-_ verted into sugar, while without it 25°20 per cent. of starch was converted. Morphine sulphate. With this alkaloid O. Nasse* has experimented, using, however, the acetate. He found that the presence of 071 per cent. of the salt caused a slight increase in the diastatic action of saliva (109: 100). Our results with the sulphate of morphine are as follows: Total amount Starch Alkaloid salt. Wt. Cuin 4. reducing bodies. converted. 0 0°1245 gram. 0°2532 gram. 22°78 per cent. 0°05 per cent. 01415 | 0°2880 25°92 0°50 0°1605 0°3276 29-48 2°00 071428 0°2908 26°17 The stimulating action of the alkaloid salt up to 2-0 per cent. is very apparent. Quinine sulphate. With the acetate of this alkaloid, Nasse found, by the use of 0°1 per cent., an increase in the starch-converting power of the saliva (115:100). With the sulphate we obtained the following results : Total amount Starch Alkaloid salt. Wt. Cu in 4. reducing bodies. converted. 0 0°1358 gram. 0°2768 gram. 24°91 per cent. 0°05 per cent. 01475 0°3064 27°08 0°50 0°1355 0°2760 24°84 2°00 0-0981 01996 17°96 * Pfliger’s Archiv, vol. xi, p. 161. - q ie tiie tellin Sears Me ~ fie: a) os Mie: we and Towie Agents on the Amylolytic Action of’ Saliva. 73 In accord with Nasse’s result we see that 0-05 per cent. increases the amylolytic action of the ferment. This we verified by an addi- tional experiment which led to a like result, although not showing so great a difference as the preceding one; thus, while the saliva alone converted 23°72 per cent. starch into reducing bodies, the presence of 0°05 per cent. of quinine sulphate led to the conversion of 24°58 per cent. of starch. Voit, as quoted by v. Boeck,* has stated that qui- nine is without influence on the ferment of saliva. Cinchonine sulphate. With this alkaloid, previous experiments have not to our knowl- edge been tried. Our results are as follows: Alkaloid salt. Wt. Cu in ¥. PeAcetie Bodie: ee enal \. 0 0°1358 gram. 0°2768 gram. 24°91 per cent. 0°05 per cent. 071452 0°2960 26°64 0°50 0°1455 0°2964 26°67 2°00 0°1440 0°2936 26°42 Cinchonidine sulphate. This alkaloid, like the cinchonine, shows a steady accelerating action on the ferment. Total amount Starch Alkaloid salt. Wt. Cu in 4. reducing bodies. converted. 0 0°1358 gram. 0°2768 gram. 24°91 per cent. 0°05 per cent. 071505 0°3068 27°61 0°50 0°1460 0°2976 26°78 er) 01498 0°3056 27°50 The cinchona group of alkaloids thus show throughout an acceler- ating influence on amylolytic action, most pronounced in the case of cinchonidine. These alkaloids have long been known to prevent putrefaction and to check alcoholic fermentation and Binzt has demonstrated that this antiseptic action, in the case of quinine at least, is due to the poisonous influence exerted by the latter upon the fungi which are the immediate cause of the putrefactive changes. Couzen, moreover, has shown that the action of cinchonine on infusoria and on fermentation is similar to that of quinine, but weaker. Hence there is no similarity of action whatever, on the two kinds of fer- ments. * Zeitschrift fur Biologie, vol. vii, p. 428. + Virchow’s Archiv, vol. xlvi, 1869, p. 68. Trans. Conn. Acap., Vou. VII. 10 Oot., 1885. 74 Chittenden and Painter—Influence of Therapeutic Atropine sulphate. With this alkaloid we obtained the following results: Total amount Starch Alkaloid salt. ~ Wt, Cu in %. reducing bodies. converted. 0 0°1485 gram. 0°3028 gram. 27°25 per cent. 0°025 per cent. 071460 0°2976 26°78 0°050 . 0°1410 0°2872 25°62 0°200 0°1530 03124 28°11 0:500 0°1407 0°2864 25°77 1:000 01475 0°3004 27°03 2°000 0°1245 0°2532 22°78 The main action of the smaller percentages of this alkaloid seems to be a slightly hindering one, although there are one or two irregu- larities in the results which are not readily explainable. In the presence of 2°0 per cent. of atropine sulphate there is a decided diminution in amylolytic action. In connection with this alkaloid we have to note some recent ex- periments of Stolnikow* of St. Petersburg. This investigator, pro- ducing artificial fever in dogs by the injection of putrid matter into the blood found, first, that the salivary and pancreatic secretions were for a time increased in amount and then rapidly diminished and finally entirely ceased. This latter action of the septic poison, Stolnikow found to be very persistent and he moreover states that in physiological action the septic poison resembles atropine. Fur- thermore that artificial fever, produced as described, exercises a decided influence on the content of ferments in the pancreatic gland; that in fevers of short duration (2-10 hours) the eXx- tract of this gland has a more energetic ferment action than the normal extract, while in fevers of long duration the corresponding extract has a much weaker action. Overlooking now the physiologi- cal explanation suggested for these facts we come to the chemical one, viz: that the septic poison possibly exerts either a destructive or hindering influence on the ferment or its action. In support of this view, Stolnikow found that large quantities of the poison did weaken the amylolytic and proteolytic action of extracts from the pancreatic gland, although small quantities of the septic ferment were without action. Likewise, Stolnikow states that small quanti- ties of atropine sulphate are without action on a glycerine extract of the pancreas, but by adding to 10 ¢. ¢. of a glycerine extract, 5 ¢.c¢. of a 3°0 per cent. atropine sulphate solution and allowing the mixture to stand at the ordinary temperature for 10 hours, then on * Beitrage zur Lehre yon der Function des Pancreas im Fieber. Virchow’s Archiv, vol. xc, p. 389, 1882. tad ree and Toxie Agents on the Amylolytic Action of Saliva. 15 4 testing the amylolytic power of the ferment its action was found to be much weaker than the control. From this fact, Stolnikow consi- ders that the septic poison acts upon the ferment outside the body in a manner similar to atropine. Now it is obvious, in view of the extreme susceptibility of the ferments of the saliva and pancreas to the action of acids and alka- lies, that the atropine solution must be perfectly neutral. Several specimens of atropine sulphate that we have examined, have had a slight acid reaction. In view of the apparent identity of the amylolytic ferments of the salivary and pancreatic secretions we have repeated in principle Stolnikow’s experiment with human saliva, using perfectly neutral atropine sulphate. To 10 ¢.c. of the dilute, neutral, saliva hitherto used, 0°3 gram of pure atropine sulphate was added (=3-0 per cent. of the alkaloid salt, while Stolnikow’s mixture contained but 1°0 per cent.) and the solution allowed to stand for 18 hours at the Laboratory tempera- ture. On now being added to the starch solution, diluted up to 100 c.c. [0°3 per cent. atropine sulphate] and placed at 40° C. for 30 minutes, the starch paste quickly became clear and it was found on examination that 29°16 per cent. of starch had been converted, while the control, in the presence of 0°2 per cent. of atropine sul- phate, showed a conversion of 28°11 per cent. of the starch. Hence there had been no destruction of the salivary ferment by even 3°0 per cent. of pure atropine sulphate, although as our previous experi- ments show very much smaller percentages may, by their presence, hinder the uction of the ferment. Strychnine sulphate and brucine sulphate. O. Nasse has previously studied the influence of 0:1 per cent. strychnine acetate on the diastatic action of saliva and has noted a slight increase in amylolytic action in the presence of the strychnine [109:100]. Our results with the two alkaloids are as follows: Total amount Starch Alkaloid. Wt. Cu in 4%. reducing bodies. converted. 0 0°1485 gram. 0°3028 gram. 27-25 per cent. Strychnine sulphate. 0:050 per cent. 071444 0°2936 26°42 0°250 0°1448 0°2936 26°42 0-500 01462 0:2976 26°78 Brucine sulphate. 0°050 per cent. 0°1503 0°3060 27-54 0°500 071524 0°3100 27-90 1-000 0°1505 03060 27°54 76 Chittenden and Painter—Influence of Therapeutic With the brucine salt a slightly increased action is noticed in all three of the experiments; while with strychnine a constant diminu- tion in amylolytic action is to be seen. . In this connection it is to be remembered, that a trace of free acid in the alkaloid salts would introduce an appreciable error into the results, and therefore all of the alkaloid salts experimented with, were especially purified for this purpose, any adhering acid being removed by repeated crystallization, etc. The following table shows the relative acceleration and retardation of the various salts (the percentages more generally used) compared with their controls expressed as 100. Table showing relative amylolytic action. 0:0008 0°0005 | 0001 0°002 | 0:005 | 0°010 | 0°025 0°05 | O1 | OS | 10 | 20 " p.c. | p.c. | p.c.| p.c. | p.c. | p.c! | p.c. } p.c: | p.c: | pie. pic.) pace iam —————» ily soe] |i Hg Gls eA ie. ae 94:3) 50°8/ 38:2] 16-7) 0 | -...|---. relu||-.tab eget) eer lelal sine esse Sy) les 2 Pee t85°9| Bog Ole cuas See ae eves) lot) ceo) See eee eee ipl, a0 2s ene eer eae A) Vso) tpi eet dg Be | eee re He(CN)ao- 25) 222 L.02-| s---|106:2/010-%), 96-9)4 2 | Sse) el Blo ye oe CuSO. woi Ol= sane on oe| (84:0) 2222) 313 )/222 5). W502 ee ee ee pe Pb(C2H302)2+3H20 ---|100°7/100°3/100-3) 97-7] 85-2) 85-7) ... | 99°5| 98-6] 88-9) 78-5) ____ AsOs oO ee E 102°2}104"4/100:0/10676) .94:2| 22..| -25-| -2-.fecee) aoe een Finis Oe soeeee setae ce Bees L00%5 93-0 2258 O'S), Se Sse Ol] Sa eee eee eee (NH sAROue ce orto _---|106°2]107-0] _.__|109°6) ....|111-4! 85:8] 66-6] 105] 0 | __.. K(SHOWO pH aO nee 5 ze, k liens | ...-[104°5| __._|107°9)114°5] ___.]114°5]120-2/168-3}101-6} 79:4 SiGe fee ee eee TOTS) .- 0], 08 | eee oo e) see ee oe pee WnsO@ihnOssceeee s: 99°7/101°3| 985 96:3) 90-9) 84-5) ... | 52-8) 47°5) 0 P__._| ._.. RpeGls sex eewioneah ack s sewn! 91:5) coe) 255) 22) OBE) Oy | W242 oe WeSO{+- 1H OG. 2 ee Bees |8s2 sels | eee ool. 52. eee Re K.Mn.20g meso edo os S455 Jee) ~---| --~--| ---- G8:6| S522 0 ool boos | Se) Ha le | nn MgSO,+7H.0O Soascsess||/ esse | -- -- SESE || SS95]) socis TOS:b gee -..| 35°. | aa RON SE Nese 8 sea 86°9) 72°23) 00.) .-.| 9222] 528) 205) oan ee rr Re Po(O Ns Ses Omene) cont) eee f Sere Pe. SS eS) SOB SIL ly OOS ee KeFeo(ON)i2 wewe cows we| ce ee| ce ea| o= =e | mewn] sean] aoe 107°0) =---- 91:3 ee ee RO Ree ee eee. ("he ale epee ep eS ee MAME Nec 100°9| 966] ._.. CLO sie Rene tee! FT oo Ae ees wee! ous} nacsl eal |: Aleks OES OR Vath Soe 2 ithe hei ee ae ee <2 | occ} saen| =<] c--| ---. | +22 0S TIGEN eet ds gdh Sa wien] eene| tote] oot eee el ele ge] eal s- =.) Sen eet rr NaGieeo = san 7 td 1uy3 sais] Nal Lee ERO tee pee ae ee .-..| .---]105°5/103°3}103-4 Na.B,0, + 10H,0.-.-..- Steck co. | pee Nose en Reese cere eee |. O | . 2S) 2555 ee (Nib )s seGSOg PO nc- =| sen | enne aw, (ase cal A 113°7| ....|129°4) eee COP ppa SEAS OPE is G1 0a ore] eaoaem Wemeineemy Pere r ed (trees) = ES | [108-6| .-..| 99°%) (2) (ia EsOgere tn Ota oe pe _ | 0) coe eal eee etme 106°9] ....|107°0] _...]106°6} .2 (Ci dine), . H.SO,+3H,0} .---| ----| - Seseil* See eee eee 110°8| ....|107°5}' 22.) Soa 9 (At), sea sOs canes Sl ok.. Ae | po 20 | Rees pee eee _--.| 98.2} 94:0] _...| 94°5] 99-1) 83°b) lam MSt)s HaS0.4+-6Hs0. 22.) ---.| ---- cy) Se ee --2.|-<-.-- . 0 0-1 0-1 These three mixtures were warmed at 40° C. for 24 hours; then to A was added 0°025 gram HgCl, dissolved in 25 c. c. 0°2 per cent. HCl, to B 25 c.c. 0:2 per cent. HCl and to C 25 ¢.c. 0-4 per cent. HCl. The three solutions were now exactly alike; in 6, however, the ferment had been exposed to the action of 0-1 per cent. HgCl, in an acid solution for 24 hours, in C' to the action of the same percent- age of the mercury salt in an aqueous solution, while A served as * Abstract in Jahresbericht fiir Thierchemie, 1875, p. 168. 88 Chittenden and Allen—Influence of various Salts a control. 1 gram of fibrin was added to each of the mixtures, which were then placed at 40° C. for 2 hours. £ and C digested the same amount of fibrin as A, consequently the mercuric chloride could have exerted no destructive action whatever on the ferment. Wassilieff,* in Hoppe-Seyler’s laboratory, found by comparative ) Pl Mi y> ¥: p experiments that mercurous chloride (calomel) has no effect on the proteolytic action of pepsin. Mercuric bromide, Mercuric iodide and Mercurie cyanide. These three salts of mercury were experimented with, only so far as to compare the action of small quantities, with the action of like quantities of mercuric chloride. In using the bromide and iodide it was necessary, on account of their insolubility, to dissolve them with the aid of an equal weight of sodium chloride, consequently these two salts of mercury were doubtless present in the digestive mix- tures, in part at least, as double salts. Marle, however, found that the action of mercuric chloride with small quantities of sodium -chloride was not different from that of mercuric chloride alone, and doubtless the same is true of the iodide and bromide of mercury. Following are the results we obtained: Mercury Undigested Fibrin ; Relative proteo- salt. residue. digested. lytic action. 0 0°3590 gram, 64°10 per cent. 100°0 HgBr. 0°005 per cent. 03731 62°69 97°8 0025 0°3980 60°20 93°9 Hel, ; y 0°005 03114 68°86 107°4 0-025 0°3904 60°96 95°1 He(ON)z 0005 0°3105 68°95 107°5 0°025 0°3985 60°15 93°8 0°100 0°3183 68°17 106°3 From these it is evident that mercuric bromide is less vigorous in its hindering action than mercuric chloride; the iodide still less so, while mercuric cyanide, in similar percentages, appears to cause an increase in proteolytic action. The iodide, likewise, in the smallest percentage experimented with, causes increased proteolytic action. None of these salts then, approach mercuric chloride in the intensity of its hindering action on gastvie digestion. * Ueber die Wirkung des Calomel auf Gihrungsprozesse und das Leben von Mikro- organismen. Zeitschrift f. Physiologische Chemie, vol. vi, 113. Li ee Me a aed Bit ei on the Proteolytic Action of Pepsin-hydrochloric Acid. 89 Stannous chloride. This salt shows marked action in retarding gastric digestion; its retarding effect increasing directly with the amount of stannous chloride added. SnCly.’ vreskdue, digested. “iytie actions 0 0°2576 gram. 74°24 per cent. 100°0 0-025 per cent. 02728 72°72 97°5 01 0:4826 5174 69°6 0°5 07332 26°68 35°9 1:0 0°8155 18°45 24°8 2°0 : 0°9010 9°90 13:3 Arsenious oxide. This substance might naturally be expected, in view of its well known antiseptic properties, to hinder proteolytic action, more or less. It is known to hinder putrefaction and to prevent also the fermentative action of yeast. Contrary to our expectations, however, the action of arsenious oxide, so far as it is to be seen, is an acceler- ating one, causing increased proteolytic action. The following results were obtained : Undigested Fibrin Relative proteo- As ,03. residue. digested. lytic action. 0 0-2111 gram. 78°89 per cent. 100°0 0:05 per cent. 071872 81:28 103°0 0-1 0-2160 78°40 BEES) 0-2 0°1900 81:00 102°6 0°5 0-1707 82°93 105°1 The stimulating action is slight, still it is plainly recognizable. Drs. Schifer and Béhm* have previously studied the action of arseni- ous acid on the digestion of albumin by artificial gastric juice, and they came to the conclusion, using 0°02 and 0°04 gram As,O, respec- tively, in 34 c. c. of fluid containing egg-albumin, that arsenious oxide is without influence on the decomposition of albumin by the gastric juice ferment. Our results, though not so large in number as theirs, would indicate a slight accelerating action. Arsenic is known, when administered in small, repeated doses, to act as a tonic; the history of arsenic-eating, indicates that the sub- stance has some positive tonic influence over nutrition, and Dr. * Jahresbericht fiir Theirchemie, 1872,,p. 363. Ueber den Einfluss des Arsens auf die Wirkung der ungeformten Fermente. TRANS. ConN. AcAD., Vou. VII. 12 Oct., 1885. 90 Chittenden and Allen—Influence of various Salts W ood* states, “‘ there is much reason for believing that it acts largel ) § g as a direct stimulant to nutrition.” The results obtained in our ex- periments certainly accord with this statement. Arsenic acid. The experiments tried with arsenic acid, tend to confirm the accelerating action noticed with arsenious oxide. In the first series of experiments the following results were obtained: Undigested Fibrin Relative proteo- H3As04. residue. digested. lytic action. 0 0°2696 gram. 73°04 per cent. 100°0 0-2 per cent. 0°2614 73°86 101°1 0°5 01514 84°86 1161 2°0 0°2583 74-17 101°5 5-0 0°3915 60°85 83°3 The accelerating action is here so very pronounced, that a second series of experiments was undertaken by way of confirmation. These give in a general way the same results, although with 0°5 per cent. the stimulating action is not so pronounced as in the first experiment. These two series of experiments illustrate another point, which it is well to mention here, namely: that definite percentages of any par- ticular substance do not invariably give precisely the same result, even when compared with their respective controls. They do, however, generally point in the same direction, and although not always giving exactly the same numerical expression, they show clearly the nature and extent of the action. Undigested Fibrin Relative proteo- H3As0Oq4. residue. digested. lytic action. 0 0°2490 gram. 75°10 per cent. 100°0 0-2 per cent. 0°2401 75°99 101°2 05 0°2367 76°33 101°6 1:0 0°2335 76°65 102°0 2°0 0°2622 73°78 98-2 5:0 03176 68°24 90°8 0 071493 85°07 100-0 10-0 0°4207 51°93 68'1 Plainly then, arsenic acid in small percentages does accelerate the proteolytic action of pepsin-hydrochloric acid, while in large percent- ages (5-10) it causes a diminution in the action of the ferment. Arsenic acid tends to make the fibrin become very gelatinous. * Therapeutics, Materia Medica and Toxicology, p. 390. on the Proteolytic Action of Pepsin-hydrochloric Acid. 91 Zine sulphate. With this salt, no experiments appear to have been hitherto made. Our results show a decided diminution in proteolytic action, even in the presence of 0-01 per cent. of the salt, while with a few thou- sandths of one per cent. the figures indicate a slight accelerating action. Three distinct experiments were made as follows: ZnS044+7H 0. Se eiaes, diccstee pe as 0 0°1744 gram. 82°56 per cent, 100-0 0°001 per cent. 071609 83°91 101°6 0°005 071617 83°83 101°5 0°010 . 0°2053 79°46 96°2 0°025 0°2573 74-27 89°9 3°000 0°8400 16°00 19°3 0 0°1630 83°20 100°0 01 0°4848 51°52 61°9 0°3 0°7133 28°67 34°4 Os5S i) 0°7382 26°18 31-4 0°8 O'T671 23°29 27-9 15 0°8202 17-98 21°6 0 071493 85°07 100°0 10 0:7683 23°17 27-2 A glance at these results, shows plainly a gradual decrease in pro- teolytic activity. It is to be noticed that in the presence of the larger percentages of these metallic salts, the fibrin does not swell up in the 0-2 per cent. acid. Manganous chloride. In small fractions of one per cent. this salt gave such irregular results that it is doubtful if they can be relied upon as expressing any particular action. With 0°3 per cent. the retarding action of the manganese salt commences to be very pronounced. Following are the results: . Undigested Fibrin Relative proteo- MnCly. residue. digested. lytic action 0 0:1923 gram. 80°77 per cent. 100°0 0-001 per cent. 0°2022 79°78 98°7 0-010 0°1815 81°85 101°3 0°025 0°2066 79°34 98-2 0-050 071855 81°45 100°8 0 071880 81:20 100-0 0°3 0°3687 63°13 tian 0°8 0°6438 35°62 43°8 15 0:6612 33°88 41:7 3°0 0°7400 26°00 32°0 92 Chittenden and Allen—Influence of various Salts Ferrous sulphate and Ferrie chloride. The salts of iron, doubtless on account of their physiological importance and their great therapeutic value, have been experimented with by several observers. It has been a prevalent opinion that iron salts tend to produce disturbances in gastric digestion. Petit,* how- ever, states as a result of experiment, that preparations of iron, in small quantities, do not hinder the action of pepsin, but in large quan- tities they retard the action of the ferment, doing so according to Petit, by the hydrochloric acid of the gastric juice displacing the acid of the iron salt, thus forcing the pepsin to act with a less energetic acid. Diisterhoff,t dealing with the same question, came to the con- clusion that iron salts of the organic acids, exercise the greatest retarding effect on pepsin digestion, and moreover, that ferrous salts are better adapted to the organism than ferric salts. Diister- hoff also concludes that while the retarding action of iron salts is doubtless due, in part, to the setting-free of the acid of the iron salt by the acid of the gastric juice, there is in addition a specific action of the iron preparation of an unknown nature, prejudicial to digestion. Lastly, Bubnow { found that moist ferric hydroxide in small quanti- ties (not weighed) causes a scarcely recognizable diminution in pro- teolytic action, while the presence of 1 per cent. of ferrous chloride and ferrous sulphate causes marked retardation, as does also an excess of ferric hydroxide. The most intense action was observed on the addition of 5 per cent. of ferrous sulphate. No quantitative results, that is, percentages of albumin digested were, however, obtained. Our experiments were made only with crystallized ferrous sulphate and ferric chloride. It appears superfluous to try the action of ferric hydroxide, which must necessarily, if in sufficient quantity, neutralize the acid of the gastric juice and thus prevent digestion by with- drawal of the free acid. * Quoted by Bubnow in Zeitschrift fiir physiologische Chemie, vol. vii, p. 316; also abstract by Herter in Jahresbericht fiir Thierchemie, 1880, p. 309. + Ueber den Hinfluss von Kisenpraparaten auf die Magenverdauung. Jahresbericht fiir Thierchemie, 1882, p. 257. ¢ Ueber den Einfluss des Hisenoxyhydrats und der Hisenoxydulsalze auf kiinstliche Magenverdauung und Faulniss mit Pancreas. Zeitschrift fiir Physiologische Chemie, vol, vii, p. 315. aa. <“ — eet alts a ee on the Proteolytic Action of Pepsin-hydrochloric Acid. §3 Undigested Fibrin Relative proteo- FeS04+7H20. residue. digested. lytic action. 0 0°1835 gram. 81°65 100°0 0-001 per cent. 0°1916 80°84 99°0 0°005 0°2241 1759 95°0 0:010 0°1895 81°05 99°2 0:025 0°2573 74:27 90-9 0:050 02773 72°27 88°5 0 0°1935 80°65 100-0 0-1 0°3467 65°33 81°0 0°3 07274 27°26 33°8 0°38 0°8080 19°20 23°8 15 0°8447 15°53 19:2 Here, with the ferrous salt, we find pronounced diminution of pro- teolytic action, commencing even with 0°001 per cent. With ferric chloride, the following results were obtained. Undigested Fibrin Relative proteo- FeegClg. residue. digested. lytic action. 0 0°1842 gram. 81°58 per cent. 100°0 0-001 per cent. 0°2111 78°89 96°7 0°005 0:2059 79°41 97°3 0-010 0°2165 78°35 96-0 0°050 0°2332 76°68 93°9 0 0°1961 80°39 100°6 0°3 0°6526 34°74 432 0-5 0°8035 19°65 24°4 0:8 0°8794 12°06 15:0 3°0 0°9582 418 5°2 A comparison of the two series of results, shows no pronounced and constant difference in the amount of action between the two iron salts; both retard proteolytic action about equally; although with the larger amounts, as with 0°5 per cent. and beyond, ferrie chloride _ appears the most injurious. Comparing the results with those obtained with the manganese salt, which of late has been recom- mended as a therapeutic agent where iron cannot be taken, we see that the manganese is throughout, far less injurious than the two salts of iron. As to the manner in which the iron salts produce their retarding effect on proteolytic action, it is evident that it cannot be due to a simple displacement of the acid of the iron salt, by which the pepsin is made to act with a less compatible acid, since ferric chloride acts similarly to the sulphate, in which case there could be no such injurious replacement, 94 Chittenden and Allen—Influence of various Salts Magnesium sulphate. Pfeiffer * alone appears to have studied the influence of magnesium sulphate on gastric digestion. He found that retarding action com- menced in the presence of 0°24 per cent. of the salt and was very great in the presence of 4:0 per cent. Our results show decided retarding action, even in the presence of 0°005 per cent. of the crys- tallized salt. At the same time, it is to be remembered throughout, that probably differences in the strength of gastric juice, would cause some variation in the amount of retardation, produced by any given percentage. Undigested Fibrin Relative proteo- MgSO4+7H,O. - residue. digested. lytic action. 0 0°1081 gram. 89°19 per cent. 100-0 0005 per cent. 01910 80:90 90-7 0010 0°2330 76°70 86°0 0°050 0°3260 67°40 (N55) 0°100 0°4428 bb12 62°3 0 0°2605 73°95 100°0 0°3 0°7551 24°49 33'1 0°5 07886 21:14 28°5 0°8 0°8250 17°50 230 15 0°8891 11:09 15:0 3°0 0°8894 11°06 14:9 It is noticeable here, that while the retarding influence of the salt, becomes more and more pronounced as the percentage is increased, there comes a point (1°5 per cent.) when further addition does not materially influence the action of the ferment. : Potassium permanganate. This salt, as with the amylolytic ferment of the saliva, shows very energetic action. Its influence is, without doubt, due to rapid oxida- tion and consequent destruction of the ferment ; indeed, the (at first) bright red color of the solution became almost immediately bleached out and the solution, at the same time, completely deprived of pro- teolytic power. The following results testify to its extreme activity. Undigested . Fibrin Relative proteo- Ko MnoOg. residue. digested. lytic action. 0 0-1951 gram. 80°48 per cent. 100°0 0°005 per cent. 0°8278 17°22 21°3 0°010 0°9949 0°51 0°6 It is thus more active in preventing proteolytic action, in gastric juice of the strength used, than in hindering the development of * Abstract in Centralbl. med. Wiss., 1885, p. 328. on the Proteolytic Action of Pepsin-hydrochloric Acid. 95 bacteria; although doubtless, the action of any one percentage is de- pendent in part, upon the amount of organic matter present. Marcus and Pinet* found that the permanganate in 0:1 per cent. would pre- vent the development of bacteria and in 1°5 per cent. would kill the fully developed organisms. Potassium dichromate. A single experiment with this salt gave the following results; showing a decided retarding action on gastric digestion. Undigested Fibrin Relative proteo- KeCrg07. ‘ residue. digested. lytic action. 0 0°2028 gram. 79°72 per cent. 100°0 0-01 per cent. 0°2476 15°24 94-4 0°10 0°6383 36°1LT 45°3 Potassium cyanide. Potassium cyanide we found very active in diminishing the diges- tive power of pepsin; due in great part doubtless, to decomposition of the cyanide by the hydrochloric acid of the gastric juice with conseqent formation of pepsin-hydrocyanic acid. Our first results were as follows: Undigested Fibrin Relative proteo- KCN. residue. digested. lytic action. 0 0°3255 gram. 67°45 per cent. 100°0 0°25 per cent.. 0°9687 3°13 4°6 0°50 0°9912. 0°88 is} Here, the fibrin did not swell at all, indicating the probable ab- sence of free hydrochloric acid, although of course the potassium cyanide might, per se, prevent swelling. With very much smaller percentages of cyanide, we obtained the following results: Undigested Fibrin Relative proteo- KCN. residue. digested. lytic action. 0 0°3098 gram. 69°02 per cent. 100°0 0-005 per cent. 0°4376 56°24 815 0:025 0°3750 62°50 90°5 Potassium ferrocyanide. With this salt, the results are practically the same as with potas- sium cyanide; almost complete stopping of proteolytic action, even in the presence of small fractions of one per cent. * Action de quelques substances sur les bactéries de la putréfaction, Compt. rend. Soe. de Biolog., 1882, p.718. Abstract in Jahresbericht fiir Thierchemie, 1882, p. 515. 96 Chittenden and Allen—Influence of various Salts Undigested Fibrin Relative proteo- K4Fe(CN) 6+3H20. residue. digested. lytic action. 0 0°3717 gram. 62°83 per cent. 100°0 0°05 per cent. 05969 40°31 64°71 0°10 0°7922 20°78 33°0 0°25 0°9585 4:15 6°6 0°5 1:0 0 0 0 0°3098 69°02 100°0 0°005 0°3562 64°38 93°3 0°025 0°3471 65°29 94°5 Potassium chlorate and Potassium nitrate. These two oxidizing agents produce almost exactly the same effect on pepsin-hydrochloric acid digestion; a retarding action directly proportional to the amount of salt present. Undigested Fibrin Relative proteo- KC103. residue. digested. lytic action. 0 0°2683 gram. 73°17 per cent. 100°0 0°3 per cent. 0°4565 54°35 74°3 0°8 07019 29°81 40°7 1°5 0°8173 18°27 25°0 3°0 68707 12°93 17°6 KNO3. 0 0:2870 gram. 71°30 per cent. 100°0 0°3 per cent. 0°5370 46°30 | 64°9 0°5 0°6247 37°53 52°6 0°8 07158 28°42 39°8 15 0°8148 18°52 25°9 30 0°8907 10°93 15°3 With potassium chlorate no experiments have been previously tried; with potassium nitrate, however, Wolberg* experimenting with quantities varying from 0°5 to 8°0 per cent. found in every instance, diminution in the proteolytic action of his pepsin solution. This, however, amounted to but little, except in the presence of 8 grams (8 per cent.) of the nitrate, where there was a diminution in the amount of fibrin digested, equal to 49-0 per cent. Even with 6 per cent. of the salt, Wolberg found after 24 hours, only a diminution of 6°8 per cent. in the fibrin digested. In quantity, therefore, our results do not accord at all with Wol- berg’s, since as the table shows, even 0°3 per cent. of potassium nitrate caused a diminution in the quantity of fibrin digested, amounting to 351 per cent., when compared with the control (100); while the pres- —— * Pfliiger’s Archiv, vol. xxii, p. 300. Ueber den Hinfluss einiger Salze und Alka- loiden auf die Verdanung. on the Proteolytic Action of Pepsin-hydrochloric Acid. 97 ence of 3 per cent. of the salt caused a diminution in proteolytic action amounting, in the quantity of fibrin digested, to nearly 85 per cent. The only apparent explanation of this difference in the results [unless due to difference in the amount of ferment] is in the length of time the mixtures were warmed at 40° C.; in Wolberg’s 24 hours, in ours 2 hours. This, if the true reason of the difference, would imply on the part of the ferment, ability to gradually overcome the influence of small amounts of the substance and thus eventually to digest an equal quantity of proteid matter. This, however, would in turn imply that the object sought for, viz: the influence of different quan- tities or percentages .of a substance on the action of the ferment is lost sight of. The length of time best adapted to the experiment, is naturally that which will bring out most clearly and decisively all differences of action. Sodium tetraborate (Borax) and Boracie acid. Sternberg’s experiments* with both of these substances, have shown that, although possessed of no germicide value, they prevent the multiplication of bacterial organisms and are thus valuable anti- septics. Wolberg, in experiments made with artificial gastric juice, found that in a 24 hours digestion, 0°5 gram (0°5 per cent.) of borax caused a slight acceleration in proteolytic action (0°4 per cent.), while with 1 per cent. of the salt, retardation occurred to the extent of 23°3 per cent., and in the presence of 4:0 per cent. almost complete stopping of proteolytic action. Our results, however, fail to show any stimu- lating action on the part of the borate, although retardation is very pronounced. Undigested Fibrin Relative proteo- Na2B407+10H20. residue. digested lytic action. 0 0°3610 gram. 63°90 per cent. 100°0 0°05 per cent. 0°3852 61°48 96°2 0°20 0°4080 59°20 92°6 0°5 ~ 0°7710 22°90 35°8 1-0 0°9899 1°01 15 Doubtless, the retarding action of this salt is due wholly to the liberation of boracic acid and the consequent neutralization of the hydrochloric acid of the gastric juice. Boracic acid itself, offers no obstacle to the proteolytic action of pepsin-hydrochloric acid; on the contrary it increases it, but pepsin-boracic acid has little digestive * Amer. Jour. Med. Sciences, April, 1883, p. 335. TRANS. Conn. AcaD., Vou. VII. 13 OctT., 1885. 98 Chittenden and Allen—Influence of various Salts power. The influence of boracic acid on pepsin-hydrochloric acid is seen from the following experiments: Undigested Fibrin. Relative proteo- H.;BO3. residue. digested. lytie action. 0 0 2395 gram. 76°05 per cent. 2 10050 0°) per cent. 0°2232 77°68 102°1 0 0°2049 79°51 100-0 0°5 01875 81°25 102°2 3°0 01729 82°71 104-2 6:0 0°1445 85°55 107-6 Evidently then, the action of borax consists simply in withdrawing from the pepsin the hydrochloric acid of the gastric juice. The low digestive power of pepsin and boracic acid is shown by the following experiment : - A, B C. D H,0 sol. pepsin _---- 50 ce. 50 c.¢. 50 cc. 50 cc. Hl 0:2 per cent. _.-. 50 0 0 0 He BO See en nace 0 0-2 gram. 0°3 gram. 0°5 gram. ORS a Ze 0 50 ce. 50 cc. 50 ¢. 100 100 100 100 01¢HCl 0:2 %H BO, 0:3%H;BO, 05 % HBO; To each, was added | gram of purified fibrin, after which the mix- tures were warmed at 40° C. for 2 hours. Following are the results. A. B. C. D. Wt. of undigested residue.--..- . 0°1180 09615 0°9705 0°9620 Per cent. digested ......-.------ 88:20 3:85 2:95 ~ 3-80 Ammonium oxalate. With this salt the following results were obtained : Undigested. Fibrin Relative proteo- (NH4)2C204+2H20. residue. digested. lytic action. 0 0°3254 gram. 67°46 per cent. 100°0 0-610 per cent. 0°3579 64°20 95°1 0°025 0°3627 63°73 94-6 01 0°3920 ; 60°80 9071 0°5 0:9049 : 9:51 14:1 0 0°3098 69°02 100°0 1:0 0°9958 0 42° 6°6 As to the cause of this retarding action, it is probable, that, as in the case of borax, the oxalic acid of the salt is displaced and the ferment compelled to act with the acid thus liberated. But if we compare the results of the present series with those of the preceding, ° on the Proteolytic Action of Pepsin-hydrochloric Acid. 99 we find that, with like percentages of the two salts, ammonium oxal- ate has far the greater retarding power, and yet it is well known that the ferment acts well when combined with oxalic acid. More- over, our experiments show further on, that ammonium chloride has no greater retarding power than sodium chloride. Hence, there must be some reason, other than the one mentioned above, to account for all of the retarding action manifested by the oxalate. Petit* states, that the maximum digestive action of oxalic acid, with 0°2—-0°4 per cent. of his pepsin, is attained with 0°5—4:0 per cent. of the acid, according to the amount of pepsin. The comparative digestive power of pepsin-oxalic acid and pepsin- hydrochloric acid is shown by the following experiments :+ A. B. @ H,0 sol. pepsin --.-- ni) ares BOKeHC: 50 ce. 0°2 per cent. HCl..-. 50 0 0 Cr One arse eens 0 0-5 gram, 1-0 gram. Om ees fs sea 0 50 ¢@.c. 50 ce. 100 100 100 01% HCl 0°5 % CoH20,4 1:0 % C.H.O0, Warmed at 40° C. for 2 hours with 1 gram of pure, dry fibrin, the following results were obtained : A, B. o Wt. of undigested residue _..-_ 0°2170 04450 04371 Per cent, digested i212. ---.-- 78°30 55°45 56°29 A second series, with larger amounts of oxalie acid, gave the fol- lowing results: A. B. C. H.0 sol. pepsin--- 50c.c¢. 50 c.e. 50 ¢. e. 0-2 per cent. HCl. 50 0 0 Or OW aa S252 0 1°5 grams. 2-0 grams. ET Ope et 0 50 ¢c. ¢. 50 c¢. ¢. 100 100 100 0-1 per cent. HCl 15 per cent. C.H.0,4 2-0 per cent. C,H204 Undigested residue 0°1085 gram. _ 0°3090 gram. 0°3640 gram. Per cent. digested 89°15 69°10 63°60 These four results being placed on the same basis, i. e. compared with their respective controls (100) show as follows: * Jahresbericht fiir Thierchemie, 1880, p. 309. | Made in this laboratory by Mr. R. W. Pinney, 100 Chittenden and Allen—Influence of various Salts Relative proteo- Per cent. of acid. lytic action 071 HCl 100°0 0°5 CoH.0, 70-7 1:0 Toler 1°5 eo 2°0 wales This shows;maximum action, with our amount of pepsin, in the presence of J°5 per cent. of oxalic acid and shows, moreover, that the retarding influence of ammonium oxalate on proteolytic action, is not fully explained by the suggested neutralization of the hydrochloric acid of the gastric juice, unless it be that the ammonium chloride, formed by the reaction, effects pepsin-oxalic acid differently than it does pepsin-hydrochloric acid. Sodium chloride. With this salt we obtained, by our method, the following results : Undigested Fibrin Relative proteo- NaCl. residue, digested. lytie action. 0 0°2936 gram. 70°64 per cent. 100°0 0:005 per cent. 0°2824 71:76 101°6 0-010 0°2896 71:04 160°5 0°025 0°3441 65°59 R 92°8 0°050 0°3511 64°89 91°8 0-100 0°3744 62°56 88°5 0 0:2669 73°31 100:0 / 0°3 0°4953 50°47 68°8 05 0°6175 38°25 52°1 0°8 0°6898 : 31°02 42°3 Is5 0°7825 AWE) 29°6 3°0 0°8249 Lib 23°83 Alex. Schmidt* has recorded the retarding effect of sodium chlo- ride on the proteolytic action of gastric juice; Petit+ likewise, has stated that small quantities of sodium chloride interrupt the action of the ferment, and Wolberg{ has recorded the same result with varying percentages of the salt; noting in addition, that very small quantities cause a slight acceleration of. proteolytic action, amounting in his experiment to 2°5 per cent. With 0°005 per cent. of the salt, we found as the results show, acceleration amounting to 1°6 per cent., larger amounts causing a gradual and proportional diminution, in proteolytic action. * Pfliiger’s Archiv, xiii, p. 98. } Jahresbericht fir Thierchemie, 1880, p. 309. } Pfliger’s Archiv, xxii, p. 298. on the Proteolytic Action of Pepsin-hydrochloric Acid. 101 Pfeiffer* has also called attention to the retarding action of this substance. Potassium chloride. With this salt our results are as follows: KCl. vrestiee. digested. Miytie action. 0 071552 gram, 84°48 per cent. 100-0 0-005 per cent. 071817 81°83 96°8 0°025 0°1550 84°50 100-0 0:050 0°2565 74°35 88°0 0°1L00 0:2097 79°03 93°5 0 0°1930 80°70 100-0 0°3 03997 60°02 74:3 15 0°6815 31°85 39°4 3°0 0°7282 27°18 33°6 The only noticeable difference between the action of this salt and the preceding, is the absence of any acceleration on the part of the potassium chloride and with larger percentages, a less vigorous retard- ‘ing action. Ammonium chloride. For the sake of comparison a few experiments were tried with this salt, with the following results : Undigested Fibrin Relative proteo- (NH4)C1. ; residue. digested. lytic action. 0 0°1880 gram. 81:20 per cent. 100°0 0-3 per cent. 0°4649 53°51 65°9 0°8 0°6615 33°85 417 3°0 0°6970 30°30 37°3 By looking at the table of comparisons, we see there is little con- stant difference in the amount of retardation caused by the ammo- nium, potassium and sodium salts of hydrochloric acid. This result, however, is quite different from that obtained by Wolberg, who~ found that ammonium chloride influenced proteolytic action but very little, while both potassium and sodium chloride caused great retard- ation. This difference in result, may be due to difference in strength of gastric juice or to difference in length of time the mixtures were warmed at 40° C.; certainly in our experiments, with pure anhydrous salts and 2 hours digestion, very little difference in digestive action is noticeable ; throughout, sodium chloride causes a little less proteolytic action than the corresponding potassium salt, while of the ammonium salt, little is to be said except that it causes equal retardation. * Centralbl. med, Wiss., 1885, p. 328, 102 Chittenden and Allen—Influence of various Salts W olberg in speaking of the relative retarding action of the three sodium salts of hydrochloric, nitric and sulphuric acids, states that the sulphate retards the most, then the nitrate and lastly the chloride. We have noticed the same fact in our work and take it as additional evidence of the liberation of the acid of the salt added, the retarding influence of the salt being dependent, in part, on the digestive power of the pepsin-acid formed. The following experiments,* showing the relative digestive power of the several acids in conjunction with. pepsin, testify to the proba- bility of this view. All of the solutions contained 50 c. c. of an aqueous solution of glycerine-pepsin, each having a total volume of 100 c. c. and differing from each other only in the nature and percentage of the acid present. In testing the digestive power of the solution, 1 gram of pure fibrin was added and the mixtures warmed at 40° C. for two hours, after which the amount digested was determined in the usual manner. Percentage of fibrin digested in the presence of the different per- centages of acids. Control. 0:05-% O14 0:24 O38 G O4L% 0°5% 01% HCl HNOst---- 9°20 48°75 73°65 67°20 46°00 87°65 LES OVALS Sree 19°50 24°70 25°80 DD bo 24°95 88°05 CoH,O9---- 3°70 2°30 87°50 Comparing these results with their respective controls, figured as 100, we have the following values for sulphuric and nitrie acids, ex- pressive of their relative proteolytic power when combined with pepsin, as compared with 0-1 per cent. hydrochloric acid under simi- lar conditions. Per cent. acid. HNO3. H2SO4. 0°05 10°5 oer 0-1 5b°D 22:1 , 0-2 84-0 28°0 0°3 T6°7 29°] 0-4 52° 2 25°6 0°5 anne 28°2 From this it is evident, that nitric acid is most active, with our amount of pepsin, in a 0°2 per cent. solution, while sulphuric acid attains its maximum action in 0°3 per cent. ; moreover, nitric acid is * Made in this laboratory by Mr. R. W. Pinney. + The acids are calculated as pure HNOs, H.SO,, ete, ieee si thay a eT 7 7 ® eC ee ae eae ee ee eo \ant o“aa Ye er AS on the Proteolytic Action of Pepsin-hydrochloric Acid. 163 more than four-fifths as active as 0:1 per cent. hydrochloric acid, while sulphuric acid is only a little more than one-fourth as active as the hydrochloric acid; acetic acid being practically worthless. Henee it is obvious, that, the base being the same, acetates, borates and other salts, the acids of which are not capable of working with pepsin, will most readily retard gastric digestion; then of the other salts experimented with, sulphates and lastly nitrates. A glance at the table of comparisons, shows here and there, results which manifestly accord with this view, notably lead acetate, cupric sulphate, zine sulphate,* magnesium sulphate and potassium nitrate. That this withdrawal of free hydrochloric from the pepsin, is only a partial explanation of the mode of action of neutral salts is plain from the fact that chlorides exert very pronounced retarding action; moreover the action of these latter as well as of the others cannot be due merely to mechanical causes, viz: the semi-saturation of the digestive fluid, since 3-0 per cent. of boracic acid stimulates instead of retarding, and even the presence of 10 per cent. of arsenic acid causes retardation amounting to only 32 per cent. According to Petit,+ moreover, saccharose to the extent of 16 per cent. does not interrupt gastric digestion. Again, that the base entering into the composition of the salt has much to do, in many cases, with its re- tarding proteolytic action, is apparent when we compare the action of ferric chloride, manganous chloride, sodium chloride, mercuric chloride and other metallic salts. That this action is due in part to -eapability of combining with the proteid matter, thus rendering it non-digestible, is unquestionable. Acceleration, produced by neutral salts has been noticed by other observers, but no explanation of the cause has been offered. It seems probable, however, that in the case of pepsin-hydrochloric acid, one plausible reason, at least, may be suggested. If, as has been mentioned, many of the neutral salts are decomposed by the acid of the gastric juice, it would follow that the addition of very small amounts of the salts would diminish slightly the percentage of free hydrochloric acid, while the acid liberated from the salt, would be entirely without deleterious action. Nearly every experimenter in this direction has employed in the preparation of gastric juice 0-2 per cent. hydrochloric ~ acid, which is well adapted for the purpose, but the action of the acid is dependent in part on the amount of ferment. With our solution of pepsin, the following results, expressed in the percentage of fibrin * Compare Petit, Jahresbericht fiir Thierchemie, 1880, p. 309. . + Jahresbericht fir Thierchemie, 1880, p. 309. 104 Chittenden and Allen—Influence of various Salts digested, were obtained with different percentages of hydrochloric acid; the amount of pepsin being the same in each ease. Percent; ii@leneees oe O05 O-1 O°2 O38 O-4 Per cent. fibrin digested 73-8 SI'3 84-0 81°7 63°8 This shows plainly, that, with the amount of pepsin employed in our experiments, acceleration of proteolytic action on the addition of neutral salts, might in some instances be due to slight diminution of free hydrochloric acid. That this is not the sole cause, however, is plain from the fact that closely related salts do not act alike. Potassium bromide and Potassium iodide. With these two potassium salts, the following results were ob- tained : Undigested Fibrin Relative proteo- KBr. residue. digested. lytic action. 0 0°3837 gram. 61°63 per cent. 100°0 0005 per cent. 0°3205 67°95 110°2 07025 073035 69°65 113°0 0°10 0°4.204 57‘96 94-0 0°50 0°5690 43°10 70°0 1-00 0°6203 37-97 616 Kl. 0 0°2572 gram. 74°28 per cent. 100°0 0°005 per cent. 0°2755 72°45 97-5 0025 0°3421 65°78 88°6 0710 072841 71°59 96-4 0°50 06367 36°33 48°9 1°00 0°7586 24°14 32°5 By comparison of these two series of results, we see that there is a very decided difference in the action of the .two salts; potassium bromide in very small quantity, causes a decided acceleration in pro- teolytic action, which is wholly lacking with potassium iodide. Again, there is a very great difference in the amount of retardation produced by the two salts ; thus by comparison with their respective controls we see, that while 1 per cent. of potassium bromide causes retardation in digestive action, amounting to 384 per cent., the same percentage of iodide causes retardation amounting to 67°5 per cent. It is to be supposed, naturally, that in the presence of large amounts of the salts, the ferment must act wholly in combination with hydrobromic and hydriodic acid respectively; that is to say, the hydrochloric acid of the gastric juice must have replaced these two acids in the potassium salts, and thus new pepsin-compounds formed, of which pepsin-hydrobromic acid is the more active digestive agent. q 3 ~ on the Proteolytic Action of Pepsin-hydrochloric Acid. 105 Putzeys,* indeed, found that he obtained practically the same proteolytic action [retarding] with pepsin and hydriodic acid, as with potassium iodide and pepsin-hydrochloric acid. The following results, showing the relative digestive action of pepsin with hydro- bromic and hydriodic acid respectively, were obtained by Putzeys, = Digestive pro- HI. HBr. ducts formed. 0°625 gram. a 15°86 per cent. 0°937 spice 31°33 3°310 nets 2°33 SSReE 0°882 gram. 26°40 sen3 2°200 45°60 BOSE 3°309 46°60 It is thus evident, that both hydrobromic and hydriodic acid can, to a certain extent, replace the hydrochloric acid of the gastric juice, although they are much less active than the latter acid. Moreover, hydrobromic acid is much more efficient than hydriodic in connec- tion with the ferment, for in comparatively large doses the latter acid will completely stop proteolytic action. As a practical result, Pntzeys suggests, that for therapeutic purposes, potassium bromide and iodide should be given $ to 1 hour before meals. Our results are plainly in accord with Putzeys’ observations. The following table shows the relative influence on the proteolytic action of pepsin, of the various inorganic salts experimented with, compared with the controls, expressed as 100. * Jahresbericht fiir Thierchemie, 1877, 279. De l’influence de l’iodure et du bro- mure de potassium sur la digestion stomacale. TRANS. Conn. AcAD., VOL. VII. 14 OctT., 1885. a al SR ee oot ee eee oa eP Le oa OG lee SRA eee ORG Mg See ee nae me he Ta Bee Sal se le HOT. ee” OO eas eee eee ORC eee OTs Gairewt een cs ue 1g SPS eke Fy eS LEY IP ee loge elnnare eras (ae etteeee ee ame ea Wine hc ee 10("HN) 7777 | TTT 18-88 | 777 [9-68 | 7777 [BBP ILS [889 |~-~~ 19-88 [8-6 [8-26 /9-001/9-10T)"~"~ |-~77 Ton 108N rT om Hesee | PBR ASO”) Tl CMe pele Ween OS OMe eS BOON ke ee tox BS eal ee aie Sle | 3°" neaozy: — (BGS D129 |GerOmp TA eae [ele at Ta eae "ONM re OR Ore [kee OS" Pear See ee age eee ee wc Mbt es ‘OI BS SO) Ga en or ROL | ah CO eee Syl aoe eee a, © ‘od'H Ce Sr s[RC8. Peo Oa i a Geo iee Jee ices or ener S O°HOL+*0'a*®N eel ele el OO ee eer [sera OG NOS, (Saal) ke ee ORR ODF EIN) ee (2 | 0 ee OSGeo" SRG mee Oia ete ae O* Hs + °(NO)0a"H meen eae ceed eM ay ese rae NS roe ae hele seca ae 19.0 Sang ees rear ea sor ‘OU NY Se eleet |6-RE-| = 0-90 ee" > 2-60 19-82 |T-8S |" =" GCOS (Gah i = (OOS. h-Oere FS O*HL+*0S3N eeniwe Oss is) Aeekh |r 2 ciSserope Habit = ie (SHO MiG 86 1E10%) So rue BG i Se ee ee "100K ae ra | : : : 5GG.\| = Bo mae ek O°HL + OSH Bee Soha les oes he SILO LSPS, (Geral as Heo san GseOl sr [0:06=|S-86 IAG a eo oe anes "10°80 aQm GME oe C.6 4 sO. COkia -- O-1Ollai i GeEOL oo a [ce ysl Te Seca ecules epee earn | ‘OSsV°H a eee |e cag ee Wee oie

|-~-= foreoT|6-1Otle-oore-FOL. ~~ OfHE + "COR O)Id SE ied kel ese €@ |F-9% |G-18 |"~-~ [2-19 |9-98 |T-98 |0-46 |8-201|8-POT|-~7- ~~ ~~~ OHS + 'OSng Sie ae claae Slee Oia. 18°66) GOL Tee eoea ce oe “NOSE eee eee eae ate ese Het ees ae eee catagtens he ees ST SG |". a IPOD) cul ok oe "13H econ ~ene | ee-e A= demo ecee | ing with tance, except in every ins . Alkaloid salts, Wolberg, however, found that morphine chloride, 106 Chittenden and Allen—Influence of various Salts ' N lor) ‘ ic.2) ES nN ' ice) oF ioe) oO re oe) uw a io 2) lor) So lor) nN or) lor) i=) 1D for] i=) > for) "w010n duhijoaj01d aaynjas Hurnoys 29D], d solution. According to Petit, alkaloids do not interrupt the action of pepsin in aci strychnine, quinine sulphate and narcotine, used in very small quantities to 100 c. ¢.), caused Iphate, retardation amounting on an average to nearly 3 0°56 grain quinine su ( on the Proteolytic Action of Pepsin-hydrochlorie acid. 107 _ per cent.; with quinine sulphate, slight acceleration was obtained. Strychnine and narcotine, in very small quantity, gave slight accel- eration. Dr. H. v. Boeck* states, that quinine is without influence on the activity of pepsin. In our experiments, using larger amounts of the alkaloid salts, retardation is very pronounced, doubtless due in part to replacement of the sulphuric acid of the salts by the acid of the gastric juice. Undigested Fibrin Relative proteo- Alkaloid salt. resiaue. digested. lytic action. 0 0°3555 gram. 64°45 per cent. 100°0 (Sr), . H,SO, + 6H,0. 0°5 per cent. 0°6825 BLD 49°2 1-0 0°8213 17°87 27°7 (Br), . H,.SO, + H,0. 0°5 0°5685 43°15 66°9 1:0 07700 23°00 35°6 (At)» . HeS0,. 0°5 0°6412 35°88 55°6 (Q)..H,SO, +7H,0. 0°5 0°6606 33°94 52°6 (Ci) + H.SO, +2H,0. 0°5 06885 31°15 48°3 ~ (Mo). H,SO, +5H,0. 0-5 0°6225 alano 58°5 (Na), . H,SO,+H,0. 05 06365 36°35 56-4 Percentage retardation of the alkaloid salts (0°5 per cent.) SURV Ch Gee awe Eee mee staf 2 oS. oe See eee DUS IS THULE py re So se So RS es Ree LE 1) Ed et 33°71 PRU C ee net wea ee Rae wes SEI.) 2s a PE 44-4 Oourinesay sists sss. 25. a2 whe tes ee ee 47-4 Fin CHORIN Geass Soe sc Soro sete te ee ae he By ler MGEDIING Ese: ete ses ee ys. 2 os SUES EM 41°5 INGnCOUN Gs eee cee eet ae 43°6 * Zeitschrift fiir Biologie, vol. vii, p. 428. VIL.—InNFLuence or Various THERAPEUTIC AND Toxic Sups- STANCES ON THE PROTEOLYTIC ACTION OF THE PANCREATIC FER- MENT. By R. H. Cuirrenpen anp Geo. W. Cummins, Pu.B. Wirsn the proteolytic ferment of the pancreatic juice, few sys- tematic experiments have been tried to ascertain the influence of those substances the frequency of whose use, as therapeutic or toxic agents, renders their action on this ferment a matter of no small con- sequence. The well known experiments of Heidenhain, Kiihne and Schmidt have been confined to the action of sodium carbonate, sodium chloride and kindred salts of physiological importance. Pfeiffer* has indeed, in addition, experimented with a few of the sulphates of the alkalies and alkali-earths, but of the large number of metallic and other salts in common use, few have been tried, while the action of alkaloids and the gases occurring in the intestinal canal, has not been studied at all. Method employed. It is a characteristic of the proteolytic ferment of the pancreatic juice, that it exercises considerable digestive power in a neutral solution.t In view therefore of the fact that the ferment, while in the intestinal canal, may be obliged to act in an acid-reacting medium (due to acid-proteids) as frequently as in an alkaline or neutral one, it seemed advisable in the experiments, to employ a neutral solution of trypsin. Moreover, it becomes necessary to use such a solution, if salts of the heavy metals are to be experimented with, since in alkaline solutions, carbonates or oxides of the metals would be formed and thus affect the results; still, again, the action of all substances can be best studied in neutral solution, since under such conditions, no replacements or decompositions of any kind take place to compli- cate the action of the substance, other than the direct action on the ferment or the proteid matter. Hence, a neutral solution has in every case been employed as being the most satisfactory, while only such substances have been experimented with, as contained no free acid or alkali, the destructive action of which is well known. * Centralbl. med. Wiss., 1885, p. 328. + See Chittenden and Cummins, Amer. Chem. Jour., vol. vii, p. 46. Also Trans. Conn, Acad., vol. vii. Chittenden and Cummins— Action of the Pancreatic Ferment. 109 The solution of trypsin was prepared according to Kiihne’s* method from dried ox pancreas freed from fat; 40 grams dry pan- creas in 500 c. c. 0°1 per cent. salicylic acid, neutralized and diluted to 2 litres.t In each digestion experiment, 25 c. c. of this neutral trypsin solution were used, to which was added 25 ¢. c. of water containing the substance to be experimented with, or in the control 25 ¢. c. of water alone, making the volume of the digestive mixture in each case 50 ¢.c. In testing the proteolytic action of the different solutions, 1 gram of pure dry, pulverized fibrin, described in the preceding article, was added and the mixtures warmed at 40° C. for six hours, after which the undigested fibrin was filtered on weighed filters, washed thoroughly and dried at 100-110° C. until of constant weight. In this work as in the study of the salivary ptyalin and pepsin, it has been the effort mainly to ascertain the relative action of small quantities of the various salts, rather than the percentages necessary to completely stop the action of the ferment, this to our mind being much the more important. Mercurie chloride. With small percentages of this salt, the following results were obtained : Undigested Fibrin Relative proteo- HgClo. residue. digested. lytic action. 0 0°4495 gram. 55°05 per cent. 100-0 0°002 per cent. 0°4465 55°35 100°5 0-003 0°4405 55°95 101°6 0-005 0°4562 54°38 98°7 0°025 05076 49°24 89°4 0-100 07753 22°47 40°8 As seen from the table, 0-1 per cent. mercuric chloride diminishes the proteolytic action of the ferment more than one-half. Its action in this percentage is more energetic than on pepsin, although the smaller quantities are proportionally far less active; in one case (0-003 per cent.) even causing acceleration. Wassilieff { has studied the influence of mercurous chloride on pan- creatic digestion and finds that calomel does not affect the action of the proteolytic ferment, while it does prevent the formation of _ putrefaction products, viz: indol, phenol, ete. * Untersuchungen aus der physiolog. Inst. d. Universitat Heidelberg, vol. i, p. 222. + The solution was kept from putrefaction by the use of a little 20 per cent. alcoholic solution of thymol; enough of which remained dissolved in the fluid to pre- vent putrefaction also during the six hours digestion at 40° C. ¢ Zeitschrift fiir physiol. Chemie, vol. vi, p. 112. 110 Chittenden and Cummins—Influence of various Substances Mercurie iodide and Mereuric bromide. These salts dissolved in sodium chloride in such proportion that the ultimate solutions contained like percentages of both salts, gave the following results : Undigested Fibrin Relative proteo- Helo. residue. digested. lytic action. 0 0-4707 gram. 52°93 per cent. 100°0 0°005 per cent. 0°4780 52°20 98°6 0°025 0°5085 49°15 * 92°8 0-100 0°5994 40°06 75°6 0-200 0°6580 34:20 64°6 HgBrg. 0 ; 0:4707 gram. 52°93 per cent. 100-0 0:005 per cent 0°4400 56°00 - 105°S 0:025 0:4840 51°60 97-4 0°100 0°5721 42°79 80°8 0°200 0°6548 34°52 65°2 Aside from a slight acceleration, noticed with 0°005 per cent. of bromide, the two salts act very much alike, causing retardation of proteolytic action; less pronounced however, than that caused by mercuric chloride. Mercurie cyanide. The action of this salt is somewhat peculiar, causing at first when in small quantity, noticeable retardation followed in the presence of larger percentages by increased proteolytic action, though still below the action of the normal solution of trypsin. In a general way, its action on this ferment accords very nearly with its action on the amylolytic ferment of saliva and the proteo- lytic ferment of the gastric juice. Undigested Fibrin Relative proteo- Hg (CN). residue. digested. lytic action. 0 0°2668 gram. 73°32 per cent. 100°0 0:005 per cent. 0°3192 68°08 92°8 0°025 0°3209 67-91 92°6 0°050 0°3244 67°56 92:1 0-100 03308 66°92 91°2 0 0°3675 gram. 3°25 per cent. 100°0 0°3 per cent. 0°4094 59°06 93°3 0°5 0°3880 61°20 96°7 1°5 073932 60°68 95°9 Its action is to be attributed mainly to the hydrocyanic acid radical, judging from the action of potassium cyanide on the one hand and the action of mercury salts on the other. There is, how- ever, without doubt a close connection between the action of these salts and their power of combining with proteid matter in general. GES on the Proteolytic Action of the Pancreatic Ferment. 111 Cupric sulphate. With this salt a more energetic retarding action is to be noticed, than in the case of the proteolytic ferment of gastric juice; 0:1 per cent. of the salt causing a retardation in proteolytic action of 65°7 per cent. as compared with a retardation of 38°8 per cent. in the case of pepsin. Undigested Fibrin Relative proteo- CuS0O4+5H20. residue. digested. lytic action. 0 0°5035 gram. 49°65 per cent. 100°0 0°005 per cent. 0°5027 49°73 10071 0°025 ; 0°5320 46°80 94°2 0-050 0°5856 41°44 83°4 0-100 0°8295 17°05 34°3 Lead acetate. With this salt the following results were obtained, agreeing essen- tially in the smaller percentages, with those obtained with cupric sulphate. In the presence of 0:1 per cent., however, retardation is far less than with the same percentage of cupric sulphate; 0°5 per cent. completely stops proteolytic action. . Undigested Fibrin Relative proteo- Pb(CyH302)0+8H20. residue, digested. lytic action. 0 0°4562 gram. 54°38 per cent. 100°0 0-005 per cent. 0°4655 53°45 98-2 0:025 05391 46:09 84:7 0°050 0°5660 43°40 79°8 0-100 0°6410 35 90 66°0 0°500 iO 0 0 Stannous chloride. This salt, in conformity with its action on the amylolytic ferment of saliva, causes very decided retardation in the proteolytic action of trypsin, requiring but a very small amount of the salt to com- pletely stop the action of the ferment. Undigested Fibrin Relative proteo- SnClg. residue. digested. lytic action. 0 0°3875 gram. 61°25 per cent. 100°0 0°0005 per cent. 0°4495 55°05 89°8 0°005 0°5370 46°30 75°5 0°025 1¢0 0 0 Arsenious oxide. Schifer and Bohm have experimented with arsenious acid, study- ing its influence on the proteolytic action of a glycerine infusion of * Abstract in Jahresbericht fiir Thierchemie, 1872, p. 363, 112 Chittenden and Cummins—Influence of various Substances pancreas. They find that arsenious acid is without influence on the action of the ferment. We do not know whether they added arsen- ious acid to a neutral or alkaline solution of the ferment, as we have seen only an abstract of their paper, but we find that the addition of very small amounts of arsenious oxide to a neutral solution of . trypsin, does produce a slight retardation in the proteolytic action of the ferment. Owing to the comparative insolubility of arsenious oxide in water, only small percentages could be employed, but these show, as might be expected from the acid character of the substance, a decrease in proteolytic action. - Undigested Fibrin Relative proteo- As2Ozg, residue. digested. lytic action. 0 0°4598 gram. 54°02 per cent. 100°0 0°001 per cent. 0°4630 53°70 .99°4 0-005 0°4513 54°87 101°5 0°025 04710 52°90 97°9 0:050 0°4723 52°71 97°6 Arsenic acid. This substance, in conformity with its more decided acid character, causes greater retardation than arsenious acid; which fact tends, by analogy, to make the retarding action of the latter still more proba- ble. The results are as follows: Undigested Fibrin Relative proteo- Hg AsO4. residue. digested. lytic action. 0 0°4598 gram. 54°02 per cent. b00°0 0°005 per cent. 0°4563 54°37 100°6 0-025 04887 51°13 94-6 0°050 0°5264 47°36 87°4 0°100 0°6340 36°60 67:7 Ammonium arsenate. This salt in small amount, appears to increase the proteolytic action of the ferment, which fact would indicate that the retarding action of the two preceding arsenic compounds is due more to their acid character than to the arsenic contained in them. Undigested Fibrin Relative proteo- (NH4)3A804. residue. digested. lytic action. 0 0°4598 gram. 54°02 per cent. 100°0 0°050 per cent. 0°4620 53°80 ; 99°6 0100 0°4442 55°58 102°8 0°3675 63°25 100°0 0°5 0°4071 59°29 ’ 93°7 on the Proteolytic Action of the Pancreatic Ferment. 113 Potassium antimony tartrate. This compound of antimony fails to produce with trypsin the marked acceleration, noticed with the amylolytic ferment studied. This difference in action, since both ferments were in neutral solu- tion, indicates a specific difference in the nature of the two ferments. Following are the results obtained : Undigested Fibrin Relative proteo- K(SbO)C4H40¢. residue. digested. lytic action. 0 0°3978 gram. 60°22 per cent. 100°0 0-2 per cent. 0°4105 58°95 97°8 0°5 0°4129 58°71 97°4 1:0 0°4258 57°42 95-3 1:5 0°5406 45°94 76:2 Ferric chloride and ferrous sulphate. With these two salts of iron the following results were obtained : ‘ Undigested Fibrin Relative proteo- FeoClg. residue. digested. lytic action. 0 0°5215 gram. 47°85 per cent. 100-0 0°005 per cent. 075431 45°69 95°4 0°025 0°6243 Slow 718°5 0:050 0°7457 25°43 53°] 0°100 10 0 0 FeSO4+7H,20. , 0 0-4075 gram. 59°25 per cent. 100°0 0:005 per cent. 0°4548 54°52 92-0 0°05 0°6225 Sano 63°7 0°10 OT1T7 28°23 47°6 0°25 0°7104 28°96 48°8 0°50 °* 0:7219 27°81 46:9 1-00 00-7675 23°25 39°2 1°50 0°7861 21°39 36°1 Ferric chloride is seen to be far more energetic in its hindering action on the proteolytic ferment, than the ferrous salt. A like result was obtained with the amylolytic ferment of the saliva and to a lesser extent with pepsin-hydrochloric acid. Bubnow* has tried experiments with both of these salts and found, as might be expected, that when present to the extent of 5 per cent. they prevented the appearance of putrefaction products (indol, phe- nol, etc.) but did not interfere with the action of the unorganized ferment. As no quantitative results were obtained, we can make no direct comparisons. In our experiments, it is to be remembered that - putrefaction is prevented by thymol. - * Zeitschrift fiir physiol. Chemie., vol. vii, p. 327. Trans. Conn. ACAD., Vou, VII. 15 Oot., 1885. 114 Chittenden and Cummins—Influence of various Substances Manganous chloride. Following are the results obtained with this salt : Undigested Fibrin Relative proteo- MnCl. residue. digested. lytic action. 0 0°5215 gram. 47°85 per cent. 100°0 0-005 per cent. 0°5201 47-99 100°3 0°050 0°5139 48°61 101°6 0°100 075483 45°17 94°4 0500 0°6807 3193 66°7 Comparing the influence of this salt on the proteolytic action of trypsin, with the results obtained with the closely related iron salts, it is seen that the manganese salt has a far less retarding influ- ence than the latter; a fact which agrees with what was observed in studying its action on pepsin-hydrochloric acid. Consequently, manganese salts, in so far as they are adapted to replace iron salts as therapeutic agents, are apparently less liable to interfere with normal digestive action. : Zine sulphate. Undigested Fibrin Relative proteo- ZnSO4+7H20. residue. digested. lytic action. 0 0°5035 gram. 49°65 per cent. 100-0 0°005 per cent. 05175 48°25 97-2 0-025 0°5805 41°95 84:5 0°050 0°6802 31°98 64°4 07100 0°8301 16°99 34:2 The action of this salt on trypsin is, both in character and extent, similar to its action on the amylolytic ferment of saliva. Com- pared with pepsin-hydrochloric acid, however, the action of the salt is seen to be more energetic on the pancreatic ferment. Barium chloride. The following results were obtained : Undigested Fibrin Relative proteo- BaCl,+2H,0. residue. digested. lytic action. 0 0°3710 gram. 62°90 per cent. 100°0 0°05 per cent. 0°3515 64°85 1031 0°5 0°3885 61°15 97°2 30 0°4986 — 60°14 197 With this salt a slight acceleration in proteolytic action is to be seen with 0°05 per cent., while the larger amounts cause noticeable retardation. Magnesium sulphate. Undigested Fibrin Relative proteo- MgS0O4+7H20. residue. digested. lytic action. 0 03495 gram. 65°05 per cent. 100°0 0°05 per cent. 03541 64°59 99°3 0°5 O'3810 61°90 95°1 3°0 04.706 52°94. a 81°3 ie Pe ae on the Proteolytic Action of the Pancreatic Ferment. 115 Here retarding action is similar in extent to the action of barium chloride. Compared with zinc sulphate, the difference in action on trypsin is about the same in extent as the difference found in the action of the two salts on the amylolytic ferment of saliva. The retarding action of this salt on pancreatic digestion has been previously noticed by Pfeitfer.* Potassium permanganate. The retarding action of potassium permanganate is very pro- nounced. There is, however, a noticeable difference between the action of the salt on trypsin and its action on pepsin and ptyalin, the two latter being much more readily affected by the permanganate; that is, by much smaller percentages. Undigested Fibrin Relative proteo- KgMn20z3. residue. digested. lytic action. 0 0°4562 gram. 54°38 per cent. 100-0 0°001 per cent. 0°4570 54°30 99°8 0°003 04608 53°92 99-1 0-010 0°4833 51°67 95-0 0 0°3675 63°25 100-0 0°1 0°4487 55°13 87-1 075 00-7769 22°31 35°2 10 1:0 0 0 Potassium dichromate. Undigested Fibrin Relative proteo- KeCrv07. residue. digested. lytic action. 0 0°3978 gram. 60°22 per cent. 100°0 0-05 per cent. 0°4032 59°68 99-1 0°2 0°4245 57°55 95°5 0°5 04693 53°07 88°] 1-0 0°5525 44-75 74:3 15 0°6321 36°79 61°0 The retarding action of this salt is proportionally greater than ; that of the other potassium salts, indicating that the acid has a specific action of its own. Moreover, being an acid salt, the acid radi- cal is present in larger amount than in the neutral potassium salts experimented with. Potassium cyanide. The very pronounced acceleration in proteolytic action caused by the larger percentages of this salt is very interesting, especially when we recall the fact, that the retarding action of the same salt on the * Centralbl. med. Wiss., 1885, p. 328. 116 Chittenden and Cummins Influence of various Substances amylolytic ferment of saliva and on the proteolytic ferment of gastric juice was equally decided, even with very small pecentages. Undigested Fibrin Relative proteo- KCN. residue, digested. lytic action. 0 0-2668 gram. 73°32 per cent. 100°0 0°005 per cent. 0°2790 72°10 98°3 0°025 0°2840 71°60 97°6 0°050 02678 73°22 99°8 0°100 0°2600 74:00 100°9 0 0°3675 63°25 100°0 0°3 0°2076 79°24 125°2 0°5 0°1985 80°15 126°7 10 0:2697* 73°03 115-4 ibs) 0°3315* 66°85 105°6 Potassium ferrocyanide and Potassium ferricyanide. With these two salts the following results were obtained : Undigested Fibrin Relative proteo K4Fe(CN)5+8H,0. residue. digested. lytic action. 0 0°3045 gram. 69°55 per cent. 100°0 0°005 per cent. 0°3363 66°37 95°4 0°50 0°3293 67:07 96°4 2°00 0°3753 62°47 89°8 KFe(ON), 2 0-005 per cent. 0°3268 67°32 96°8 0°05 0°3370 66°30 95°3 2°00 i 0°3912 69°88 87°5 Here, unlike the cyanide, there is no acceleration in the proteolytic action of the ferment, neither on the other hand is there very pro- nounced retardation; less indeed than is produced by a like per- centage (2°0) of sodium chloride. Sodium tetraborate. With this salt we obtained the following results : Undigested Fibrin Relative proteo- NagB407+10H20. residue. digested. lytic action 0 0°4116 gram. 58°84 per cent. 100°0 0°05 per cent. 0°3729 62°71 106°5 0°2 0°3260 67:40 114°5 0°5 0°2332 76°68 130°3 10 0°1860 81°40 138°3 2°0 0°1663 83°37 141.7 3°0 0°2276 17:24 131°2 5-0 0°3141 68°59 1165 * Tt was impossible to wash these completely, consequently the weights are undoubtedly too high, on the Proteolytic Action of the Pancreatic Ferment. 117 Here, unlike the action of the salt on the two preceding ferments studied, there is to be seen a gradual increase in proteolytic action, which is very pronounced in the presence of 2 per cent. of the salt, then gradually diminishes ; although even with 5 per cent. of the crystallized salt, the action of the ferment continues to be increased far above that of the normal juice. Sodium sulphate. Following are the results obtained : : Undigested Fibrin Relative proteo- Naz, SO4+10H,20. residue. digested. lytic action. 0 0°3495 gram. 65°05 per cent. 100°0 0°05 per cent. 0°3602 63°98 98°3 0°5 0°3558 64°42 99°0 20 0°3938 60°62 GBH 5-0 0°4360 56°40 86°7 The retarding action of this salt is quite pronounced, although it is noticeable that its retarding action is not equal to that of mag- nesium sulphate nor indeed to that of sodium chloride. Pfeiffer* has also noticed the retarding action of this salt. Weiss,+ however, has stated that sodium sulphate in very small quantity increases the proteolytic activity of a pancreas extract. As we have not seen the original article we do not know whether definite per- centages were employed or not; with 0°05 per cent. of the crys- tallized salt, under the conditions of our experiment, there was certainly no acceleration in proteolytic action. Potassium chlorate and Potassium nitrate. Undigested Fibrin Relative proteo- KC1O3. residue. digested. lytic action. 0 0°3662 gram. 63°38 per cent. 100-0 0:05 per cent. 0°3991 60°09 94:8 0°50 0°3743 62°57 98-7 2°00 0°4101 58°99 93°0 KNO3. 0 0°3710 gram. 62:90 per cent. 100-0 0-05 per cent. 03751 62°49 99°3 0°5 0°3804 61°96 98°5 2°0 04080 59°20 94°1 50 0°4473 55°27 87°8 * Loe. cit. + Jahresbericht fiir Thierchemie, 1876,%p. 177. 118 Chittenden and Cummins—Influence of various Substances The effects of these two salts on the proteolytic action of the fer ment are very similar, with the exception that potassium chlorate appears to be a little more energetic in action than the nitrate. Weiss* has stated that a very small quantity of potassium nitrate added to a pancreas infusion, causes slight increase in the proteolytic — activity of the ferment. This, however, is not observable in our ex- periment with 0°05 per cent., although retardation is very slight. Potassium chloride. This salt, unlike sodium chloride, appears to cause retardation of: proteolytic action, and is without any accelerating influence what- ever. The extent of its retardation, however, is not so great as in the case of sodium chloride. Following are the results obtained. Undigested Fibrin Relative proteo- KCl. residue. digested. lytic action. 0 0°3662 gram. 63°38 per cent. 100°0 0:05 per cent. 0°3740 62°60 98:7 0°5 0°3672 63°27 99°8 2°0 0°4331 56°69 89°4 5:0 0°4723 52°77 83°2 Sodium chloride. With this salt many previous experiments have been tried; Heid- enhain,+ after noting the accelerating action of sodium carbonate, tried the influence of sodiam chloride and found that it increased the proteolytic power of the ferment. E. Pfeiffer, however, states that he found sodium chloride to exert a retarding influence on the proteolytic action of the pancreatic ferment. Pfeiffer{ moreover states that he found sodium carbonate to exert its accelerating action only when present in 0°5 per cent., not in 0°24 per cent. as found by Heidenhain. Recent results§ obtained by us in another connection, however, fully substantiate Heidenhain’s statements on this point. Lastly, Lindberger|| has found that sodium chloride, which in a neu- tral or alkaline solution of trypsin accelerates its proteolytic action, causes in an acid solution (0°018 per cent. HCl, presumably as acid- proteid) marked retardation of ferment action, without, however, * Loe. cit. + Beitrage zur Kenntniss des Pancreas, Pfliger’s Archiv, vol. x, p. 578. t Loe. cit. § Chittenden and Cummins, Amer, Chem. Jour., vol. vii, p. 46-47. Also Trans. Conn, Acad., vol. vii. || Jahresbericht fiir Thierchemie, 1883, p. 281. on the Proteolytic Action of the Pancreatic Ferment. 119 causing destruction of the ferment, as after dialysis the ferment: was again active. Our results accord in the main with Heidenhain’s; acceleration, followed by retardation of proteolytic action as seen from the follow- ing figures. Undigested Fibrin Relative proteo- NaCl. residue. digested. lytic action. 0 0°2993 gram. 70°07 per cent. 100°0 0:05 per cent. 0°2635 73°65 105-1 0°2 0°3001 69°99 99°8 0°5 0°3372 66°28 94:5 10 0°3923 60°77 86:7 2°0 0°4352 56°48 80°6 3°0 0°436] 56°39 80°4 5°0 04740 52°60 75-0 Potassium bromide and potassium iodide. The action of these two salts is wholly an accelerating one; more pronounced, however, in the case of the bromide than in the iodide. Following are the results of the experiments : Undigested Fibrin Relative proteo- KBr. residue. digested. lytic action. 0 0°3881 gram. 61°19 per cent. 100°0 0°05 per cent. 03773 62°20 101°6 0°5 0°3681 63°19 103°2 2°0 0°3795 62°05 101-4 5°0 0°3278 67:22 109°8 KI. 0 0°3881 gram. 61°19 per cent. 100°0 0°05 per cent. 0°3575 64°25 » 105°0 0°5 073915 60°85 99°4 3°0 0°3897 61:03 39ui Influence of gases on the proteolytic action of trypsin. Podolinski* in Heidenhain’s laboratory, has shown that oxygen gas has the power of converting the zymogen of pancreas into the active ferment and that carbonic acid is without such power; more- over, that carbonic acid added to a sodium carbonate solution of pancreatin (trypsin) retards the proteolytic action of the ferment, because the normal carbonate is thus changed into acid carbon- ate which does not favor the action of the ferment so well as the simple salt. Hydrogen was found to be without such action. Podo- linski further found that while oxygen favored the conversion of zymogen into the ferment, it did not influence the proteolytic action * Pfliiger’s Archiv, vol, xiii, p. 426. 120 Chittenden and Cummins—Influence of various Substances the ferment. Hydrogen was also found to be without marked action on the ferment, but carbonic acid retarded decidedly the proteolytic action of the ferment in an alkaline solution. We have tried the influence of three gases, such as. the ferment would naturally be brought in contact with in the intestinal canal, and find that they exert a marked influence on the activity of the ferment. We employed, as in the preceding experiments, a neutral solution of trypsin, and kept the solutions saturated with the gas during the experiment, by passing a constant current through the fluid contained in a small flask. As a control, air was passed through one digestive mixture, which served to keep the powdered fibrin in an equal state of agitation and thus make accurate com- parison possible. Following are the results : Undigested Fibrin Relative proteo- residue. digested. lytic action. IAL Pe RRO ets tec eraira ee 0°4218 gram. 57°82 per cent. 100-0 Hydrogen (H) --.--..--- 0°3670 63°30 10971 Carbonic acid (CO,) ----- 0°5665 43°35 74:9 Hydrogen sulphide (H,8)_ 0°4884 51°16 88-5 From this it is seen that hydrogen gas causes a slight acceleration in the proteolytic action of the ferment, while carbonic acid causes marked retardation, which fact agrees with Podolinski’s results, and shows moreover, that the action of the gas is the same in neutral and alkaline solutions. Hydrogen sulphide also causes retardation, although not so marked as carbonic acid. Influence of alkaloid salts on the proteolytic action of trypsin. So far as we know, no previous experiments have been tried on this subject. Our results show, so far as our experiments extend, a greater susceptibility on the part of trypsin to the action of alkaloids than the amylolytic ferment of saliva. Moreover, with trypsin, the alka- loids in only one case cause acceleration of proteolytic action. Com- pared with pepsin-hydrochloric acid, however, we find that neutral solutions of trypsin are not so readily affected as the former ferment, except by one alkaloid, viz: narcotine, where retarding action is very — pronounced, All of the alkaloids experimented with were in the form of pure sulphates. Morphine sulphate. ; This salt exerts but little retarding action. The results are as follows: on the Proteolytic Action of the Pancreatic Ferment. 12] (Mo), .H280,45Hg0. > reelues Gigeated. ‘fytte actions 0 0°3875 gram. 61°25 per cent. 100-0 0°05 per cent. 0°3952 60°48 98.7 0-5 0°4105 58°95 96:2 2°0 0°4542 54°58 89°L Atropine sulphate. This alkaloid is very similar to morphine in its action. Undigested Fibrin Relative proteo- (At)2.HgSO4. residue. digested, lytic action. 0 0°3875 gram. 61-25 per cent. 100-0 0°05 per cent. 0°3909 60°91 99°4 0°5 0°4078 59°22 96°6 2-0 : 0°4619 53°81 7 87°8 Stolnikow* has stated that a very small amount of atropine sul- phate is without influence on the ferment power of a glycerine ex- tract of pancreas, but that large amounts of the alkaloid salt, con- siderably diminish the proteolytic action of the ferment. Strychnine sulphate and Brucine sulphate. These two alkaloids produce more marked effects on the ferment, than the preceding. Of the two, as the results show, strychnine is more powerful in preventing proteolytic action. J Undigested Fibrin Relative proteo- Alkaloid salt. residue. digested. lytic action. 0 0°4511 gram. 54°89 per cent. 100°0 (Sr), . H,S0, + 6H, 0. 0°05 per cent. 0°4760 52°40 95-4 Soap OAD 05792 42:08 76°6 2°0 0°6882 31:18 56°8 (Br), . HySO,+H,0. 0:05 0°4915 50°85 92°6 0°5 0°5480 45°20 82°3 2°0 0°5452 45°48 82°8 Narcotine sulphate. Undigested Fibrin Relative proteo- (Na)e.H2gS04+H20. residue. digested. lytic action. 0 04511 gram. 54°89 per cent. 100°0 0°05 per cent. 0°5205 47°95 87:3 0:5) 0°9943 0°57 1:0 2°0 yalgy) 0 0 This alkaloid causes a complete stopping of proteolytic action ; nearly so, even when present to the extent of only 0°5 per cent. * Virchow’s Archiy, vol. xe, p. 435. Trans. Conn. Acap., Vou. VII. 16 Oct., 1885, 122 Chittenden and Cummins—Influence of various Substances uinine sulphate, Cinchonine sulphate and Cinchonidine sulphate. ) 7 The following table of results shows the action of these three salts: Undigested Fibrin Relative proteo- Alkaloid salt. residue. digested. lytic action. , 0 0°4679 gram. 53°21 per cent. 100°0 (Q).. H,SO,+7H,0. 0°05 per cent. 0°4811 51°89 97°5 0°5 0°6310 36°90 69°3 . 2°0 : 0°7600 24°00 45°1 4 (Ci)2 -H.S0, + 2H.0. 0°05 0°4868 51°32 96°4 0°5 0°6409 35°91 67:4 2°0 : 0°8327 16°73 314 (Cidine),. H,50,+3H,0. 005 0°4467 55°33 104°0 0°5 05977 40°23 75°6 2°0 0-7 707 22°93 43:0 Cinchonidine, in the smallest percentage, causes a slight accelera- tion in proteolytic action; retardation is also less marked with 0°5 per cent. than in the case of the other two alkaloids.- Otherwise the results are much alike. On the opposite page is a table showing relative action of the various salts on the proteolytic power of the ferment, compared with the action of the controls expressed as 100. O. Nasse,* by a study of the influence of various salts (inorganic) on fermentation, particularly their influence on the amylolytic action of the salivary ferment, pancreatic ferment, and the invert ferment of yeast, came to the conclusion that there is a manifest and import- ant dependence on the part of the ferments in question, in their action, on the presence of salt molecules, and moreover, that this dependence is specific for each individual ferment; that the quantity, as well.as the quality of a salt, exercises a specific influence upon each ferment. x Our own results, with still different ferments, and with a larger number of substances, both related and more varied in character, all testify to the truth of this statement, viz: that the unorganized fer- ments are much influenced by the presence of salts, and moreover, that there is no distinct relationship among the ferments in question in their behavior towards the various salts experimented with, as a study of the three tables of comparisons show. ‘Thus one and the same salt may affect two ferments in quite a different manner, as seen — ———— * Pfliiger’s Archiy, vol. xi, p. 157. of the Pancreatic Ferment. “ore on the Proteolytic Act 8-601 0-94 G:E8 8-18 1-98 GOTT eeee On mine Clee Om a4 a ¢ HOOD sH 10 02 (o8) ~ 2) 1-68 1-66 |7--" F-08 F101 9-08 ¥-68 1-46 0-86 1-86 G-LEL LIFT €-18 h-6) | G18 8-68 jeee- /0-19 6-96 9-GOT|F-SLT Fk | 6-96 €-G8 9-9), 9-9) F-19 6-69 0-1 9.96 6-96 F-66 6-€0T G.76 8-66 G.86 1-86 0-66 €-0€1 ¥-96 L-9@T 1-88 GSE 1-46 6-16 1°99 6-9P P16 1&6 1-26 G-SE1 6-66 --2- 9-79 6-9 6-00T| 1.18 GPE ¥-F6 9-LP N20) 8-201 L.L9 0.99 EVE 6-16 9-GL 8-08 8.07 ‘od £0 "Oo -d 6-0 — Grayato 10 02 T6655 a= 9-TOT/¢-00T Day! 0-0 Cay rol $20.0 ‘od ‘od ‘Od | ord ot0-0 | £00-0 | §00-0 | 200-0 —029/09204A 30212076 PS meaoice Pee (opera osaah(ifay)) Sake Ne O*H9 + "OS" * (48) 7S salle le ee ee OMG OS THE: GurT) L HULNOYS 2IQDT, O'H? +09" 0) Sea ee ee a Se OUR Er “ORCS (Cy qe Se 2 eer -- O° H +*0S"H °2(eN) Tees ca YOSPE GV) Sons See OSE OS * es On) Sas eGel Ds: oa ale pseetaae||0)530 rae ONION CS a Tmt at OOD OF HOT + 'OS*eN San OSMOL + -OracCeN ">= 8T( NO) o 7° o O*HE + °(N0)2a* a NOM EMEAH ET CO kA iG f eee ozs eS See Soto: Caan pS aes eee ~""" O°H@ + 10a tee OSM La tOSUZ "> OU. pee es ges mar TO HceuO eke pate fessor $s oe ee Se MELO San wi Se oo Sees cepe: ame TOsV*(THN) ES OOO OOF Te LEONE AL ee ae eS bee = a NS an fOrsV oa ee 4 “eee oT Ge tr Seo SNe SSS Shier ae O'HE+ *(F0"H50)4d OF SSS a ae o* Hs + Fosno ry thes saccade cas Sar oe CN ery ao oy ies amiesiatech tes eh ea gs ONT sj Saget ar ae GT ae eae See iahsha) int ek ee! Sah lee Sypercans ar eee Ty 124 Chittenden and Cummins—Influence of various Substances, ete. in the action of sodium tetraborate on the salivary ferment and on trypsin. It is moreover, evident, from a comparison of the results obtained with the three ferments, that under. the conditions of dilution, ete., with which our experiments were tried, the salivary ferment is the most sensitive to the action of the various salts, while of the other two, trypsin is as a rule most readily affected. Still, it is hardly possible to draw a direct comparison between the two proteolytic ferments, since they act under such different conditions; the fact that pepsin acts only in an acid medium and that both the strength and nature of. the acid affect the activity of the ferment, intro- duces an additional factor which makes direct comparison in the case of the two ferments impossible. The possible reason for the slight acceleration of proteolytic action produced by several’ neutral salts, in the case of pepsin-hydrochloric acid, has been already referred to; but why neutral salts, which in large percentages show retarding action, should in smaller percentages added to a neutral solution of the ferment (ptyalin or trypsin) produce acceleration, can only be conjectured. We might assume a simple stimulation of ferment action by mere contact. ‘That it may become very pronounced, is evident from the action of borax, potassium cyanide and _potas- sium bromide in the case of trypsin and mercuric cyanide, ammonium arsenate, ferrous sulphate, potassium chlorate, sodium chloride, tartar emetic, and alkaloid salts in the case of the salivary ferment. As to the manner in which the various salts produce retardation of ferment action, it would appear as if many of the results obtained, could be accounted for only by assuming, in addition to the views already offered, a direct influence in many cases, either destructive or hindering, on the ferment itself. Those substances, which are particularly injurious to animal cells show in many cases no retarding action whatever, on the unformed ferments; this is particularly noticeable in the case of the arsenic compounds, which affect the ferments only as the solutions become acid. On the other hand, certain of the metallic salts are alike inju- rious to both and doubtless for the same reason, viz: on account of their power of combining with albuminous matter, which fact applies with equal force to the vegetable organisms (organized ferments). Nearly all germicides act injuriously on the unformed ferments. Many salts, however, well known as antiseptics, are without injuri- ous action, except when present in large quantity; notably borax in the case of trypsin or boracie acid in the case of pepsin-hydrochlori¢ acid. ae VILI.—InFuvence or TEemperaAture on THE RELATIVE AMYI- oLyTIc ACTION OF SALIVA AND THE DraAsTASE or Maur. By R. H. CuirrenpEn anp W. E. Martin, Pu.B. Ir has long been known that the ferment of saliva and the diastase of malt, differ from each other in the temperature best adapted to their amylolytic action. Paschutin* states that saliva ten times dilu- _ted, converts starch into dextrin and sugar most rapidly at 38° to 41° C., while the strongest action of the malt ferment occurs at 70° C. ; rising slowly from 50° C. up to this temperature. According to Kiihne,t the amylolytic action of salivary ptyalin is most energetic at 35° C., while the action of malt diastase is most rapid at 66° C.; ptyalin being destroyed at 60°C. Kjeldahl} states that the amylolytic power of diastase rapidly increases with increase of temperature up to +50° C. By 54° C., ferment action is more vigorous than at 50° C., while maximum action lies between +54° C.and +63°C. Above +63° C. amylolytic action rapidly diminishes with increase in tem- perature. Exposing diastase for a long time to a temperature below 63° C. does not, according to Kjeldahl, weaken perceptibly the action of the ferment. Higher temperatures, however, cause a diminution in amyl- olytic power proportional to the length of time the ferment is heated ; thus Kjeldahl states that long continued warming at + 66° C. produces the same effect upon diastase as heating for a shorter time at + 70° C. For the ptyalin of saliva, Kjeldahl finds the temperature most favor- able for amylolytic action to be about +46° C. From this tempera- ture, amylolytic action diminishes on both sides, although somewhat more rapidly by increase in temperature. O’Sullivan§ and Brown and Heron|| have also studied the in- fluence of temperature on the amylolytic action of malt extract, more however with a view to ascertaining the relative proportion of products (maltose and dextrin) formed and the nature of the change involved, than any comparison of the effect of temperature on the energy of the ferment. * Quoted by Hoppe-Seyler, Physiologische Chemie. p. 187. + Lehrbuch der Physiologischen Chemie, p. 20-21. ¢ Abstract in Jahresbericht fiir Thierchemie, 1879, p. 381-383. § On the action of malt-extract on starch, Journal Chem. Soc., 1876, ii, p. 125. || Beitrage zur Geschichte der Starke und der Verwandlungen derselben. _ Liebig’s Annalen der Chemie, vol. excix, p. 213. Also Journal Chem. Soe. 126 Chittenden and Martin—Influence of Temperature on the Hiippe* likewise, has made a study of the effect of high tempera- tures on the two ferments, in manner similar to the experiments of Bull, Hiifner and Salkowski, not, however, to ascertain the effects of definite temperatures on ferment action, but rather to ascertain the extent to which the dry ferments can be heated without destroying their peculiar properties. It has been our purpose to obtain by quantitative methods, definite expressions of the influence of temperature on the relative amylolytic action of the two ferments. Our method of determining amylolytic action, is based upon the gravimetric determination of the cupric oxide-reducing power of the solution, resulting from the action of the ferment upon starch paste, according to the method of Allihn.t+ This gives very concise and definite results of admitted accuracy. The cupric oxide-reducing power of a solution, resulting from the amylolytic action of these two ferments, must necessarily express the degree of intensity of ferment action, since the more energetic the action, the larger the amount of sugar (maltose and dextrose) formed, with higher reducing power; while the weaker the action, the larger the amount of dextrins with lower reducing power. The amylaceous material employed in the experiments, was purified corn starch. In each experiment 1 gram of the starch was made into a paste with 50 ¢. c. of boiling water, then 40 c. c. more water were added, and lastly, when everything was in readiness, 10 ¢. c. of the ferment solution, either saliva or malt extract ; thus making a volume of 100 c. ¢. containing 1 per cent. of starch. In every case, the fer- ment was allowed to act upon the starch at the desired temperature for exactly thirty minutes, when further ferment action was at once stopped by the addition of a definite quantity of dilute acid. The ferment being thus destroyed, the solution was neutralized by adding an amount of sodium hydroxide equivalent to the acid, after which the solution was concentrated, then made up to exactly 100 ¢. ¢., and in 25 ¢.¢., or one-fourth of the filtered fluid, the reducing bodies were determined. From the weight of metallic copper so obtained, the reducing bodies are, for the sake of comparison, calculated as dex- trose, from which in turn is calculated the percentage of starch con- verted. Naturally the amount of starch digested, is larger than the figures indicate, since the reducing power of maltose. and the dex- trins is much smaller than that of dextrose, but the above method of calculation is most convenient and for comparison quite sufficient. * Jahresbericht fiir Thierchemie, 1881, p. 446. Ueber das Verhalten ungeformter Fermente gegen hohe 'l’emperaturen. } Zeitschrift fir Analytische Chemie, xxii, p. 448. | Relative Amylolytic Action of Saliva and Diastase of Malt. 127 The saliva employed in the experiments was filtered, human mixed saliva, carefully neutralized and then diluted’; 20 ¢. ¢. saliva to 80 c. c. water. As 10 c. ¢. of this fluid were used in each experi- ment, 1 gram of starch was exposed to the action of 2 ¢.”c. of normal saliva in a dilution of 1:50. The malt extract was prepared from coarsely ground, malted barley by extracting 10 grams with 200 c. c. water at 40° C. for 3-4 hours; then filtering, neutralizing and diluting to 500 ¢. ¢.,a few drops of thymol being added to prevent acid fermentation. The amylolytic power of 10 c. c. of this diluted extract was a little more than that of a similar quantity of the dilute saliva. ach series of experiments, however, was made with different extracts, each of which showed considerable variation in amylolytic power. A. Amylolytic action at definite temperatures. In all of these experiments, the 90 c.c. of fluid containing the starch paste was brought to the desired temperature by immersion in a large water-bath carefully regulated, the thermometer being immersed in the vessel containing the starch paste. In a similar manner, the ferment solution was quickly brought to the same tem- perature, care being taken in the latter case, that the fluid did not go beyond the requisite point. When the temperature became con- stant, 10 c. c. of the ferment solution were added and the action con- tinued for thirty minutes. The results are clearly expressed in the following series of tables: SERiEs [.*—SaLIva. Total amount Starch Temperature. Wt. Cu in 4. reducing bodies. converted. 10° C. 0'0935 gram. 0°1904 gram. 17°19 per cent. 20 071227 0°2496 22°46 40 01410 0°2872 25°83 50 0°1003 0°2040 18°35 Series IT.—Sativa. Total amount Starch Temperature. Wt. Cu in ¥. reducing bodies. converted. 20° C. 00891 gram. 0°1816 gram. 16°34 per cent. 30 0°0945 0°1924 ei 40 071203 0°2448 22°03 50 0°1419 0°2888 25°99 55 0°1129 0°2296 20°66 60 0°0451 0°0936 8°42 65 0°0125 0°0282 2°53 * It is of course understood, that the results in any one series are obtained with the same ferment solution. 128 Chittenden and Martin—Influence of Temperature on the Series ITI.—Sattva. Temperature. Wt. Cu in ¥%. noamene Bout. eto erthas 20° C. 071177 gram. 0°2396 gram. 21°56 per cent. 30 : 071273 0°2592 23°32 40 071285 0°2616 23°54 45 0°1174 0°2392 21°52 50 071131 0°2300 20°70 55 0°1029 0°2092 18°82 60 0°0883 01800 16°16* 65 0°0354 0°0748 6°73 70 0°0017 SERIES ITV.—SaLIVvA. Temperature. Wt. Cu in 4. aoe Reales. acre 25° C. 0'0748 gram. 0°1528 gram. 13°75 per cent. 30 0-0897 0°1828 16°45 40 0°1070 0°2180 19°62 45 0°0990 0°2016 18.18 50 . 0:0806 0°1644 14:79 55 0-0715 01460 13.14 60 00278 0°0596 5°36 65 0°0142 0°0328 72-95 SERIES Y.—SALIVA. Total amount Starch. Temperature. Wt. Cuin ¥%. reducing bodies. converted. 40° C. 0°1062 gram. 02164 gram. 19°47 per cent. 2 0'0611 0°1252 11°33 Sertes VI.—Matt Extract. : Total amount Starch Temperature. Wt. Cuin 4. reducing bodies. converted. 309 (O} 01374 gram. 0°2800 gram. ~ 25.20 per cent. 40 071520 0°3100 27°90 50 0°1606 0°3280 29°52 60 0°1408 0°2864 25°77 70 0°0544 071124 10°11 80 0°0127 070296 2°66 SERIES VII.— Matt EXTRACT, Total amount Starch Temperature. Wt. Cuin 4. reducing bodies. converted. 355 C. 01573 gram, 0°3208 gram. 28°87 per cent. 45. 071552 0°3168 28°51 50 071575 0°3212 28°90 55 01617 0°3300 29.70 65 0°0585 071200 10°80 75 0°0366 0:0768 6°91 * See remarks further on, in regard to this high result. "i AP a Oe te a a a Kelative Amylolytic Action of Saliva and Diastase of Malt, 129 Serres VIII.—Matr Exrracr. Total amount Starch Temperature. Wt. Cu in 4. reducing bodies. converted. 30° C. 071301 gram. 0°2648 gram. 23°83 per cent. 40 071471 0°2996 26°96 45 0°1542 03148 28°33 50 01488 0°3036 27°31 55 01320 0°2688 24°19 60 0°0691 0°1412 12°70 65 0°0654 0°1340 12°06 Series 1X.—Matt Extract. Total amount Starch Temperature. Wt. Cu in ¥%. reducing bodies. converted. 30nC. 0°1283 gram. 0°2612 gram. 23°50 per cent. 35 071435 0°2924 26°31 40 0°1507 0°3072 27°64. 45 0°1562 0°3188 28°69 484 071573 03208 =- 28°87 50 071588 0°3244 29°19 55 0°1445 0°2944 26°45 60 0°0742 071516 13°64 65 00561 071152 10°36 SERIES X.—MALT EXTRACT. Total amount Starch Temperature. Wt. Cu in 4. reducing bodies. converted. 40° C. 0°1419 gram. 03042 gram. 27°36 per cent. 2 0°0299 0°0636 5°72 By a study of these results, it is evident that in the case of the salivary ferment, variations in amylolytic action are not very great between the temperatures of 20° and 50°, or even 55° C. With the temperatures experimented with, however, amylolytic action appears to reach its maximum, in the case of saliva, at 40°; although in one single instance, for some unaccountable reason it appeared to be greater at 50° C. With the diastase of malt on the other hand,- amylolytic action reaches its maximum at 50° C., although in one in_ stance it appeared somewhat greater at 55° C.; great variations, how- ever, are not to be observed between the temperatures of 30° and 55° C. Brown and Heron,* working with extract of malt and pure potato starch at different temperatures, obtained results by determination of both specific rotary power and cupric oxide-reducing power, which point to the same conclusions as those obtained by us. Thus at 40° C., the malt extract having been previously heated at the same temperature for 20 minutes, these investigators found at the end of 30 minutes, as * Liebig’s Annalen der Chemie, vol. excix, p. 221. Also Journal Chem. Soc., 1879. Trans. Conn. AcaD., Vou. VII. 17 Oct., 1885. 130 Chittenden and Martin—Influence of Temperature on the a result of the action of the malt extract on the starch (a)j =163°3°, while at 50° C. under the same conditions (a)j = 162°7° and at 60° C. (a) j = 164:1°, or in a second experiment, (a) j = 163°7°. These re- sults show at 50°C. the formation of a little more maltose than at 40°, although the difference is very slight; while at 60° C. the amount of maltose formed, is less even than at 40° C. Evidently then, the maximum amylolytic action of diastase of malt takes place at tem- peratures far below 60° C.; even below 55° C. At very low temperatures, there is a corresponding difference in the action of the two ferments, as is apparent from the results ob- tained at 2° C.; the ferment of saliva being comparatively far more active at this Parrctatnre than the ferment of malt. Hence it is apparent throughout, that diastase requires a higher temperature than the salivary ferment, in order to act with equal vigor ; at the same time it is evident that at the body temperature, say 40° C., the difference in action between the two ferments is not very great. At 80° C. the diastase of malt still acts upon starch, although only slightly ; the salivary ferment, however, under the conditions of our experiments, does not act at all at 70° C. and only slightly at 65° C. With these higher temperatures, it makes con- siderable difference in the ultimate result, whether the ferment solu- tion is quickly brought to the desired temperature or not, and whether it remains long at the temperature in question, before being added to the starch solution. Thus, in the action of saliva at 60° C., if the ferment be warmed quickly to nearly 60° C., say 59°°C., and. then added to the starch paste at 61° C., as was done in the case of Series III, amylolytic action is considerably greater than when the saliva is actually brought to 60° and kept there for a moment or so to be sure of its constancy. Some variation in the length of time, required to bring the ferment solution to the desired temperature, was unavoidable, and doubtless, slight variations in the results at higher temperatures, occur from this cause. It was not, however, our purpose at this time, to heat the ferment in order to induce a change in its character, but simply to prevent any alteration in the temperature of the starch mixture on addition of the ferment, so that the action of the ferment on the starch might take place at a constant temperature, Relative Amylolytic Action of Saliva and Diastase of Malt. 131 The effects, on amylolytic action, of exposing the ferment of saliva to different temperatures for varying lengths of time. Brown and Heron* state that a malt extract, warmed quickly to 66° C, and then added at once to starch paste at the same temperature, differs but little, in the first stages of its action, from a malt extract heated at 60° C.; if, however, the malt extract be warmed for say 10 or 15 minutes at this temperature, previous to adding it to the starch paste, its amylolytic action is very much weakened. Evidently then, under the influence of the increased temperature, a portion of the fer- ment is destroyed or else changes are induced, by which the action of the ferment is modified. Results of like nature were previously obtained by O’Sullivan,+ with malt extract. With saliva, we have tried the following experiments, designed originally to throw light on the comparative destructibility of the — ferment. SERIES XI.—SALIvA. The saliva was exposed to the designated temperature for the speci- fied time, then added to the starch paste at the same temperature and its amylolytic power determined. Temperature. eenetne: Wt. Cu in ¥. Hi ear ag ee éesaerett 60° C. 0 min. 0:0409 gram. 0-0852 gram. 7°66 per cent. 60 15 0°0213 0'0464 417 60 30 0°0210 0°0460 4°14 At 60° C. therefore, the coagulating point of albumin and the tem- perature at which ptyalin is supposed to be destroyed, it is apparent that destruction of the ferment is not complete even by 30 min- utes exposure to this temperature. The peculiarity of the results, moreover, make it doubtful whether we have to do with destruction at all. If the reduced amylolytic action is due to simple destruc-_ tion of the ferment, we should expect less ferment action after 30 minutes exposure than after 15 minutes; as it is, the action in the two cases is the same. A certain time, however, is required to produce the change in the character of the ferment. Similar results are shown in the following series of experiments, conducted in the same manner as the preceding, only at different temperatures. * Loe, cit., p. 227. + Loe. cit., p. 143. 132 Chittenden and Martin—Influence of Temperature on the SERIES XII.—SALIVA. Time of Total amount Starch Temperature. exposure. Wt. Cu in reducing bodies. converted. 50° C. 15 min. 0:0773 gram. 0°1576 gram. 14°18 per cent. 50 60 0°0765 071560 14:04 55 30 00474 00984 8°85 55 60 : 0°0414 0°0864 (igi | Comparing these results with those obtained at like temperatures in Series I.-IIL., it is seen that a few minutes exposure at the desig- nated temperature, lowers materially the amylolytic power of the solution, while doubling the time of exposure does not materially affect the result; a fact which is not consistent with the view that diminution in amylolytic power, under these conditions, is due to gradual destruction of the ferment. The following series of experiments, also with saliva, throw ad- ditional light on the action of high temperatures on this ferment. In these two series, the saliva was exposed to the designated temper- ature for the specified time, then cooled to 40° C. and added to the starch paste a@ a like tenperature. SERIES XIIJ.—SatIva. Time of Total amount Starch Temperature. exposure. Wt. Cuin 4. reducing bodies. converted, 40° C. 0 min, 0:1081 gram. 0°2200 gram. 19°80 per cent. 50 30 0°1026 0°2088 18°79 55 30 0°0986 0°2008 18°07 60 30 00279 00596 5°36 . Series XIV.—SALIva. Time of Total amount Starch Temperature. exposure. Wt. Cu in %. reducing bodies. converted, 40° C. Q min, 0°1062 gram. 0-2164 gram. 19°47 per cent. 55 180 0°0798 0°1628 14°65 It would appear from these results, that by exposure of the saliva to 50° or 55° C., in the latter case for even 3 hours, and then cooling to 40° C. and testing the amylolytic power of the ferment at that temperature, less diminution of ferment action is to be observed, This speaks still more strongly against destructive action, by simple coagulation, and at the same time suggests that not only does expos- ure to say 55° C. affect the character of the ferment, but also that the action of the ferment so treated, is in a given time different at that same temperature from what it is at 40° C., or the temperature of maximum action. This latter point, however, which is contrary to the law laid down by Brown and Heron* for malt extracts at tem- * Liebig’s Annalen der Chemie, vol. excix, p. 221. Also, Journal Chem. Soe., 1879. a? a Relative Amylolytic Action of Saliva and Diastase of Malt. 133 peratures above 50° C. we reserve for further investigation. Owing to the great difficulty in rendering saliva perfectly neutral, it is possi- ble that the observed low result at 55° C. in Series XII. may be due to the presence of a trace of either acid or alkaline carbonate. Finally, it is to be observed that the majority of the results ob- tained, indicate that the influence of different temperatures, on the amylolytic action of the salivary ferment, is due rather to change in the character of the ferment, than to the direct influence of the various degrees of heat upon the cleavage of the starch molecule ; similar in character to that indicated by the work of O’Sullivan, and also of Brown and Heron in the case of malt diastase. IX.—Iyevvence or Bitz, Binz Satts anp Bite Acips on Amyto- LYTIC AND Protrrotytic Action.* By R. H. CarrrenpEN AND Gro. W. Cummins, Pu.B. Tux influence of bile and bile acids on the digestive processes of the intestinal canal has long been considered an important one, still few experiments have been made to determine the exact influence of these substances by themselves on ferment action. The form in which the main constituents of the bile exist in the intestinal canal depends naturally upon the reaction of the contents of the intestines. If these have an acid reaction, bile acids must be present; if alkaline, salts of these acids; and it is fair to presume that under these two conditions the presence of bile may be productive of different effects on ferment action. Recorded observations tend to show that ordi- narily the contents of the intestines possess a distinct acid reaction ; thus Schmidt-Miilheim+ has found that in dogs fed on albuminous matter, the contents of the small intestines are invariably acid, — although the mucous membrane sometimes possesses an alkaline reac- tion. It is evident that in such cases the alkali of the bile must have combined with the acid of the chyme, which would be followed by liberation of the bile acids and partial precipitation of the same in combination with the proteid matters of the chyme. Moreover, the recorded observations of Schmidt-Miilheim tend to show that this acid condition of the contents of the intestines persists through- out the entire length of the intestinal canal. Uffelmann{ has like- wise found, in corroboration of the above, that the faeces of infants naturally nourished possess a weak acid reaction, while, on the other hand, Nothnagel,§ as a result of 800 observations, finds that human excrement, in the case of adults, varies decidedly in its reaction, being generally alkaline, more rarely acid or neutral. It is hardly proper, therefore, to conclude that it is only necessary to study the * Also published in the American Chemical Journal, vol. vii, p. 36. + Archiv fiir Physiologie, DuBois Reymond, 1879, p. 56. + Jahresbericht fiir Thierchemie, 1881, p. 305. § Jahresbericht fiir Thierchemie, 1881, p. 309. - = ae o* Chittenden and Cummins—Influence of Bile. 135 influence of the bile acids in their free condition on ferment action, since in the passage of the ferments through the intestinal canal there are times, doubtless, when the reaction of the mass is more or less alkaline, especially in the small intestines, for some distance be- yond the opening of the bile and pancreatic ducts. In either case it is an interesting point to ascertain whether the bile salts have an action at all analogous in kind or extent to that of the free acids. Many observations* are recorded concerning the duodenal precip- itate formed in the duodenum by the action of bile on the acid-re- acting chyme. The precipitate itself has generally been supposed to consist of a mixture of syntonin, peptone and bile acids, but recent experiments of Maly and Emicht with pure bile acids tend to show that only the non-peptonised albuminous bodies are precipitated, viz : coagulable albumin and syntonin, and these only by taurocholic acid, while peptone and “ propeptone” remain in solution. This fact lends favor to the view advanced by Hammarsten, that the object of the precipitation of albuminous matter on the walls of the intestines is to prevent its too rapid passage through the intestinal canal, thus giving ample opportunity for the action of the pancreatic juice. The addition of taurocholic acid to a solution of peptone, Maly and Emich find, is followed by the formation of a distinet opal- escence or fine dust-like precipitate, slowly changing to fine droplets. This precipitate, however, which is doubtless the same as observed by Hammarsten and Briicke on the addition of bile to portions of a digestive mixture, does not contain according to Maly and Emich, any peptone, but consists of taurocholic acid, possibly in a modified form. Both of these precipitations, however, would tend to mechanically throw down, to a greater or less extent, any ferment present, and thus diminish ferment action; but, as Maly points ont, the main reason for a diminished action, in the case of pepsin, is to be sought for, not in a precipitation of the ferment, but in the formation of a compound of albumin with the bile acid, not digestible by pep- sin-hydrochloric acid. But since this precipitation, as a normal reac- tion in the animal body, must take place in the intestinal canal, it is equally important to ascertain the extent of its digestibility in pan- _ creatic juice, or, in other words, to ascertain the exact influence of eres bile and its several constituents on the proteolytic action of trypsin * See Maly in Hermann’s Handbuch der Physiologie, vol. v, p. 180. + Monatshefte fiir Chemie, vol. iv, p. 89. 136 Chittenden and Cummins—Influence of Bile as well as on the action of pepsin and on amylolytic action. The only data bearing on these points are the recent experiments of Maly and Emich, who have found that 0:2 per cent. taurocholic acid hin- ders the digestive action of pepsin-hydrochloric acid, while 1 per cent. of glycocholic acid is without influence. The same investigators likewise state that 0°1 per cent. taurocholic or glycocholic acid stops the amylolytic action of the pancreas ferment, and that 0-2 per cent. taurocholic acid or 1 per cent. glycocholic acid will completely stop the amylolytic action of the salivary ferment. Our experiments on this subject were commenced before the above results were published, and we have continued them, since we wished to ascertain likewise the influence of the bile salts, and also the effects of both salts and acids, as well as the bile itself, on the proteolytic ferment of the pancreas. The results of Maly and Emich, moreover, not being quantitative, do not express the relative effects of the various percentages of bile acids used, but simply the percentage of acid necessary to stop ferment action under the conditions de- scribed by them. 1.—Influence on Amylolytic Action. As amylolytic ferment, we have employed filtered human mixed saliva made neutral and then diluted to a known volume. In study- ing the influence of the various percentages of bile salts and acids on the action of the ferment, we have used a digestive mixture (50 or 100 ce.) containing 1 per cent. of starch previously boiled with water, and 2 per cent. of saliva, together with the given percent- ages of bile salts or acids. The extent of diastatic or amylolytic action under the varying conditions was determined in each case by estimating the amount of reducing substances, maltose and dextrose, formed during 30 minutes warming at 40° C. Further diastatic action was at once stopped by boiling the digestive mixtures, after which they were diluted to a known volume, and the reduc- ing substances determined in a given portion of the diluted fluid by Allihn’s gravimetric method.* The reducing substances are in each instance calculated as dextrose, and the diastatic action is expressed in the percentage of starch converted into sugar. We first tried the influence of crystallized ox bile, since bile itself contains a small amount of a diastatic ferment. A 1 per cent. solu- tion of nicely crystallized ox bile was made, with which the following results were obtained : * Zeitschrift fiir analytische Chemie, xxii, 448. — on Amylolytic and Proteolytic Action. 137 Crystallized Total amount Starch bile. Wt. Cu in 4.* reducing bodies. converted. 0 per cent. 0:0643 gram. 0°2636 gram. 23°72 per cent. 0°01 0°0630 0°2584 23°25 0:02 0°0686 0°2804 25°23 0°03 0°0693 0°2836 25°52 0°05 0°0656 0°2688 24°19 0-10 00734 C-3000 27°00 0°20 0:0665 0:2724 24°51 0°35 070447 0°1860 16°74 Here it is plain that a mixture of sodium glycocholate and tauro- cholate, in such proportion as they are contained in crystallized ox bile, exerts no appreciable retarding influence on amylolytic action until present to the extent of 0°35 per cent. On the contrary, smaller percentages unmistakably tend to increase the diastatic action of the ferment. The solution of crystallized bile had, how- ever, a slight acid reaction, and possibly this may have had some in- fluence in giving the latter results. The saliva and starch were both neutral. Experiments were next tried with sodium taurocholate alone, and also with sodium glycocholate. Following are the results: Sodium Total amount Starch taurocholate. Wt. Cu in 4. reducing bodies. converted. 0 per cent. 0-0787 gram. 0°3212 gram. 28°90 per cent. 0°3 0°0030 070146 1°51 0°5 0°0023 00112 1:00 Sodium glycocholate. 0°5 0-0783 0°3196 28°76 It is thus plainly evident that sodium taurocholate has a very decided action on the amylolytic ferment of saliva, while the same percentage of glycocholate is entirely without effect. The retarding action of crystallized bile is thus, without a doubt, due wholly to the taurocholate. Moreover, even smaller percentages of sodium’ taurocholate retard amylolytic action with almost equal energy. The following results were obtained under like conditions as the preceding, except that the 2 per cent. of saliva employed was not neutralized. Sodium Total amount Starch taurocholate. Wt. Cu in ¥. reducing bodies. converted. 0 per cent. 0°0590 gram. 0°1212 gram. 21°81 per cent. 014 00079 070192 3°45 Sodium glycocholate. 0°20 0°0758 0°1548 27°86 * One-eighth of the entire digestive mixture. Trans. Conn. Acap., Vou. VII. 18 Oct., 1885. 138 Chittenden and Cummins—Influence of Bile Thus even 0°14 per cent. of sodium taurocholate under these conditions almost entirely stops amylolytic action. The smaller per- centage of glycocholate, however, causes the same increased amyl- olytic action observed with the smaller percentages of crystallized bile. With the bile acids the following results were obtained. The glycocholic acid used was a nicely crystallized specimen prepared from ox bile, while the taurocholic acid, prepared from the same source, was amorphous: bile carne Wt. Cu in 4. Soe Eds Padre 0 ; 0°0694 gram, 0°1420 gram. 25°56 per cent. 0°01 taurocholic. 0:0753 0°1538 27°68 0°05 0°0783 0°1598 28°76 0°10 0:0060 0:0146 2°63 0°20 0 0:05 glycocholic. 00523 0°1082 19°47 0°10 0:0095 0°0234 4:21 0°20 0°0056 0°0136 2°44 0°50 trace 1°00 0 It is thus seen that 0°1 per cent. taurocholic acid prevents amyloly- tic action almost entirely, while 0°2 per cent. does not allow the con- version of any starch into sugar. This agrees exactly with the results obtained by Maly and Emich.* These same investigators, however, found only a trace of amylolytic action in the presence of 0:05 per cent. taurocholic acid; a result which does not agree with what we have found, working, however, under somewhat different conditions. The presence of 1:0 per cent. glycocholic acid entirely prevents the conversion of starch into sugar, while 0°5 per cent. allows only the smallest amount of diastatic activity. Maly and Emich likewise found that 1:0 per cent. of glycocholic acid stopped the diastatic action of saliva. We have repeated the last series of experiments in part, using, however, normally alkaline saliva instead of neutralized. Per cent. Total amount Starch bile acid. Wt. Cu in 4. reducing bodies. converted. 0 0:0590 gram. 0°1212 gram. 21°81 per cent. 0-1 glycocholie. 0°0107 00258 4°64 0°2 00057 0°0139 2°50 0°1 taurocholic. 00052 0°0126 2°26 * Loc. cit., p. 118. a on Amylolytic and Proteolytic Action. 139 These results agree exactly with the preceding, and both together plainly show that only small percentages of bile acids are required to entirely prevent the amylolytic action of saliva. Assuming that the amylolytic ferment of the pancreatic juice is similar in its nature to the ferment of saliva, it would follow from our experiments that whether the contents of the intestines are acid or alkaline, the pres- ence, beyond a certain percentage, of taurocholic acid, either as free acid or as a taurocholate, would tend to diminish amylolytic action. Very small percentages, however, would have little, if any, retarding effect, indeed might increase amylolytic action. As to glycocholic acid, the free acid is much more powerful in its action on the amylo- lytic ferment than the sodium salt of the acid. Considering these results in the light of a possible application to changes in the intestinal canal, it becomes an interesting point to ascertain whether bile itself exerts the same influence on amylo- lytic action as the bile salts. Moriggia and Battistini* state that while bile mixed with chyme gives a precipitate which, among other things, contains mucin, bile acids and pepsin, thus hindering gastric digestion, it does not, on being mixed with saliva, hinder its amylo- lytic action. ‘This they found to be the case both with bile contain- ing mucin and with bile from which the mucin had been removed by acidifying. We have, therefore, made the following experiments with fresh ox bile containing 7°46 per cent. of solid matter. The digestive mixtures contained as before 1 per cent. of starch, 2 per cent. of neutral saliva, and were warmed at 40° C. for 30 minutes: Total amount Starch Ox bile. Wt. Cu in \&. , reducing bodies. converted. 0 per cent. 0°0753 gram. 0°3072 gram. 27°64 per cent. 2°0 0°0875 0°3568 32°11 50 0:0690 0°2824 25°41 10°0 00719 0°2944 26°50 20°0 0-0770 0°3144 28°30 Here in close accord with what has been found before, the presence of a small percentage of bile causes increased amylolytic action; larger percentages, however, have little, if any, effect ; certainly not such an effect as would be expected from the known action of the bile salts. The bile itself possessed to a slight extent, diastatic action; 20c.c. of the bile (20 per cent.) converting 4°53 per cent. of the starch into sugar in 30 minutes. This, however, could hardly account for the increased amylolytic action noticed above in the pres- * Jahresbericht fiir Thierchemie, 1876, p. 196. 140 Chittenden and Cummins—Influence of Bile ence of 2 per cent. of bile. Wittich* and also Hofmann have noticed the occasional diastatic action of bile, Wittich even extracting the ferment from human bile by his glycerine method. Gianuzzi and Bufalinit have shown that the action varies considerably im bile | from different animals and individuals, and without any apparent dependence upon the nature of the food. Ewaldt{ states that the diastatic capacity of bile appears to be slight in all cases, and is not found in bile which has stood for some time. We have found, how- ever, in bile from several animals considerable diastatic power; thus in one sample of fresh sheep’s bile, 25 ¢.¢. (25 per cent.) converted 24°33 per cent. of starch into sugar in 30 minutes at 40° C. We have likewise found great variation in diastatic power, varying, expressed in the percentage of starch converted into sugar under the conditions described, from 4 to 24 in the case of herbivorous animals. We have also noticed in bile from sheep and oxen the presence of a small amount of sugar, or at least a substance capable of reducing Fehling’s solution. In one instance the amount was not inconsider- able; 25 grams of ox bile yielding, by Allihn’s method, 0:040 gram metallic copper, equal to 0°0209 gram dextrose or 0°08 per cent. Naunyn, we believe, has already claimed the presence of sugar in bile. While we know then that bile acids and bile salts by them- selves retard very decidedly the amylolytic action of ptyalin, it would appear that the retarding influence of the latter may be, in part at least, counteracted by other substances naturally present in the bile. 2.—Influence on the Proteolytic Action of Pepsin. It has long been known that bile has a retarding action on pepsin digestion, and Maly and Emich have recently shown the percentages of bile acids necessary to bring the action of pepsin to a standstill. We have, however, in addition, experimented with bile itself, and as in the case of the amylolytic ferment, have endeavored to study the influence of the bile acids quantitatively. The method employed for measuring proteolytic action is one frequently used in this labora- tory, and which has invariably given satisfactory results. The only feature which calls for description is the preparation of the proteid matter to be digested. The material consists of carefully selected * Jahresbericht fiir Thierchemie, 1872, p. 243. + Jahresbericht fir Thierchemie, 1876, p. 197. { Lectures on digestion, Amer. ed., p. 77. on Amylolytic and Proteolytic Action. 141 and thoroughly washed blood fibrin. All soluble matters are re- moved by successive extraction with boiling water, cold and boiling alcohol, and finally with cold and warm ether. The fibrin is thus obtained in a perfectly friable condition and can be easily ground to a coarse powder. It is then dried at 100-110° C. This materia! is well adapted for quantitative experiments with pepsin-hydrochloric acid; the residue remaining after a digestion can be rapidly filtered with the aid of a pump, and can be easily freed, by washing, from peptones and other soluble products of digestion. The. gastric juice employed in the experiments, consisted of a hydrochloric acid solution of a glycerine extract of the mucous mem- brane from a pig’s stomach, in the proportion of 10 grams glycerine extract to 1 litre of 0:2 per cent. hydrochloric acid. 50 or 100 cc. of this pepsin-hydrochloric acid were employed in each experiment, to which was added 1 or 2 grams of the dried fibrin (2 per cent.), - together with the given percentage of bile or bile acids. We first tried the influence of bile itself, using fresh ox bile, | slightly alkaline in reaction and containing 10°02 per cent. of solid matter. The digestive mixtures were warmed at 40° C. for two hours, then filtered at once, and the undigested residue washed thoroughly,* and dried at 100° C. until of constant weight. Following are the results of the first series of experiments, with 2 grams of fibrin and =m 100 c. c. of gastric juice. Bile in Weight of Fibrin digestive mixture. undigested residue. digested. 0 per cent. 0°1957 gram. 90-21 per cent. * : 0°25 . 0-1890 90°55 0°50 0°2050 89°75 1:00 0°2234 88°83 3°00 0°5453 72°13 5:00 0°7642 61°84 * In all of the pepsin-hydrochloric acid digestions the presence of bile or bile salts naturally causes more or less of a precipitate, dependent in amount upon the percent- age of bile and also upon the amount of digestive products. In washing the undi- gested fibrin it was of course necessary to remove this precipitate. This was accom- plished by pouring over the precipitate on the filter 50 c. c. of 0°5 per cent. potassium hydroxide and then washing with water until the alkali was wholly removed. The following experiment shows that under these conditions the alkali affects the swollen fibrin bvt little, if any. Two portions of fibrin of 2 grams each were warmed with 100 c.c. of 0-2 per cent. HCl for 30 minutes, then filtered and one washed with water alone, the other with water and alkali. The first gave 1:9272 grams dried residue, the other 1:9155 grams. 142 Chittenden and Cummins—Influence of Bile A second series, tried under the same conditions, but with larger percentages of bile gave the following results : Bile in Weight of Fibrin digestive mixture. undigested residue. digested. 0 per cent. 0°1979 gram. 90°10 per cent. 0°25 0°2456 87°72 0°50 0°1927 90°36 9°00 1°1955 : 40°22 13°00 1°6611 16°94 16°50 1°7812 10°94 20°00 19241 3 29 From these two series of experiments it is evident that the pres- ence of bile, from 1 per cent. upward, causes diminished proteolytic action, the retarding effect being proportionate to the amount of bile present. 20 per cent. of bile stops the action, under these con- ditions, almost completely. It is fair to presume, therefore, that the reflux of but a small amount of bile into the stomach would be pro- ductive of a diminished proteolytic action. These results, therefore, agree with the older statements of Briicke, Hammarsten and others, to the effect that bile added to a gastric digestion has the effect of bringing the proteolytic action to a stand- still. We next tried the influence of the individual bile acids with the following results : Taurocholic Weight of Fibrin acid. undigested residue. digested. 0 per cent. 0°1311 gram. 86°89 per cent. 0°025 01461 85°39 : 0-050 0°2200 78:00 0°100 0°2421 TO9 0:200 0°2668 13°32 0°500 0°3579 64°21 Here it is seen that the smallest percentage of taurocholic acid added, produces a distinct effect on proteolytic action, and in the next series of experiments still smaller percentages of acid cause an equally marked effect. In both series of experiments, the mix- tures were warmed at 40° C. for 1 hour and 30 minutes. Taurocholic Weight of ; Fibrin undigested residue. digested. 0 per cent. 0°1499 gram. 85°01 per cent. 0-010 01819 81°81 0-015 0°1900 81:00 0°020 0°2947 70°53 0°050 03110 68°90 Adding taurocholic acid to the digestive mixture in the form of a sodium salt has the effect of diminishing still further the action sana on Amylolytic and Proteolytic Action. 148 of the ferment; doubtless, due in part to the percentage of free hydrochloric acid being diminished by decomposition of the tauro- cholate. Taurocholic Weight of Fibrin acid. undigested residue. digested. 0 per cent. 0°2059 gram. 79°41 per cent. O01 0°6198 38°02 0-2 0°6426 36°74 0°5 0°6475 35°25 Maly and Emich found that 0:2 per cent. taurocholic acid entirely stopped the action of pepsin; in our experiments, however, ferment action was still manifest even in the presence of 0°5 per cent. of the acid. Whether this difference in result is due to difference in the acid used, or to difference in method, we cannot say. Glycocholic acid we found to be entirely without influence on the action of pepsin, as did also Maly and Emich. 3.—The Proteolytic Action of Trypsin in Neutral, Alkuline and Acid Solutions. The trypsin solution was prepared according to Kiihne’s method,* from dried pancreas freed from fat; the solution after neutralization always contained some sodium salicylate, sufficient to prevent putre- faction during short digestive periods. According to Kiihne,t tryp- sin acts quite energetically, both in neutral and in salicylic acid solu- tions, but most energetically when the pancreatic solution contains 0-3 per cent. sodium carbonate. According to Heidenhain,{ the action of definite percentages of sodium carbonate varies with the amount of ferment. 5 We have tried quantitative experiments as a preliminary to study- ing the influence of bile, with the following results ;§ the mixtures were warmed at 40° C. for 3 hours and 40 minutes, and contained 2 per cent. of fibrin. Reaction of Weight of Fibrin the fluid. undigested residue. digested. neutral 0:2312 gram. 76.88 per cent. 01 per cent. NazCO; 071570 84°30 02 0°0925 90°75 0°3 00772 92°28 0°4 0°0426 95°74 0°5 0°1038 89°62 0°1 pr. ct. salicylic acid 0°5651 43°49 * Untersuchungen aus der physiolog. Inst. d. Universitaét Heidelberg, vol. i, p. 222. + Ibid, p. 223. ¢ Pfliiger’s Archiv, vol. x, p. 576. § The pancreatic juice was prepared from 20 grams dry pancreas, and finally diluted to 1000 c.c. 50. c. were used in each digestion with 1 gram of pure fibrin, 144 Chittenden and Cummins—Influence of Bile With a larger percentage of fibrin and a longer period of diges- tion the results are somewhat different. The following were obtained with 4 per cent. of fibrin in 6 hours and 40 minutes at 40° C.: Reaction of Weight of Fibrin the fluid. undigested residue. digested. neutral 0:3785 gram. 62°15 per cent. 0°1 per cent. Na.CO; 02581 74°19 0°2 0°1395 86°05 0:3 01588 84°12 0-4 01629 83°71 0°5 0°1318 86°82 0-1 pr. ct. salicylic acid 0°4728 52°72 An average of the two series of results plainly shows that there is but little difference in digestive action in the presence of 0°2—0°5 per cent. sodium carbonate, although in a given solution a change in the percentage of alkali is at once manifest, to a slight extent, in the amount of fibrin digested. Greatly increased percentages of alka- line carbonate materially diminish the action of the ferment, as the following series of experiments indicate; the mixtures were warmed for 2 hours at 40° C.: Reaction of Weight of Fibrin the fluid. undigested residue. digested. neutral 0°5863 gram. 41°37 per cent. 0°5 per cent. Na.CO, 0°1584 84°16 1-0 0°3760 62°40 2°0 0°7010 29°90 3°0 0-7892 21:08 4-0 0°8373 16-27 ; 5:0 0°8608 13°92 The difference in action between a neutral trypsin solution and a solution containing salicylic acid is quite noticeable, at the same time it is evident that in the acid-reacting fluid the ferment simply acts more slowly, and if time be given, the action will approach more closely to that of the neutral solution. It is of course understood that the salicylic acid in the above experiments does not exist in a free state, but in combination with the proteid matter present, and doubtless in most of the experiments recorded, where trypsin has been exposed to the action of small fractions of a per cent. of acid, no free acid has been present, but only varying percentages of acid- proteids.* Kiihnet+ has pointed out that hydrochloric acid above 0°05 per cent. is injurious to the action of trypsin, and Heidenhain{ has * See Danilewsky. Centralbl. med. Wiss., 1880. + Verh. Naturhist. med, Vereins zu Heidelberg, 1877, p. 193. { Pfliger’s Archiv, vol. x, p. 578. Oe ee ee ee ee ee ee Bear. ad = sy. on Amylolytic and Proteolytic Action. 145 shown that the addition of 0°1 per cent. hydrochloric acid to an aqueous extract of the pancreas stops its action. C. A. Ewald* how- ever, found that while pepsin-hydrochloric acid destroyed trypsin, trypsin could digest fibrin in the presence of 0°3 per cent. hydrochlo- ric acid. Mayst likewise found that trypsin digestion could take place in the presence of 0°3 per cent. hydrochloric acid, but only when a relatively large proportion of fibrin was present, and in cor- roboration of Kiihne’s statement, he showed that trypsin could be destroyed both by pepsin and dilute hydrochloric acid. Engesser{ found that a pancreatic juice did not lose its tryptic power by two hours warming with a gastric juice containing 0°5 per cent. hydro- chloric acid. Langley,§ on the contrary, has shown that a glycerine extract of the pancreas loses a very appreciable amount of trypsin when warmed for two and a half hours with 0°05 per cent. hydrochloric acid. Lindberger,|| working with an alcohol precipitate from a glyc- erine extract of ox pancreas, in which there would naturally be present but a small amount of proteid matter in addition to the tryp- sin, found that in the presence of 0°1 per cent. hydrochloric acid the ferment was entirely without action, and even in the presence of 0-012 per cent. hydrochloric acid, fibrin was much more slowly dis- solved than by a neutral trypsin solution. Lindberger, moreover, found that weaker acids, as acetic and lactic, had a much different effect than the stronger hydrochloric acid; thus with dilute acetic acid, digestion of the fibrin was almost as rapid as with a neutral solution of trypsin, while with small amounts of lactie acid, fer- ment action was even more energetic than in a neutral solution. There is, however, no guarantee that in these experiments free acid was present.4 We have found that free acids, even when present in small per- centages, completely stop the proteolytic action of trypsin, and that. when considerable albuminous matter is present, the action of tryp- sin is much hindered by the addition of acid to a neutral solution, even before the proteid matters present are saturated with acid. 0:1 per cent. free salicylic acid, in the presence of proteids already satu - * Jahreésbericht fiir Thierchemie, 1880, p. 297. + Untersuchungen a. d. physiolog. Inst. d. Univ. Heidelberg, vol. iii, p. 378, 1880. ¢ Jahresbericht fiir Thierchemie, 1880, p. 297. § Journal of Physiology, vol. iii, No. 3. || Jahresbericht fir Thierchemie, 1883, p. 281. | We have seen only the abstract of Lindberger’s paper, so cannot speak positively on this point. TRANS. Conn. ACAD., Vou. VII. 19 OcT., 1885. 146 Chittenden and Cummins—Influence of Bile rated with the acid, allows no proteolytic action whatever. Further- more, a sufficient amount of proteid matter just saturated with hydrochloric acid, no free acid being present, will almost completely stop the action of trypsin. Proteid matter, however, only partially saturated with acid has a much smaller retarding action. This, doubtless, was the condition of the mixtures in Mays’ and Enges- ser’s experiments above referred to, for, as Mays states, the ferment could act in the presence of 0°3 per cent. hydrochloric acid only when a relatively large proportion of fibrin was present. A pancreatic juice prepared from 20 grams of dried pancreas by warming it at 40° C. with 200 ¢«. 0-1 per cent. salicylic acid, ete., was finally made exactly neutral and diluted to 500 c.c.; 25 ¢.c. of this solution required 7°5 ¢.c. of a 2°0 per cent. solution of salicylic acid to completely saturate the proteids present,* the excess of free acid necessary to give the tropzolin reaction being deducted. Three digestive mixtures were made as follows: 1. 25 c.c. of the neutral pancreatic solution +50 c.c. water. 2. 25 c.c. of the same pancreatic solution+7°5 c.c. 2:0 per cent. salicylic acid solution +17°5 c.c. water. The mixture was acid to test papers, but gave no reaction with tropzolin 00. It therefore contained no free acid, but 0°3 per cent. of combined acid. 3. The same as No. 2, but 2°5 ¢.c. more of 2°0 per cent. salicylic acid, so that the solution contained, in addition to the acid proteids, O°1 per cent. free salicylic acid. One gram of fibrin was added to each of these and the mixtures warmed at 40° C. for 8 hours and 40 minutes. No. 1 digested 88°34 per cent. of the fibrin, No. 2, 13°44 per cent., while No. 3 had no action whatever. Much smaller percentages of combined salicylic acid cause an equally diminished proteolytic action; thus, in the case of a carefully dialyzed juice where the proteid matter was much diminished, the digestive mixture, with its proteids wholly saturated, contained but 0:060 per cent. of combined salicylic acid; yet this mixture, in 15 hours at 40° C. digested but 17°10 per cent. of fibrin, while the same amount of the neutral trypsin solution digested 57°80 per cent. * Tested by tropolin 00 according to the method of Danilewsky (Centralbl. med. Wiss., 1880). One drop of a solution containing 0-028 per cent. free salicylie acid gives a reddish-violet color, which is, however, not permanent as in the case of hydro- chlorie acid, but transient. With hydrochloric acid, one drop of a 0-003 per cent. solution will give the reaction. on Amylolytic and Proteolytic Action. 147 Combined hydrochloric acid has a greater hindering action than salicylic acid, as the following results show : Fibrin digested Pancreatic solution of trypsin. in 18 hours. neutral 57°80 per cent. 0-034 per cent. combined HCl+no free HCl 3°90 0034 =| Y HCl1+0'C05 per cent. free HCl 2°31 0-034 uf “ HCl+0:010 = a 0°87 It is thus evident that in an ordinary digestive mixture, or even where albuminous matter is present only in limited quantity, the addition of hydrochloric or salicylic acid to a neutral solution of trypsin reduces its proteolytic action to a minimum before any free acid is present. 4.— Influence of Bile, Bile Salts and Bile Acids on the Proteolytic Action of Trypsin. The addition of bile to a neutral pancreatic juice causes but little change in its proteolytic action, as is seen from the following results obtained with ox bile containing 8°3 per cent. solid matter :’ Bile. ai ectealcenliue, ae 0 per cent. 04118 gram. 59°82 per cent. 1-0 ; 0°3907 60°93 10:0 0°3938 60°62 A slightly increased action is the only effect produced on the trypsin.* The addition of bile to an alkaline pancreatic juice does not produce any very different results. The following were obtained with a pancreatic juice containing 0°3 per cent. sodium carbonate and fresh ox bile containing 10°02 per cent. solid matter : Weight of Fibrin Bile. undigested residue. digested. 0 per cent. 0°3056 gram. 69°44 per cent. 0°25 0°3074 69°26 0°50 0°3488 65:12 1:60 0°3633 63°67 5°00 0°3278 67°22 10°00 0°3603 63°97 Here there is no increased proteolytic action, neither is there any very great retarding effect produced. Pure sodium glycocholate and taurocholate produce results similar to bile, as the following table shows. The pancreatic juice contained 0°3 per cent. sodium carbonate : * Compare Heidenhain, Pfliiger’s Archiv,, vol. x, p. 579, 148 Chittenden and Cummins—Influence of Bile Sodium Weight of Fibrin taurocholate. undigested residue. digested. 0 per cent. 0°2308 gram. 76°92 per cent. 0°05 0°2566 74°34 0°10 0°3048 69°52 1:00 0°2832 71°68 sodium glycocholate. 0°10 0°2576 74°24 0°20 0°3154 68°46 The presence of 3:0 per cent. crystallized ox bile caused a some- what different result, increasing the proteolytic action slightly; thus, while the control, containing 03 per cent. sodium carbonate, digested 88°69 per cent. of fibrin, the same trypsin solution plus 3 per cent. of crystallized bile digested in the same time 89°73 per cent. of fibrin. While bile or bile salts have but little influence on the proteolytic action of trypsin, the bile acids, even small percentages, have a much more marked effect. The following results, obtained by the addition of the bile acids to a neutral pancreatic juice, show the extent of the action : Weight of Fibrin Bile acids. undigested residue. digested. 0 0°2516 gram. 74°84 per cent. Glycocholic, 0°03 per cent. 01993 80°07 Taurocholic, 0°10 0°3455 65°45 0°20 0°4332 56°68 0°50 0°4170 58°30 Here the retarding influence of taurocholic acid is very manifest, while, on the other hand, the small percentage of glycochoNe acid appears to increase the action of the ferment. In view of the possible acid-reacting character of the contents of the small intestines, it becomes an interesting point to ascertain the influence of bile on the action of trypsin in the presence of more or less combined acid. With a pancreatic juice in which the proteids were partially saturated with salicylic acid, 0°1 per cent. combined acid being present, the following results were obtained : Weight of Fibrin * Bile. undigested residue. digested. 0 per cent. 0°4822 gram, 51°78 per cent. 1-0 0°4858 51°42 10°0 0-409] 59°09 This increased action in the presence of 10 per cent. of bile accords with Lindberger’s results, this experimenter having found that bile in the presence of small percentages of (combined?) acetic and lactic acids tends to diminish the retarding effect produced by the acids alone. In the presence of combined hydrochloric acid, the bile salts pro- duced no effects whatever; the trypsin was entirely without action, X.—ABSORPTION OF ARSENIC BY THE Brain. By R. H. Cuirren- DEN AND Herpert E, Smita, M.D. SomE time since one of us* advanced the view that the amount of arsenic present in the brain, in cases of arsenical poisoning, is an in- dex to the form in which the poison was taken, viz: whether in a readily soluble and diffusible form, such as sodium arsenite, or in a comparatively insoluble form, as arsenious oxide or aceto-arsenite of copper. The original experiments of Scolosuboff+ on animals, with sodium arsenite, plainly showed the capability of nerve tissue for the absorption of arsenic ; yet the recorded observations of toxicologists tend to show, as a rule, the presence of but traces of this metal in cases of arsenic poisoning, either acute or chronic. Scolosuboff’s re- sults are, however, undoubtedly correct ; arsenic when taken in a very soluble and diffusible form without doubt does accumulate in the brain, but in our opinion only when in that condition, and thus in _the more common forms of poisoning with the white oxide or other insoluble forms of arsenic, but a trace of the poison is to be found in the brain at any one time. With a firm belief in the truth of the above statement, founded on personal experience and the recorded results of other workers in this field, it was maintained by one of us in a previous papert that the presence of weighable amounts of absorbed arsenic in the brain may be taken as an indication of the administration of a soluble form of the poison. Experiments on animals tend to show the correctness of the theory and the results of toxical investigations, so far as our knowledge extends, contain nothing contrary to this view. If true, we ought never to find under any circumstances an accumulation of arsenic in the brain, after the administration of an insoluble form of the poison. Hence, the study of arsenic cases, where the form of poison is known, is of great importance in this connection. We have had a recent opportunity of adding two more cases to * Chittenden, Amer. Chem. Jour., vol. vy, p. 8; and Medico-Legal Journal, vol. ii, p. 237. + Bulletin de la Société Chimique de Paris, vol, xxiv, p. 125. } Chittenden. loc. cit. 150 Chittenden and Smith—Absorption of Arsenic by the Brain. the list of those which bear testimony to the truth of the above theory ; two fatal cases of arsenic poisoning, one of which was caused by the white oxide, the other presumably by Paris green or aceto- arsenite of copper. Case A.—L. K., a middle-aged laboring man, ate for his dinner at noon a quantity of bean soup. Almost immediately after, he was seized with vomiting and purging, cramps in the legs, and all the ordinary symptoms of acute arsenic poisoning. There were no marked cerebral symptoms. At 9 P. M. of the same day the patient died in a condition of collapse, having thus lived nme hours after eating the poisoned soup. An autopsy made the following day by Dr. M. C. White of the Yale Medical School, to whose courtesy we are indebted for a description of the case, and also for the organs for analysis, revealed the following points of interest: “The mucous membrane of the stomach was very much inflamed, especially around the cardiac orifice. The duodenum was likewise much inflamed, also the lower part of the rectum, showing here as a red mottled conges- tion. The remaining portions of the intestines were normal, The brain showed marked congestion. The kidneys were normal in ap- pearance, the urinary bladder was nearly empty, and the mucous lining somewhat reddened. The lungs were normal, except the lower half of the right one, which was a little congested. The heart . normal; small fibrinous clot in the right ventricle.” In order to draw deductions of any value from the distribution of arsenic in the body of the deceased we must know positively as to the form in which the poison was taken. Fortunately, we were able to obtain the residue of the soup eaten by the deceased. Microscopic examina- tion of the sediment plainly showed octahedral crystals of arsen- ious oxide, and we were able to separate from a small portion of the solid residue 24 milligrams of the oxide. Plainly the soup was poisoned by simply mixing with it arsenious oxide in substance. As to the quantity of arsenic present in the soup we have the follow- ing data: 125 c. ¢., oxidized with hydrochloric acid and potassium chlorate, yielded 314°6 milligrams of arsenious sulphide, equal to 253°2 milligrams of arsenious oxide. As to the amount taken by the deceased we have no knowledge; we infer, however, since the soup constituted the main portion of his dinner, that a large quantity was eaten, which view we think is substantiated by the intensity of the vomiting and purging so characteristic of large doses of the poison, Here then, we have an unquestionable case of poisoning with arsenious oxide, and under conditions most suitable for rapid absorp- Chittenden and Smith— Absorption of Arsenic by the Brain. 151 tion ; a probable empty condition of the stomach, together with a large amount of the poison, a considerable portion of which must probably have been dissolved in the soup. Added to this, nine hours intervened between the taking of the poison and death. Certainly then everything favored an absorption of poison by the brain, if such is characteristic of this form of arsenic. Naturally the vomit- _ ing and purging would remove much of the poison, still the relative proportion of absorbed arsenic would not be materially altered, and thus if Scolosuboff’s results with soluble arsenites are applicable to arsenious oxide, we ought to find in this case, in conformity with his results, a larger percentage of arsenic in the brain than in the liver or kidneys. Following are the results actually found :* Liver (1259 grams) contained 76:0 milligrams As,Os;. Kidney and bladder (332 grams) contained 0°6 milligram As.O3. Brain ($=528 grams dry) contained simply a recognizable trace. Case B.—J. G., a young woman, age unknown. Regarding the details of this case we have less definite knowledge. She was last seen alive on Friday night, at which time she threatened to poison herself. The following Monday morning she was found dead, and near her an open package of Paris Green. She had evidently been dead some time, and both the condition of her room and person gave evidence of excessive purging and vomiting. An autopsy by Dr. White showed an entire absence of inflammation of the alimentary tract, and also a lack of any abnormal condition sufficient to account for death. The verdict was therefore, death by poisoning with Paris green or aceto-arsenite of copper. Through the kindness of Dr. White we were able to obtain por- tions of the body for analysis. The contents of the stomach were entirely free from arsenic, the poison having been wholly removed - by the purging and vomiting; the trace found therefore, was the amount absorbed by the muscle tissue of the stomach. Following are the amounts of arsenic found in the parts analyzed : Liver (2984 grams) contained 12°78 milligrams As.Os;. Kidneys and bladder (515 grains) contained 3°40 milligrams As.Qg. Muscle of thigh (735 grams) contained 0°97 milligram As,O3. Stomach (425 grams) contained a trace. Brain (1179 grams) contained a trace. * The method of analysis consisted in the oxidation of the tissue with nitric and sulphurie acids, the arsenic being weighed as metallic arsenic. See Amer. Chem. Journal, vol. ii, p. 235, 152 Chittenden and Smith— Absorption of Arsenic by the Brain. The trace of arsenic in both the muscle tissue of the stomach and in the brain was very small; the entire brain could not have con- tained more than 0:2 of a milligram of arsenic. These results plainly substantiate the views set forth above and lend favor to the belief that Scolosuboff’s results with sodium arsen- ite are applicable only to that form of poison, and not to the more insoluble compounds of arsenic. These two cases, therefore, are additional evidence that in poisoning with arsenic the presence of an appreciable amount of poison in the brain, is an indication amount- ing almost to proof positive of the administration of a soluble and diffusible form of arsenic. 4 : . ¢ XI.—IvFivence or Porassitm snp Ammontum BromipEs on Metasousm. By R. H. CuirrenpEn anp W. L. Cursert, rb: As a question in the physiology of nutrition, it is very desirable to be able to state something definite regarding the influence of bro- mides upon the metabolism of the body; particularly their influence upon the metabolism of proteid matter, as shown in the excretion of urea and uric acid, and in view of their special application as therapeutic agents in diseases of the nervous system, their influence also on the decomposition of nerve substance, as shown in the excre- tion of phosphoric acid. ‘Two complete investigations appear to have been made upon this subject; one in 1868 by Dr. J. H. Bill,* and one in 1883, by Dr. B. Schulze,t the results of which are more or less in direct opposition to each other. We have also seen a refer- ence to two other investigations, quoted by Dr. Wood,{ in which Dr. Rabuteau found the daily excretion of urea slightly lessened under the influence of bromide, as did also Dr. Bartholow. Dr, Bill’s investigation, which was a very thorough one, had for one of its objects to ascertain whether bromides reduce the amount of phosphoric acid excreted, like such known hypnotics as morphine ; at the same time careful examination was made of the variations in ufea and uric acid under the different conditions of the experiments. The experiments were all conducted on one person with a body weight of 160 pounds, which remained fairly constant throughout. The experiments were made in series under known conditions with uniformity of habits, diet, etc. Unfortunately, however, no data are given regarding the nature of the diet, the closeness with which it was adhered to, or whether the body was kept in a state of nitro- genous equilibrium. Each series of experiments, moreover, covers at the most, but six days; three days without bromide and three days with, consequently slight variations might easily be absorbed in an average of three results. The urea in Dr. Bill’s work was deter- * Amer. Jour. med. Sciences, July, 1868. Experimental Researches into the action and Therapeutic value of Bromide of Potassium. + Zeitschrift fir Biologie, vol. xix, p. 301. Einfluss des Bromkalium auf den Stoff- wechsel. ¢ Therapeutics, p. 337. Trans. Conn. AcapD., Vou, VII. 20 Nov., 1885. 154 Chittenden and Culbert—Influence of Potassium mined with a “mercury solution,’ phosphoric acid with uranium solution and uric acid by precipitation and weighing as such. The results obtained were as follows: With moderate doses of potassium bromide (3°0-8°0 grams per day) urea was not affected, phosphoric acid was slightly increased, uric acid likewise, though much more decidedly; with larger doses of bromide (10°0-14:0 grams per day, continued for 2-38 days) phosphoric acid was diminished in amount, but this Dr. Bill intimates could not be attributed to regular hypnotic action, since the other urinary constituents were likewise diminished, notably the urea. Both large and small doses of bro- mide increased-the quantity of urine passed in the twenty-four hours. This Dr. Bill asserts was not due to the increased drinking of water, for no thirst, not even with the largest doses, was ever present. Dr. Schulze, experimenting on his own person, obtained results quite different from these. This investigator lived on a fixed diet of the following composition : ; 220 grams fresh meat =1'13 grams N. 50 grams air-dried wheat bread==0-°92 grams N. 30 grams cocoa powder =1'14 grams N. 30 grams butter. 30 grams sugar. 5 grams salt. 1500 grams water. 9°19 grams N. . The average amount of nitrogen excreted daily was about 11 grams. Taking this amount of food daily, with uniform habits of sleep, exercise, etc., Dr. Schulze states that he soon reached a point where the daily excretion of nitrogen, sulphur and phosphorus remained fairly constant. Potassium bromide was then taken three days, in divided doses of 10 grams each day. Diuretic action was very noticeable. Phosphorus was diminished, sulphur very much in- creased, and nitrogen (on two days) apparently slightly increased under the influence of the bromide. As, however, the increased excretion of sulphur was not accompanied with a corresponding increase in the excretion of nitrogen, Schultze considers that the increase in sulphur cannot be due to increased metabolism of sim- ple albuminous matter, and seeks to show that the potassium bro- mide must have decomposed, to a slight extent, some nitrogenous phosphorized principle or principles, such as lecithin (glycerine-phos- phoric acid) and nuclein, so abundant in the brain and nerve sub- stance in general, Schulze therefore concludes that under the influ- - = Oe ee i el SS. — = and Ammonium Bromides on Metabolism. 155 ence of potassium bromide there is probably a decided diminution of metabolic activity in the nervous system, accompanied by decreased nervous irritability. By determining the nitrogen of the feces, Schulze concluded that the bromide exercised no particular influence on the digestibility of the food. In our experiments great care was taken first, to insure body equi- librium and then to obtain sufficient data by analysis, to be sure of the requisite constancy in the composition of the daily excretions. The experiments were tried throughout on the person of one of us (W. L. C.), of good physique and vigorous constitution. The diet was weighed out each day with scrupulous care and was as follows: uPopi ene dtifitmser ps ses ees) bie Soe ie tek 142-0 grams. IB GAeTHOCS Bae aie ern ee eet 2 hs Fae oe oes 283-p) att WMD CA bEDEC AC teem ee ee os 2 es 5 es es ee Se ts 2560. Chik waghilj es ee eee eee 50-0 2s ULC bee eee ee Nee nn a ake es aoe arr (yy SUE Goes yee ee a ee ane 28°3 * eset Roce 4k 2 PE A) Ss Pe tae Oe eee eee ee Orin, use NVAln] Lena ny y= Sport Pane di eet Se bre ibys T00;0 0 WVANIGINE. = ee ee ee ee eee awliyGy— This diet was commenced on the third day of April and continued for nine days before any attempt was made to ascertain the daily amounts of urea, etc., excreted. Then the urine was analyzed for nine successive days, after which doses of potassium bromide were taken. The above daily amount of food was divided into three por- tions and taken at the same time each day; at 7:30 a. m.,1 p. m., and 6 p.m. Exercise was taken regularly and in stated amounts; consisting of a walk each morning before breakfast and exercise with dumbbells just before retiring at 11 p.m. Care was taken not to exercise so freely as to induce perspiration. Throughout the day routine duties allowed of regular habits. It was thus found possi- ble to keep even the minor conditions of the experiment constant throughout. The urine was collected from 7:30 a. m. of one day to 7:30 a.m. of the next, and was at once analyzed. Urea was deter- mined by Pfliiger’s* modification of Liebig’s method, with a standard solution of mercuric nitrate. All the precautions so carefully worked out by Pfliiger; preparation of a mercuric nitrate solution of the proper specific gravity, a standard solution of sodium carbonate to neutralize the acid of the former, preliminary determination of * Pfliiger’s Archiv, vol. xxi, p. 248. 156 Chittenden and Culbert— Influence of Potassium chlorine and removal of the same, before precipitating the urea; were carried out with very satisfactory results. Uric acid was determined according to the older method of Heintz* with the modi- fication of Zablins, and being conducted each time under exactly the same conditions and with a urine of approximately the same composition, the results are to be considered as strictly comparable. Chlorine and bromine were determined in the usual manner with a standard solution of silver nitrate; the results, however, are not given as they are of value only as essential to the urea determinations. Total phosphoric acid was determined by means of a standard solu- tion of uranium nitrate.t Phosphoric acid in combination with alkali- earths was determined by precipitation with ammonium hydroxide, allowing the mixture to stand 24 hours, filtering the precipitated phosphates, washing thoroughly with diluted ammonia, then dissolv- ing in a definite amount of dilute acetic acid and titrating with uranium solution. Total amount of solid matter contained in the 24 hours’ urine, was calculated by the use of Christison’s formula. The diet specified, was commenced on the 3d day of April; on the 12th the urine was collected for analysis, the body weight taken and the investigation then carried forward without interruption. ‘Table No. I. gives the results of the analysis of the urine for nine con- secutive days, and shows the average amount of variation to be expected under the conditions of the experiment. On the 21st of April, 60 grains of potassium bromide were taken in divided doses as seen in Table No. Il. The bromide was taken about midway between the hours of eating, so that it might not affect digestion. On the 22d the dose was increased to 100 grains and then to 150, the latter amount being taken daily for three consecutive days. Table No. II. shows the effects of the bromide on the system, for the six days it was taken, On the first day, the only apparent influence of the bromide is to cause a diminution in the amount of phosphoric acid excreted; seen both in the total P,O, and in the P,O, in combination with alkali- earths. On the second day, the diuretic action of the salt is apparent, accompanied with an increase in specific gravity, and a decided increase in the amount of urea excreted, together with a slight in- crease in the amount of uric acid. Phosphoric acid was still dimin- ished in amount. On the third day, the body weight commenced to diminish and continued to do so throughout the experiment ; diuretic * Die Lehre vom Harn, Salkowski und Leube, p. 94-95, + Die Lehre yom Harn, p, 184, and Ammonium Bromides on Metaholism. 157 action was still apparent as was also increased elimination of urea, and to a slight extent of uric acid likewise. Indeed, the most notice- able effect of the bromide, next to its diuretic action is its decided influence on proteid metabolism as shown by the increased elimina- tion. of urea. As to phosphoric acid the results are not so striking, although an average of the two series shows a diminished excretion, both of total P,O, and of P,O, in combination with alkali-earths. The average difference in the two series of results is clearly shown by the following table : Average of Table : No.1, without KBr. No. II, with KBr. Total quantity of urine -.---.-_---.- 926 ¢. ©. LoOUOKenc: SOR Glee Sane Aenea an Somes ones 1025, 8 1026, 3 Total solid matters_-_------ jee eee 56°7329 grams. 63°6252 grams. Mota Oste ters St See ose 2°7540 275426 P.O; in combination with Ca and Mg- 06022 0°5452 Winicracidl Saye ae eee. se eens 2 06752 0°6858 WCE) Se ee OES Bee ae ee ee 34°8681 35-9454 Our results therefore plainly indicate, that under the influence of potassium bromide, nitrogenous metabolism is increased while the excretion of phosphoric acid is slightly diminished in amount; not however, to any such extent as would be expected by an active hypnotic agent. The bromide taken, the largest doses about 10 grams per day, produced its usual physiological effects; such as drow- siness, diminution of the circulation with accompanying coldness and paleness of the skin. Constipation was not noticed while taking the bromide, but later on it became somewhat troublesome, once or twice alternating with a slight diarrhcea. In accord with Dr. Bill, we no- ticed an increase in the acidity of the urine while taking the bromide, as also a deepening of the color. With bromide of potassium therefore, our results agree with those of Dr. Schulze in showing an increased excretion of nitrogen (urea and uric acid), although far more pronounced than he found in his experiments, while the diminution in phosphorus is less pronounced than found by Schulze. With Dr. Bill’s experiments our results agree in so far as the diminution of phosphoric acid is concerned, but are entirely different as regards the urea. Since, however, Dr. Bill retained uniformity in diet only during the days of the experiments, it is quite possible that lack of nitrogenous equilibrium may have had some influence on his results. The increased elimination of urea noticed in our experi- ments is certainly indicative of increasei metabolic activity ; it is, 158 Chittenden and Culbert—Influence of Potassium however, suggestive that potassium bromide has been recently found to exercise a very decided accelerating influence on the proteolytic action of both pepsin-hydrochloric acid* and trypsin ;+ while, there- fore, this fact may have something to do with the increased elim- ination of nitrogen, particularly as the diet used is quite rich in pro- teid matter, it seems more probable to suppose that the above changes in the excretion of nitrogen are due rather to changes in the tissue proteids; still it would have been interesting if the nitrogen in the feeces had been determined each day. Although the last dose of potassium bromide was taken on the 26th of April, the same diet was still continued and the urine carefully examined daily, until the 8th of May, at which time the amount of bromine in the urine was reduced to a minimum, Dr. bill states that bromine usually disappears entirely from the urine in ten days after the last dose of bromide. The results of the twelve days analyses are shown in Table No. Ill. In examining this table it is interesting to note how quickly the elimination of urea is changed on stopping the doses of bromide. On the 26th, the last day the bromide was taken, the excretion of urea amounted to 37°5 grams; on the 27th it fell to 31°8 grams, far below what it had been any time before the bromide was taken. It would thus appear that after withdrawal of the bromide, nutrition which had been accelerated, rebounded in proportion to the preceding acceleration. Uric acid, moreover, which had likewise been increased in amount by the bromide, was now also correspondingly diminished. Furthermore, the diuretic action of the bromide was at once stopped, and the specific gravity fell to 125°5. In the case of phosphoric acid, however, the action of the bromide appears to be continued for a day or two after its withdrawal, and indeed it is noticeable through- out, that the diminution in phosphoric acid excreted, is not at all proportional to the amount of bromide taken. In fact phosphoric acid, both total P,O, and alkali-earth P,O,, appears to be more deci- dedly diminished on those days when the amount of bromide in the blood was the smallest, notably on the 21st, 24th and 27th of April. By the 3d day after withdrawal of the bromide, the excretion of urea had gone nearly back to the daily amount, prior to taking the bromide; still it is to be seen in Table No. Ili, that the average ex- cretion of urea, uric acid and phosphoric acid is below the average excretion recorded in Table No. I. In fact after the continued doses * Chittenden and Allen. Trans. Conn. Acad., vol. vii, + Chittenden and Cummins, — [bid. . h and Ammonium Bromides on Metabolism. 159 of potassium bromide, the metabolism of the body did not fall back to its original height; but being temporarily accelerated during the exhibition of potassium bromide, it [the nitrogenous metabolism | fell back on withdrawal of the same, to a far lower level, and al- though later somewhat increased in amount, its average was still lower than recorded in Table No. I. Nutrition had evidently been disturbed; the body weight showed gradual diminution, and on the 4th and 6th of May there was slight diarrhcea accompanied with a decided decrease in the amount of urea and uric acid excreted. Dr. Bill appears to have experimented somewhat with sodium bro- mide, although we find no results recorded, aside from the fact that this salt, like potassium bromide, caused an increased excretion of uric acid, and the general statement that “‘when taken by the mouth, bromide of sodium does not produce the same effects as bro- mide of potassium.” In view of the increased excretion of urea, noticed under the influence of the potassium salt, we were interested in seeing whether ammonium bromide would have a like influence, especially in view of the fact that v. Schreeder* has shown that ammo- nium carbonate is directly convertible into urea by passage through the liver. The physiological action of ammonium bromide is stated to re- semble in many points that of potassium bromide, while in other points it differs: essentially.t As to its influence on metabolism no experiments whatever appear to have been made. On the 9th of May, 75 grains of ammonium bromide were taken, in divided doses, as shown in Table No. IV. In all, 425 grains of the salt were taken in four consecutive days. The action of the salt on the system was not as pleasant as that of potassium bromide; caus- ing a general weakness and indisposition, a slight diminution in the pulse, an occasional cold perspiration, more marked lividity of the. countenance and a parched, dry taste in the mouth. An habitual eruption of the skin was moreover much increased and accompanied with acne on the back and shoulders. Undoubtedly these disagree- able symptoms were much augmented by the temporary lassitude which was beginning to be apparent; doubtless due to the approach of warm weather together with lack of the accustomed vigorous exercise and the long continued use of the somewhat monotonous diet. * Archiv f. exp. Pathol., vol. xv, p. 364. Also Report on Progress in Physiological Chemistry in Amer. Chem, Jour., vol. v, p. 219. + Wood, Therapeutics, p. 341. 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Like the potas- sium salt, ammonium bromide caused increased acidity in the urine and a brighter color. As to its influence on metabolism, a study of Table No. IV, and comparison with No. III, plainly shows a decided accelerating influence on the excretion of urea; the average of the results, moreover, shows a very slight diminution in the excre- tion of total phosphoric acid, at the same time it would appear, in accord with what was observed with potassium bromide, that the diminution was greatest with the smallest amounts of bromide, as on May 9th and 10th and on the 13th and 14th after withdrawal of the bromide. With the largest amount of ammonium bromide, on the other hand, phosphoric acid appeared to be increased in amount, thus according with what Dr. Bill observed with like quantities of potassium bromide. The average difference in the two series of results is shown by the following table: Average of Table No. IIL o.1V without (NH4)Br. with (NH4)Br. Total quantity of urine’ -...------..- Didi: ec: 1072. ¢. ¢. SpiGriseesce a2 seo sae sees cae 1024, 8 1024, 4 Mopalisolidem abters== eee 54°3970 grams. 62°3608 grams. TotalskiO pect ic ass Coens Sees 2°5643 2°5130 P.O, in combination with Ca and Mg- 0°4972 0°5749 Heo ea eel ge eS 0°6599 06751 (Ureda ss. P< choco a Si eeeecce sens 32°8579 34°6505 It is thus seen that diuretic action is even greater with the ammo- nium salt than with potassium bromide; likewise the excretion of both urea and uric acid is greater under the influence of ammonium bromide than in the case of the potassium salt; as to phosphoric acid the table of averages shows practically nothing, but as before ob- served a study of the individual results does indicate some action of the salt, although diminution in the excretion of phosphoric acid under the influence of ammonium bromide cannot be so surely claimed as with the potassium salt. After withdrawal of the ammonium bromide, the urine was exam- ined for two days more; the results showing the same, or even greater drop in the excretion of urea, observed under like conditions with the potassium salt (Table No. V.). Hence, so far as our experiments ex- tend, the influence of the two salts on the metabolism of the body is very much alike, differing only in extent of action; the ammonium and Ammonium Bromides on Metabolism. 165 salt, as might be expected, causing the largest excretion of urea, not, however, necessarily from any greater influence on proteid metabolism, but merely as furnishing a certain amount of ammonia to be excreted as urea. Finally, our results, with both salts, fail to show that dimi- nution in the excretion of phosphoric acid to be expected from active hypnotic agents, and in this respect, therefore, our results show noth- ing antagonistic to Dr. Bill’s conclusion “that bromide of potassium in its legitimate action, is an ahzsthetic to the nerves of the mucous membranes and a depressor of their action. Its hypnotic effects are secondary.” XITT.—INFLUENCE oF CINCHONIDINE SULPHATE ON METABOLISM. By R. H. Carrrenpen anp Henry H. Wuirrnovuss, Px.B. WuiteE much attention has been paid to the physiological study of the cinchona alkaloids; quinine particularly having been experimented with by several observers, to ascertain its influence on the metabol- ism of the body ; we have not been able to find any recorded state- ments bearing on the action of the closely related alkaloid, cinchoni- dine. In physiological action, quinine is taken as a type of the group ; cinchonine is stated* to be similar to quinine but less power- ful, and that its history in the organism is parallel with that of qui- nine; cinchonidine is likewise stated to be weaker than quinine and in physiological action, apparently its equivalent when taken in doses one-third larger. Presumably, therefore, its action on the metabol- ism of the body is similar to that of quinine, although.apparently no attempt has been made to determine this point. In view of the spe- cial action of quinine on nitrogenous metabolism, we have devoted our attention mainly to a study of the influence of cinchonidine on the excretion of urea, uric acid, phosphoric acid and chlorine. The experiments were conducted wholly on the person of one of us (H. H. W.) under uniform conditions of diet, exercise, etc. The diet, weighed out accurately each day, was composed of 255 grams meat (beef). 255 grams wheat bread. 149 grams potatoes. 50 grams oat meal. 35 grams butter. 21 grams sugar. 570 grams milk. 350 grams water. This diet, divided into three definite portions each day, was taken for some time previous to the experiments, so that the system might become accustomed to it and the metabolism of the body brought to a constant point. The body weight was determined each morning; the urine collected from nine a. M. of one day to nine A. mM. of the next, making the 24 hours’ urine, the analysis being made the same * H. C. Wood, Therapeutics, p. 81. a Chittenden and Whitehouse—Sulphate on Metabolism. 167 day. Urea was determined by Pfliiger’s* modification of Liebig’s method, chlorine being removed by a standard solution of silver nitrate. Chlorine was determined by evaporating 10 ¢.c. of the urine with a weighed amount of potassium nitrate in a platinum eru- cible, igniting until the organic matter was completely removed, dis- solving in water, acidifying with nitric acid, neutralizing with cal- cium carbonate and then titrating with silver nitrate solution. Uric acid was determined by Heintz’s method as modified by Zablinst and phosphoric acid with a standard solution of uranium nitrate.t The amount of solid matter was calculated by the use of Christison’s formula. After taking the above diet for some time, the urine was analyzed for seven consecutive days prior to the exhibition of cinchonidine. The results, seen in Table No. I, show a very close agreement in the daily excretions. On the 11th of May the first dose of cinchonidine sulphate was taken. The alkaloid salt was a finely crystallized preparation ob- tained from Powers and Weightman. The daily dose was usually divided into three portions and taken in tiny gelatine capsules, about five hours apart. In view of the fact that cinchonidine is a weaker alkaloid than quinine, it was not deemed necessary to try the influ- ence of very small doses; on the first day, therefore, 15 grains of the salt were taken; on the second day 21°8 grains; on the third day 35°1 grains; and on the fourth 50 grains, making a total of 121-9 grains of cinchonidine sulphate in four consecutive days.§ The re- sults of the analyses of the four days’ urine, as well as those of the three following days, on which no cinchonidine was taken, are shown in Table No. II. Comparing these results with those in Table No. I, and in Table No. III, it is seen that cinchonidine exercises a very decided influ- ence on the nitrogenous metabolism of the body. Urea is at once affected : its excretion on the first day even, is diminished 6 per cent., while on the second day it is diminished 10 per cent., and on the fourth day when the largest dose of cinchonidine was taken, the excretion of urea was 16 per cent. less than in the normal urine. The influence * Pfliiger’s Archiv, vol. xxi, p. 248. + Die Lehre vom Harn, Salkowski and Leube, p. 94—95. { Die Lehre yom Harn, p. 184. § While taking the larger doses of cinchonidine, an intense ringing in the ears was temporarily experienced (cinchonism) together with partial deafness and slight dizziness. On one or two occasions a slight nausea was felt. Se) > :S Ss i fluence of Cinchon In Chittenden and Whitehouse— 168 ‘ANINQ) IVNAON—'T FIaVyL {86-1 F9L-0 OF0-& 6FT-9 188-99 90T ‘PPV OF9- TP 6FL-0 616-€ 991-9 198-99 $-960T ‘PPV POP: LP 891-0 OT8-6 FEE-9 696-89 g-9G0T ‘PPV 666-6F 618-0 OTL: OTS-9 696-€9 LGOL ‘PHV L8.TF ToL-0 80L-8 &6F-S TLP-8¢ LOOT “PPV F89-6P L6L-0 P¥0-& 6Gg-¢ STL-89 8601 “‘pHy SE ag Ce On ee ead “Bol ) “plow otf) “O°” [eIOL | “OuLLOTyD ee *It) ‘dg “HOTIOVIY LLOT 066 £06 0&6 "0 °O O16 oul) Ayyuenb [eo 0096S 0096S 0096S nee 0066S O0F6S OO0F6S ‘SuUIBIs ‘qys1em Apog OL “AVI 169 Sulphate on Metabolism. 0 98-68 TTL-0 108-6 989-¢ 161-79 G- LOO ‘PPV CL6 0088S Ay 0 686-C§ 999-0 068-6 860-9 616-09 ¢-960T ‘POV 0&6 OOF6S oT 0 T8P-SE 869-0 C6L-T Col-9 LOF-9G S601 ‘PPV O&0T 00609 cI OS | STT-SE GLg-0 861-6 CGB. G6G-8¢ FeO ‘PPV GOT 00009 las i Ea 4 so AOS aE| SS cee Set fe Od, T-G§ G6E-8E 661-0 660-6 CETL 86F-89 FoOT ‘PV C6TT 00009 &T 8-18 L96-LE 6242-0 669-6 988-¢ 162-19 » -960T ‘POV CL6 0066& oT cT CTL-6& STL-0 9LL-6 vP0-G OFF-6¢ LoOT ‘PPV |9 'D 086 0096¢ IT “SURI *SUIBIS “UUBIS *SUIRIS “SUIBIS “SMURLO ‘SuBIs TA yey ayeydins ; : “aug . "a1Y ay SE “ANIGINOHONIO FO SLOMAN FHL PNIMOHS—'T] AIAV Nov., 1885. 22 eS _— S S| o > A < o <= 2 oO gi - 4 < [e=f = L Ss 'S aS ~ ° Se >) S Ss > SY) ) ~> s = i H > ) > Ss = ~~ = — S S > = = D RSS S 170 | ee ee 8-SP LOF. OF COL-0 FL9-G 80L-9. FOL-69 8-L60T ‘POV LVOT ; 006¢ GG | | DS 666-0F 669-0 GE0-§ TG8-P 166-69 OS0T “prey ¢18 | 00062 =| «T@ | ES 0 PCE- SP 668-0 i mBOTe CEL G f66-L9 G.6G0T “‘PDV 0&6 0006¢ 06 0 SOP: SP FFL-0 | 610-8 OLG- PE8-69 C8301 PRY; 066 ~ 0096E 61 0 6L6-0F TOL-0. 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GOL: OF C9E- GP 60T-LE 082-LE GE Te 2 SS” Sl" ayes nae sae ei ae Bolly GOL-0 6LL-0 899-0) CTL-0 OLGOS aa See eee plow OLty) G&8-6 SIT-§ 668-6 GEL-S PESIGS. fr eae ae SS eee eye ae *O'd [401 O8L-¢ C66-¢ &IT-9 ¢c0-9 ORO: OF cee Sa ee aia SULtOTT): ‘SUIBIS [Ce.99 *‘SUIBIS BFQ.e9 "SUIR.IS 163-09 ‘SUIBIS JF0.89 ULES OP: FO: seta $10}}BUL PILOS [eyIOJ, 6B0L 88801 9-801 P-SB0T POOLE Se ok ae ary gee “I “dg ‘O "OD 196 0°O 886 700 G96 0 'O Zor 20.50 TOOT. “aes ce outin Ayuendb peqoy, “eUIpIuoOyouty “OUTpPIUOYaNIg ‘OUTpPIuOYoUly jo “OUTplUOTPUIg “OULIN [BUIIO NT VIAL noyyTAr dSOp ISET 9 (TM ‘T 8Tqey, ‘TIT PL, THT S48 L, SurMoypoy skep ¢ ‘IT PBL, ‘Tl 48h ‘SETASUY GOVUAAV ONIMOHG— ‘AT wavy, 172 Chittenden and Whitehouse—Influence of Cinchonidine of cinchonidine on the elimination of urea continues to be felt after discontinuing the use of the alkaloid; thus even three days after the last dose of cinchonidine, the elimination of urea is 6 per cent. less than in the normal urine. On the fourth day it is nearly back to the normal amount [see Table No. TI.], and on the fifth and sixth days the excretion of urea rises somewhat above the average. Uric acid does not appear to be correspondingly affected. It is only under the influence of the largest doses, or rather under the long continued action of the alkaloid that the ex- cretion of uric acid is diminished; thus on May 14th, when the final dose of 59 grains of cinchonidine sulphate was taken, the excretion of uric acid was for the first time diminished ; its diminution, how- ever, was very perceptible and it continued for the two succeeding days, after withdrawal of the alkaloid salt. Phosphoric acid was greatly diminished in amount under the influence of cinchonidine ; the diminution commencing to show, as in the case of urea, with the first dose of the alkaloid salt, then gradually increasing in amount with increase in the dose of cinchonidine until maximum diminution was reached on the day after the final dose of the alkaloid. The alkaloid salt, moreover, appears to have had a slight diuretic action. On the 21st of May, cinchonidine sulphate was again taken, the results of the analyses of the 19th and 20th showing that the urine had returned to its normal composition. Accordingly, 95°8 grains of the salt were taken on the 21st and 22d, producing the same results (Table No. ILI.) as noticed in the first series, viz: diminution ‘in the amount of urea and uric acid excreted, a like diminution in the amount of phosphoric acid and an increase in the total amount of fluid. Table No. IV. shows the average results under the different condi- tions of the experiments. It is evident then, that cinchonidine has the power of lessening very materially the elimination of nitrogen, i. e. the consumption of tissue, Ranke* first pointed out the retarding influence of quinine on the elimination of uric acid. Zuntz found by experiment upon himself that 25 grains of quinine reduced his elimination of urea nearly 40 per cent. A like diminution in the excretion of urea, under the in- fluence of quinine, was noticed by Rabuteau in experiments upon dogs and also by Hermann von Boeck.f The most interesting ex- periments, however, with quinine are those carried out. by Dr.-G. * Quoted from Dr. H. C. Wood, Therapeutics, p. 74, + Zeitschrift fir Biologie, vol, vii, p, 422, eee ssnaeeeinteaaas tee ia itlage i iRtitaa pe Mammalia Sulphate on Metabolism. 173 Kerner,* and more recently by Dr. Priort and Dr. Sassetzky.{ In Dr. Prior’s article isto be found a very complete account of the liter- ature of the subject. Dr. Kerner found that taking 9°3 grains of quinine hydrochloride per day in divided doses, for 3 days, making a total of 27:9 grains, caused a diminution in the excretion of urea, amounting on an average for the three days to 12 per cent., while the excretion of uric acid under like conditions was diminished 54 per cent. Phosphoric acid, too, was diminished somewhat ; on an aver- age about 4 per cent. per day. Diuretic action was very slight. With larger doses of quinine; 77°5 grains of the hydrochloride in divided doses during three days; diuretic action was quite pro- nounced, the average increase in volume, amounting to 200 c.c. Moreover, urea was diminished on an average 23 per cent. per day, uric acid 82 per cent. and phosphoric acid 15 per cent. Oppenheim,$ however, by a dose of 30°8 grains of quinine found an increase in the daily excretion of urea amounting to 4 grams, and he considers that Kerner’s results are due to diminution of digestive action. It is cer- tain that both quinine and cinchonidine do interfere with the pro- teolytic action of the gastric and pancreatic juices,|| but this retarding action can hardly be taken as explaining in full, the results obtained by Kerner or those obtained by us with cinchonidine. Prior, moreover, by very carefully conducted experiments with quinine, has completely corroborated Kerner’s results and has shown in addition, by a daily determination of nitrogen in the feces, that diminution of urea and uric acid is not due to lack of digestive action, as suggested by Oppenheim. Prior’s results show on an aver- age, without reference to the size of the-dose, the following effects of the quinine. Quantity of Uric Sodium Sulphuric Phosphoric urine. Urea. acid. chloride. acid. acid. increase * decrease decrease decrease decrease decrease 10°65 per cent. 19°60 % 12°29 Z 9°06 & 33°70 Z 23°38 G Sassetzky’s results with fever patients, also corroborate Kerner’s statements. * Pfliiger’s Archiv, vol. iii, p. 104. + Ueber den Hinfluss des Chinin auf den Stoffwechsel des gesunden Organismus. Pfliger’s Archiv, vol. xxxiv, p. 237. ¢ Ueber den Kinfluss fieberhafte Zustinde und Antipyretischer Behandlung auf den Umsatz der stickstoffhaltigen Substanzen und die Assimilation stickstoff-haltiger Bestandtheile der Milch. Virchow’s Archiv, vol. xciv, p. 485. § Pfliiger’s Archiv, vol. xxiii, p. 476-477. || Chittenden and Allen ; Chittenden and Cummins, Trans. Conn. Acad., vol. vii. “| Pflager’s Archiv, vol. xxxiv, p. 263, 174. Chittenden and Whitehouse—Influence of Cinchonidine v. Boeck considers that quinine owes its retarding influence on proteid metabolism to a direct action of the alkaloid upon the cells and their activity, although the alkaloid doubtless does unite with albu- min or alter its constitution so as to render it less readily decompos- able. Metallic salts, as lead and mercury, certainly form compounds with albumin difficultly decomposable, as also does arsenic, and v. Boeck suggests that these metallic poisons unite with the proteid matter of the various organs of the body, while quinine unites sim- ply with the circulating albumin, explaining in this manner the ready elimination of quinine as compared with the slow excretion of mer- cury or arsenic, the latter of which v. Boeck* has shown has little if any influence on proteid metabolism. Prior, moreover, states that diminution in the urine of the end-products of nitrogenous metabol- ism is due, not to hindering of their excretion, but to actual hindering of their formation. Comparing now Kerner’s results, with the results obtained by us with cinchonidine, we see great similarity of action but decided dif- ference in extent, particularly so far as the excretion of uric acid is concerned. With cinchonidine, the greatest average daily diminu- tion in uric acid amounts to but 15 per cent., and this after taking about 121 grains of the alkaloid during four consecutive days. Se- lecting the lowest single result, that obtained on the day 50 grains of cinchonidine were taken and comparing the diminution then, with the average normal excretion, it is seen to amount to but 26 per cent. In the case of urea and phosphoric acid, the divergence is not so great; thus for urea the average daily diminution was 11 per cent. for the three days following the last dose of cinchonidine, while the greatest diminution noticed in any one day was 16 per cent.; with phosphoric acid the average diminution for the same period amounted to 19 per cent., while the greatest diminution noti¢ed any one day was 388 per cent.; a diminution which at no time was reached in Kerner’s experiments with quinine. Thus in drawing a comparison between Kerner’s results with 7775 grains of quinine distributed through three days, and our results with 121 grains of cinchonidine extended over four days, we see two strik- ing points of difference; with quinine there is a diminution in the amount of uric acid excreted of 82 per cent., with cinchonidine an average diminution of but 15 per cent. ; with quinine there is a dimin- ution of phosphoric acid amounting to 15 per cent., with cinchoni- dine, on the other hand, a diminution of 19 per cent. Hence it is to * Zeitschrift fur Biologie, vol. vii, p. 430, Sulphate on Metabolism. 175 be seen that cinchonidine has a far less pronounced specific action on the excretion of uric acid than quinine, while on the other hand, diminution of phosphoric acid is much more pronounced with cincho- nidine than with quinine. In Prior’s experiments, however, with qui- nine, diminution of phosphoric acid is more pronounced. Is this diminution in the excretion of phosphoric acid under the influence of cinchonidine to be attributed simply to decrease of pro- teid metabolism, or is it in part due to a special action of cinchoni- dine on the metabolism of some phosphorized principles, presumably those of nerve tissue? If due to general decrease of proteid meta- bolism, we might expect to find that the addition of any non-nitro- genous principle to our fixed diet, whereby the decomposition of albuminous matter would be diminished, would cause a corresponding decrease in the excretion of phosphoric acid, or in other words that diminution of urea and phosphoric acid excreted, would be in the same ratio as noticed under the influence of cinchonidine. This question we have endeavored to answer by a study of the influence of pure glucose on the elimination of urea, uric acid, and phosphoric acid, under the same conditions of diet etc., as observed in the experiments with cinchonidine. The influence of carbohydrate food on proteid metabolism has been illustrated in many ways by various investigators, but so far as we know, no experiments with pure glucose have ever been tried. Through the courtesy of Dr. Arno Behr, of Chicago, we have been supplied with an abundance of chemically pure anhydrous glucose, which we have used in the following experiment. Before taking the sugar, the urine was analyzed for ten consecutive days, to insure an accurate average of the normal excretion under the conditions of the experiment. The results are shown in Table No. V. 200 grams of glucose were then taken daily in addition to the fixed diet, for nine consecutive days. The effect on the excretion of urea, etc., is shown in Table No. VI. At no time was sugar to be detected in the urine by Trommer’s test. A comparison of the two tables shows the usual effects of carbohydrate matter on the excre- tion of nitrogen, viz: a diminution in the amount of both urea and uric acid. The volume of the fluid excreted, appears to be consider- ably lessened by taking the glucose. The excretion of phosphoric acid is likewise diminished. we d i Chittenden and Whitehouse—In 176 fluence of Cinchon « 066- CF es 169-6 816-9 CF0-99 860T ‘PIV OL6 O0F6S OT 006-SF 166-0 Ch0-€ TPE-8 &P9-0L G- LOOT ‘PIV 3 SLOT 00F6S 6 GOT-LP CTL:0 POE-E 082-2 SCP-0L LOOT | ‘plow 060T 0086S 8 000-FF G6L-0 109-6 068-9 ae 9601 ‘POV POOL 0086S : 8LO-FF F08-0 sie 668-9 | er ¢-860T ‘Poy 066 0086S 9 T9T-&F 866-0 a 896-9 ‘ae 8601 | ‘PV 096 00009 ¢ OLL: SF 406-0 LOT-& suits i OSF-GL 6601 | ‘PPV GFOT 2 ee a L09-9F 608-0 a &&E-§ P6L-9 TOL TL 8c0T ‘PPV OLOT OOF6E § 69F-9F 890-T | ae & 080-4 C6E-6L ¢-860T ‘PPV 090T 0096S G Peleg bike Suter See ster Py eee | ae | ‘voln ‘plov ong “O%Q [vO], “OULLO[ YO ci ea “ry ‘dg | ‘nomoRexy aibeeGn ‘4qysI0M Apog Stee ‘ANINQ) IVWUON—'A AIAVY 0 COP bP 168-0 696-6 008.9 969-99 9601 “PPV 9SOT 0006S &%@ ee = fag) eg eee [ee ee = 0 6&F- CP 106-0 80L-§ 686-9 618-99 960T ‘PPV | 9LOT 00009 66 0 C&8-6& 608-0 co9-6 €9¢-9 {60-89 | ie “PPV ) ae 0096S a ; “= 6 OFL- TF 98-0 PE6-6 , 066-2 0&9-99 9601 ‘PPV ae aL0Y 00609 0G z 006 6CL-8E F89-0 LOG-G OF9-¢ G9T-T9 G-960T . Ploy £96 00009 61 : 006 008-68 L8L-0 F29-6 Fie gc0-c9 8601 “PPV OL6 0086S fi S 006 L88:-0F GP9-0 GE8-6 68 STL-89 : pau poy ruse ions | Ay s 006 GOS-6P 08-0 O&T-& OGT-L 616-02 8601 UPC; LPOT 00009 9T < 006 OL0-0F {61-0 916-6 co 6P6-99 = ¢-860T “PHV OL6 0066S cI 2 006 600-0F 699-0 OFFS 698-F OLE: LG G-8601 ‘PPV OF8 0086S las . 006 BLL-OP £69-0 TEL 860-9 &FT-09 6C0T PPV £98 0086¢ &1 . , 006 PIL-OP T6L-0 T8L-@ Le L6L-19 O&0L PDV 006 0086S 6L ee S| EL | ey oe oe. aes u — “nayey asoon[y _ fo, wy "Rad () "plow ou “OFd [840], ‘oULIO[ YO *Su9}1, BUI pos [BIOT, - ‘oul Ay “MOORE Nov., 1885. 23 TRANS. CONN, Acap., Vou. VII. 178 Chittenden and Whitehouse—Sulphate on Metabolism, These points of difference are all shown more clearly in the follow- ing table of averages : Average of Normal Urine under the 5 urine. influence of glucose, Total quantityurines--=-2sse+eeseee 1030 c.c. 938ie7c: Sp. @8. b Ate eo ode oe soe ee eee 1027-9 1028 otal solid#matters= 22. =) ee soeeeee = 68°97 grams. 63°62 grams. Chlorine: 4 So2c6 oe wee ee eee 6°78 6°31 Total: Ps Opete . abcess Mae ea ee 3°00 2:15 Uiriemeid a3 2 eee eee oe 0°88 0°72 Wreal st St ase es ee eae eee Se 45°47 40°94 Coming now to the point at issue, viz: the relation between the amounts of urea and phosphoric acid excreted, we find that under the influence of glucose the average diminution of urea amounts to 10 per cent., while the average diminution of phosphoric acid under the same conditions is 8°34 per cent. With cinchonidine, on the other hand, the average diminution of urea amounts to but 8°8 per cent., while the average diminution of phosphoric acid under like conditions is 11°9 per cent. Or, if we take the average of the three days following the last dose of cincho- nidine, when both urea and phosphoric acid reach their maximum diminution, and compare these results with the average of the nor- mal excretion we see that while the diminution of urea amounts to 10°4 per cent., the average diminution of phosphoric acid is raised to 18°97 per cent. Consequently, it would appear that while cincho- nidine lowers the rate of decomposition of proteid matter-in the body, it also has an effect upon the decomposition of some phospho- rized principles, that being the only plausible explanation of the increased diminution of phosphoric acid noticed under the influence of the cinchonidine salt. ‘ Pid a eee atid XITI.—Txse Post-mortem Formation oF SuGAR IN THE Liver, IN THE PRESENCE OF PEptonres. By R. H. Cuirrenpen AND ALEXANDER Lampert, B.A., Pu.B. CLAupE Brrnarp’s discovery in 1848, that the liver contains sugar, both before and after death, led at once to the inquiry as to the source of the sugar. This was apparently answered by Bernard’s later discovery of glycogen, an amylaceous body readily convertible into sugar by acids and various ferments. Thus, Bernard’s theory that the liver sugar resulted exclusively from glycogen has long been an accepted fact. In 1880, however, Seegen and Kratschmer in the first of a series of investigations,* state that the sugar formed in the liver does not have its origin, as supposed by Bernard, wholly in gly- cogen but that it is undoubtedly formed in part from other material. In a later communicationt the same investigators show, in corrobora- tion of their previous statement, 1. that the amount of sugar in the liver is increased very rapidly after death, in one case nearly 50 per cent. of the entire amount being formed within 10 minutes, while the whole process comes to an end inside of 24 hours; 2. that the glyco- gen formed in the liver is much more resistant to ferment action than has hitherto been supposed and that consequently the post-mortem formation of sugar by the action of a ferment upon glycogen could not take place so rapidly as the above. Moreover, direct experi- ments with dogs and with rabbits showed that in the first few hours after death, there was but little if any diminution in the amount of glycogen. Hence, Seegen and Kratschmer claim that the amount of glycogen remaining essentially the same, while the amount of sugar is greatly increased, tends to ‘show conclusively that the liver sugar must be formed from some other material than glycogen and they venture the opinion that this source, whatever it may be, furnishes all of the liver sugar. Boehm and Hoffmann,{ however, take exception to the views of Seegen and Kratschmer, claiming possible analytical inaccuracies from the methods. of procedure. They show, moreover, by experi- * Ueber Zuckerbildung in der Leber. Pfliiger’s Archiv, vol. xxii, p. 236. { Pfliger’s Archiv, vol. xxiv, p. 467. { Ueber die postmortale Zuckerbildung in der Leber. Pfliiger’s Archiv, vol. xxiii, p. 205. 180 Chittenden and Lambert— Post-mortem Formation ments on cats and dogs, that after death, contrary to the state- ments of Seegen, increased formation of sugar is attended with a cor- responding decrease of glycogen, at least within such limits as are incident to the errors of experiment ; further that in the case of a cat’s liver 32 per cent. of the liver glycogen disappeared in 24 hours after death, thus indicating less resistance to the action of ferments than would be implied by Seegen’s and Kratschmer’s results. In a later investigation,* Seegen shows that pieces of finely divided liver, kept in contact for an hour or longer with a solution of pep- tone yield a larger amount of sugar and even of total carbohydrates, than equal weights of the same liver under like conditions of treat- ment, without peptones. These results were obtained with the livers of calves, rabbits and dogs. Seegen, therefore, concludes that the liver is capable of forming from peptones, sugar and earbo- hydrates which are convertible into sugar. A study of the analytical data plainly shows that the increase in sugar and total carbohydrates in the presenee of peptone, although pronounced, is not great. The following experimentt with a calf’s liver obtained from the market shows the most marked increase. With peptone. Without peptone. Time of the Total vy iro Total No. experiment. Sugar. carbohydrates. Sugar. carbohydrates. ia 30 minutes 3:'844% 9°52 % 340% 88 & Il. 48 hours 3°56 8°92 3°70 86 Ju, 96 ~ 2°66 8-00 2°82 78 Here the increase in total carbohydrates is seen to be only 0°72 per cent. and of sugar only 0-44 per cent. after 30 minutes. In Nos. [I and III, longer standing in contact with the peptone tends to reduce the amount of sugar and to diminish the increase of total carbohy- drates. This is attended with increase of acidity and Seegen con- siders that a portion of the sugar is decomposed in this long contact with peptone with formation of acid. In a still later communication,{ Seegen reports the results of other experiments tending to confirm his theory of the formation of carbo- hydrate matter from peptones in the liver. Thus, by feeding peptones to dogs, Seegen found that the content of sugar in the livers of eight dogs was considerably greater that in the normal liver, taking for the latter value the average of a number of determinations. * Die Hinwirkung der Leber auf Pepton. Pfliiger’s Archiv, vol. XXV, p. 165. + Pfliiger’s Archiv, vol. xxv, p. 171. { Pepton als Material fiir Zuckerbildung in der Leber. Pfliiger’s Archiv, vol. xxviii, p. 99. of Sugar in the Liver, in the presence of Peptones. [81 Likewise, by the injection of peptone solutions directly into the portal circulation of dogs, Seegen found the amount of sugar in the liver increased two and even nearly three times above the normal amount. Lastly, by warming portions of freshly excised liver at 40° C., with a solution of peptone in water and some fresh, defibrinated blood, through which a constant current of air was made to pass, the amount of both sugar and total carbohydrates was con- siderably greater than under like conditions, but without peptones. The following experiment* taken from Seegen’s account, illustrates the average increase of carbohydrates under this method of treatment. Two portions of a dog’s liver taken 15 minutes after death, were mixed with 50 c.c. of water and 50 c.c. of defibrinated blood. To one portion 5 grams of peptone were added and air passed through the mixture for 5 hours. Following are the results obtained in both: Wt. of portion Liver Total of liver. Method of treatment. sugar. carbohydrates. Glycogen. 40 grams. without peptone and blood, 3°04 G 69% 2°12 4 (Dai with peptone and blood, 3°87 8-4 2°02 Other experiments indicated that peptones themselves are without diastatic action and that the blood and air (to form oxy haemoglobin) are by themselves without influence on the liver. Hence Seegen concludes that the liver cells, retained in a living condition by the action of blood rendered arterial by a current of air, are capable of forming from peptone more or less sugar; thus establishing, if true, that the animal organism is able to form carbohydrates from albu- minous material. This is certainly a very important question, for if Seegen’s views are correct they overthrow the long accepted belief in the origin of liver sugar in the hepatic glycogen. It is true. that Bernard himself, before his discovery of glycogen, thought that the liver sugar origi- nated in albumin and there have always been, up to the present time, difficulties in explaining the origin of liver carbohydrates on the dehydration theory alone. As is well known, a certain amount of glycogen is formed during a purely animal diet and in chronic cases of Diabetes the excretion of sugar is continued even on a pure albu- minious diet. Moreover, the suggestion has been before made that peptones in their passage through the liver undergo change. Thus Plosz and Gyergyait noticed that while considerable peptone was to be found in the blood of the mesenteric veins and more or less in * Pfliiger’s Archiv, vol. xxviii, p. 123. + Ueber Peptone und Ernahrung mit denselben. Pfliiger’s Archiv, vol. x, p. 536. - 4 . 182 Chittenden and Lambert— Post-mortem Formation the liver, only the merest trace was to be found in the blood of the hepatic vein, indicating thereby a decomposition of peptone in its passage through the liver. Maydl* claims that since the products of the decomposition of all forms of glycogen are the same, it follows that the glycogens them- selves are all identical, and since it is extremely improbable that the various carbohydrates with their different chemical constitutions should give one glycogen, he argues that it all must come from one source, viz: albumin. This is not the place, however, to discuss the relative merits of the dehydration and storage theories, it is enough simply to under- stand that the possible origin of liver sugar in proteid matter is one which would make clear many hitherto unexplained points. The great obstacle, has been to understand where and in what manner the liver sugar could be so formed. Seegen’s views therefore are of great importance, and are, moreover, in no sense, wholly inconsistent with previous ideas, but the question at once suggests itself whether the analytical data on which they are founded are sufficient to war- rant their adoption. The determination of sugar in organic fluids is not without diffi- culty, and where slight variations in results may cause differences of half a per cent. or more, it becomes an extremely delicate matter to determine how far such results shall be trusted. Consequently, what- ever may be said as to whether the formation of sugar in the manner indicated by Seegen is a natural or an artificial process, we need first of all to know positively whether the liver under any circumstances is able to form sugar or other carbohydrate matter from peptones. This all hinges on the accuracy of Seegen’s results, obtained by warm- ing portions of liver with peptones. If an increase of sugar and total carbohydrates is found in the presence of peptone, then we must con- clude that the latter has at least some influence on the formation ol the liver sugar. Recent experimentst have plainly shown that neutral peptone has a stimulating influence on the amylolytic action of ptyalin of saliva and diastase of malt ; both of these ferments convert more starch into sugar in the presence of peptone than without and it is natural to suppose that the presence of peptone would similarly affect the amylolytic ferment which presumably acts upon glycogen. Seegen’s results, however, appear to show that while sugar is increased * Zeitschrift fiir physiol. Chem., vol. iii, p. 196. Ueber die Abstammung des Glykogens. + Trans. Conn. Acad., vol. vi, p. 343, vol. vii, p. 44. —— of Sugar in the Liver, in the presence of Peptones. 183 in the presence of peptone, glycogen remains nearly stationary, or if diminished, not at all in proportion to the increase in sugar. Boehm and Hoffmann, however, found the liver glycogen much less resistant and that its decrease was in proportion to the increase in sugar. Delprat,* too, came to similar conclusions and could obtain no proof whatever, of the correctness of the views advanced by Seegen and Kratschmer. We have, therefore, in view of the importance of the subject, undertaken a study of the question in the hopes of throwing some additional light upon the matter. In this, however, we have limited ourselves entirely to a study of the post-mortem formation of sugar and carbohydrates by the liver in the presence of peptones. Methods employed. The animals experimented with, mainly rabbits, were killed by severing the jugular vein, the blood being collected and_defibrin- ated. The liver was quickly taken out, the gall bladder removed and the liver then converted into a fine pulp by chopping, since it is probable, as v. Wittich has suggested, that glycogen is unequally distributed through the liver. Two equal portions of the sampled and finely divided liver were accurately weighed out and placed in separate flasks ; one, with a solution of peptone and a known volume of blood, the other with an amount of distilled water equal in vol- ume to that of the two former. Both were then placed in a bath and warmed at 38-40° C. for the time of the experiment. A con- tinuous current of air was made to pass through the blood solution in order to render it arterial. At the end of the experiment, the mixtures were poured into boiling water and extracted as long as a trace of glycogen could be detected in the fluids, by the iodine test. This usually took abont two days, working on an average with 40 grams of liver. At the beginning of the extraction, the tissue was generally boiled with 400-500 ¢. c¢. of water for about fifteen minutes and then filtered through a funnel plugged with absorbent cotton. By repeating this operation four or five times, the greater portion of glycogen could be removed, but a complete extraction could be~ ob- tained only by long continued boiling with fresh quantities of water or long heating on the water-bath, the tissue being ground up occa- sionally in‘a suitable mortar. The various filtrates were evaporated on a water-bath and finally united and made up exactly to 500 «. ¢., after which the extracts were filtered through dry paper filters to * Jahresbericht fiir Thierchemie, 1881, p. 321, 184 Chittenden and Lambert— Post-mortem Formation remove any traces of suspended matter which might have passed the cotton. Of these fluids, 200 ¢. ¢. of each were used for the deter- mination of glycogen and sugar, and 200 ¢. ¢. also, for the determi- nation of total carbohydrates. Determination of glycogen and sugar.—The 200 ¢. ¢. of fluid for the determination of glycogen and sugar were evaporated to a small bulk and then, when cool, precipitated by a large volume of alcohol. After standing 24 hours the clear supernatant fluid was filtered from the precipitated glycogen and peptones. The alcoholic filtrate and washings, containing the sugar, were then evaporated, the residue dissolved in water and made up to 100 ¢.¢., in an aliquot portion of which the sugar was determined gravimetrically, by Allihn’s* improved method. The precipitate of glycogen, with its frequent admixture of pep- tone, was dissolved in water, the solution made up to 200 ¢. c. and then sufficient 10 per cent. hydrochloric acid added to make the solution contain 2 per cent. HCl. The mixture was then heated in a closed flask at 100° C. for 17 hours in order to convert the glycogen into dextrose, after which the solution was neutralized, concentrated somewhat, again made up to 200 ¢. ¢. and in an aliquot portion of this fluid, dextrose was determined by Allihn’s method, from which was calculated the amount and percentage of glycogen. Delpratt states that in attempting to determine glycogen by Briicke’s method he found the results considerably higher than when the isolated gly- cogen was converted into sugar by boiling with acid and thé glyco- gen calculated from the data obtained. In our own experiments, the frequent presence of peptone prevented entirely the use of Briicke’s method. 12 hours heating at 100° C., however, with 2 per cent. hydrochloric acid was found in our case insufficient to completely convert the glycogen into dextrose, while 17 hours was found amply sufficient for complete conversion and at the same time allowed no decomposition of the sugar formed. This is well illustrated by the following experiments : A. 0°7665 gram pure, dried glycogen dissolved in 100 c. ¢ of water, was heated at 100° C. for 12 hours with sufficient hydrochloric¢ acid to make the entire fluid contain exactly 2 per cent. The solution was neutralized, care being taken that the reaction did not become alkaline, then concentrated and finally made up to 50 ce... 14 c.c. gave 0°4215 gram Cu=0°2251 gram dextrose=0°2025 gram glycogen. 4 © 90-4933“ ~Gu=e0-2263 “! “ ==0:2036 kt * Zeitschrift fiir analytische Chemie, xxii, p. 448. + Jahresbericht fiir Thierchemie, 1881, p. 322. ." od >y RR I. ES eee of Sugar in the Liver, in the presence of Peptones. 185 The 14 c. c. should have contained 0°2414 gram dextrose, the equiv- alent of 0°2146 gram of glycogen. B. 0°6190 gram glycogen dissolved in 100. ¢. of water was heated at 100° C. for 17 hours in the presence of 2 per cent. of hydrochloric acid. Solution was then neutralized, evaporated and made up to 50 e.e. 18 c.c. gave 0°4585 gram Cu=0°2470 grain dextrose=0°2223 gram glycogen. 18 “ “ 04555 “ Cu=0°2454 uf =0°2208 i The 18 c. c. should have contained 0°2475 gram dextrose, equal to 0°2228 gram of glycogen. Hence, it is seen that 17 hours heating at 100° C. is needed for a complete conversion of glycogen into dextrose, which was the time invariably employed in the after experiments. Influence of peptone on the conversion of glycogen into sugar by 2 per cent. HCl at 100° C.—The question naturally suggested itself, in this connection whether the presence of peptone would interfere in any way with the complete conversion of glycogen into dextrose or whether the peptones by this long heating at 100° C. with the acid, would undergo any change by which reducing bodies might be formed and thus endanger the accuracy of the results. The latter point was tested by heating 2 grams of peptones in 100 ec. ¢. of water containing 2 per cent. of hydrochloric acid for 17 hours at 100° C., at the end of which time no reduction at all could be obtained with Fehling’s solution. ; The first point was tested by the following experiment: 09290 gram of pure glycogen was dissolved in 100 ¢. ¢. of water, then 2 grams of peptone were added and sufficient acid for the solu- tion to contain exactly 2 per cent. HCl, after which the mixture was heated at 100° C. for i7 hours. The solution was then neutralized, brought to a volume of 100 c. c. and the sugar determined. 10 ¢. ce. gave 01985 gram Cu=0°1017 gram dextrose=0'0915 gram glycogen. TO ces 072025, S° “Cu—0°1039 =0°0934 Ghd ‘ whereas in the 10 c. c. then should be present, according to caleu- lation 0:1032 gram dextrose, the equivalent of 0°0929 gram of glycogen. Consequently the presence of peptone does not interfere with the accurate determination of glycogen by this method. Influence of the presence of peptone on the determination of sugar by Allihn’s method.—Seegen* finds that the volumetric determina- tion of sugar with Fehling’s solution is not materially affected by the presence of peptone. By repeated experiments we have con- vinced ourselves, that in the use of the gravimetric method, the * Pfliiger’s Archiv, vol. xxviii, p. 115. TRANS. Conn. Acap., Vou. VII. 24 Nov., 1885. 186 Chittenden and Lambert—Post-mortem Formation . presence of peptone may, unless certain precautions are taken, inter- fere slightly with exact determinations. With the Allihn method, variations of 2—5 milligrams in the amount of reduced copper are liable to occur if care is not taken in regulating the length of time the alkaline copper solution is heated after addition of the sugar solution. Under ordinary circumstances results most nearly in ac- cord with theory are obtained by adding the sugar solution, as recom- mended by Allihn, to the previously heated Fehling’s solution and then heating further until bubbles just begin to break upon the surface of the liquid. If heated longer, even only half a minute, a slight increase in the amount of reduced copper will generally be observed. Now whenever peptone is present to any extent in the sugar solution, we have found by experience that complete reduc- tion does not take place quite so rapidly ; the loss is not great, some- times but a milligram or so, still the difference is appreciable. This, however, can be avoided by simply allowing the standard copper solution to boil for about 45 seconds after the addition of the sugar solution. Under such conditions, repeated trials have shown us, that the presence of peptone does not offer the slightest obstacle to aceu- rate determinations of dextrose. Whenever, therefore, in the follow- ing experiments the solution to be tested contained peptone, the above rule has been invariably followed. Determination of total carbohydrates.—For this purpose 200 e. ¢. of the liver extract were heated in a closed flask at 100° C. with sufficient 10 per cent. hydrochloric acid to ensure a content of 2 per cent HCl, for 17 hours. The solution was then nearly neutralized, care being taken that the fluid did not become alkaline, concentrated and finally brought to a volume of 200 ¢. ©, in an aliquot portion of which the total carbohydrates in the form of dextrose were deter- mined in the usual manner. Seegen* states that in the determination of total carbohydrates, the fluid, after heating with acid, always became very dark, which occasionally interfered somewhat with the determination of sugar, Delprat,t however, states that in bis experiments the solution, under like conditions, became brownish yellow and generally deposited a flocculent brownish black precipi- tate of organic matter. Moreover, in some cases, particularly with the livers of dogs, cats and calves, the cuprous oxide, in determining total carbohydrates, would remain dissolved to a great extent, thus interfering with the accuracy of the volumetric determination, some- * Pfliiger’s Archiv, vol. xxviii, p. 121. + Jahresbericht fiir Thierchemie, 1881, p. 323-324. of Sugar in the Liver, in the presence of Peptones. i87 times to the extent of even 1-2 ¢. c. of the sugar solution. In our experiments the acid solution was usually yellow or yellowish brown, and invariably at the end of the 17 hours contained the flocculent precipitate described by Delprat. By nearly neutralizing the solu- tion, the amount of this precipitate was considerably increased and on then filtering the fluid, after having made it up toa volume of 200 ¢. c., considerable organic matter was removed, This, we found, had a decided influence on the accuracy of the determination, since alkaline solutions of this neutralization precipitate appeared to deci- dedly retard separation of the cuprous oxide. By paying attention to this point, we had no difficulty in obtaining fairly concordant results, by the use of the Allihn gravimetric method. Experiment 1. A large sized rabbit was killed, the blood collected and defibrina- ted, the liver quickly removed and finely chopped. Two portions of 40 grams each were weighed out and treated as follows : A. B. 40 grams liver. 40 grams liver. 50 c.c. of a 10% solution of peptone. 95 ¢c.c. of water. 25 e.c. of blood. 20 ec. c. of water. These were placed in flasks, and warmed at 40° C. for two hours. The liver was in contact with the peptone 40 minutes after the death of the animal. A continuous current of air was kept passing through A. Following are the analytical results: Glycogen ; total volume of the resultant sugar solution 200 c. c. Sugar; total volume of the solution 100 c.c. Total carbohydrates; volume of the resultant solution 200 ec. ec. Glycogen A. Volume Equivalent Equivalent : used. Weight Cu. in dextrose. in glycogen. Total amt.* Per cent. 25 ¢.¢. 0°2345 gram. 071209 gram. 01088 gram. 0°8704 gram. 544 25 0°2360 071217 0°1095 0°8760 5°47 Glycogen B. 25 cc. 0°2675 gram. 0°1386 gram. 0°1247 gram. 0°9976 gram. 6:23 25 0°2659 01377 07123 0-9912 6°19 Sugar A, 25 ¢. ¢. 0°2260 gram. 01164 gram. ——----- 0°4656 gram. 2°91 25 0:2255 UISTLTG SA Ae ee ho 0°4652 2°90 * Total amount of glycogen, dextrose or carbohydrates calculated as dextrose, contained in the above volume (100 or 200 ec. c.) and representing, therefore, the amount contained in two-fifths of the 40 grams of liver. 188 Chittenden and Lambert— Post-mortem Formation Sugar B. Volume Equivalent used. Weight Cu. in dextrose. t Total amt. Per cent. 25 @. C. 0°2135 gram. 0°1097 gram. 04388 gram, 214 Total Carbohydrates A. 12°5 c.¢. 0°2160 gram. O-1111 gram. 17776 grams. 11°10 20 0°3367 01768 17680 11°05 Total Carbohydrates B. 25 ¢. Cc. 0°4030 gram. 0°2146 gram. 1'7168 grams. 10°73 25 04042 0°2153 1°7224 10°76 The following table shows the average percentage results : Amount of = Total liver taken. Method of treatment. Glycogen. Sugar. carbohy@rates. 40 grams. With peptones and blood (A), 5°46 J 2°91 % 11:08 4 40 Without peptones and blood (B), 6°21 2°14 10°75 —0°75 +0°17 +0°33 From this it is seen that while in the presence of peptone and blood there is a slight increase of both total carbohydrates and sugar, there is also a more than corresponding decrease in the percentage of glycogen. . Hxperiment II. / Liver of a rabbit, removed directly after death and treated in the same manner as in Experiment I. A. B. 40 grams sampled liver. 40 grams sampled liver. 50 c.c. of a 10 per cent. peptone solution. 145 ec. c. of water. > 25 grams blood. 70 e.c. of water. Warmed 2 hours at 40° C., with a current of air passing through A. Glycogen A. Volume Equivalent Equivalent Total Per used. Weight Cu. in dextrose. in glycogen. amount. cent. 25 ©. ¢. 03170 gram. 0°1659 gram. 0°1493 gram. 11944 grams. 746 Glycogen B. 25 cc. 0°3420 gram. 0-1798 gram. 0°1618 gram. 1°2944 grams. 8:09 Sugar A, 25 ¢. ¢. 0°2520 gram. O2S03 cram) eee 0°5212 gram. 3°26 Sugar B. 10. ¢; ¢. 0-0865 gram. 0:0443. cram, = eee 04410 gram. 2°75 Total carbohydrates A, MOEN: 0°2205 gram. 0234 grams 99 ee 2°2680 grams. 14°15 Total carbohydrates B. UOKeNC; 0°2110 gram, O;LO84ipram? = 9 = eeee- 2°1680 grams. 13°55 - Ss of Sugar in the Liver, in the presence of Peptones. 189 Amount of . Total liver taken. Method of treatment. Glycogen. Sugar. carbohydrates. 40 grams. With peptones and blood (A), 746 % 3°26 % 14°15 % 40 Without peptones and blood (8B), 8:09 20D 13°55 — 0°63 +0°5] + 0°60 Experiment I. A small rabbit, treated in the same manner as the preceding : A, B. 25 grams sampled liver. 25 grams sampled liver. 50 c. c. of a 10 per cent. peptone solution. 125 c. c. of water. 50 grams of blood. 25 c.c. of water. Reaaed for 2 hours at 40° ©, with a constant current of air passing through A. Glycogen A. Volume Equivalent Equivalent Total Per used. Weight Cu. in dextrose. in glycogen. amount. cent. 25 ¢c. ec, 0°0435 gram. 0:0226 gram. 0:0203 gram. 0°1624 gram. 162 25 90455 00236 0°0212 0°1696 1:69 Glycogen B. 25°C. C. 0°0425 gram. 0°0221 gram. 00198 gram. 0°158+4 gram. 1:58 25 0°0405 0-0211 0°0189 0°1512 1:51 Sugar B.* 25 ¢. ¢. 0°1413 gram. O:0/e Oh oram yes a= = 0°2876 gram. U 25 01400 © 00713 4 stages Ge 0°2852 2°85 Total carbohydrates A. 25 ec. 0°1560 gram. O09 Gjerames eee 0°6368 gram. 6°36 25 071585 005095 2 eee 0°6472 6°47 Total carbohydrates B. 25 ©. ¢. 0°1445 gram. O;0cS6rerams 7 asa —= 0°5888 gram. 5:88 25 01427 0-0726 eS 0°5808 5°80 Following are the average percentage results: 3 Amount of Total liver taken. Method of treatment. Glycogen. Sugar. carbohydrates. 25 grams. With peptones and blood (A), 1-65 % Leas 6°42 % 25 Without peptones and blood (B), 154 2°86 % 5°84 +0°11 +0°58 In this experiment there is the same slight increase of total carbo- hydrates in the presence of peptone noticed in the two preceding experiments. The increase, however, is not great, and it suggests at once the question, whether the airenicer although constant, are * Sugar A was lost, 190 Chittenden and Lambert— Post-mortem Formation beyond the ordinary limits of error. This question we have en- deavored to answer in the next experiment. Experiment IV. A rabbit’s liver removed from the body immediately after death, was prepared in the usual manner. Two mixtures, exactly alike, were then made as follows: A. B. 25 grams of liver. 25 grams of liver. 100 c. c. of water. 100 c.c. of water. These were im the bath 23 minutes after the death of the animal and were warmed at 40° C. for 2 hours. The two portions were then extracted and analyzed as in the preceding experiments; the object being to see how great a variation would be* obtained by this like treatment of the two portions of sampled liver. Following are the results : Glycogen A. Volume Equivalent Equivalent Total Per used. Weight Cu. in dextrose. in glycogen, amount. cent. 25 ¢. ¢. 0°1575 gram. 00802 gram. 0°0721 gram. 0°5768 gram. 5°76 Glycogen B. 25 ¢. ¢. 0°1585 gram. 0°0809 gram. 0°0728 gram. 0°5824 gram. 5°82 Sugar A. MORCHC: 0°0433 gram. 0:°0225 gram. .. ----- 0°2250 gram. 2°25 Sugar B. . l0vexe: 0°0430 gram. 0:0224 sram; 5) eee = 0°2240 gram. 2°24 Total carbohydrates A. 25 ¢. ¢. 0°2340 gram. 0°1207 gram. aces 0°9656 gram. 9°65 Total carbohydrates B. 25 ©. c. 0°2335 gram. OAL20SseTaTitas ese 0°9624 gram, 9°62 Percentage results. Glycogen. Sugar. Total carbohydrates. A 5°76 per cent. 2°25 per cent. 9°65 per cent. B. 5°82 2°24 9°62 —0:06 +001 +0°03 These results plainly show that when the conditions of the exper- iment are exactly the same, the average variation in results will be considerably less than 0:1 per cent. Consequently variations greater than this must have their origin in something other than the ordinary errors of analysis. Hence, in the three preceding experiments we have to account for an average increase of about 0°5 per cent. in of Sugar in the Liver, in the presence of Peptones. 191 total carbohydrates in those cases where peptone and blood are both present. A comparison of Seegen’s results* show that while an aqueous solu- tion of peptone alone in contact with fresh liver increases somewhat the percentage of both sugar and total carbohydrates, the addition of blood, kept arterial by the passage of a current of air through the fluid, appears to still further increase the percentage of sugar aud carbohydrates. Seegen, moreover, shows by a blank experi- ment, that blood alone in contact with the liver has no more influence on the formation of carbohydrates than distilled water. Experiment V. This experiment was tried mainly to see what influence peptones by themselves in the absence of blood, would have on the forma- tion of sugar and total carbohydrates. ‘Two portions of sampled liver from a largé rabbit were treated as follows : A, B. 50 grams liver. 50 grams liver. 50 c. c. of water containing 2 grams of peptones. 50 c.c. of water. The solution of peptone was poured over the liver just 45 minutes after the death of the animal. The mixtures were placed in a bath at 40° C. for 3 hours, after which they were allowed to stand at the temperature of the room for 21 hours. They were then extracted and analyzed in the usual manner, with the following results : Glycogen | 9G-TS | TL68-0 | 06-9 | 0868-0 WALF-0 II $ eh vee hie Se gies Sects Nese "TTT fare fares | os" | @@-TG] 2998-1 | 28:9 | SPPF-0 | 688h-0 | 1 3 s - ‘meds 2 60) aks cd eat q ce 2°90 2 mei a mes “TURIS ~ a “punoj 5 qian uosnp | 5 a punoj “5 panoy ‘pesn ‘ON < ee aoe 'Oseg | ‘punoj X *09 OEE ag! eae a 212 Nithne and Chittenden— Globulin and Globulose Bodies. Globulose Bodies. After removal of the above mentioned coagulum, the solution re- mained perfectly clear at all temperatures up to 100° C., even when rendered more strongly acid and also on subsequent neutralization. On the addition of nitric acid, however, to the cold solution, a pre- cipitate was formed which disappeared on the application of heat, reappearing as the mixture became cool. Crystals of salt alone, produced a heavy precipitate in the solution, while salt and acetic acid gave a still further precipitate, and when these reagents failed to cause any further precipitation, nitrie acid or metaphosphorie acid would still give a noticeable turbidity. In order to separate the globulose bodies from one another, the entire solution was concentrated on a water bath to the consistency of a thin syrup and then rubbed up in a mortar with salt in sub- stance (the fluid being perfectly neutral), complete saturation being insured by long standing with an excess of salt crystals. The pre- cipitate so formed being separated by filtration, the filtrate was par- tially precipitated by the cautious addition of 30 per cent. acetic acid saturated with salt, whereby a mixture of proto- and deutero- globulose was separated. After removal of this precipitate the filtrate was finally treated with more of the above acetic acid until nothing further was precipitated. The various precipitates were then subjected to strong pressure to remove as much of the salt-saturated fluid as possible, then dissolved in water and dialyzed for the complete removal of the salt and to separate heteroglobulose. Protoglobulose. This body, precipitable by sodium chloride alone, was purified by saturating the first dialyzed solution again with salt, then dialyzing a second time under repeated changes of reaction, by the alternate addition of acetic acid and sodium carbonate and finally by neutral reaction, until all chlorine was removed from the solution and the admixed heteroglobulose completely separated. The clear filtered fluid was then concentrated, after which the protoglobulose was pre- cipitated with alcohol, washed with alcohol and ether and so ob- tained as an almost white powder. The substance, so prepared, gave when rubbed up with cold water, a filtrate not quite clear and with a noticeably alkaline reaction. It differed from a solution of proto- albumose from fibrin in one respect, viz: that on boiling in the pres- ence of a small amount of sodium chloride it beeame quite turbid, Kiihne and Chittenden— Globulin and Globulose Bodies. 213 the turbidity, however, disappearing completely as the solution be- came cool, (the opposite of the albumose reaction : compare Zeitschrift fiir Biologie, Band xx, p. 45), We think it may be assumed that this single deviation from the reactions of fibrin-protoalbumose is not due to the presence of impurities (heteroglobulose, ete.), because no heteroglobulose whatever separated from a portion of the sample, even after dialyzing a week longer. If the solution was made even very slightly acid or alkaline, the turbidity did not then occur on heating. Further, the small content of ash (0:4 per cent.), which con- sisted. only of calcium sulphate with a trace of ferric oxide, testifies to the purity of the preparation and the completeness of the dialysis. The preparation was analyzed with the results shown in the accom- panying table. Deuteroglobulose. This body is about as difficult to purify from the preceding one as deuteroalbumose from protoalbumose. We succeeded, however, in separating it by rejecting the first portions of the precipitate pro- duced by acetic acid and sodium chloride and using only the last portions precipitated; or after the acetic acid failed to give any further precipitate, by using the small! precipitate produced by the moderate addition of alcohol. This last precipitate naturally enclosed considerable sodium chloride, but the deuteroglobulose was obtained perfectly pure after removing the salt by dialysis, since the globulose solution, even when noticeably acid, gave no turbidity whatever on the addition of salt in substance. The quantity, however, was unfortunately too small for analysis. It suf- ficed only for determining the reactions, which agreed with those of the substance obtained by the later precipitation with acetic acid except in one particular, viz: that the latter preparation in a neutral or slightly alkaline solution showed an extremely slight turbidity on the addition of crystals of sodium chloride. All other reactions were identical and corresponded so completely with those of deutero- albumose that it is only necessary to call attention to the latter (see Zeitschrift fiir Biologie, Band xx, pp. 26-28, or Amer, Chem. Jour., vol. vi, pp. 46-47) and to especially mention the non-precipitation of deuteroglobulose in a solution free from salt, by nitric acid in any quantity and at any temperature. Since no heteroglobulose whatever separated from the solution of the last acetic acid precipitate during dialysis, even with repeated ‘change of reaction, the substance was therefore prepared for analysis, Kithne and Chittenden— Globulin and Globulose Bodies. 214 00-001 IT&E ~ ve a . aN ae TA. O 06-6 16-6 06-6 26 pals va coy oe ae S - 60-91 ee ae OT-9T 60-91 aoa ee ae apy Bidar || keer eo are Oy aes 9EFL-O A 3 eo — ee §0-91 | PI-8GL | 6-61 | $-6E | ~~~ 4 ee ee &1EP-0 | Al — aan py se GO-9F | 9-892 | 8-8T | 8-6 es ane eet | eer: 86EC-0 Ill Ek i.) rae wes Sa 7H ~~ | ~""" | 8F-TS | 0086-0 | @6-9'| 0208-0 9667-0 II | | Le: one ors Rioy ee Sa Renee Saar ST-TS | LLGG-T | 16-9 TISP-0 4049-0 | I of mes of “TRIS of “wu 40) al) <5) 0 of mana of meis ‘weds | ‘i anne n “ONS + HOW 2 aINssalg ap ay Fi sacar z “panos eae ON wy [wy | s | aeose | 8 [omy my | 9 | "00 | # | OF | conto | “ASOTAAOTNOLOUd dO SISATVYNY & i Kiihne and Chittenden— Globulin and Globulose Bodies. 215 simply by concentration of the solution freed from chlorine, pre- cipitation with alcohol and washing with ether. The ash (1°17 per cent.) consisted only of calcium phosphate and a trace of sulphate. LTeteroglobulose. This body was obtained from the gummy precipitate which sepa- rated, during dialysis, from the solution of the first precipitate thrown down from the neutralized digestive fluid by salt alone. The sticky mass was separated from the sides of the parchment tubes, dissolved in sodium chloride of from 3 to 5 per cent., repre- cipitated by saturation of the solution with salt, the precipitate again dissolved in dilute salt solution and the substance finally separated by long continued dialysis in running water. After thorough wash- ing with water, alcohol and ether it appeared as a light, white powder, not unlike heteroalbumose in general behavior and reac- tions. After each precipitation and treatment with dilute sodium chloride, heteroglobulose -left a residue, which like dysalbumose was readily soluble only in dilute acids. From the following analysis it is to be seen that the preparation, in spite of its long continued and repeated dialysis, contained 2°03 per cent. of ash, which con- sisted mainly of calcium carbonate with a small amount of phos- phate and sulphate. The composition of the three globulose bodies shows the same slight differences as noticed in the case of the various albumose bodies (from fibrin). Unlike the latter, however, the content of car- bon in the globulose bodies never falls below 51 per cent., and fur- thermore it is always higher than that of the globulin from which the globulose was derived. The percentage of nitrogen, which in the albumose bodies was found a little higher than in fibrin, exceeds that of the globulin more yet, in some cases by more than 1 per cent., and the same holds true of the percentage of sulphur. In contrast to the albumose bodies, the percentage composition of the globulose bodies gives no grounds whatever for the assumption that they arise {rom the digested globulin by simple hydration. It must not be for- gotten, however, that the digestion of globulin by gastric juice is a process quite different from that of fibrin digestion and one hitherto much less clearly understood, since besides the globulose bodies there is formed a large quantity of a substance which is separated by boiling and which resembles ordinary coagulated albumin. Some- thing similar, indeed, has been known ever since Briicke’s study of fibrin digestion, but it has long been accepted that the coagulum Kithne and Chittenden— Globulin and Globulose Bodies. 216 00-.00T CLES 98-1 16-91 G6-9 6G-1¢ ‘O.0B.10A VW ‘asojngojbouajnaq aawf-yso fo wouprsodmood aboywaolag TL-T 66+ GT 96-9 9g-T¢ ¥6-9 8F-T¢ OWAno LL00-0 9200-0 68-1 69-1 C6-1 “URI % “panoy TSV S *mRIs "ONM + HOM Vim uoIsny 1aqye 'Ogug 1879-0 0618-0 88-9 6FT6-0 98-9 0166-0 meoters We OINSSII J 5 L “‘punoy N mes *punoy “00 of “"meI3 “punoj H O*H L¥P9-0 OFg9-0 IITA GLF9-0 ITA PEIS-0 IA 919F-0 A 90TS-0 SO0FS-0 Til S9Fs-0 II ILLP-0 I “mei3 ‘pesn ‘ON aouRysqng “ASOTOATOTONONALOANG AO SISNIVNY Ohi 00-001 zs a 89-66 San as 9) - aie a) = S IEG S16 06-6 a ee ea re wa Bl S 80-91 a Es. TL-9T 20-91 ei ge 7, § 86-9 = eee a. > GOL c6-9 Ff = OL -é¢ fot ie 3 a Boos C0-6¢ st’eg OD S ‘o3vI0AV S ‘asopnqopbowajzazy aatf-ysp fo uor1rsodwmos abpyUadaq & Ss S 00-6 8600-0 | ~~~ aia 8 peti || eens EG hws, | ear aa wea Ss Lesy-0 «| TIA S 90-6 OGEO-0L | 5 a5 a es al ar eke | aang oP oat Ea a Se ae Tr8¢-0 =| ITA S Siar i a es 80-6 9160-0 sated || See Ss Bate pte eon) li ae S| hPa cp09-0 | IA ~S * ie Sse CLG 6680-0 (aS i a Se ea ae re eee ——s i eae mee LOGG-0 A S Sean Peas ie ST a eer ets 6L-CT | L2-F9h | 9-6T | 40-97 | | <1 aie et 8e7rs-0 | AT B g S <5 ge S| (Name i eae GL:ST | PL-79L | 0-08 | FP-8F)| | Ss otal osama ce9s-0 =| TIT 3 a SS Sa a -s= | ---1- Je-= | /og.g0| oogo0 | 28-0 | opto | oueo | © eS" : : : 5 K a ae tae Pte highs aa ae hg ee ee SLOPES Si Gs0n | 0820) eShesd o0e¢e-0 =| I g en 3 SS ee es —— } = Sy : “TUT “1 ia * : Ss wis weds resona) || OO, “ces ae S “weI8 a = ? ‘punoy Ya ou ee ee L p “punoj ig ‘punoj ‘pasn ‘oN S af mice : ae ‘OSes x ; “punoy N 500 ora cael Z ; ee oe a ‘ASOTOSOTOOUELAY HO SISATYNY 218 Kiihne and Chittenden— Globulin and Globulose Bodies. obtained by Briicke on boiling the neutralized digestive fluid, arose from the globulin present in the fibrin employed, which had not been previously washed with salt water. Globulin, moreover, yields this body in much greater quantity, even after several days’ exposure to the action of an energetic gastric juice and it was still found abund- antly among the products of a second digestion of the first neutral- ization precipitate. | Comparison of the Analyses. ae ; a Sex am Ev | | a | Z BUS os os =e) oa E 2 30 2 ae ee) o5 ong re g wee ae) 50 oo || O26 S| o | ves Me | Ab eh, Aes. ro C 51°14 52°03 DlEDil | DILb 2 52°10 | a, | 50°88 52°68 H 700 6:93 | 6°98 6°95 698 Ht 6°89 6°33 N 14°64 15°89: | 16:09 15°94 16°08 | N 17 08 16°91 Ss 1°67 18 0n 2220 1°86 2°16 S | 1-23 1:10 O 20D Piya) i RPI 23°73 22°68 | O} =| 23°92 22°48 In this review of the composition of globulin and of the products of its digestion we have included also an analysis of fibrin, of a fibrin-albumose and of hemialbumose from the urine of a person with osteomalachia. We call attention again to the latter because its surprising corre- spondence, especially to heteroglobulose, appears to confirm the be- lief, expressed in our former paper that the difference in the albumose from urine and that from fibrin depends on the formation of the former from an albuminous body, whose digestion, at least as regards the formation of albumose bodies, was then unknown, and for which we had already turned to globulin, In order to gain further information concerning the cleavage of globulin in the process of digestion, the remaining material was used in the following experiments. 1, Heteroglobulose dissolved in 0°3 per cent. sodium carbonate and warmed at 40° ©. for fourteen days with pure trypsin (with the addition of thymol, as usual) remained perfectly clear, even after neutralization, and failed to yield afterwards any body resembling antialbumid. Among the products of the digestion, there was found in addition to an abundance of antipeptone, only a trace of leucin, no tyrosin whatever, while with bromine water the alcoholic extract, * Compare Zeitschrift fiir Biologie, Band xix, p, 202. + Ibid., Band xx, p. 40. + According to Hammarsten. Kiihne and Chittenden— Globulin and Globulose Bodies. 219 which had been dissolved in water after driving off the alcohol, became simply a little darker, but not rose-colored or violet. Hence heteroglobulose is to be considered as belonging to the anti group. 2. Protoglobulose, which still contained some heteroglobulose, when treated in the same manner with trypsin, behaved similarly but afforded besides an abundance of leucin, also some tyrosin and an extract which became deep violet on the addition of bromine water. Hence protoglobulose gives evidence of belonging to the hemi group. Finally, we submitted to the digestive action of trypsin the third and fifth precipitates (so-called parapeptone) which were separated . in continually decreasing quantities by neutralization, after renewed, energetic pepsin digestion of the original globulin. Both tailed to yield any coagulum during their digestion with 0°3 per cent. sodium carbonate, and after the trypsin had acted for fourteen days, neu- tralization with acetic acid yielded a heavy precipitate, while consid- erable antipeptone was found in the solution. Although the diges- tion of the third neutralization precipitate still afforded a trace of leucin and tyrosin without giving any reaction with bromine, no leucin, tyrosin or a substance colored by bromine water could be obtained from the precipitate separated after the fifth pepsin diges- tion. Hence globulin, like fibrin and other albuminous bodies, yields dur- ing pepsin digestion at the last only bodies of the anti group, which are peptonized, though slowly, by trypsin, but yield no further cleay- age products. XV.—Perronrs. By W. Ktune anv R. H. Cuirrenpen. Suyce there has been discovered in neutral ammonium sulphate a means for the complete precipitation of the albumose bodies, we have been induced to take up anew our former investigations on the behavior and composition of peptones. As these latter bodies are not precipitated by the ammonium salt, we had expected to obtain peptones free from the primary cleavage products of albumin and thereby advance another step in our knowledge of the definite prod- ucts of the proteolytic action both of pepsin and of trypsin. Re- newed investigation was demanded by the probability that hitherto pepsin-peptones entirely free from albumose have never been ob- tained, for such peptones as are to be found in commerce or in the hands of the most careful investigator of gastric digestion can readily be shown to contain albumose by saturating a solution of the preparation with ammonium sulphate. There will result an abundant precipitate of albumose and a surprisingly small residue of non-precipitated pep‘ ones or the entire absence of such a residue. Only antipeptone obtained by trypsin digestion will occasionally form an exception, and even then in most cases we cannot but doubt that the peptones so formed are wholly free from albumose. In order to be certain of the presence of peptones in a digestive fluid, it must be made slightly acid with acetic acid, rubbed up with ammonium sulphate till saturated and then filtered from the excess of salt and the albumose precipitate. If the filtrate is thereupon treated with a large excess of strong sodium hydrox- ide and then a few drops of very dilute cupric sulphate be added, the appearance of the rosy red color of the biuret reaction will indicate the presence of peptones. If peptones are absent the fluid will be pure blue without a tinge of violet, since the solution can contain no other albuminous body. Even after an apparently energetic pepsin digestion the latter result is not at all rare, and a heavy precipitate by the ammonium salt is so frequently seen, that it is still to be doubted whether there is a pepsin-acid digestion which causes the disappearance of all albumose. On the contrary, the albu- mose precipitate after a sufficiently long and energetic trypsin diges- tion is very slight and peptone is to be found abundantly in the solution. Kiihne and Chittenden— Peptones. Oe ix pa We have endeavored to prepare pure peptones in quantity from the solution saturated with ammonium sulphate. For this purpose the solution was first freed from the greater part of the salt by con- centration and crystallization. During this process a small amount of a nitrogenous substance separated, perhaps albumose formed again from peptones when the solution was vigorously boiled and the temperature rose to 110° C. The mother liquor, after suitable dilution, was boiled with hot saturated baryta water until all ammonia was expelled, during wiich operation the precaution was taken to use no excess of barium hydroxide and thus decompose the peptones. From time to time, therefore, portions were filtered, tested for sulphate, and when this became small in amount the last portions of sulphuric acid were removed by barium carbonate. From the fil- trate, which always contained much barium, the latter was entirely removed by dilute sulphuric acid, either immediately or after a pre- ; vious purification of the barium peptone compound. The peptones were then precipitated with alcohol and occasionally further purified with phosphotungstic acid. Naturally the large amount of ammo- nium sulphate to be removed formed a correspondingly troublesome quantity of barium sulphate, which could be handled only in large filtering bags, and occasioned a large loss of peptone in spite of a most careful washing of the precipitate with boiling water and the application of pressure. On evaporating the peptone solution, which contained but little salt, no resinous precipitate resembling albumose was to be seen. 1. Amphopeptone. We have designated as amphopeptone the end product of the digestion of albumin by pepsin and acid. The first attempt to obtain : this peptone free from albumose and in a quantity in some degree ; proportionate to our wants, showed us that there was needed not only the most active digestive fluid possible and long exposure to a temperature of 40° C. but also a very large amount of pepsin. Such a quantity of the ferment could be procured, however, only by first dissolving considerable quantities of the mucous membrane of the stomach in acid; quantities which must be taken into consideration, in addition to the fibrin to be digested, since something is formed in the self-digestion of the mucous membrane which necessarily remains mixed with the peptone. It is known that mistakes have already been committed by not distinguishing the products arising from the material of the mucous membrane, from those derived from the digested substance. For example, Hoppe-Seyler’s erroneous asser- 222 Kiihne and Chittenden— Peptones. tion that pepsin digestions yield leucin and tyrosin, rests wholly upon this circumstance, for since the digestion of the mucous mem- brane always commences with the disappearance of a mucilaginous substance, the derivatives of the latter must necessarily be expected in the resultant solution. Probably for this reason, artificial gastric juice which has been prepared from mucous membrane and is no longer mucilaginous, gives a precipitate when,treated with alcohol which differs much from the precipitates of albumose and peptone in being almost as elastic as rubber and, as a rule, forming when shaken, a single ball in which the pepsin is then ordinarily inclosed. We have not yet examined this substance closely, since in the course of the investigations to be described, another more suitable method for precipitating and isolating the ferment has been discovered. We shall designate this elastic body for convenience, mucin-peptone. This mucin-peptone might possibly conceal the whole amount of pep- tone expected from the digested fibrin, or remain mixed with the latter in considerable quantity. In spite of this objection, which we at no time lost sight of, we prepared a quantity of fibrin-peptone without attempting to remove or to prevent the mixture in question. The observations made by Dr. Pollitzer* in the Physiological Insti- tute at Heidelberg, on the influence of pepsin-peptone free from albu- mose on coagulation of the blood, were performed with such ampho- peptone, which is not perfectly pure. Supported by the following analyses of this peptone in our pre- umption that it was rendered impure by mucin-peptone, we sought a process that would exclude this impurity. This was found almost of itself after we had noticed that ammonium sulphate invariably precipitated from the acid solutions, in addition to albumose, the entire quantity of active pepsin. While, therefore, nothing capable of digestion with acids could in any way be obtained from the ‘fil- trates, an exceedingly active juice was formed by dissolving the pre- cipitate in dilute hydrochloric acid. Hereafter, we accordingly pre- pared the strong pepsin solution, by simply precipitating large quan- tities of very concentrated gastric juice containing 0°5 per cent. hydrochlorie acid with ammonium sulphate and dissolving the resin- ous precipitate, which did not contain an objectionable quantity of albumose bodies, in fresh dilute acid. By this means the mucin-peptone was gotten. rid¢ of, since it could not be precipitated by ammonium sulphate and thus a new method was found for prepar- ing and isolating pepsin, which we shall enter upon at another time. * Verhandl, d. Naturhist. med. Verein zu Heidelberg, N. F. III, p. 293, 4 Kiihne and Chittenden—Peptones. Dy bo wo 4 1. Amphopeptone prepared with ordinary gastric juice. Gastric juice, prepared from 145 grams of isolated mucous mem- brane from the fundus of pigs’ stomachs by two days self-digestion in two litres of 0°4 per cent. hydrochloric acid, was added to 585 grams of well washed and boiled fibrin, previously swollen in four litres of acid of the same strength and the whole warmed for two days more at 40°C. The thin fluid-like mixture so obtained, was neutralized with sodium hydroxide and then filtered from the undis- solved residue of the mucous membranes (nuclei of the gland cells) and the slight neutralization precipitate. After being made slightly acid with acetic acid, the fluid was heated to boiling, evaporated to two litres, then saturated with neutral ammonium sulphate, separated from the slight coagulum and precipitated albumose, again concen- Bee ee ee trated to one litre and freed from a large portion of the ammonium sulphate by crystallization at 0° C. In order to still further separate the salt, the solution was treated With one litre of absolute alcohol, again placed in the cold, and finally strained through linen to remove the fine powdery salt, which was wholly free from precipitated pep- ———— ee SS Ieee ee ee tone. After having been freed from alcohol, by vigorous boiling and concentration to the consistency of syrup and from much salt by crystallization, the thick fluid was filtered by suction, boiled after much dilution with a large amount of barium carbonate until the odor of ammonia had vanished. From the solution, separated from the barium sulphate and again much concentrated, alcohol precipi- tated the peptone as a barium compound which could be freed from salts (especially sodium chloride) by repeated precipitation and_boil- ing with alcohol. Finally the barium-peptone was decomposed as much as possible with dilute sulphuric acid. As was seen later from the concentrated peptone solution, there remained dissolved a trace of sulphuric acid, but only enough to make the fluid assume a slight opalescence after boiling with barium chloride and hydrochloric acid. An attempt was made to purify the isolated peptone by evaporating, precipitating with alcohol, dissolving in water and reprecipitating with alcohol. This did not succeed well, as shown later by the high percentage of ash. By drying first on a water bath, then in an air bath at 105° C. with frequent stirring, which destroyed the firm resinous surface, the peptone gradually became solid, and changed to a puffed up mass. The resulting product could be ground, when cold, to a light, very hygroscopic powder and weighed in this condi- tion 25 grams. pale > A ieee ; > 4 , | 4 : 224 Kiihne and Chittenden—Peptones. In an attempt to dry the substance for analysis, during which the temperature was allowed to rise to 110° C. it was found impossible to obtain a constant weight, perhaps on account of decomposition setting in, as suggested by an unpleasant odor which had begun to develop while on the water bath. Portions of 8-10 grams lost daily 0°03-0°04 gram. The analyses were accordingly made only after drying many days. Portions purified with alcohol, dissolved in boiling water with addition of hydrochloric acid, gave no reaction to be distinguished when heated with barium chloride. Carbon, hydrogen and nitrogen were determined as before, the sulphur by a method already used by us to some extent,* viz: by fusion with potassium hydroxide and potassium nitrate according to the method distinguished by Hammarsten as 1a.t+ The results of the analysis (Amphopeptone A), shown by the following table, were hardly satisfactory and the low percentage of carbon, particularly, was quite a surprise to us, hence we proceeded at once to the previously mentioned preparation of a peptone, which would probably be rendered less impure by derivatives of the mucous membrane and which would, moreover, be easier to purify further. 2. Amphopeptone prepared with purified pepsin. Preparation of the pepsin.—1220 grams of isolated mucous mem- brane from tbe fundus of ten pigs’ stomachs were warmed at 40° C. with seven litres of 0°5 per cent. hydrochloric acid for six days. The mixture was then saturated directly with ammonium sulphate, by which a resinous precipitate, with large, sticky lumps was formed, easily collected on a cloth filter. After pressing out the salt solu- tion as much as possible and washing with water, the gummy mass was dissolved in five litres of 0°4 per cent. hydrochloric acid and warmed again at 40° C. for a few days. Then for the first time it was filtered through paper. As preliminary experiments had shown that gastric juice which contains small quantities of ammonium sul- phate molds easily, the second digestion and the following fibrin digestion were carried on in the presence of 0°25 per cent, of thymol, which wholly prevented the formation of mold. The mass sub- mitted for the second time to self-digestion, gave now with the ammo- nium salt a much smaller precipitate, which contained only a very * Compare our earlier papers. Zeitschrift fiir Biologie, vols. xix and xx, + Zeitschrift fiir physiol. Chem., vol. ix, p. 288. a 00-001 > &G-TE oe ie” ss ie a" ee Free age), 2 é4+0 81-0 99-0 ge ae) a: att gone =u a &4-9T ae: +. a 8L-9T 69-91 “ae ee Toma am NL e 67-9 aa Ao = i 8P-9 oP-9 89-9 HH A 89:97 “ap -" ae — CG. FP 69-PP CrtF DO ‘OBBIOA VW ‘goungsqns datf{-ysp ayy fo uorpsodumods abnywadtag x ; "he | ae al eae 6-0 GeO SGG0FO ss ae Ss eG ag At ae oa: ee [ae ell ea eli” ae og6r-0 | IIIA ™ Ss iStes inne ae Soo 0p a & OES OSPOsO she Ut eae aes Bee Ape SRS Sao SNE SS He OGOR 0) ei Das ES add el he a | eae &6-8 | 9670-0 | ~~ ee | ie aS ae ae Pe eat ao Ee ere) oe Jee ae aN S ae oe eee a ay cea Fog | Renae ae CPST | 6-F9L| 9-F | 6-08 aes a oa Sa em Ge! eT | GPOF-0 | A Ss raha 2 yates. ES Ml neko ares is pce ater ||| pee Sop er ees ees 5 Pg. bees AU om S FE-eT | TLL) 6-F | 0-04 4 " | | 669¢-0 | AT ss S 7 a ae fee wallop oe ee ime 2) peer a aaa ee ee RCO Ova OLReLO | 96-¢ | co08-0 | 109¢-0 | IIT < ia S | eee ee aes Ge iee oa SS Se ee be ee ta eee) ee ROSES OGHOLO nae L9GG-0 69GF-0 | IL ie ‘ < pea alias gama Maris Eerie vee es Sor NE ah A ae ee 27 eS OF | SOP SD) 120088 s2000 8-0) SGT ORO I = “uIBls x aie 0. 3 a a a g % yse bat % Yse Jo was “TURAD cl SUE rt lie ae “meds ‘ues | oe 5 Od Hee Sayon 2 *puno 3 “pun 2 “Sold 2 no % BEST Stone. es F | oy, mos |SCTOMPEP) P J | ysy | oo hala. 9 jess J n ee aoUR}s N a $8 rogrg TO}FE S OS® UsV “punoy N OO O 1st -qug = Es ‘(V) TNOLAGAOHAN VW 226 Kithne and Chittenden— Peptones. small amount of albumose bodies, while a portion digested as a test for the third time, gave in the filtrate from the precipitate produced by the ammonium salt, so faint a biuret reaction for peptones that it was plainly evident, that the slight residue of albumins from the mucous membrane now remaining, could be overlooked without danger. Digestion of the fibrin.—3800 grams of washed but not boiled fibrin were digested with the twice precipitated pepsin, which was dissolved in ten litres of 0°4 per cent. hydrochloric acid. To obtain as little albumose and as much peptone as possible, the mix- ture was allowed to remain at 37°-40° C. for two weeks. At the end of that time, filtered portions gave only slight precipitations by neu- tralization, but a heavy precipitate was obtained with ammonium sulphate, with sodium chloride, with sodium chloride and acetic acid, and still further by sodium chloride and nitric acid or metaphos- phoric acid. Nevertheless the filtrate saturated with ammonium sulphate contained much peptone.* Preparation and purification of the peptone.—F or this purpose the filtrate was neutralized with sodium hydroxide, filtered through linen, especially for removing the impurities of the fibrin, the filtrate slightly acidified with acetic acid, concentrated to about four litres, precipitated with an excess of ammonium sulphate, filtered and pressed, the solution boiled with barium hydroxide and finally with barium carbonate and a large quantity of water, until ammonia could no longer be detected. The barium sulphate was then removed by filtration through cloth bags which were repeatedly washed and pressed, the solution evaporated to about four litres, the barium- peptone decomposed with a very slight excess of sulphuric acid, the new precipitate of barium sulphate filtered off, the solution concen- trated to two litres, the free acid neutralized with ammonia and after cooling, six per cent. English sulphuric acid (previously diluted) was added; then the sulphuric acid-peptone solution was precipitated with a large excess of phosphotungstic acid, the precipitate washed first with six per cent. sulphuric acid, then with a large quantity of water, after which the compound was decomposed with excess of barium hydroxide and the excess completely removed from the fil- * Later experiments have shown that pepsin acts much more energetically if the ammonium sulphate is completely removed by dialysis, before each new solution and digestion of the pepsin-containing precipitate in hydrochloric acid, and further, that nearly pure pepsin becomes wholly inactive by being warmed with dilute hydrochloric acid in the presence of even small quantities of ammonium sulphate. 5S tail ieee ES a a ae as See ys ; : =, a: 3 : Kihne and Chittenden— Peptones. 227 trate with sulphuric acid. The peptone solution thus obtained had a distinctly acid reaction, and strange to say, contained hydrochloric acid, which was hardly to be expected after the very careful washing which the precipitate had received. The solution was neutralized with ammonia to render the acid harmless on concentration. Then we succeeded in obtaining the evaporated residue free from ammo- nium chloride by repeated precipitation and boiling with alcohol. As already mentioned, the method gives rise to much loss and the same holds true of the otherwise excellent precipitation of peptone by phosphotungstic acid according to the method of Hofmeister, for so far as our experience extends, peptones cannot be completely pre- cipitated in this manner. There arises in the filtrate, containing excess of phosphotungstic acid, not* only additional turbidity and precipitation due to peptone, but considerable quantities of peptone are still found in the liquid, which has perhaps remained clear for months, if treated with barium hydroxide ;—a circumstance which we were not able to prevent even by strongly acidifying the solu- tion to be precipitated with phosphotungstic acid, with either sul- phuric or hydrochloric acid. Behavior of the peptone.—This peptone was also difficult to con- vert into a dry state, although we did succeed ultimately in bringing it to a constant weight as a fine, exceedingly hygroscopic powder, by heating for some time at 105° C. im vacuo. The first difficulty was found in commencing the drying, for although we treated the glue-like mass repeatedly with absolute alcohol, then for a long time with ether and finally boiled it again with alcohol, thereby changing it into an almost dry, crumbling condition, we were compelled at last to stop its further direct drying, since at 100° C. the preparation took on the consistency of pitch and formed a bulky foam trom which alcohol vapor continually escaped. Therefore the alcohol was first driven out by thorough boiling with water and the latter removed as’ much as possible at 100°C. ‘This was also a tedious performance for, although the substance no longer foamed up so violently, it did not become dry until after many days of stirring and breaking the cover- ing which continually bubbled up. The same proceeding was re- peated, although in a less degree, on transferring the substance to the air bath at 105° C., and only the single portions taken for analysis could be brought to a constant weight without puffing up further at 105° C. While drying, the unpleasant odor noticed from amphopeptone A was also observed here, although only in a slight degree. 228 Kiihne and Chittenden—Peptones. The peptone thus obtained appears (when dried at 105° C.) asa dry, light yellowish powder. It can be preserved in this form only when most tightly stoppered. In the air it soon forms large balls, becomes sticky like pitch and melts to a tough mass which does not become visibly thinner. What is truly surprising is the behavior of the peptone towards water. freed from fat. Digestion of the fibrin.—300 grams of dry fibrin, purified by wash. ing and boiling with water, then with alcohol and finally by extrac- tion with ether were softened with boiling water (when the weight amounted to 970 grams after squeezing with the hands), then Warmed at 40° C. with 3 litres of 0°25 per cent. sodium carbonate containing 0°5 per cent. of thymol, ‘To this was added the whole Ei Kiihne and Chittenden— Peptones. 23) infusion obtained from the 88 grams of self-digested pancreas, after which the mixture was continued at 40° C, for six days. At the end of the first day nearly all of the fibrin had disappeared, although a considerable portion appeared to float on the surface of the fluid. When examined more closely, however, this residue proved to be extremely light, hollow, easily crushed and with a somewhat greasy feeling. A similar residue, the amount of which we did not deter- mine, remained at the end of six days and consisted mainly of antialbumid with much tyrosin. Preparation of the antipeptone.—The solution resulting from the above digestion was made slightly acid with acetic acid, boiled, passed through a filtering bag, concentrated to 1 litre, and freed from a large amount of leucin and tyrosin by crystallization and fil- tration. The resultant, brownish-looking syrup was treated with alcohol until peptones began to precipitate, and after the latter had been redissolved by boiling the solution, it was placed aside for crystallization. The filtrate, which now contained only a small amount of amido acids, was freed from alcohol by boiling, diluted with a saturated solution of ammonium sulphate, which had also served for washing out the mass of crystals on the filters, and then completely saturated with the ammonium salt in substance. After separating the slight precipitate so formed, in which some leucin and tyrosin was detected, the greater portion of the ammonium salt was removed from the filtrate by repeated concentration and crystalliza- tion, while the remainder was gotten rid of, as before, with barium hydroxide and barium carbonate. Since in this case, precipitation with phosphotungstic acid could not yet be employed, we attempted to purify the peptone as much as possible from other products of digestion (amido acids), first, as a barium compound by repeated precipitation and boiling with alcohol, after which the barium-pep- tone was exactly decomposed with sulphuric acid and the free pep- tone purified in a similar manner by repeated precipitation and extrac- tion with alcohol, once or twice in the presence of a little acetic acid. The peptone thus obtained, when dried at 105° C., weighed 120 grams. Assuming tbat albuminous bodies by complete typsin digestion, split up into 50 per cent. of products arising from the wholly decompos- able hemipeptone and 50 per cent. of antipeptone not further changed by typsin, then the amount obtained—120 grams—agrees with this so far as it is possible, with the unavoidable losses which the treatment of large quantities in this manner implies. The 388 grams of dry albumin (300 grams of fibrin and 88 grams of self- 232 Kiihne and Chittenden—Peptones. digested material from the pancreas) would have had to yield 194 grams of antipeptone if there were no loss. The loss, however, of 74 grams noticed in our experiment is sufficiently explained by the noticeable solubility of peptone in the water contained in alcohol, and by the conversion of a portion of the peptone into antialbumid. Behavior of the antipeptone.—This peptone was still more difficult to dry than the amphopeptone formed by pepsin digestion, and it could only be accomplished after the removal of all alcohol by thorough boiling with water. As the solution became very concen- trated on the water-bath, hydrogen sulphide, as shown by reaction with lead acetate, was given off together with a strong odor of valeri- anic acid, which was also evolved quite noticeably at 105° C. In order to obtain a constant weight it was necessary to dry the mass at 110° C, The analysis of the product is shown in the accompanying table. Antipeptone (D). The behavior of the preceding preparation while being dried, nat- urally suggested the suspicion that the substance was either decom- posable at 100° C. or less in the air, or else that it contained some decomposable admixture. We therefore attempted a further parifi- cation of antipeptone and at the same time a more cautious method of drying. For this purpose another preparation of antipeptone was made in the following manner. 230 grams of commercial dry pancreas, somewhat less active than that employed in the preceding preparation, were warmed at 40° C, for three hours with 1200 ¢. c of 0-1 per cent. salicylic acid, after which the mixture was neutralized with sodium carbonate and to it was added directly 1920 grams of boiled, moist fibrin, 82 grams of dry sodium ‘arbonate and 32 grams of thymol. This mixture was warmed at 40° C. for seven days, at the end of which time the residue of the pancreas, the antialbumid produeed, and considerable sepa- rated tyrosin, formed a noticeable sediment, which was filtered off and pressed, after the residue had been thoroughly washed with water warmed at 40° C. The filtrate was made slightly acid, heated to boiling, and as this produced only aslight precipitate it was imme- diately concentrated to about three litres. On cooling, an abundant slate-colored precipitate separated, composed almost entirely of tyro- sin. After removing this by filtration, the solution was saturated with ammonium sulphate and the resultant filtrate treated as in the _00-001 It -86 cay Tee eee 64-0 FL-0 TL-0 ahs 4 “tac Ce 68-91 oe cl 68-91 78-91 «om ie £49 5 a ia REN Wie 6-9 PL:9 08-47 re Pkt: ae =e TS-L7 0&8: LP ‘OSRI0AV 2338 OMARO Nov., 1886. ‘a0UDpsSgns datf{-yso ayy fo worrsod wos abyzuaIag =o = 5, ES | OO 2 PABLO OBO eB eee pam a | eet Ree a ee a ee ee a - 30 | ; oS te OSTQQ =| S150 = \ 4 Ss a ee aE CL Sn en ee a i oe ee Se ee ee Ge 7 ay ae ; (QO) ANOLGHdLINV -- = Pp. ; eo : N e , 2 aia 234 Kithne and Chittenden—Peptones. previous case; that is, a barium peptone compound was formed, puri- fied with aleohol and this exactly decomposed with sulpburic acid. In order to separate the free peptone, the solution was concentrated at a gentle heat with the addition of a little ammonia, precipitated and boiled with alcohol, the almost liquid precipitate dissolved in water, the solution acidified with acetic acid, concentrated again, pre- cipitated and extracted hot with alvohol, repeatedly washed and kneaded with ether, kept for a long time under ether and then slowly dissolved in as small an amount of cold water as possible. When filtered, a small residue of tyrosin appeared in the preparation. The new solution was concentrated at a gentle heat, precipitated again with alcohol, the peptone boiled and washed with alcohol, allowed to stand for some time with a large quantity of absolute alcohol, again treated with ether as before, and as it had now become friable, it was immediately dried in vacuo over sulphuric acid. On attempt- ing todry it at 100° C. the melting mass foamed so much that it had to be dried in the air. When this had been done with frequent stirring on the water bath for several days, the mass was finally dried com- pletely im vacuo, first at 100° C., then at 105° C. A few weeks of this drying were needed to bring the substance to a constant weight, and in order to prepare the various quantities for analysis they had to remain in vacuo over sulphuric acid and at 105° C. for some time. Probably in consequence of the thorough treatment with ether, the preparation when warmed gave much less odor than the former one. . Composition of Antipeptone (D). I. 0°5061 gram substance gave 02875 gram H,O = 6°31 per cent. H and 0°7937 gram CO, = 42°76 per cent. C. II. 04449 gram substance gave 0°2527 gram H,O = 6°31 per cent. H and 0°7024 gram CO, = 43°05 per cent. C. III. 0°5010 gram substance gave 0°2870 gram H,O = 6°36 per cent. H and 0°7885 gram CO, = 42°91 per cent. C. IV. 0°4414 gram substance gave 56:0 c.c. N at 21°0° C. and 759°2 mm. pressure = 14°91 per cent. N. V. 0°5870 gram substance gave 765 c.c. N at 21°6° ©. and 758°0 mm. pressure = 15°18 per cent. N. VI. 0°6930 gram substance gave 0:0694 gram ash = 10°01 per cent. VII. 05017 gram substance gave 0:0504 gram ash= 10°04 per cent. VIIL. The ash from 05017 gram substance gave 0°0325 gram BaSO, =0°'89 per cent. S calculated on the original substance. a. Kiihne and Chittenden— Peptones. 235 On account of the large percentage of ash and the large amount of sulphates, the sulphur of the organic matter was not determined. Percentage composition of the ash-free substance. Average. Cee 47°52 47°83 47°69 —— ime 47°68 ler see 701 7-01 1:07 Aeeigt ee 7°03 ) BAe Be Seine 16°57 16°80 16°68 Antipeptone (E). In order to obtain a still purer preparation and especially to free it from the large percentage of ash, a portion of antipeptone (D) was dissolved in boiling water after the first treatment with ether, then when cold the solution was acidified with sulphuric acid to such an extent that it contained 6 per cent. of acid and precipitated with a large excess of phosphotungstic acid. The precipitate, after the manner already described under amphopeptone (B), was washed thor- oughly with dilute sulphuric acid and then with water, finally de- composed with baryta, the barium-peptone precipitated with alcohol, washed, the alcohol driven off by heat, the aqueous solution of the compound exactly decomposed with sulphuric acid, the solution con- centrated after the addition of a few drops of ammonia, precipitated with alcohol, dissolved again in water, concentrated with the addi- tion of a little acetic acid, again precipitated with alcohol, and the product so obtained treated thoroughly with alcohol and ether in the same manner as preparation D, and finally dried in the same manner as that. The substance so prepared, was lighter colored than the preceding, not quite so hygroscopic, and in drying gave scarecly any odor. The analysis of the product is shown in the-accompanying table. Antipeptone (F). (Gland peptone.) This peptone was obtained as a bye product in a preparation of trypsin from 1,000 grams of dry pancreas and was formed wholly from the albuminous bodies of the gland substance, after extraction with alcohol and ether. It is not probable that the peptone con- tained, in any considerable quantity, any products from the digestion of elastin, since the elastic tissue could have been but little altered under the conditions in which the self-digestion of the gland took place during the preparation of the infusion, and furthermore there would have b»en needed for solution in the latter, a finer subdivision ‘ at Kiihne and Chittenden—Peptones. 236 00.001 hhehG 49-0 86-8T 69-9 69-97 ‘OSBIOAY 99-0 CBSE 16-81 "86-81 19-9 ov: OF ‘aounysqns aatf-ysp ay? fo Uoiprsodwos abpyUdaolaq 0010-0 £9-0 €9-0 92-0 bL-0 0060-0 00¢0-0 b yse ot WOIy § “mUeRIs “yse oly WOT ‘osead “ys Fo g Buyonpop: IOYL g | | *mUBIs “punoy rosrad 9-LGL |P-1s LLL |P- 16 L-9CL |8-66 69-96 0-061 89-6¢ UsV “TUBIS “punoz ysyv 00ST TE OF80-T | 6868-0 | 8888-0 SF-9 | 861e- 1 | x 8EFC-0 XT 0616-0 see9-0 961-0 | THA | LA IA 6129-0 A 0064-0 | AT 1168-0 2689-0 TI —-19¢9-0 I } “WRIs “panoyz (0/0 ‘(q) ANOLAAdIINY “mes *punoj H. | Fa | O°H “TRIs ‘pesn | e01R4s “qns ON Kiihne and Chittenden—Peptones. 23 ~T and long continued action. However this may be, the albumins of the pancreas naturally cannot be classified with the substances ordi- narily used in digestion experiments, such for example as fibrin, without further investigation, for although there may be substances in the gland cells like serum-albumin, globulin and myosin, there are also many bodies quite different from these, as, for example, the leu- coid precipitated by excess of acetic acid, zymogen and trypsinogen, _all of which are decomposed by self-digestion and yield amido acids and peptones. So long as trypsin digestions are not ordinarily con- ducted with pure trypsin, it is of especial interest to find out the composition of the gland peptones, which, as a rule, have invariably been mixed in greater or less quantity with the antipeptones hitherto investigated. Preparation.—1,000 grams of dry pancreas were warmed at 40° C, for twelve hours with five litres of 0:1 per cent. salicylic acid and 0°25 per cent. of thymol, filtered through muslin, the residue warmed another twelve hours with two litres of 0°25 per cent. sodium carbonate and 0°5 per cent. of thymol, again filtered and pressed, the two fluids united, brought up to an alkalinity of 0°25 per cent. of sodium carbonate and then warmed at 40° C. for three days. After filtering through paper, the whole solution was slightly acidi- fied with acetic acid and then saturated with five kilos. of ammo- nium sulphate, by which means there was precipitated a little albu- mose and all of the trypsin, the further treatment of which is of no interest here, while the gland peptone remained in solution. It is to be noticed in the separation of this peptone that it was treated exactly like preparation (C), excepting that the second purification with ether could be omitted. The preparation, after drying for some time, left a small residue of tyrosin when dissolved in cold water. It was therefore precipitated from this solution with alcohol, then freed from alcohol by boiling with water, dried directly over a water-bath and finally im vacuo at 106° C. until a constant weight was obtained. The analysis of the product is seen in the following table. Antipeptone (G). (Gland peptone.) . This preparation was obtained from the preceding product by the following process; the solution of the peptone was acidified with 6 per cent. of sulphuric acid, precipitated with a large excess of phos- photungstic acid, the precipitate carefully washed, then decomposed with barium hydroxide, the latter exactly removed with dilute sul- Kihne and Chittenden—Peptones. 238 00-00I 68-08 eer i oe a a i 08-0 08-0 19-0 Aes eos warceaee Gtr Ae ate le. te ie ——— see eee R. H. Chittenden—Dehydration of Glucose. ~T bo or Third experiment.—A rabbit in a condition of hunger was killed, the stomach divided longitudinally along the curvatures, and one-half after being cleaned and finely divided, was placed in contact with 0200 gram of glucose dissolved in 70 c. c. of water, and warmed at 48°8° C. for one and one-half hours. The mixture was then heated to boiling with the addition of some crystals of sodium sulphate, the tissue and coagulated albumin filtered off and the residue washed with about 300 c.c. of hot water. The fluid was concentrated, brought to a volume of 100 c.c. and then treated as in the preced- ing experiments. Following are the results obtained : : STOMACH. a. Before treatment with sulphuric acid. 25 c.c. gave 0°0834 gram Cu =0-0425 gram dextrose x 4=0°1700 gram dextrose. b. After treatment with sulphuric acid. 25 c. c. gave 0°0798 gram Cu==0-0407 gram dextrose x 4=0°1628 gram dextrose. Here, as before, there is no evidence of any change in the charac- ter of the glucose; still in spite of the comparatively large volume of wash-fluid used, the sugar was not wholly recovered. Fourth experiment.—A cat killed in full digestion was employed in this experiment. One-half of the stomach, finely divided, was placed in contact with 0°200 gram of glucose in 75 c.c. of water. A portion of the small intestine was also treated with a like amount of sugar, in the same manner. Both were warmed for three hours at 48°8° C., then treated by the same method as used in the preceding experiments. + STOMACH. a. Before treatment with sulphuric acid. 25 c.c, gave 0°0879 gram Cu =0-0449 gram dextrose x 4=0°1796 gram dextrose. b. After treatment with sulphuric acid. 25 c. c. gave 0°0886 gram Cu = 0°0452 gram dextrose x 4==0°1808 gram dextrose. _ INTESTINE. 3; a. Before treatment with sulphuric acid. 25 c. c. gave 0°0668 gram Cu =0°0342 gram dextrose x 4=0°1368 gram dextrose, b. After treatment with sulphuric acid. 25 c. c. gave 0°0684 gram Cu =0°0350 gram dextrose x 4=0°1400 gram dextrose. Here again, there is no evidence whatever of any change in the reducing power of the sugar solution. Pavy has also pointed out that the stomach and intestine of the rabbit, as well as of other animals, have a transformative action on saccharose as well as on dextrose. The transformative energy how- TRANS. Conn. AcaD., VoL. VII. 33 Nov., 1886. bo or io 2) R. H. Chittenden—Dehydration of Glucose. ever of the intestine, is much greater in this case than that of the stomach; which, according to Pavy, accounts for the early discovery by Bernard of the well known action of the intestine on cane sugar. i Bernard supposed dextrose to be formed, but Pavy shows that maltose or a body resembling maltose in reducing properties, is the usual product and that glucose or dextrose is formed only in the presence of considerable ferment. The ferment which produces this change, unlike the ferment which acts upon glucose, is situated on the surface of the mucous membrane and thus frequently the contents of the stomach are likewise found to possess transformative power. Fifth experiment.—A rabbit with stomach partially filled with food was killed, the stomach rinsed with water, then minutely divided and separated into two equal parts. One portion was placed in con- tact with 0°195 gram of glucose in 75 ¢. ¢. of water, while the other portion was mixed with a like amount of pure saccharose, also in 75 c.c. of water. A portion of the small intestine was likewise finely divided and one portion placed in contact with 07195 gram of glucose in 75 ce. c. of water and the other portion with 0°200 gram of saccha- rose dissolved in 75 c. c. of water. All four mixtures were warmed at 48°8° C. for-two hours, then heated to boiling with the addition of sodium sulphate and finally each brought to a volume of 100 ¢. ¢. 25 c.c. of the saccharose solution, which had been in contact with the stomach tissue, gave no reduction whatever with Fehling’s solu- tion. 25 ¢.¢. of the saccharose-intestine solution, however, gave 0:0726 gram Cu, equivalent to 0:0371 gram dextrose. ; With the glucose solutions, the following results were obtained : STOMACH, a. Before treatment with sulphuric acid. 25 c.c. gave 0°0906 gram Cu =0°0462 gram dextrose x 4=0°1848 gram dextrose. b. After treatment with sulphuric acid. 25 e.¢c. gave 00889 gram Cu =0°0454 gram dextrose x 4=0°1816 gram dextrose. INTESTINE. . Before treatment with sulphuric acid. > ¢c.c. gave 0°0712 gram Ok u =0°0364 gram dextrose x 4=0°1456 gram dextrose, b. After treatment with sulphuric acid. 25 c.c, gave 0°0689: gram Cu =0'0353 gram dextrose x 4==0'1412 gram dextrose. With glucose, the same results are to be observed here as in the preceding experiments; the only variations in reducing power, before and after treatment with sulphuric acid, being such as would come within the ordinary limits of error. In one single case, a transforma- R. H. Chittenden—Dehydration of Glucose. 259 tion of saccharose was noticed when the sugar solution was warmed for two hours with a portion of stomach tissue from a rabbit killed in full digestion. The reduction with Fehling’s solution was quite strong. With glucose, however, many experiments have been tried in addi- tion to those given above, and invariably with the same negative result. The conditions of the experiments, moreover, are in many cases identical with those of Pavy’s except in the method of deter- mining reducing power. There seems, therefore, to be no plausible explanation of the results obtained, other than that in the above experiments there was no dehydrating ferment present. Pavy states that the ferment in question, or rather “the active principle con- cerned in the transformation of glucose is susceptible of being de- stroyed by the agency of gastric digestion,” so that there is the pos- sibility of such destructive action having taken place in the stomach of the animals experimented with. It is further stated, however, that the converting principle is situated in the underlying portion of the mucous membrane, so that destruction could hardly be expected, except perhaps in the slow self-digestion occurring after death. Cer- tainly, the carefully rinsed tissue could not have retained sufficient gastric juice to affect the results. Furthermore, such decomposition would apply only to the stomach mixture and not to the intestines, unless sufficient proteolytic ferment from the pancreatic juice should adhere to the walls of the intestines to exert destructive action; but in the last experiment given, it is to be noticed that the saccharose ferment, which is presumably equally sensitive, showed vigorous action while the glucose was unaffected. It seems strange, therefore, if such a dehydrating ferment is norm- ally present in the alimentary tract, that we have not been able to obtain some tangible evidence of its presence, either in the stomach or intestines. Since the above was written, the writer has noticed that M. Ogita,* experimenting with dogs, has also been unable to confirm Pavy’s results, both in the dehydration of glucose and in the inver- sion of saccharose. *See Jahresbericht fiir Thierchemie, xv, 275, a ve RARE juitigas ene ath) hea hy rey tie neg a ns eet Ae acts neto. artan ii ee i ee T Bie y' 4) sia asl: A e = * gh free ea RAL iar be GA OT i ay iva tat re key ae? | any Take! aY 4 ¢ x! ia tytn} seg, ‘ : . : . Pai ey PR NTIE. nd ites / eer pon neaem ¢ ‘ oe q é ' «| . Logs eahera i i ey ; vie wary, j i tan apy yA ae 5 4 ’ 7 + in’ Wr’ Sslistoteaitad vv ¥} wee Lites) ors . tis , > alla SANT Oi ‘i ey be abe “al ea ee haven 309 , ; MRR tL : iPastelin ‘ $ rel eer Al ; pda gh (=r) j g che ' Ma il i Sr tk » Ia ‘ i ri { f h tj z ‘ BY \ ‘ ' ~ sad, o> 4 » += wed ; fn ' f \ \ j V7 hy f i , \ , ‘ t ye i ' ; - by ro ; ; ee ee 7 a. * Z , 4 2 : ty hee * ‘ 5 Y . oa . i sii bulee VE PRG IN ee ' i , ; t , } Ps j ; ~ eh : - Ss . «; f Wu pe ~ ‘ P 1mMo vt ’ Ow : \ t a é ; pat ey aie > ' ] é iy Fag feo M > | ‘ bP tae, Fe iy ’ a ‘ wi? ‘ wy > i . IV Unique TH Two Fors oy, 8543 XI Two Forms y . 7652 Bh, XIV Unique mt z OS UI Two Forms (pe S00 = a a 2 VII Six Forms 7esy Cf, VIII Five Forins L.S.Punderson.Photo. Lith.New Haven. 7 . Ue 4 | iene Bi ’ | 3 Seirans. Conn Acad. Vol. VIL. : z : » b i 5 FLO ) # JOOS OF | ice RS 1S: & X Two Forms . Bos TA «Cj, 5452 1542”), 4'g'2" a a ARs, Z soc vA, se obey pool oe ison é 8 : 2 é xe Sy C} 2 , 3 : iO) Oe bp 5 Q 5 74520 (Cu, aa2 L.S.Punderson,Photo.Lith.New Haven. Se N pate: Del. ‘oe i) Pius we at .> 4 Trans. Conn. Acad. Vol. VIL. | PLATE IIL 4 = XXHI - Forms Bie) (ee) Ce) (De) rs) ee ee SK Pe Ee RR 6 =" ( Bee) Lr ce LY) 8) LED. LS ‘ 74°32 Cv, «= 4#5*2 65432 C*by 49382" 5453) Cf, 48592" 74°32) Cw, «492897 74932 Cw, «= 4342" 74932 Cw, 53928 65422 C%w, 47392" ‘ ia (1 XXV Unique XXVI BH HSS ee ee Ca) UN LB 4*372? 65432 C%a, 47392 65: —-= on PEVIOvE | a eee) Co.) Oe Ud) La Neen FS ED PB a | a ‘ AHO ae 64*5* XXXI ere lag o> ai > | | = = XXNU Four a z | aaa Q 3 gagge Y 3093 e442" Ota, 493828 493928 6s? 0%, 5342? sea w see amram Rae | a) AAI | OR OS LS f a 0) () ee) | ‘a i r) y) s 2 1.3. Punderson,Poto, Lith.New Haven. as : . a SS Ser Se es , . 7 , Trans.Conn.Acad. Vol. VIL. / PLATE IV | XXXV Unique XXXVI Six Forms GE) Cs 493*2? oata* 43628 5932, C4732" 59492 fg = 4342" 574P2 C4 48828 L.S.Punderson,Photo.,Lith.New Haven. ae kr i‘ n> a ae Vy Fae amt > a , ¥ ' Ms : 7 ‘Ua ‘+d > ¥ . yA -< : . } 4 ’ i ban * > . . , ~ a, A 4 © Traus.Conn. Acad. Vol. Vi LVI Unique LIX Two Forms 1 { BO) ( BO ig Bex) Uy 243" 503g 483828 LXIV Four Forms (2 85327 De, 54°32" BI) (82) ( Sel oe JL US lta Rd Ae ie tT” aed ‘ a ‘ ie? a or my 2s os Conn. Acad, Vol. VIL. PLATE. VL (BE ) So) Se eae A E . ‘ | fe ei \) * 1 #) (an yy ao) 64972" Da, 53¢ 643922 Du, 64372 643%2* 64372 643"2* Du, 47392 642922 i g ae ~@ ’ ray | eh fe | : 1) L2) LS) USS USD De) Ups 3 a Soy eee | eS | FRB SIS (8 felis LA) LED) Les CU ad : ry «() a Co, 7, cS) EY, ~) Ws} (63 |) |B 8 |} |g ee ¢) S S a 73 (ERIS BL) lO) cf) 16s <> | <> GT Y 2) (a) Ge) | { ee) { Sy D O71 CK Maal) |e) ey | j @) eta ws J) 69382" D%), Amph = 53392 D1, 64392 59992 Dim 547322 693928 Dt 648g2® =—«-«sSASG2# D8p, SA8S2F 5 4agat D* ag ca me SS CN) { GAS 2 ee By, 2 x) : rine sap” ee 49332 4ea2e f Amph 54892" wast 54°32" sat SS a3" INI ittle, Del - L.S.Punderson, Photo. Lith.New Haven. LP — ‘e/a Trans. Gonn.Acad. Vol. VIL. PHELERE BRRESE W) 18) V8) US) IS US AS (212 (B(R (et OG) LS) be) LD U8) UP 2 ( (N(R (8S BL) UD) LS igs US) US = 2 © - ERERDE oh GH Le) LS) Lea) US 54392» Dig Amph + N 2 Little ,Del. L.S. Punderson,Photo, Lith.New Haven. de oan » . Lae Plate VIE > CS > me} w 3) <= i= = ° O a c 3 — = “‘SNINWOC ) NV NaC INALLIHO—'S Atv mn NOILV? LVYldsay | l it nt ee sil aa 4 le Nl ans. Conn. Acad. Vol.VIL | [HED erton from nature. LS.Punderson &Son,Photo -Lith NewHaven. DICTYNA. trea Trans. Gonn.Acad Vol. VIL. tia | Ss a HEmerton from nature. L L.S.Punderson &Son,Photo Lith NewHaven. Ta. AMAUROBIUS — TITANOECA . 4 rans.Conn.Acad.Vol. VIL. fem hy | Mili : === z lays iff nm UMW Ys Wy = ORG 7 SM Ty UWS aN. MY a IN htt i-thirk Te rT SNL LLU —— — SMEs is Avaya ee ee ee ee eee 4 i. 4 J Em erton from nature. LS.Pundersan &Son,Photo tith New Haven ULOBORUS -HYPTIOTES. XVII.—InFLUENCE oF Uranium Sats on THE AmyLotytic AcTION OF SALIVA AND THE ProtEotytic AcTION oF PrEpstn AND Trypsin. By R. H. Cuitrenpen anv M. T. Hurtcs- inson, Pu.B. Lirrce is known regarding the physiological, or even toxical action of the uranium salts. In 1825, Gmelin* reported upon the results of some experiments on the toxic action of uranic nitrate, but aside from the work done at that time, little is known regarding the action of uranium. It is our purpose, therefore, to carry out in this Lab- oratory, as opportunity offers, a series of experiments on the physio- logical and toxical action of uranium salts, and we have commenced the work by endeavoring to ascertain the influence of the above salts on the amylolytic and proteolytic action of the ferments occurring in the digestive fluids of the body. In this connection we wish to ex- press our obligations to Professor H. Carrington Bolton, of Trinity College, for his kindness in supplying us with an abundance of chemically pure uranium compounds. 1. Influence on the amylolytic action of saliva. The method employed in determining the extent of amylolytic _ action was much the same as that previouslyt used by one of us, except that the amounts of reducing substances formed under the different conditions of the experiments, were determined volumetri- cally by Fehling’s solution, instead of by Allihn’s gravimetric method. The experiments were made in series, in which one diges- tion of each series served as a control for comparison. The volume of each digestive mixture was 100 ¢. ¢. and contained | 1 gram of perfectly pure potato starch, previously boiled with a por- tion of the water, 10 c. c. of a diluted neutral saliva and a given per- centage of the uranium salt to be experimented with. The mixtures were then warmed at 40° C. for 30 minutes, at the end of which time, further ferment action was stopped by heating the solutions to boiling. The saliva employed in the experiments was human mixed saliva, freshly collected, filtered and made as neutral as possible with 0°2 per cent. hydrochloric acid, then diluted with water in the pro- * Kdinb. Med. Surg. Gaz., xxvi., 136. + Studies from this Laboratory, vol. i, 1884-5, p. 2 and 53. TRANS. Conn. Acap., Vou. VII. 334 Nov., 1886. 262 Chittenden and Hutchinson—Influence of portion of 1:5. Hence, each digestive mixture contained 2 c. ¢. of undiluted saliva, The amount of reducing substances, which for the sake of conven- ience are calculated as dextrose, were, as already mentioned, deter- mined volumetrically and from the data so obtained, the percentage - of starch converted was likewise calculated. Uranyl nitrate. With this salt the following results were obtained : Total amount Relative U0.(NO3)2+6H20. reducing bodies. Starch converted. amylolytic action. 0 0°4135 gram. 37°21 per cent. 100-0 0:0001 per cent. 04083 36°74 98°7 0-0003 0°3873 34°85 93.6 0-0005 0°3698 33°28 ~ 89-4 0-001 , 0-3612 32°50 87°3 0-003 0°3131 28°17 75°5 The inhibitory action of the uranyl salt is plainly manifest in these results. A second series of experiments, with still larger per- centages of uranyl nitrate, show the retarding action still more plainly. Total amount Relative UO.(NOs3)2+6H.0. reducing bodies. Starch converted. amylolytic action. 0 0°4066 gram. 36°59 per cent. 100-0 0-001 per cent. 0)°4000 36°00 98°3 0-002 0°3880 34°92 95°4 0-003 03034 27°30 46 0-004 02545 22°90 62°5 0-005 01550 13-99 38-2 0-008 trace. The largest percentage of the salt used (0°008 per cent.), is seen to | almost entirely prevent the action of the ferment, thus showing how extremely sensitive the salivary ferment is to the action of this salt. Comparing the two series of experiments, it is seen further, that a given percentage of the salt, say 0°001 per cent., is much more active in one case than in the other, indicating that the action of the salt is not constant. This is undoubtedly true to a limited extent. The action of a given percentage of the salt is constant only under dike conditions. In the above series of experiments, the saliva is different in the two cases, and the real explanation of the difference in action is to be sought for in the amount of proteid matter contained in the saliva. Undoubtedly the retarding action of the uranium salt is checked, in part at least, like that of mercuric chloride,* by the * Studies from this Laboratory, 1884-85, p. 71. Uranium Salts on Ferment Action. 263 proteid matter of the saliva. Uranium is a well-known precipitant of albuminous matter and hence the larger the amount of albumin and - globulin contained in the saliva, the weaker the retarding action of the uranium salt. Previous experiments have shown that saliva varies somewhat from day to day in its content of proteid matter, as well as in the amount of ferment, and although the former difficulty is obviated as much as possibie by diluting the saliva, still this point must be borne in mind in making comparisons of the different series of experiments. Uranyl acetate. This salt appeared somewhat more inhibitory in its action than the nitrate, due possibly to its greater acidity, The two following series show the extent of action: Total amount Relative U0.(C:H;02)2+H.O. reducing bodies. Starch converted. amylolytic action. 20) 0:4049 gram. 36°44 per cent. 100-0 0-001 per cent. 02305 20°74 56°9 0-002 0°1698 15°28 41°9 0-003 trace. 0 04032 36°28 100:0 00003 0-4331 38:97 107-3 0:0005 0°3322 29°89 82-4 0:0008 0°3192 28°89 79°6 0-0010 02882 25:93 71:4 The presence of 0003 per cent. of the salt almost entirely stops the action of the ferment, while 0°0003 per cent. decidedly increases amylolytic action. ‘his latter influence is similar to that exerted by many other metallic salts in very small fractions of one per cent. and is doubtless to be attributed, in part at least, to the stimulating action of the acid-proteids formed, or in part, as suggested by Duggan,* to a more complete neutralization of the digestive fluid. Ammonio uranous sulphate. With this salt the following results were obtained : U(SO4)o + (NA4)s Total amount Relative O,+ HO. reducing bodies. Starch converted. amylolytic action. 0 0°3384 gram. 30°45 per cent. 100-0 00003 03935 30°41 116°3 0-0005 -. 0°3798 34:18 112-2 00008 03563 30°40 99°8 0-001 0°3550 30°28 99-4 0-002 02951 26°55 87-2 0-008 00805 7°24 23°7 *See Amer. Chem. Jour. vol. viii, p. 211. 264 Chittenden and Hutchinson—Influence of Here there is to be seen both stimulating and inhibitory action, both quite pronounced; and further, the salt is perfectly neutral, so that such action as is exerted must be due to the salt itself. Sodio uranic sulphate. This salt, which like the preceding was exactly neutral to test papers, Shows both stimulating and retarding action, but in extent somewhat smaller than that of the uranous salt. Following are the results obtained : Total amount Relative UO.SO, + NaeSO,. reducing bodies. Starch converted amylolytic action. 0 0:4049 gram. 36°44 per cent. 100-0 0-0003 per cent. 0:4100 36°90 101°3 0:0005 0:4000 36°00 98°8 0:0008 0°4100 36°90 101-2 0-001 0°4117 37°05 101°7 0-002 0-25380 22°77 62°5 0-003 0°2000 18:00 49-4 0:005 trace. Potassio uranic oxychloride. UO.Cl. +2KCl Total amount Relative +2H,0. reducing bodies. Starch converted. amylolytic action. 0 04032 gram. 36°26 per cent. 100-0 0:0005 per cent. 0°3951 35°55 98-0 0-0008 04016 36°14 99°6 0-001 04083 36°74 101°3 0-002 0°1881 16-92 46°6 0-003 0:1078 9°65 26°6 0-005 trace This salt had an acid reaction and its retarding effects are seen to be somewhat more pronounced than that of the two preceding neu- tral salts. Ammonio uranie citrate. (UO2)s(C5H507)2 + Total amount Relative (NH4)sCe6H50;7. reducing bodies Starch converted. amylolytic action. 0 0:4135 gram. 37°21 per cent. 100-0 0-0008 per cent. 04298 38°68 1039 00005 0°4016 36°14 . 97-1 0:0008 04049 36°44 97°9 0-001 0°3873 34°85 93°6 0-002 04016 36°14 97°1 0-008 0°3843 83°86 89-6 Oranium Salts on Ferment Action. 26 Cr (UOz)3(CgHs507)2+ Total amount Relative (NH4)3C5H;0;. reducing bodies. Starch converted. amylolytic action. Ole; 0°4117 gram. 37:05 per cent. 100-0 0:004 per cent. 0°2378 21°40 57:7 0-005 0°2144 19-29 52°0 0-006 0°2816 21°39 577 0-007 0°1845 16°60 44°8 0-008 0°1765 15°88 42°8 0-010 trace The action of this salt is mainly a restraining one, but the action is pronounced only with the larger percentages. As to the way in which these uranium salts diminish the amylolytic action of the ferment, we cannot say definitely. What has previously* been written regarding the action of other metallic salts, under like conditions, is doubtless true here. Loss of amylolytic power is due in part, no doubt, to partial direct destruction of the ferment, as well as to change in the reaction of the fluid. Coupled with this destructive action, however, there must be in addition something in the mere presence of these salts, dependent on chemical constitution, that controls the action of the ferment. The following table shows the relative acceleration and retardation of the various salts, compared with their respective controls expressed as 100. 2. Influence on the proteolytic action of pepsin-hydrochloric acid. The influence of uranium salts on the proteolytic action of pepsin- hydrochloric acid, was determined by ascertaining the amount of fibrin digested or dissolved in a given time, by a definite volume of a standard, artificial gastric juice, in the presence of varying amounts of the uranium salts. The gastric juice was made by dissolving 10 c.c. of a glycerin extract of pepsin in one litre of 0°2 per cent. hydro- chloric acid. The volume of each digestive mixture was 50 cc. ; composed of 25 c.c. of the above mentioned artificial gastric juice and 25 c.c. of 0:2 per cent. hydrochloric acid, containing the neces- sary amount of uranium salt. The proteid material consisted of purified fibrin, coarsely powdered and dried at 100°C. One gram of _ fibrin was used in each experiment. The digestive mixtures were warmed at 40° C. for one hour and then the undissolved residue was collected on weighed filters and finally dried at 100-110° C. until of constant weight. The amount of fibrin dissolved is taken as a meas- ure of the proteolytic action. * See Studies from this Laboratory, vol. i, 1884-5, pp. 70-75. Trans, Conn. Aoap., Vou. VII. 34 Nov., 1886 400-0 | | 200-0 100-0 8000-0 > i) Ss) = = T i~ S yh xg ‘=> < — S ss = 3 § Ss <= D $ <= 5 66 ‘NOILOY OILATOTANY AO 1-26 | 6-201 0.86 | vice 8-86 | &-10I GBI | $-O1T 728 | SLOT 7-68 | 9-86 000-0 000-0 -10 ormem or1rmoumuly Rear Sa aplaoryo -kxo o1meim o1ssejog --ageydyns ormeam Orpo0og ----gq[¥Bg Jo a8equI.I0g NOILVGUVLEY GNV NOILVHETHOOY FAILVTEY ONIMOHS WIdV | Uranium Salts on Ferment Action. 26 ~T Following are the results obtained with the various salts : 4 Uranyl nitrate. Relative U0.(NO;)2+6H.20. Undigested residue. Fibrin digested. proteolytic action. 0 071853 gram. 86°47 per cent. 100°0 0)°025 per cent. 0°1365 86°35 99°8 0-050 0°1378 86°22 99°7 0-100 02397 76°08 87°9 0-500 0-5003 49-97 578 1-000 06638 33°62 38°8 Uranyl acetate. U0.(C2H302)2 Relative +H,0. Undigested residue. Fibrin digested. proteolytic action. 0 0°1453 gram. 85°47 per cent. 100-0 0-025 per cent. 0°1581 84°19 98°5 - 0050 01867 81:33 95-1 0-100 02052 79°48 92°9 0-500 0°7507 24°93 29-2 1-000 1:0050 eae 0 It is to be noticed here, that the retarding action of the acetate, as with saliva, is far greater than the nitrate, a fact which is doubtless dependent in this case on the nature of the acid united with the ura- nium. Further, it is to be noticed, that the action of uranyl sulphate falls about midway between the action of the nitrate and acetate. These facts accord with views previously* expressed, and show plainly that the extent of the retarding action of salts in general is dependent in part, on the liberation of the acid of the salt and the digestive power of the pepsin-acid formed. Experiments have shown that nitric acid of appropriate ‘strength, united with pepsin, is about four-fifths as active as hydrochloric acid, while sulphuric acid is only a little more than one-fourth as active as hydrochloric of the same strength and that acetic acid is practically inactive. Hence, the base being the same, acetates, citrates, and other salts, the acids of which are not capable of working with pepsin will most readily retard gastric digestion. This view being correct, uranyl nitrate, sulphate and acetate should retard gastric digestion in just such relative pro- portion as our experiments show they actually do. —_—_—— poet eae = *See Studies from this Laboratory, 1884-85, p. 94-95. 268 Chittenden and Hutchinson-—Influence of ; Uranyl sulphate. Relative U0.S80, +3H,0. Undigested residue. Fibrin digested. proteolytic action. 0 0°1832 gram. 81°68 per cent. 100-0 0:025 per cent. 02545 74:55 91°3 0-050 0°2673 73°27 89-7 0-100 0°3155 68°45 83°8 0°500 ()6084 59°16 479 1:000 08225 17°75 21°7 Ammonio uranous sulphate. USO, + (Ntf,)2S04 Relative +H,0. - Undigested residue. Fibrin digested. proteolytic action. 0 0-1833 gram. 81°67 per cent. 100-0 0-025 per cent. 02859 71:41 874 0-050 0°31438 68°57 83°9 0-100 0°3742 67258 76°6 0500 0°9113 8°87 10°8 1-000 10070 0 0 } A comparison of the action of the two last salts, shows plainly that the ammonio uranous compound has a far greater inhibitory action than the simple urany] sulphate. Amimonio uranic citrate. (UO2)s(Cg5H;07)2 Relative + (NH,4)sCeH;0;. Undigested residue. Fibrin digested. proteolytic action. 0 0:1747 gram. 82°53 per cent. 100-0 0025 per cent. 0-1795 82°05 99-4 0-050 0°2102 78°98 95°7 0100 02180 : 78°20 94°7 0500 09055 9°45 11-4 1-000 0 0 0 Sodio uranic sulphate. : U0.S0,+ Na.S0, Relative > +2H,0. Undigested residue. Fibrin digested. proteolytic action. 4 0 02624 gram. 73:76 per cent. 100-0 0):025 per cent. 02666 73°34 99°4 0-050 0-3688 63°12 85:5 : 0-100 04438 55°62 75:6 ; 0500 08131 19°69 26°7 1-000 09891 1:09 15 In the two last series, the ammonio uranic citrate is noticeable for not causing a gradual diminution in the proteolytic action of the ferment; but on the contrary, it gives rise to a sudden and rapid fall- ing off in proteolytic action, when a certain percentage of the salt is added. The same thing is to be noticed in the case of the uranyl - ai hieieideieict a coadt ee eer o % audits Uranium Salts on Ferment Action. 269 acetate and the reason doubtless lies in the fact that the acid in these two salts is wholly incapable of forming an active compound with pepsin, and thus when a percentage of the salt is added sufficient to use up all of the hydrochloric acid of the gastric juice, digestive action comes to a full stop. Potassio uranie oxychloride. UO.Cl, + 2KCl Relative +2H,0. Undigested residue. Fibrin digested. proteolytic action. 0 0°3063 gram. 69°37 per cent. 100-0 0-025 per cent. 0:2472 75°28 108-0 0-050 ; 0°2123 73°77 113°5 0-100 0°2582 74°68 107°6 0-300 02648 732 105°9 0-500 0°3578 64°22 92-5 With this salt, unlike any of the preceding, there is to be seen a direct stimulating action on the ferment. Only in the presence of 0°5 per cent. of the salt is there any retarding effect produced. This naturally suggests that possibly uranium per se, at least in small fractions of a per cent., has really a stimulating effect on ferment action, but that owing to its combination with acids, in the forma- tion of salts, its apparent effects in the case of pepsin-hydrochloric acid are those due to combination of the normal acid of the gas- tric juice and liberation of the acid of the uranium salt. In this way only, can we explain the noticeable difference in action of the oxychloride and the other uranium salts. Thus 0°5 per cent. of the former causes but slight diminution in proteolytic action, while ; with all the other salts, the same percentage causes on an average, a diminution in proteolytic action of at least 50 per cent. This would apply, of course, only to small percentages of uranium, for larger ‘ amounts of oxychloride would cause the formation of an indigestible - a Ae —_- uranium-albumin compound. ~ Here, however, as in the case of all the salts, the action of any given percentage is constant only under definite conditions. Dimin- ish the amount of ferment, for example, and the amount of dissolved proteid matter consequent thereto, and then the retarding action of _ the same percentage of uranium salt will be correspondingly in- creased. This is well illustrated in the following series of experiments with potassio uranic oxychloride. Using the same percentages of salt as employed in the preceding series, with the same strength of acid, but with onlv half the same content of pepsin extract, and the fol- lowing results were obtained ; 270 Chittenden and Hutchinson—Influence of Relative Uranium salt. Undigested residue. Fibrin digested. proteolytic action. 0 0:1652 gram. 83°48 per cent. 100°0 0-025 per cent. 0°1982 80°18 96-0 0-050 0:2192 78:08 93°5 0-100 0:2569 74°31 89°2 0300 03486 65°14 78:0 Increasing the percentages of the oxychloride still further, either with this strength of pepsin or the preceding, and there is seen a gradual diminution in the action of the ferment. Compared, how- ever, with the action of the preceding salts, retardation is seen to be quite slow; thus even 2:0 per cent. causes a diminution of pro- teolytic action amounting to only 50 per cent. The following table of comparisons shows the relative acceleration and retardation of the various salts compared with their respective controls expressed as 100. ; TABLE SHOWING RELATIVE PROTEOLYTIC ACTION. Percentage of Salts_---._---| 0-025 | 0:05 Of) O83) Od | 1:0 | 2.0 Paneremenmecc Uranyl nitrate 2--£-o2e 2". 99°8 | 9977 "87-0 4 ars | 38:8 Uranylacetate -....... --.. 98-5| 95-1 92-9/ | 29.9) 0 | Uranyl sulphate __..._...-- 91:3 | 89:7 | 83°8| _..- | 47-9 | Vg eee: Ammonio uranous sulphate., 87°4, 83°9 766) ..-. | 108) 0 Sodio uranic sulphate --- . ty 99-4) 85:5 | 75°6| __-. | 26-7 | 1:5 Ammonio uranic citrate..-.| 99°4) 95°7 94:7) _... | 11°4 0 Poem. Potassio uranic oxychlo- ) 1 108-0 | 118°5 | 107°6 | 105-9 | 92:5} _ - | -.-- Hie ts, eee J2 96-0 93:5 s92| 78-0] 81:0 66-2) 49-4 In retarding proteolytic action, the uranium salts act in part by combining with the proteid matter to be digested, forming a uranium- albumin compound, which is indigestible. Further, in a solution at all concentrated, the uranium salt is liable to precipitate mechanic- ally a portion or all of the pepsin along with the albuminous matter. In addition to this, however, retardation is also due, as already ex- pressed, to liberation of the acid of the salt by the hydrochloric acid of the gastric juice and to the subsequent formation of a pepsin-acid only partially, or not at all, capable of digestive action, ae Uranium Salts on Ferment Action. 271 3. Influence on the proteolytic action of trypsin. The method employed in determining the extent of proteolytic ac: tion in this case was much the same asin the preceding. The trypsin solution was made as neutral as possible and was prepared from dried ox pancreas, previously extracted with alcohol and ether; 20 grams dry pancreas, extracted with 200 c.c., 0°1 per cent. salicylic acid and ultimately diluted to 1 litre. » 2, EXPERIMENT I. Hypodermic injection of a solution of tartar emetic. 0120 gram of tartar emetic, dissolved in a little water, was intro- duced under the skin (right thigh) of a cat weighing 1262 grams. Trans. Conn. Acap., Vou. VII. 36 Nov., 1886, 282 Chittenden and Blake—Distribution of 3:10 p. m., solution injected. 3:16 ‘* vomited copiously. 3:20 ‘ y again, simply mucus. eee ge ye mucus and bile. Boo lon. ay and purged. 3:45 “ partially paralyzed. 3:54 ‘* vomited again. 4:30 ‘“ much prostrated. 5:10 ‘* dead.°* The various organs were then separated and the absorbed antimony determined, according to the method already indicated. Following are the results: Sb per 100 Total weight, Weight of Sb, grams of tissue, grams. milligrams. milligrams. hi vier S27 BRS See eer ae 52:0 6°35 12°21 Brain 422 - th Cet eee 27°5 0°60 2°18 Heart and lungs .__------2.-: 32°0 0°70 2:18 Kisineys--8eo2 s- o Lee peel 2:0 0-15 1°25 Stomach and intestines _-_-_-_- 74:0 0-80 1:08 Muscle*from back 2) 222s5542 138-0 1°25 0°90 335°5 9:85 These figures show the greatest absorption by the liver; the brain stands next, while the muscle tissue appears to have absorbed but a relatively small amount of the antimony. In this connection it must be remembered that two hours only, intervened between the intro- duction of the poison and the death of the animal, hence it is evident that the brain tissue must have a decided tendency to hold absorbed antimony. The antimony, however, was introduced in the form of a readily soluble salt and under conditions directly favoring rapid and wide-spread distribution. That there is certainly some decided selec- tive action, is evident from the fact that the muscle tissue, which must truly have had as good an opportunity as the brain tissue, retained per 100 grams of substance far less of the poison. Elimin- ation had evidently commenced, for the kidneys contained a decided amount of antimony. Experiment II. Hypodermic injection of a solution of tartar emetic. In this experiment, a smaller amount of tartar emetic was used than in the preceding; and further, the poison was introduced in three distinct doses, thus allowing longer time for absorption. In fact, the animal lived 22 hours after the first dose, hence the experi- Mn eS exe PTR Ie KEP awh = we = — ory silts Seteesdalbsaen: beets A Pig i a Antimony in the organs and tissues. 283 ment stands in striking contrast to No. 1, in which the animal lived but two hours; while the results, contrasted with the preceding, show plainly the influence of time on the distribution of the poison. Following are the results of the experiment on a rabbit weighing 1295 grams. Mar. 31, at 5:20 p.m., injected under the are of leg, 0°012 grm. tartar emetic. April 1, ‘* 8:45 a.m., ve : 0-035 Ce April 1, ‘‘12:45 p.m., Be a 0:085 a Total, 0-082 Animal died at 3:05 p. m. Following is the distribution of the antimony: Sb per 100 Total weight, Weight of Sb, grams of tissue, grams. milligrams. milligrams. Hergmeys Sook nk 11°5 0-60 5°21 ILIA SIPs eee eae 63°0 1°50 2°38 Brann ese aw ele ee 2 8 ke 9-0 0:20 2°22 Stomach and intestines-_-_--_- 98-0 2-00 2°04 Heart.and lungs -_-_-_-.-_--.-- 17°0 0°25 1:47 Miuscleyfrom: back = 2-2-2... _- 106-0 0-70 0°66 304°5 5°25 As might naturally be expected, the results indicate a more ‘eyen distribution of the poison than in the preceding experiment. Although two- thirds as much antimony was used as in experiment No. 1, the liver contains a far smaller proportional amount of the poison than in the preceding experiment, while the kidneys stand first in their content of antimony. Between the brain and the liver, there is but little difference and the experiment plainly substantiates the preceding in showing the tendency of brain tissue, under these conditions, to absorb and retain antimony. In the muscle tissue the percentage of absorbed antimony is almost exactly the same as in No. 1, that is, proportional to the amount of antimony introduced. The animal had evidently lived long enough to admit of a fairly complete distribution of the poison, and elimination having been going on for some time, those parts which had originally contained the most, particularly the liver, had been drawn on to the greatest extent; so that at the time death intervened, the excretory organs, notably the kidneys, were the richest in poison. This fact further indicates that the elimination of absorbed antimony proceeds some- what rapidly. 284 Chittenden and Blake—Distribution of Experiment III. Hypodermic injection of a solution of tartar emetic. In this experiment, a cat weighing 1613 grams, had injected under the skin of its hind leg 0°150 gram of tartar emetic in one dose. There was some purging and vomiting, and the animal died in 4} hours after the administration of the poison. The object sought in this experiment, which is virtually a repetition of No. 1, with a some- what larger dose of antimony, was simply to see whether there would be found the same relative absorption of antimony by the liver and kidney as in No. 1, and if by chance there should occur a longer interval of time between the introduction of the poison and death, what then would be the relative amounts of antimony in the two organs. As stated above, the animal lived 4} hours after the administration of the poison, or 24+ longer than the cat in No. 1. Following are the results of the analysis of the two organs: A, — Reps Se nap Sb per 100 Total weight, Weight of Sb, grams of tissue, grams. milligrams. milligrams. ba Vers Pee oe eer a oe ae 62:0 2°50 4°03 PROG YS "2 -eeee gee ee 14:5 0°25 1:72 — emit ory These confirm to a certain extent the results of No. 1, while at the same time the smaller difference between the amount of antimony * = ee gs contained in the liver and kidneys, as compared with the difference found at the end of two hours (see experiment I), would-seem to indicate that the liver had already absorbed its maximum amount, 4 and that at the time of death, elimination was well under way; or in H other words, that the removal of the absorbed antimony from the liver had already commenced. EXPERIMENT IV. (a.) Hypodermic injection of a solution of tartar emetic. (b.) Injection of a solution of tartar emetic per rectum. These two experiments were undertaken to ascertain whether the avenue by which the poison was introduced, would influence materi- ally the relative absorption of the antimony. The results, however, although interesting, do not definitely answer the question, Absorp- tion by injection per. rectum is so much slower than by hypodermic injection, or the effects produced are so much slower in manifest- ing themselves, that it is impossible to have the conditions exactly alike in the two cases, Either the time required to produce a given ac natelinaiedaadadiaianiai aa Se ee dl ~— 2 wey caatinhs Antimony in the organs and tissues. 285 effect, in the case of injection per rectum, will be longer than by hypodermic injection, or else the amount of poison must be corre- spondingly increased; either of which introduces an objectionable element into the experiment. (a.) Rabbit weighing 1485 grams had injected under its skin 0°80 gram of tartar emetic dissolved in a little water. Injection made at 11:35 a.m. At 3:45 p. m., 4 hours and 10 minutes after the first injection, 0°08 gram more was injected in the same manner. At 4:05 p- m. the animal died. (6.) Rabbit weighing 1512 grams had injected per rectum 0-08 gram of tartar emetic dissolved in a little water. Injection made at 11:45 a.m. At 3:50 p.m. the animal apparently not being affected at all, whereas rabbit (@) was strongly under the influence of the poison, 0°160 gram more of the salt was injected per rectum as before. At 5:30 p. m. the animal was still alive, but evidently feel- ing the effect of the poison. The animal died during the night. Following are the results of the analysis of the parts from the two rabbits: Raspit (a) hypodermic injection. Sb per 100 Total weight, Weight of Sb, grams of tisssue, grams. milligrams. milligrams. UECIR HATS (G1 7 a ae a 10:2 0°65 6°34 STE T0j ae A a A ee 5:5 0°20 3°63 ARGO. 8 ee ee eee ee 54:0 1°30 2°40 Mearhand lunes 2.9!) oS 15°5 0-30 1:93 Stomach and intestines -_----- 174:0 1°60 0°92 Minmelanes Sipe trl U2 Los 110°0 0°50 0:45 369°2 4°55 Rassir (6) injection per rectum. Sb per 100 Total weight, Weight of Sb, grams of tissue, grams. milligrams. milligrams. Stomach and small intestines... 172 8°89 15°30 ‘STenTi CS 2 re 9 0-40 4-40 Rectum and adjoining intestine. 18 0°55 3°05 ILAMASER alesse Aas ted lie apa 54 1:60 2°96 Mebane: Pb 13 0:25 1:92 Museled=2 26 wiser 2: eget syns V1 100 1-10 ala TOGHIATE)S is te ae ene 20 0°20 1:10 eartand lungs)... 2. -+-- 17 trace 286 Chittenden and Blake— Distribution of Comparing first, the results obtained from rabbit (a) with those of the three preceding experiments, we see at once that the distribu- tion of the antimony is much the same as in No. 2, in which, how- ever, the animal lived nearly 22 hours after the introduction of the first dose of poison and somewhat over two hours after the last. — The fatal dose, moreover, in this case was nearly the same in amount as the first dose in experiment IV a. The conditions, however, of this experiment (IV @) do not exactly accord with any of the preceding, hence close comparisons cannot well be made. The brain, as in all of the experiments with tartar emetic, contains a proportionally large amount of antimony, while the muscle contains a very small amount. The only thing in this experiment not exactly in accord with the preceding experiments, is the proportionally large amount of poison in the kidneys, as com- . pared with the liver. The only apparent explanation seems to be that, the first dose being small, the liver had quickly reached its maximum absorption and elimination had been rapidly going on; so that at the end of the four hours intervening between the first and second doses of the poison, the kidneys had drawn extensively from the liver, rapidly diminishing its content of the poison. Further, - after the second dose of poison, the time before death was so short that the additional absorption by the liver was not sufficient to make up the deficiency, and hence the results found. In this connection, it must be remembered that tartar emetic is very readily soluble and diffusible, and that being injected in solution directly under the skin, its absorption must necessarily be very complete and rapid. In Rabbit (4) the conditions are wholly different from those of the preceding experiments. In all, 0:24 gram of tartar emetic, dissolved in water was introduced into the rectum and 8-10 hours, at least, must have intervened between the administration of the first dose of — the poison and death. ‘That the stomach and small intestines should contain the largest proportional amount of antimony is perhaps not at all strange, since the antimony solution would naturally pass rapidly by osmosis through the entire alimentary tract. That this, however, is not the full explanation, is evident, when we compare the amount found in the large intestine with the former. If due simply to osmosis, the percentage amount of antimony would be about the same all through the intestines; hence we must look to some selective action for explanation of the increased amount found in the small intestines. In all of the preceding experiments, the amount of antimony found in the stomach and intestines has been blah ccias iain Ilia nc dela ach oti 2 ety ® wre iy Sachisthniriththderthk- 4s dhaletheniaad-aesemmnartateke Oe ATS es; 5 Antimony in the organs and tissues. 287 considerably greater than in the muscle tissue. Undoubtedly, the greater vascularity of the former has much to do with its greater content of the metal, but even this is not sufficient to account for all of the antimony found; for whenever blood itself has been analyzed, the amount of antimony has not been large. Unquestionably then, we must assume special absorptive action on the part of the epi- thelial cells of the stomach and small intestines. In this connection it is well to notice the work of Brinton, who proved that when tartar emetic was injected into the vein of an animal, it was very freely and rapidly eliminated by the stomach. This was also cor- roborated by Dr. Richardson who, in addition, found that a simi- lar elimination followed the inhalation of antimoniuretted hydro- -gen.* In addition, it may be that absorption of antimony from the alimentary tract goes on slowly and that hence only a por- tion was removed. This idea has considerable to support it, when we consider the distribution of the absorbed antimony. Remembering that in this experiment, a larger amount of anti- mony was used than in any of the preceding ones, and that there was apparently ample time for absorption, one cannot help but think that the content of antimony in the remaining tissues and organs is very small. This is very evident, and must be due to one of two causes; either there has been a lack of absorption or else elimination has been going on very rapidly. The brain contains a noticeable amount of antimony, larger than found in any preceding case, while the liver and kidneys both contain a comparatively small amount. The amount of antimony in the kidneys and particularly the amount in the urine, plainly indicates that elimination was going on rapidly; but the fact that the percentage content of antimony in the liver is greater than in the kidneys, would perhaps indicate that at the time of death, absorption was not completed. Such being the case, the_ only inference to be drawn from the two preceding experiments, is that the introduction of tartar emetic into the rectum leads simply to a much slower absorption and distribution of the antimony than by hypodermic injection, but that there is no essential difference in the relative distribution of the poison under these two conditions. * Quoted by H. C. Wood, Therapeutics, p. 159. 288 Chittenden and Blake—Distribution of EXPERIMENT V. (a.) Tartar emetic in substance, introduced into the stomach. (b.) Antimonious oxide (Sb,O,) introduced into the stomach. In this experiment there were two objects in view; one was to see the effect of tartar emetic in substance, as compared with the action — of the same salt introduced into the system by hypodermic injection or per rectum; the second, to compare the absorption of an énsoluble compound of antimony (Sb,O,) with that of the more soluble tartrate. In this experiment two dogs were used and the poison was fed to them at regular intervals, in small doses, for a period, in each case, of 17 days. The animals were then killed and the various parts anal- yzed. The two experiments were exactly alike in a respect, except in the amount of poison administered. (a.) Dog weighing 7°75 kilos was fed 0°762 gram of tartar emetic during a period of 17 days, in two or three doses daily, the individ- ual doses being small enough not to induce vomiting. The first two days, the dose was 0°016 gram per day, the third 0-020 gram, the fourth 0°030 gram and so on, increasing each day until the last daily dose was 0°085 gram of the poison. The dog was then killed by chloroform, just six hours after the last dose of poison was adminis- tered. ; (0.) Dog weighing 14:2 kilos was fed 2°073 grams of antimonious oxide, during a period of 17 days, in two daily doses of from 0°032 to 0°125 gram per day. The dog was then killed by chloroform, 18 hours after the last dose of antimony was given. Following are the results of the analysis of the various parts: Doe (a) with tartar emetic—(0°762 gram). Sb per 100 Total weight, Weight of Sb, grams of tissue, grams. milligrams. milligrams. WAV OT. aioe ses pe eee eee = 304 17°80 5°85 Salivary elandsi2e-s- 26-4 eee 11 0°25 2°27 Keidnieys' iiiits 2s Sake = 5B 1°25 2°15 Braise Sule. te eee ees 7 1:15 1:51 WOnPMG\-feilccere pate gaat 36. 0-40 dtd Marseler(ibig bh) 22 sean see 150 1°60 1:06 (sj 6) Ce”) ipa aL = 19 O15 0°80 HiGartes-ss-. 2-5 eee 77 0°50 0°66 PMG Peete oie OS ee 140 0°50 0°36 Bone (femur and tibia)---..--- 43 0:10 : 0°23 BOS ee eed oro 3 eee 130 0:20 0°15 ReBUGS eee ee hes woe 12 trace Pancrens sees oot eae 23 trace 1079 24°05 A 9 AM tag RRP I oN WRT RG ei he >= SA eae he poles Sn tw? ~ Antimony in the organs and tissues. 289 Doe (6) with antimonious ovide—(2°073 grams). Sb-per 100 Total weight, Weight of Sb, grams of tissue, grams, milligrams. milligrams. itv elgg meres ee tee 452 23°70 5°24 nein ee es Be Si! at i! 140 1°80 1:28 Musele (foreles)): 24) 225255. 157 1°20 0-76 penile renew fe oe RS) SL 79 0-40 0°50 Migscte(thich).<. 22.0... 2)... 200 0-90 0°45 rite Alesse LS LSS. 2. | 82 0-10 0-12 BIST ge ee ee ee oe 117 trace Blood ~ 222. ne pa Shack a ih eee 440 trace 1667 28°10 In considering these results, we notice first that in dog (a) the dis- _ tribution of the poison is much the same as in the preceding experi- _ ments with tartar emetic, viz: the liver, kidneys and brain stand first in their content of antimony. That the liver should contain _ more per 100 grams than the kidneys, although the animal lived full eight hours after the last dose of poison was taken, is here to be expected, since absorption as a whole would naturally be slower _ than in some of the preceding experiments; and, further, in this case b probably all of the antimony would be absorbed through the portal - circulation. In the case of dog (4), the conditions are different from any heretofore; we have here an insoluble form of antimony con- _ trasted with a readily soluble and diffusible salt. Solution must necessarily be somewhat slow in this case, but the acid juices of the - stomach unquestionably do dissolve and render diffusible, at least a _ portion of, this form of the poison. We notice first that the total amount of antimony administered, is fully three times as much as the amount of tartar emetic given, and yet the amount of antimony recovered from the different tissues and ' organs is but 4 milligrams more than in the case of tartar emetic. _ This suggests that either considerable antimony is excreted by the kidneys (more than in the case of tartar emetic) or else that consid- erable passes through the alimentary tract unabsorbed. The dog being confined in a cage of suitable construction, the 24 hours’ urine was collected on several occasions and the amount of antimony deter- Mined. Thus on one day, when 0-097 gram of antimonious oxide had been administered, following after a daily dose of 0-064 gram, the 24 hours’ urine contained 13°5 milligrams of antimony (Sb). Later, at a time when the daily dose was 0°130 gram of the oxide, the 24 honrs’ urine contained 22°5 milligrams of antimony. Hence it is plain that Trans. Conn. Acap., Vou. VII. 37 Nov., 1886. 290 Chittenden and Blake—Distribution of considerable of the antimony given was being absorbed; but bearing in mind that this latter amount was the largest excreted by the kid- neys in any one day, and further that the daily dose of antimony was being increased each day rather than diminished, it is also plainly evi- dent from the amount of absorbed antimony found, that a certain por-— tion must pass through the alimentary canal unabsorbed. Further, the small amount of antimony found in the kidneys supplements this view, as does also the noticeably small amount of absorbed antimony found throughout the body, aside from the liver. One of the main objects in trying this last experiment was to see what influence the form of the poison would have on its absorption by the brain. With arsenic, it has been plainly demonstrated by one of us,* as well as by other workers in this field, that soluble and readily diffusible forms of arsenic are absorbed by the brain in | appreciable quantities, while arsenious oxide for example, no matter whether taken in large or small doses, single or oft-repeated, is never found in the brain other than in mere traces. With antimony we had expected to see something of the same kind. The results, how- ever, although tending in that direction, are not quite as decisive as we should have liked. The antimony found in the brain in the anti- monious oxide case is, to be sure, considerably smaller in amount than that found in (a), although the dose of antimony given in the former was much larger than in the latter case. But it is also to be seen in the antimonious oxide case, that the amount of absorbed antimony in the brain, although very small, is still greater than the amount found in the kidneys or muscle. We attempted another experiment in the same direction with rab- bits, but as the amounts of antimony found in the brain in both ani- mals were hardly more than mere traces, the results do not give us any additional light on the matter. In spite of the fact that the experiment was a failure, so far as its main object was concerned, we venture to describe it, since it well illustrates in other respects, the greater virulence and diffusibility of tartar emetic. Two rabbits of nearly equal weight were selected, and to one potassium antimony tartrate was fed in gradually increasing doses for a period of 17 days, at the end of which time the animal died with all the symp- toms of antimoniacal poisoning. To the other rabbit, antimonious oxide was fed for the same period of time, in doses the same as given to the first rabbit; that is, doses equivalent to the antimony (Sb) contained in the tartar emetic. Each rabbit, therefore, received * See Studies from this Laboratory, vol. 1, for the year 1884-85, p. 141. Antimony in the organs and tissues. _ 291 _ twice a day, the same equivalent of antimony, and at the end of the 17 days the one rabbit had taken 2°34 grams of tartar emetic, the other 1°08 grams of antimonious oxide. While each rabbit had taken the same amount of antimony, the one which had taken it in the form of potassium antimony tartrate was much more severely affected by the poison. In this case there was severe purging and finally death on the 17th. day. In the case of the rabbit fed with antimonious oxide, the only apparent effect of the poison was a loss ‘of appetite and great thirst. This animal was killed with chloro- form on the death of the first rabbit. Both forms of antimony were administered as powders, by way of the mouth, in small gelatin capsules. Following are the results of the analysis of the various parts from the two rabbits: Rassit (a) fed with tartar emetic. Sb per 100 Total weight, Weight of Sb, grams of tissue, grams. milligrams. milligrams. Liye SS ee eee ee 50°0 4:8 9°60 LATOR 6-7 0-5 ; 7°40 Heart and. lnngs --_: -.-.2.-=-- 18:0 0-4 2°22 Muscle from back --_---------- 550 0-5 0-91 Muscle from leps:_----..+------ 79-0 0°3 0°38 rains sere ao SS Fa he ae ie trace 216°4 6°5 Rassir (6) fed with antimonious oxide. Sb per 100 Total weight, Weightof Sb, grams of tissue, grams. milligrams. milligrams. DiGi. ue 57:0 1:3 2°28 Muscle’ from back | _..-.---.-- 77-0 0-7 0:90 Muscle from legs ------------- 100-0 0-7 0:70 z Lin Sie) 721) Se 8-0 trace ear and lungs _2.-..--...-- 16-0 trace BTC a a ee 8:5 trace 266°5 at Looking at these results and remembering that each animal re- ceived the same amount of metallic antimony, it is evident that tartar emetic is much more completely absorbed than the oxide. - With tartar emetic, however, the results are not exactly in accord with the previous ones, obtained with this salt; thus the amount of antimony absorbed by the brain is far smaller proportionally than 292 Chittenden and Blake—Distribution of Antimony,?ete. found hitherto. To be sure, the compound was not in the previous | experiments introduced into the stomach of a rabbit in the form of powder, and it is possible that the reason for the difference -in the amount of antimony found in the brain in this case and that in the — brain of the dog similarly treated, lies in the fact of a slower - absorption from the stomach of a herbivorous animal. XLX.—INFLUENCE or ANTIMONIOUS OxIDE on METABOLISM. By R. H. CairrenpDEN anv JosepuH A. Buakns. Tue physiological action of antimony has been studied mainly with potassium antimony tartrate, the form in which antimony is most commonly used therapeutically. No experiments, however, appear to have been made, even with this salt, to ascertain the influence of an- timony on the metabolism of the body. Giithgens, however, as quoted by Dr. H. C. Wood,* found in some incomplete experiments an increase in the elimination of urea after repeated non-toxic doses of antimony. It is further reported+ that antimonic acid or other preparations of the metal, when taken in half gram doses daily for about two weeks, cause a diminution in the amount of glycogen in the liver and even a total disappearance of it, and that the liver, kid- neys and heart undergo fatty degeneration. Grohe and Moslert have confirmed the latter and state that in the production of the famous fatty livers, a certain amount of the white oxide of antimony is fed to the geese daily. Aside from these facts, there appears lit- tle definite regarding the action of antimony on the physiology of nutrition. What we have, therefore, endeavored to ascertain in the present experiment is the influence of antimony on metabolism; or particu- larly, on proteid metabolism as manifested in the excretion of nitro- gen, sulphur and phosphorus. Previous experiments§ have shown that potassium antimony tartrate has a noticeable retarding action on pancreatic digestion; we have not, however, deemed it best in the _ present experiments to use tartar emetic, as the ready solubility and diffusibility of the compound might cause too rapid absorption and thus lead to speedy toxic action. In spite, therefore, of the fact that we have not made any experiments on the influence of antimonious oxide on digestive action, we have preferred to use the latter in the present experiments, because of its probable slower toxic action and also because it has been so extensively used as a means to induce, or _toaid in the production of, fatty degeneration. * Therapeutics, Materia Medica and Toxicology, p. 156. + See Virchow’s Archiv., 1865, Band xxxiv, p. 78. + Compare H. C. Wood. Therapeutics, p. 161. § Studies from this Laboratory, 1884-85, p. 105. 294 Chittenden and Blake—Influence of Our experiments were made on a setter dog, weighing 12°6 kilos. The animal was confined in a suitable cage, so that the excretions could be collected daily without loss. The food consisted of fresh beef and crackers, together with a suitable amount of water. The beef was prepared as follows: About 40 lbs. of fresh beef, freed from . fat, tendons, etc., was finely divided by passing through a sausage machine and then dried at a low temperature until it had lost about 5 per cent. of water, and was in a condition suitable for preservation. 50 grams of this preserved meat, together with 75 grams of the sam- pled crackers, soaked in 300 c.c. of water, were fed to the dog twice daily. The meat, as determined by Kjeldahl’s method, contained 12-4 per cent. of nitrogen, while the crackers contained 1°9 per cent. Hence the dog was fed daily 15:25 grams of nitrogen. é On May 11th, the dog was put upon this diet and from the 17th on, the 24 hours’ urine was collected daily and analyzed. After a neaed of two weeks, during which daily analysis of the urine had shown a fairly constant composition, antimonious oxide was added to the diet in the quantities indicated in the table of results; the diet of course continuing the same throughout the length of the experiment. We deemed it better, as well as more accurate, to measure the in- fluence of the antimony by a daily determination of the total nitro- gen, sulphur and phosphorus of the urine, rather than to attempt a determination of urea, uric acid, phosphoric acid, ete. Nitrogen, we determined, according to the method of Kjeldahl,* modified slightly as suggested my Dr. E. H. Jenkins, of the Agricultural Experiment Station, viz: 5 c.c. of the acid urine were placed in a long pear-shaped bulb and evaporated to dryness quickly on a water bat The resi- due was heated directly over a small flame with 10 c.c. of pure con- centrated sulphuric acid and 0°7 gram of oxide of mercury, until oxi- dation was almost complete. Then, a little finely powdered potassium permanganate was added, to render the oxidation quite complete. The solution was then diluted, an equivalent amount of potassium sulphide added to convert the mercury into sulphide, and lastly a standard solution of sodium hydroxide, after which the ammonia was driven off by boiling and collected in standard acid. Total phosphorus and sulphur were determined as follows : 50 ¢.c. of urine were evaporated in a capacious silver dish with 10 grams of potassium hydroxide and 10 grams of potassium nitrate and the resi- * Neue Methode zur Bestimmung des Stickstoffs in organischen Kérpern. Zeitschrift fiir analytische chemie, xxii, 366. - Antimonious Oxide on Metabolism. 295 due heated carefully until the organic matter was completely oxidized. The fused mass was then dissolved in water and diluted to 250 c.c. Of this, 100 c.c., representing 20 ¢.c. of the original urine, were used for the sulphur, while the second 100 c.c. were used for the phosphorous, determination. For sulphur, the 100 ¢.c. were acidi- fied with hydrochloric acid and evaporated to dryness on a water bath in order to remove all nitrate and nitrite. The residue was then dissolved in water acidified with hydrochloric acid, and the sulphuric acid precipitated with barium chloride in the usual manner. For phosphorus, the 100 ¢.c. were acidified with nitric acid, evaporated to dryness, the residue dissolved in water, acidified with nitric acid and the phosphoric acid precipitated with molybdenum solution. This precipitate was then dissolved in a dilute solution of ammonia, the phosphoric acid reprecipitated as ammonio-magnesium phosphate, and the phosphorus finally weighed as magnesium pyrophosphate. Chlorine was determined volumetrically in the usual manner, with a standard solution of silver nitrate, after destruction of the organic matter by fusion with potassium nitrate, etc. The results, expressed in grams per 24 hours, are shown in the ac- companying tables. The 24 hours’ urine represents the quantity passed from 9 A. M. of one day to 9 A. M. of the next. As, however, the ani- mal was not always regular in its passage of urine, it frequently happened that the quantity on one day would be very small, while on the next it would be correspondingly increased, without any change in specific gravity, and with. a daily average corresponding to the normal, as for example on May 25th and 26th. In order, therefore, to obviate the difficulty which this irregularity tends to introduce into the results, we have added to the table a daily average of each three days results; a study of which shows plainly that antimonious oxide, in the present experiment at least, does not have any noticeable influence on the excretion of any of the ele- ments determined. Numerically, there is .a slight increase in the amount of nitrogen excreted during the taking of the antimony, but the increase is noticeable only in the grand average and is altogether too small to be of much significance. Further, it is to be noticed that the average for the two series does not show any corresponding in- crease in sulphur. If antimony causes an increased excretion of nitrogen, it means an increase in proteid metabolism, which should in turn give rise to an increased excretion of sulphur and phosphorus. It is to be noticed in the daily results, that the excretion of sulphur nd phosphorus runs parallel with the excretion of nitrogen; an in- Chittenden and Blake—Influence of 296 ‘SOT SBM OULIN 9Y} JO UOTA10d & PZZ oY} UC ~ T8640 8TG4-0 418-0 bE%-6T T-SGOL Gale Ol oe eee ase1oae ATE 6086-0 6016-0 9686-1 ¥66-9T ¢- LOT 8F9 “‘proe "96 6L8L-0 6619-0 6699-0 oTS-6 0-LE0T 66& “‘proe “GG PLOP-0 P299-0 9699-0 c6F- TE 0) 0801 907 ‘pre ‘ye | AR 6169-0 G9L9-0 9689-0 689.11 9:9601 1 ey | ee ae eseieae Apred 9cTS-0 8ELL-0 9F9L-0 966-ET £-6601 89F ‘pre "EGx C6LE-0 6969-0 8TTL-0 F06- TT 0-L601 997 ge ‘Ts 0GL9-0 909S-0 9TLS-0 869-6 ¢-8201 OLY ‘ouryex[® ‘03 «= Av T£49-0 1969-0 8899-0 VOL IT 9.9601 08f = 7 -asereae ATrEd TL8S-0 9TL9-0 ccsl-0 868-TT ¢- L601 697 IBIS" 3) ABE G0GL-0 6869-0 1899-0 | 676-01 | C-FE0T | OTS ‘PRPs ‘ST i 6E6L'0 68L¢-0 6619-0 96-01 | C-FE0T GOP ‘proe =| ‘zr ARH ‘Suede “meIs ‘WURIs “WBIS ‘SUUBIS | 72°0 | | ee “euLIO[YO, ‘anydyng ‘snaoydsoyg | ‘MOBOIYN uy ‘dg “OTUINTO A “mOyoRoy | “aye ‘KNONILNY LAOHLIA, 297 Oxide on Metabolism. tmontous Ant ‘ ‘ogyjedde z00d Ul PUR ]JOAUN JRIYMoTMIOS pouoes Sop oy sv ‘pezATVUR JOU SBA oUTIN oY} OUNL Jo pg oY} pue Av JO 44GZ 04} UooMyog + 2695-0 8049-0 TS6h-0 9508-0 S119-0 GLGL+O c-0 C.0 t 6986-0 P8s8c-0 6099-0 6668-0 6609-0 EGLL-0 8L8T-0 0989-0 6978-0 Th95-0 6999-0 &669-0 T1¥S-0 Loc9-0 Tope-0 GCPS-0 6h69-0 6419-0 1619-0 8889-0 6LEL-O *SULBIS “TRL “URIS “URLS ANS RAO “OUTLOTYO, sanydng “‘snaoydsoyg: jo yunouty “panur7zU0I—ANOWLINY LOOHLI AA SULIT §-L60F 997 -*“SoLtos oY} LOZ VSR.IOAY 098-61 84601 OG me Nn eae cae ase10ae ATE | BOL TT ©. 8601 VIP ‘pre g POLO G.LE0T BRP “pre a PLS-ET. C-L80T 00g ‘prow ‘g foune TLG-TE 8601 ry digas Wt. me asvroae ATE POR- TT 08601 cep ‘pre 63 €69-01 08601 PIF ‘pre |. 8s 889-11 €-6801 OS ‘poe "Le ART ‘SUUBIS OI) UOSOI}IN 19 ‘dg -‘umnjo A ‘mOTOVEYy * ‘aye Nov., 1886. 38 _ Trans. Conn. Acap., Vou. VII. ra /- eS NW oe ae Sy dt aie ot wet - “—— Coles iy 2.4 - es Chittenden and Blake—Influence of 298 jo qunomy T8670 eases ele 20 t OCFE-0 £0 t 8689-0 O-T ut t | LOCP-0 LE8S-0 £0 t 1989-0 £0 t LO9F-0 c.() t 12e9-0 area. ee ‘meye} §O*qS “euLLOTYyO T9T9-0 LL6S-0 CLO9-0 6889-0 6199-0 O8LL-0 810-0 €F19-0 TBS anqdyng 8964-0 TETL-0 8902-0 9LSL-0 9OL4O 9F18-0 196-0 1106-0 “meds ‘snaoydsoyg GOL -6T 9.8601 sy eG Nii ae cea aseioae ATE 068: EL 0-860T PCP “proe ‘IL 8&8: LT @-LOOL ULV ‘plow ‘OL L8¢-6L 00-0801 CEP “prow 6 oun’ 780-61 8-8E01 OY oe a SF hoe advrcae ApTreq 966-81 ¢.9801 09g ‘poe 8 090-01 0-0801 OSs “poe *h 896-61 0-080L O6P ‘pro’ 9 oun ‘suuBIs ay 4) “MWOSOTYIN 9 ‘dg *auIN]O A “MOORE ‘aye ‘AdIXQ SQOINONIINY HLTA. 299 Antimonious Oxide on Metabolism. "woye} "O*qQg jo qunowy eee O£0G-0 189-0 6664-0 8G0-6IT 9-8G0L EGY ~-“SoLIOS OY} LOF OSRIOA TITG-0 6679.0 4649-0 969-11 $-8GOI ort “rors ss" 95B10A8 ATE 9CEF-0 1669-0 LOLG-0 C86-01 0-LG0T G&P “plow ur ESFS-0 FIF9-0 F99L-0 ELL-OL 0-8301 PSP ‘plow ‘OT €9¢¢C.0 8819-0 6089-0 TLP- TT C0801 FOF *prloe ‘ct oune 6987.0 9969-0 8974-0 L68-81 ¢.8601 Gof Sian ~-----9seioae ATTed TLGE-0 T8€8-0 O6FL-0 006-61 G-820T SLF ‘ouTT VATS FL GL6E-0 LOSS-0 0689-0 CF9-O1 0-L30L OTF ‘prow ‘ST 98¢8-0 G1BL-0 O0Sg8-0 8F9-8T 0-0801 FOS ‘prow “ep oune “URIS TURIS “TRIS OER Ra} 2) 7) “aULOTY.O | ‘rnqding “‘snioydsoyg “UOSO.UIN 19 ‘dg “ounyO A. “MONOvOY aye] ‘PONUYUOI—AGIXO SQOINOWIENY HLTA, 300 Chittenden and Blake—Influence of Antimonious Oxide, ete. crease in the latter is always accompanied by an increase in the two former. Inthe grand average of the results, however, the slight in- crease in nitrogen is not accompanied by a corresponding increase in sulphur. In fact, the two series of results, indicate plainly that the antimony was without any material action. The total amount of an-° timony given, 16 grains of the oxide during 13 days, was certainly sufficient in quantity to have exerted its peculiar influence if possessed of any. The antimony was certainly absorbed, and that too in con- siderable amount. Thus on the 11th of June the 24 hours’ urine con- tained 13°5 milligrams of metallic antimony; on the 17th, 22:4 milli- grams; on the 18th, 17°6 milligrams and on the 20th of June, 15:1 milligrams of metallic antimony. These quantities of absorbed anti- mony would certainly indicate the presence of sufficient antimony for some decided influence on metabolic action, if any existed. The- amount of nitrogen excreted daily, is seen to be considerably below the amount of nitrogen ingested. We did not make daily examina- tions of the fecal matter, but such as were made showed plainly that the deficiency in nitrogen was contained mainly in the feces; thus on the 5th of June the 100 grams of feces excreted, contained 2°42 grams of nitrogen. At that date, the average amount of nitrogen excreted by the urine was 12°36 grams per day ; this amount, added to the fecal nitrogen makes a total of 14°78 grams excreted, against 15°25 grams ingested; a difference to be found mainly in the hair thrown off, and in part, in the ordinary errors of analysis. | We must conclude, therefore, that small repeated doses of anti- monious oxide are without influence on the excretion of nitrogen, sulphur and phosphorus, and that consequently this compound, at least when taken in non-toxic doses, has no action on proteid meta- bolism. oe J XX.—On Some Merattic Compounps or ALBUMIN AND Myosin. By R. H. Currrenpen anp Henry H. Wurirrenouse, Pu.b. Ever since Lieberkiihn, in 1852, attempted to establish the molecu- lar weight of albumin by preparing and analyzing the copper com- pound resulting from the action of a soluble copper salt on a solution of egg-albumin, various investigations have been published bearing on the nature and composition of the compounds of albumin with the heavy metals. Before this time even, F. Rose, in 1833, had published an analysis of a copper compound of albumin in which he had found from 1°50 to 1°70 per cent. of cupric oxide, and Mitscherlich, in 1837, published an analysis of a similar albumin compound, in which he found from 2°8 to 3°3 per cent. of cupric oxide, while Lieberkiihn’s compound contained 4°6 percent. CuO. Compounds of albumin with other metals have also from time to time been prepared, such as zinc, lead, silver and mercury, and in one or two cases provisional formule have been given. The results, however, are to be considered as quite uncertain. With platinum chloride a compound appears to have been formed* of somewhat more certain composition. Aside from the more recent experiments of Ritthausent on the vegetable albumins (gluten-casein, legumin, etc.), egg-albumin has been the chief albuminous body experimented with, and copper the main metal. Recent work by one of us (C) on the albumose and globulose bodies, together with work on the products formed from casein and myosin, has led to a partial study of the metallic compounds of these bodies. As a preliminary, however, we found it necessary to study a few of the compounds of egg-albumin, and as the results thus obtained were not in accord with the more recent results of Harnack{ we have continued our work with egg-albumin and with myosin, the results of which we now present. * See Commaile, Moniteur Scientifique, 1866, and Fuchs, in Annalen der Chemie, vol. cli, p. 372. + Die Eiweisskorper der Getreidearten, etc. Journal fir prakt. Chem., vol. xii, p. 361. ¢ Untersuchungen iiber die Kupferverbindungen des Albumins. Zeitschrift fiir phy- siologische Chemie, vol. v, p. 198. 302 Chittenden and W hitehouse—Metallic I. EGG-aLBuMIN. (a) Copper Compounds. In looking over the literature of the subject, it becomes evident at once that the older investigators, owing either to the nature of the . compound, to adherent impurities or to faulty methods, were not able to obtain concordant results, since the copper compound of egg- albumin, as prepared and analyzed by six distinct investigators, was found to contain from 1°50 to 5°19 per cent. of CuO. In all of these cases the preparation of the copper compound was essentially the same; a solution of egg-albumin was precipitated with a solution of a copper salt, the precipitate collected, washed thoroughly with water, dried, and the copper determined by simple ignition. Naturally this method, as suggested by Harnack, might be expected to give too high results, since the copper precipitate would unquestionably re-— tain considerable of the inorganic matter of the albumin. Treated in this manner, however, F’. Rose,* as already stated, found the copper compound to contain from 1°50 to 1°69 per cent. of CuO. Mitscher- lich,t who held that the copper precipitate was a compound of egg- albumin with the copper salt, found in his preparations 2°8—3°3 per cent. CuO, while Bielitzki,| who demonstrated that the precipitate was an actual compound of albumin with cupric oxide, found in his preparations 4°75—-5'20 per cent. of CuO. Lassaigne, as quoted by Harnack, found 4:95 per cent. of CuO, Mulder§ 4°44 per cent., while Lieberkiihn’s| preparation contained 4°6 per cent. of CuO. Further, Ritthausen’s copper compounds of the vegetable albumins were found to contain from 11°5 to 17°0 per cent. of CuO. These results collectively, would therefore seem to indicate that when egg-albumin is precipitated by a soluble copper salt, the resulting compound does not contain a defi- nite proportion of albumin and cupric oxide, or else that there are a large number of albumin-copper compounds. More recently, how- ever, E. Harnack,* from analysis of fifteen separate preparations, comes to the conclusion that there are two distinct. compounds of albumin with copper; one containing 1°35 per cent. of Cu, the other 2°64 per cent. of Cu, indicating as Harnack suggests, a copper albu- minate in the first case of the formula C,,,H,,,N,,O0,,5,Cu, in which Cu 204 $20 66> 2 replaces two atoms of hydrogen in the albumin moleanis and in the second case, an albuminate of the formula C,,,H,,.N,,0,,5,Cu,, in— 204 * Poggendorff’s Annalen, vol. xxviii, 1833. : Miiller’s Archiv. for 1837, p. 91. + Dissertation, Dorpat, 1853. § Physiologische Chemie, 1844-51. || Poggendorfi’s Annalen, vol. Ixxxvi, 1852. §{ Loc. eit. ~ S ies = Compounds of Albumin and Myosin. 303 which two atoms of Cu replace four of hydrogen. The results ob- tained by this investigator would certainly seem to warrant this con- clusion, for the analytical data of the different preparations show but slight variations ; 1°34—-1°37 per cent. in the one case, and 2°48—2°74 per cent. in the other. Further, Harnack worked with nearly ash-free preparations, the compounds after their first precipitation and wash- ing being dissolved in sodium carbonate and reprecipitated by care- ful addition of acid. By repeating this process several times, the ash of the preparation was almost entirely removed, while the relative proportion of copper and albumin was not affected, As to the condi- tions which determine the formation of one or the other compound, there seems to be little definite other than that in general, the com- pound with smaller content of copper was obtained when the precipi- tation took place in the presence of a slight excess of albumin, and the compound with larger content of copper when in the presence of an excess of the copper salt. In no case were the copper salt and albu- min solutions mixed in definite proportions, yet in every case one of the two compounds was formed; further, Harnack states that when an amount of copper salt exactly sufficient to form the albuminate is added to a given quantity of albumin, no precipitate results; in other words an excess of the copper salt is necessary to insure a separation of the compound. Harnack’s results, therefore, differ from those of the preceding in- vestigators in that definite compounds appear to have been formed in every case, and further, in that the compounds contain a lower per- centage of copper than found by any other investigators aside from F. Rose.’ This latter, it will be remembered, found 1°50-1°69 per cent. of CuO ; 1°69 per cent. being equal to 1°34 per cent. of Cu, one of the percentages found by Harnack. Harnack further states that the average of the analyses made by other investigators, aside from Rose,_ show about 4-4 per cent. of CuO, and assuming that the various prep- arations contained an amount of ash equivalent to about | per cent. (which amount Harnack found in his preparations before purification) the percentage amount of cupric oxide would be reduced to about 3-4 =2°7 per cent. Cu, or the amount found by Harnack in his highest cop- per compound. But as Rose’s preparation was made by the simple ad- dition of an aqueous solution of egg-albumin to the copper salt and the copper determined as oxide by simple ignition, it would seem neces- sary to make the same deduction of 1 per cent. also in this case, which would make Rose’s compound contain far less CuO than found 304 Chittenden and W hitehouse— Metallic by Harnack. Further, Rose* found that the serum of ox blood yielded a similar compound with cupric sulphate, which contained only 1°14 per cent. of CuO or 0°88 per cent. of Cu. Hence there would seem to be little in these earlier investigations to substantiate the results obtained by Harnack. Mérner,+ however, working with © alkali-albuminate, found that on precipitating a solution of alkali-al- buminate with cupric sulphate, in the presence of an excess of alkali, he obtained a copper albuminate containing a percentage of cupric oxide corresponding closely with that found by Lieberkiihn. When, on the other hand, he precipitated a nearly neutral solution of alkali- albuminate with cupric sulphate, then the percentage of copper in the copper albuminate amounted to only one-third that found by Lieber- kiihn, or an amount about equivalent to that found by Harnack in his lowest copper compounds. Moérner further found that by precipitat- ing a calcium albuminate solution with cupric chloride, the albumin-— ate combined on an average with 2°33 per cent. of CuO, or just one- _ half the amount required by Lieberktihn’s formula, and considerably less than the amount contained in Harnack’s largest copper com- pounds. Preparation of the albumin solution.—In our experiments it was thought best, as far as possible, to avoid exposing the albuminate to the action of alkalies, hence especial care was taken to prepare the egg-albumin as free from salts as possible, so that it would not be necessary to purify the albuminate by reprecipitation. The whites of a large number of eggs were finely divided by scissors and by shaking with glass, then mixed with an equal volume of water and thoroughly shaken with air, after which the solution was strained through cloth. Globulin was then precipitated by the addition of dilute acetic acid (the acid added as long as a precipitate formed), the solution finally filtered through paper, after which the filtrate was made exactly neutral with sodium carbonate and again filtered. The fluid so obtained was then dialyzed in running water for many days, a little thymol being added to prevent putrifaction. The fluid finally obtained was perfectly neutral, clear and contained but a small amount of inorganic salts. In forming the albuminate we employed both cupric acetate and cupric sulphate, using in each case the same volume of albumin solu- tion, but varying the amount of copper salt. The copper salt was generally added as long as a precipitate formed. The albumin- * Loc. cit., p. 139. + Jahresbericht fiir Thierchemie, 1877, p. 8. Compounds of Albumin and Myosin. 305 ate was washed on a pump with water, until no reaction could be _ obtained with potassium ferrocyanide for copper or with acetic acid and potassium ferrocyanide for albumin. The preparations were dried at 100° C., then powdered and further dried at 110° C., until of - constant weight. The percentage of copper was first determined by simple ignition and weighing as cupric oxide. The oxide was then dissolved in dilute nitric acid, the copper precipitated as sulphide with hydrogen sulphide and weighed as subsulphide by ignition in hydro- gen gas with a little sulphur. Each series was made from a distinct preparation of albumin and nearly every compound made, was analyzed in duplicate. Following are the analytical results : Seriss I. With CuSO “ No. Am’tsub.taken. Wt. CuO. Per cent. Cu. Wt. Cu.8. Per cent. Cu la 0°5621 gram. 0°0081 gram. 1:18 0:0070 gram. 0°97 pee 107016 0:0078 1:19 0:0065 0:98 With Cu(C,H,0,),. 2a 9°5479 0:0088 eo Bele eee b 0:5708 0:0091 1:26 0:0078 © 1:08 Seriss II. With CuSO, la 05273 gram. 0.0067 gram. 1:00 0-0053 gram. 0°79 b 07075 0:00838 0:93 0:0068 0:76 With Cu(C,H,0,),. 2a 0°5697 0-0081 112 0:0071 0:98 b 0-5005 0:0073 1115 0-0061 0°95 Sertss III. With CuSO, la 0°6235 gram. 0:0085 gram. 1:07 cui oP. b 0°6705 * . 070091 1:07 0:0088 gram. 1.04 With Cu(C,H,0,).. 2a 0:8302 0:0130 1°24 fea pm b 0-9400 0°0151 1°27 nae ets Trans. Conn. AcaAp., Vou. VII. 39 Nov., 1886. 306 Chittenden and W hitehouse—Metallic Serizs IV. With CuSO,,. No. Amt. sub. taken. Wt. CuO. Per cent. Cu. Wt. Cu.S. Per cent. Cu. la 0:3621 gram. 0:0047 gram. 1°02 0:0038 gram. 0°82 b 03478 00046 1:03 00039 0:89 2a 0°4318 0-0060 1-08 00040 0-71 b 03288 0°0047 1°12 00033 0-78 3a 04083 00060 1:15 0:0047 0:90 b 04332 00064 1:17 0:0053 0-96 4a 03874 00047 1:09 00042 0:97 b ()°4289 0-0062 1°14 00050 0°90 With Cu(C,H,0,).. 5a 06358 00090 ad 00073 0°91 b 05147 00071 1:08 00064 0-99 6a 05321 00075 1:12 00065 0°95 b 0°5125 0-0073 1:13 ee sere 7a 0°7260 0:0125 1°36 ue sees b 06916 00120 1:37 00100 1:15 Series V. With CuSO,. i la 0:4838 gram. 0:00883 gram. 1°36 00074 gram. 1°21 b 05083 00091 1°41 With Cu(C,H,0,).,. 2a 0°5044 0-0085 1°32 - ee b 05042 00084 1°32 : From the analyses of these 15 preparations it is to be seen that the percentage amount of metallic copper, determined as oxide by simple ignition, amounts on an average to 1°17 per cent. When, however, the copper is determined as subsulphide, by precipitation with hydro- gen sulphide, and thus obtained free from ash, the percentage amount of copper falls on an average to 0°94 per cent. Cu. The preparations thus contain 0°23 per cent. of ash. We were not able to obtain any copper compounds with a much smaller content of ash than this, except by the use of methods which appear to affect the composition of the compound. Compounds of Albumin and Myosin. 307 Harnack states* that he was able to obtain the copper albuminate _ quite free from ash by dissolving the freshly precipitated albuminate, after it had been thoroughly washed, in sodium carbonate, filtering and reprecipitating the compound by careful addition of acid. By repeating this process several times the adhering inorganic matter was entirely removed. It seemed questionable, however, whether this treatment might not induce some alteration in the compound. The two following series of experiments were tried with the inten- tion of throwing some light upon this point. Series VI. With CuSO. No. Am’t sub. taken. Wt. CuO. Per cent. Cu. Wt. Cu.s. Per cent. Cu. la 0°6320 gram. 0°0091 gram. 1°15 0:0080 gram. 0°99 b 06980 0:0099 1:13 00086 0:98 2a 05428 00077 1°12 0-0070 1:01 b 0°5121 00077 1:19 0-0061 0°95 3 0°3146 0-0080 2°08 0-0068 aay (| With Cu(C,H,0.,),. 4a 0°6105 00085 jes ig 00075 0-96 b 05836 0:0078 1:06 00072 0-99 5a 05996 00087 1:15 00081 1-06 b 05592 0-0082 1:16 0:0069 0-99 6 0:4139 — 00131 2°51 00115 2.19 In this series of experiments all of the preparations, as before, were made from the purified albumin. Nos. 1, 2, 4 and 5 were, after pre- - cipitation, simply washed with water until the washings gave no reac- - tion either for copper or albumin. No. 3, after being washed in a similar manner, was dissolved in very dilute sodium carbonate and re- precipitated by neutralization with dilute hydrochloric acid. No. 6 _ was dissolved up twice in this manner and both preparations were _ finally washed free from chlorine. A glance at the analyses shows q plainly that this treatment has tended to increase the percentage amount of copper in the albuminate; due, doubtless, either to with- _ drawal of a portion of the albumin by the sodium carbonate, or else _ to a partial dissociation of the compound by the long continued wash- ing with water. _ The first reprecipitation has apparently increased the amount of ~ copper in the compound fully 0°7 per cent., the second reprecipita- _ tion 0°5 per cent. more. * Loe. cit., p. 202. 308 Chittenden and W hitehouse—Metallic Series VII. With CuSO,. No. Am’t sub. taken. Wt. CuO. Per cent. Cu. Wt. Cu.S. Per cent. Cu. la 06326 gram. 0°0091 gram. 1°13 = ae ae b 0-6156 0-0086 1:10 ss bbe 2a, 0°4054 0-0081 1:60 0:0063 gram. 1°28 b 0°4995 0-0099 1°58 00074 119 3a 0°3870 00068 1°39 0-0049 1-02 b 03702 0)-0066 1°40 ore epee» With Cu(C,H,0,),.- 4a 0-6175 00085 1:08 0-0074 0°95 b 0-6069 00082 1:07 0-0077 a 200 5a 0°3282 0-0072 1°73 0°0055 1°34 b 0°3755 0-0088 1°75 ae 2 segs 6a 0°7503 0-0132 1:39 0°0104 1:10 b 07016 00121 1°38 This series was prepared in the same manner as the preceding. Nos. 1, 3, 4 and 6 were simply washed with water, while No. 2 was reprecipitated once and No. 5 twice, and both ultimately washed free from all soluble matters. The results show here the same increased percentage of copper, although not so marked as in the preceding series, when the albuminate is dissolved in sodium carbonate and reprecipitated. Further, the percentage of ash is not, as a rule, materially changed by this process; thus in No. 2, where the albu- minate was reprecipitated once, the difference in the percentage of copper as determined by simple ignition and by precipitation as sulphide, amounts to 0°37 per cent., while in No. 3a, where the compound was not reprecipited at all, the difference is exactly the same, ° In precipitating the albuminate, there is formed in the fluid a small amount of either sulphuric or acetic acid. Harnack, to avoid this, states that it is better, after adding the necessary amount of cupric sulphate to the albumin solution, to exactly neutralize the mixture with sodium carbonate. If, however, the greatest care is not exercised and excess of cupric sulphate avoided, even partial neutralization of the fluid will result in the precipitation of a portion of the copper and thus show an apparent increase in the copper of the albuminate. So far, however, as our results show, the small amount of sulphuric acid libera- ted in the formation of the albuminate does not atfect the character of the compound. In the following series, after each precipitation, Lf , ‘tsi Compounds of Albumin and Myosin. 309 the mixture was made as near neutral as possible with dilute sodium carbonate and the compounds then filtered and washed thoroughly with water. The copper in this series was determined simply by ignition. Series VIII. With CuSO, No. Am’t sub. taken. Wt. CuO. Per cent. Cu. la 0:4273 gram. 0.0068 gram. 1°26 b 0°3867 0:0063 1:29 2a * 03167 0-0049 1-25 b 0°3535 0-0052 1°18 3a 0°2418 0:0030 1:00 b . 0°3079 0:0039 1:00 4a, 06210 0-0138 1°78 b 0:7091 0:0158 1:78 The results plainly show no appreciable difference in the composi- tion of the albuminate under this change in the conditions, unless in No. 4 where a larger amount of copper is found than usual. It is our opinion, however, that the small amount of acid liberated by the re- action is not sufficient to cause any especial change in the character of the albuminate; neither, probably, does very dilute sodium carbon- ate in itself change the substance to such an extent that on neutrali- zation it is not precipitated in nearly its original form, or at least that the action in this case is not any greater than that produced by water alone. In fact we are much inclined to the view that the long continued action of water will gradually but surely affect the composition of the _ albuminate, and that doubtless the change in the composition of the compound noticed in our experiments on solution of the substance in sodium carbonate and reprecipitation is due to the combined action- of the alkaline fluid and of water. Harnack states that week-long treatment of the freshly precipitated albuminate with water will gradually cause dissociation of the compound, but that it can be easily _ and thoroughly washed without any decomposition whatever. _ Our experience, however, leads us to question the correctness of _ this view. Ordinarily, it has taken us an entire day to completely wash the freshly precipitated albuminate, so that the wash-water should give no reaction whatever for copper or albumin. In precipi- tating the albumin solution with cupric sulphate, the albumin never “appears to be completely precipitated and at the same time, as Har- _ nack has observed, it is necessary to add more than the proportional 310 Chittenden and Whitehouse—Metallic amount of copper salt to obtain any separation of the albuminate. As a result, the filtrate contains considerable albumin and copper, but even after several hours washing onthe pump (the filtration is slow at the best) the precipitate still gives up traces of albumin, as shown by acetic acid and potassium ferrocyanide, long after all traces of copper have disappeared. It is not impossible to wash the compound and reach a point where the wash-water contains neither copper nor albumin, but when the washing goes on slowly and the water remains more or less in contact with the albuminate for 24 hours, then fre- quently the washings will show traces of albumin continuously, with- out our being able to reach a point where the test fails te give any reaction whatever, or to show any special change in the intensity of the reaction. The following series of experiments would appear to substantiate this view. The first six were washed for about twelve hours, when no copper reaction could be obtained in the washings and only the slightest reaction for albumin. The last six were washed for sixteen hours, and finally stood over night on wet filters with more or less water on them. At the end of this time, the washings continued to show a reaction for albumin with acetic acid and potassium ferro- cyanide, and indeed the reaction appeared to increase rather than diminish in intensity on further washing. The washings contained no copper. Following are the results of the analyses : . Serres IX.— With CuSO. No. Amt. sub. taken. Wt. Cu0d. Per cent. Cu. 1 0:4207 gram. 0-0052 gram. 0-99 2 0:4676 0:0058 0-98 3 0:4881 0-0062 1:00 4 0-3162 0-0087 0-91 5 ():2882 0:0032 0-89 6 01857 0-0024 1:02 q (2892 0-0050 1°34 8 0-4137 0-0060 1.18 9 0:4077 0-0060 1,18 With Cu(C,H,9,),. 10 06067 0°0126 1°64 11 06889 0-0111 1°27 12 0°7126 0:0109 1°22 Compounds of Albumin and Myosin. 311 While the difference is not very great, it is a constant difference, and it is to be remembered that the last six compounds differ in no respects whatever from the first six, except in being subjected to the longer action of water. In comparing now these different results, it is seen that we have not been able to obtain a copper albuminate with a higher content of Cu than 2°19 per cent., and this only as a result of two reprecipi- tations; a condition, which, from our experience, tends to alter mate- rially the composition of the original precipitate. The average of the results obtained by simple precipitation, show a content of 0°94 per cent. of Cu. A study of the individual results, however, shows too great a variation to believe wholly in the existence of a single, stable copper albuminate. Either there are one or two definite com- pounds, which, being more or less unstable, are prone to change under varying conditions and thus give rise to the variations in the content of copper noticed, or else there are a number of definite compounds liable to be formed as the conditions are varied, all of which, how- ever, must be more or less unstable. Glancing over the individual results, it is plain that an amount of Cu approximating to 0°96 per cent. is found altogether too frequently to be the result of chance. Doubtless this figure represents most closely the content of copper in the ordinary copper albuminate obtained by simple precipitation, while the majority of the variations from this figure are due mainly to dissociation. Taking Lieberkiihn’s formula of albumin, the following copper albuminates would be possible: (C72Hi12N1sSO22)3 + Cu— H»=1°29 per cent. Cu. (CroHi2N18Oo3),+Cu—H.=0'96 “« “« « (C72Hi12NisSOe2); +Cu—H.=0-77 “ <* a For the first, in which Lieberkiihn’s formula for albumin is treb- led, the percentage of copper corresponds nearly to the lowest results obtained by Harnack, while in the second formula the percentage of copper accords closely with the average of our results. Whether the weight of the albumin molecule is represented more nearly by the second formula than by the first we have not sufficient data to determine, but certainly our results with the copper albuminate show a lower percentage of copper than would correspond with the first formula. Further, it would appear that the copper albuminates are readily prone to change under slight provocation and that this point, in part, undoubtedly explains the reason for the great variation in the results obtained by so many workers, 312 Chittenden and W hitehouse— Metallic (b) Lead Compounds. Lieberkiihn* states that the lead salt of albumin cannot be obtained pure; that the insoluable precipitate formed by the addition of either lead nitrate or basic lead acetate to a solution of albumin, is simply a . mixture. With basic lead acetate, Lieberkiihn obtained a precipitate containing 17°86 per cent. of lead oxide, while the precipitate formed with lead nitrate contained 12°78 per cent. of lead oxide. With protein, Muldert obtained precipitates on the addition of neutral lead acetate and lead nitrate, which contained respectively 12°45 and 12.68 per cent. of lead oxide, while basic lead acetate gave a precipitate containing 30°63 per cent. of lead oxide. Berzeliusf states that neutral lead acetate precipitates both albumin and blood serum, but that the greater portion of the albumin remains dissolved in the fluid united with acetic acid. Basic lead acetate on the other — hand precipitates the albumin completely. These last statements accord with our own results; with a neutral lead salt only a small precipitate was obtained, the compound being soluble apparently in both excess of the lead salt and of albumin, while ‘ith basic lead acetate the albumin seemed completely precipi- tated. Further, Berzelius|| states, on the authority of Mulder, that if a solution of potassium albuminate be made as neutral as possible with acetic acid and then precipitated with lead nitrate, the lead albu- minate so obtained contains on thorough drying 5°84 per cent. of lead oxide. : Following are some of the results of our analyses. The compounds were made from thoroughly dialyzed albumin and were washed free from both lead and any excess of albumin. The preparations were dried at 110° C. until of constant weight and the lead was deter- mined first by simple ignition, with addition of a little ammonium nitrate. The lead oxide, after being weighed, was then dissolved in dilute nitric acid, the solution evaporated to a small volume, the lead precipitated with a little sulphuric acid, two volumes of alcohol added, aud the lead sulphate finally filtered and washed with 95 per cent. alcohol. The sulphate was then ignited with proper precautions and from the weight obtained, the percentage of lead again calculated. * Poggendorff's Annalen, Band Ixxxvi, p. 124. + Lehrbuch der Chemie, Berzelius, ix, p. 29. | { Lehrbuch der Chemie, ix, p. 43. || Lehrbuch, ix, p. 49. ) od ‘ by, wi Compounds of Albumin and Myosin. 313 Series I. With neutral lead acetate. No. Amt. Sub. taken. Wt. PbO. Per cent. Pb. Wt. PbSO;. Per cent. Pb. la 0:4972 gram. 0°0188 gram. 3°49 0°0209 gram. 2°85 b 03992 0:0150 3°48 0:0166 2:83 SERIES ge With neutral lead acetate. la 0°5841 0-0183 3°16 0:0216 2°75 b 0°53881 0:0198 3°40 0-0222 2°80 With basic lead acetate. 2a 05832 0:0460 7°30 0:0580 6°77 b 0:5008 0-0391 1-22 0:0487 6°62 Series III. With neutral lead acetate. la 0:°8057 0:0262 301 0:0274 2°32 b 0°6522 0:0211 2°98 0°0226 2°36 With basic lead acetate. 2a 0°7290 0:-0593 y 95) 9-0615 5°74 6b 0°7630 0-0631 7°66 0:0609 5°45 Series IV. With neutral lead acetate. la 0-4121 0:0152 8°42 0:0136 2°25 b 0°4823 0:0176 ool 0-0178 2°50 With basie lead acetate. 2a 0°7234 0:0651 S84 eee ess We 0°5365 0-0482 Sar oS - eae aaG ‘ Series V. With a large excess of basic lead acetute. la 0°6822 ; 0-2119 28781" 49 Tae pre. b 0°5429 0-1714 ab-28:.1' ih ee te: 2a — 0°5836 071923 30°56. 52 ares en ee b 0°5913 0-1960 30:7626 eee Bt 3a 0-5478 0-1896 BOE) ee eas ee eee '* b 0:5427 0°1878 SPOS AeA wyet ese = wae ste a TRANS. Conn. ACAD., Vou. VII. 40 Nov., 1886. 314 Chittenden and W hitehouse—Metallic These results plainly indicate that more than one compound of lead is formed, especially so with basic lead acetate, the composition being dependent in this case on the amount of lead salt added. With neutral lead acetate, the variations in composition are not so marked and as it is hardly possible to prepare a lead albuminate free from salts, or to eliminate them wholly in the calculations, it is question- able how far the cone should be trusted, except in a general way. The formula (C,,H,,,N,.80,,),4+-Pb—H, would require 3°10 per cent. Pb, while (C, wH,,,N,.S0,.), + Pb—H, would require 2°50 per cent. Pb, In the case of the albuminate formed with basic lead acetate, it is to be noticed that the compound made by the addition of a large excess of the lead salt, contains about five times as much lead as the ordinary basic lead compounds. (c) Lron Compounds. F. Rose* has made iron albuminate, both from egg-albumin and from the serum of ox-blood, by the simple addition of ferric chloride to the albumin solution. Two preparations made from ege- -albumin yielded respectively 2°79 and 2°88 per cent. of ferric oxide. Rose found the albuminate, when freshly precipitated, easily soluble both in excess of ferric chloride and in excess of the albumin solution, Our preparations were made wholly from dialyzed albumin, and when so prepared and thoroughly washed the compound was found almost wholly free from adhering salts, so much so that after a few trials we deemed it unnecessary to make the determinations of iron other than by simple ignition and weighing as ferric oxide. Following are some of our results: Serizs I. With Fe,Cl,. No. Amt. sub. taken. Wt. Fe203. Per cent. Fe.O;. Per cent. Fe. la 0°7033 gram. 0 0094 gram. 1°33 0°92 b 0°6430 0:0086 1°33 0°98 Srriss II. la 0:4683 0:-0052 Si kil 0:76 b 03854 0:0042 1:08 0:75 * Poggendorff’s Annalen, xxviii, p. 140, 1833. Compounds of Albumin and Myosin. 315 Series III. No. Amt. sub. taken. Wt. Fe.0s. Per cent. Fe,0;. Per cent. Fe. 1a 0°4710 0-0071 1°50 1°04 b 05329 0-0083 1:55 1:08 2a 0°7387 0-0100 1°36 0°94 b 06355 00087 1°36 0°94. Series IV. la 0-4862 0-0063 1:30 0-90 b -0°5558 0:0073 1:31 0-91 2a 0-5115 0-0070 1°36 0°95 b 0-4954 0-0066 1°33 0°92 3a 04505 0)-0063 1°39 0:97 b 0°4610 00066 1:43 0:99 4a 0-4817 0-0071 1:47 1-01 b 04571 00065 1:44 0-98 5a 04819 0-0065 1°34 0-93 b 03846 0-0051 1°33 0-93 6a 04086 0-0059 1°45 1:00 b 0°3392 00048 1-41 0-97 Ta 03814 0-0050 1°32 0-91 b 0°4107 0-0053 1°29 0-90 8a. 04452 0-0058 1:30 0-91 b 04833 0-0065 1°34 0-92 These results show a fairly close agreement with one single excep- tion, in which case the percentage of iron is nearly 0°25 below the average. The average percentage, moreover, of ferric oxide is just about one-half that found by Rose. Further, the average percentage of iron (Fe) corresponds very closely with the average percentage of Cu in the copper albuminate. Eliminating one compound with only 0°75 per cent. of iron, the average content is seen to be 0°95 per cent. . (C72Hi12N1.SOe2), + Fe —H;=0°86 per cent. Fe. (C72H112N:.8O22)3 + Fe—H;=1'14 per cent. Fe. As the iron was determined by simple ignition it would be ex- pected that the amount found would exceed the theoretical amount somewhat; hence the first formula, assuming Lieberkiihn’s formula to be correct, would be more closely in accord with our results. The results obtained indicate further, that the iron albuminate is a much more stable compound than the copper albuminate, less liable to change and less readily affected by water. 316 Chittenden and W hitehouse— Metallic (d) Zine Compounds. With zine we made but a few experiments and those mainly to see whether the low percentage of iron found in the iron albuminate — would be substantiated by a corresponding percentage of zinc in the zine albuminate. Lieberkiihn has prepared and analyzed a zine albuminate, made by the action of zinc sulphate on a neutral solu- tion of alkali albuminate, and he found the compound to contain 4°66 per cent. of zinc oxide. Our preparations were made by the action of a similar zine salt on a solution of -purified and dialyzed albumin. Following are the results obtained with two preparations made from two distinct lots of albumin : No. Amt. sub. taken. Wt. ZnO. Per cent. ZnO. Per cent. Zn. ia 02424 gram. 0:0031 gram. 1°27 0-98 b 02838 000384 1°21 0:97 2a 0°2166 00023 1:06 0°83 b 02354 0:0025 1:08 0:86 The average of these two results shows a composition proportional to that found in the case of the iron albuminate and suggests plainly that if we have to deal in these cases with a single albuminate of constant composition, the percentage amount of metal is much smaller than formerly was supposed. Further, the percentage of zinc found accords closely with the theoretical amount for a zinc albuminate formed on the type of the copper compound. (C2, 12NisSOoe), +Zn—H.=0'99 per cent. Zn. (e) Uranium Compounds. N. Kowalewsky* has recently called attention to the use of uranic or uranyl acetate as a reagent for albuminous matter, and has shown that it is not only a good precipitant of albumin at ordinary temper- atures, but also that it is an extremely delicate one. Further, Kowalewsky states that the uranyl-albumin compound on ignition leaves a dark, olive green ash, composed of the green uranoso-uranic oxide, U,O,. A determination of the amount of this ash in several preparations showed 12°09 to 13°4 per cent., presumably of U,O,. * Hssigsaures Uranoxyd, ein Reagens auf Albuminstoffe. Zeitschrift fir Analy- tische Chemie, 1885, p. 551. } ni, —S— ee Compounds of Albumin and Myosin. 317 Our preparations were made by adding uranyl nitrate to the pre- pared albumin solution and washing the precipitated albuminate until all excess of uranium was removed. The uranium in the dried preparation was determined as uranoso-uranic oxide (U,O,) by simple ignition. The results show a fairly close agreement, but they are undoubtedly somewhat too high, owing to a small amount of adherent ash. With UO,(NO)),. No. Amt. sub. taken. Wt. UsOsz. Per cent. U;0O,. Per cent. U. la 0:5980 gram. 0-0824 gram. 5°41 4:59 tes 0:°5193 00281 5°41 4°59 2a 08219 0:0428 5:20 4°41 b 0-8081 . 0:0418 Deli 4°38 3a 0:4892 00251 5:71 4°84 b 0:°5330 0:0303 5°68 4°81 4a 0°8183 0:0427 5:22 4°43 b 0:°7576 0-0394 5:20 4:4] 5 0°6985 00367 5:25 4°46 6a 04269 0):0247 5°78 4:90) b 0:°5496 00313 5°70 4°83 These results plainly do not accord at all with Kowalewsky’s. On the other hand they do agree fairly well with each other, and would seem to indicate a reasonably constant composition of the uranyl- albumin precipitate. The average of the results obtained, accords most closely with the formula (C72H112NisSOo2)3 + U—H,g which requires 4°73 per cent. U. (f) Mercury compounds. By the addition of an excess of mercuric chloride solution to an aqueous solution of egg-albumin, an albuminate of mercury is formed, insoluble in excess of the mercury salt. The compound can be easily filtered and admits of thorough washing with water. Rose first proved that the precipitate formed as above, is a compound of mercury with albumin, instead of a compound of the mercury salt with albumin as supposed by Bostock and Orfila. We have made a few preparations of the albuminate by adding a moderately strong aqueous solution of mercuric chloride to portions of the dialyzed albumin solution and washing the precipitates thoroughly with water. The mercury in the albuminate was deter- 318 Chittenden and W hitehouse—Metallic mined by ignition in a combustion tube with quick lime, with a pos- terior layer of calcium carbonate and sodium bicarbonate.* The mercury distilled, was collected in water and after thorough washing with alcohol to remove hydrocarbons, etc., was dried and weighed. Following are the results of our analyses of the several prepara- tions made. oa No: Amt. Sub. taken. Wt. Hg. Per cent. Hg. la 08050 gram. 0:0226 gram. 2°80 b 08732 00274 3°13 2a 0°7637 0-0215 2-82 b 0°7357 00201 2°73 3a 05088 ()-0150 2°96 b 06261 00167 2°66 4a 0°9152 00300 3°28 b 08503 0-0270 3°17 5a 08492 0:0218 2°56 b 08610 00237 2°75 6a 09674 00284 2°93 The average content of mercury is 2°89 per cent. The theoretical amount for (C,,H,,,N,.SO,,),+ Hg—H, is 3°00 per cent. (g) Silver compounds. Silver nitrate is a well known precipitant of albumin, and Lfeber- kiihn,} many years ago, assigned to silver albuminate a definite formula, calling for 6°67 per cent. of silver oxide. The preparation made by him from egg-albumin was found to contain 6°55 per cent. of silver oxide = to 6°27 per cent. of Ag. Mulder,} likewise, work- ing with alkali-albuminate, found that by neutralizing the solution as nearly as possible with acetic acid, and then precipitating with silver nitrate, the silver albuminate so prepared contained 6°14 per cent. of silver oxide. Fuchs§, using ordinary egg-albumin instead of alkali-albuminate, found only half as much silver (3°28 per cent. Ag), while O. Loew,}} working with purified egg-albumin, found still smaller percentages of silver in the albuminate made by him. Using an albumin * See Fresenius, Quantitative Chemical Analysis. + Poggendorff’s Annalen, 1852, vol. elxii, p. 123. t See Berzelius’ Lehrbuch der Chemie, vol. ix, p. 49. § Annalen d. chem. u. Pharm., Band cli, p, 372. || PHiiger’s Archiv fir Physiologie, Band xxxi, p. 393; Ueber Hiweiss und Pepton. = ~,i-~ I ee Compounds of Albumin and Myosin. 319 solution purified simply by three days’ dialysis, Loew found that a 1 per cent. solution of silver nitrate gave no precipitate whatever in a 5 per cent. solution of albumin. On adding a little dilute sulphuric acid to the albumin solution, however, and then pouring the mixture into the silver solution a precipitate was obtained, which on thorough washing and drying was found to contain 2°17 per cent. of Ag; while a second preparation made by using a little less sulphuric acid contained 2°40 per cent. of Ag. By precipitat- ing the albumin solution directly with a 5 per cent. solution of silver nitrate, without the addition of any acid, the albuminate was found to contain in one case 4°39 per cent. Ag, in a second case 3°91 per cent. Ag. Dissolving the freshly precipitated albuminate formed in this manner, in dilute ammonia and then reprecipitating it by the addition of dilute sulphuric acid to slight acid reaction, the albumin- ate was found to contain 4°64 per cent. of Ag. Loew sees in these results a confirmation of Harnack’s views as to the copper albuminates, and an assurance that the molecular weight of albumin corresponds to Lieberkiihn’s formula three times enlarged. Using an albumin solution purified as in our previous experiments and adding to it a 10 per cent. solution of silver nitrate as long as a precipitate was formed, four distinct series of albuminates were made* representing four distinct preparations of egg-albumin. These were all washed free from silver and also from any adhering albumin, dried at 110° C. until of constant weight and the silver determined by simple ignition. Series [,. No. Amt. Sub. taken. Wt. Ag. Per cent. Ag. la 0:5900 gram. 0:0242 gram. 4:10 b 05625 00280 4:08 2a 0:5760 0-0230 4-11 - b * 07548 0-0305 4:04 3a 0-9005 0:0362 4-02 b 0-7973 0-0325 4:07 Serres II. la 0-5859 0-0245 4°18 b 06967 0-0290 4°16 2a 0°9473 0-0385 4:06 b 06621 0-0270 4:07 3a 0-6455 00266 4°12 b 0-7000 * 0:0285 4:07 * The silver compounds were all made and analyzed by Mr. T. S. Bronson of this laboratory. 320 Chittenden and W hitehouse—Metallic Seriss III. la 0°5860 00239 4:07 b 06949 0°0284 4-08 2a 0-7090 0-0290 4-09 b 0°9053 0-03874 4:13 3a 05980 0:0247 4:13 b 08000 9-0828 4:10 Series IV. la 06217 00300 4:88 b -— 0°6810 0-0331 4:86 2a 0°5509 0-0270 4:90 b 05626 0-0278 4:86 3a 06955 00396 5°69 b 0-7515 0-040 5°72 The figures show a far smaller content of silver in all of the prep- arations than found by Lieberkiihn or Mulder. In three of the series, there is seen a constancy of composition which is quite notice- able and, further, a close agreement with the second result obtained by Loew on adding a 5 per cent. solution of silver nitrate to the albumin. In the last series, however, the percentage of silver is somewhat higher, possibly owing to incomplete dialysis of the chlorides and phosphates from the albumin solution. These figures, however, are not much higher than the highest figures obtained by Loew. A silver salt of albumin, of the composition (C,,H,,,N,.SO,,), + Ag,—H, would contain 4°28 per cent. of Ag, and while our results certainly approximate to this figure, there is variation enough to indicate an equal possibility of a mixture of two or more com- pounds. With a molecule of the size of the albumin molecule, it is possible by doubling or otherwise, to obtain a formula corresponding to almost any percentage of metal found. And inasmuch as every variation* in the method of preparing the albuminate tends to alter its composition, it seems worse than useless at present, to lay much stress on the exact constitution of the silver albuminate. A large number of albuminates are of course possible, but until we know more definitely how to separate one from another, we have no guaran- tee of the simple nature of any one. * Loew states that he has preparéd a silver albuminate containing 10°7 per cent. Ag, corresponding nearly to 6 atoms of silver, and that it is possible to prepare album- inates still richer in silver. “) Compounds of Albumin and Myosin. 321 Examining now, all of the results obtained, we find the following average composition of the albuminates studied : Copper compound, 0-94 per cent. Cu Tron compound, O;9b 5 ‘tos Be* Zinc compound, QsOile <¢ ory 40) Lead (neutral salt) compound, 2°56 * ged 24 5, Uranyl compound, -4:60 “ wrt 18) Silver compound, 4:09 * OO sear Mercury compound, Patek’ Je oe EL Accepting Lieberktihn’s formula of albumin as correct, then the following formule accord most closely with the above percentages. (Cy2Hi12NieSO22), + Cu—Hy, requires 0°96 per cent. Cu (Cz2H12NisSOe2)4 + Fe—H; + 0°86 ds Fe (Cr2Hi12Ni2SO22), + Zn— Hye Hs 0:99 Mk Zn (C72Hi12NisSO22); + Ph—H. Be 2-50 oe Pb (Cz2Hii12Ni.SO22)3 + U—He sC 4°73 UC U (C72H, 12N18SOe2)s = Ag..—H, ef 4°28 os Ag (CHEGUN BOR eB, 0h 000!) «Hg We do not, however, lay much stress upon the accuracy of these formule. The results obtained in our study of these metallic com- pounds do by chance accord with them, and inasmuch as Loew and Harnack are disposed to treble Lieberkiihn’s formula for albumin, on the basis of the composition of the copper and silver albuminates, made by them respectively, we present our results as evidence that there are equally good grounds for quadrupling the above formula. We believe, however, that with the majority of these albuminates it is possible to form a large variety of compounds with the same metal, by simply modifying the conditions of precipitation. This is evidenced by Loew’s results with silver albuminate and our own with lead and copper, and since a great variety of compounds are possible, it is equally possible that in many cases we may have to do with mixtures of such compounds, which would account for the great variability in composition noticed in some of the albuminates and for the lack of agreement in the results obtained by different workers. Coupled with this, in some cases, is the undoubted tendency of the compounds to dissociation. Il. Myosin. The myosin employed was prepared from ox flesh, by extraction with a 15 per cent. solution of ammonium chloride, after the tissue * Excepting one very low result. | Excepting the last series of compounds. TRANS. Conn. AcAD., VoL. VII. 41 Nov., 1886. 322 Chittenden and W hitehouse— Metallic had been thoroughly freed from salts and soluble albumin by long continued extraction with water. The myosin was separated from the ammonium chloride solution by dialysis, being obtained in this man- ner as a semi-gelatinous mass, readily soluble in salt solutions. In order to form compounds with the various metals, it was found best to use a solution of myosin in 5 per cent. ammonium chloride, the metallic compound when formed being washed with water until the washings gave no reaction for chlorine with silver nitrate. The com- pounds ‘were then dried, first at 100° C., then at 110° C., until of constant weight. Control experiments with the metallic salt and ammonium chloride alone, invariably failed to give any precipitate whatever. No systematic attempt has apparently been made to study any of the metallic compounds of myosin; in fact, few statements are to be found regarding the existence of such compounds. Danilewsky* some time ago, showed that myosin would combine with free mineral acids, uniting with them so that with tropzolin 00 no reaction for free acid could be obtained. With strong bases, however, according to Danilewsky, myosin does not probably combine, and the state- ment is further made that a small amount of calcium oxide ordinarily exists loosely combined with myosin, which calcium by coagulation of the myosin is liberated. Further, Danilewsky found that on adding platinum chloride in excess, to a dilute hydrochloric acid solution of myosin, a myosin-platinum chloride compound.was pre- cipitated, which after washing with water and alcohol and then drying at 100-105° C., contained 9°46 per cent. of platinum and 7°26 per cent. of chlorine. With copper, iron and similar salts we have not been able to obtain any precipitate in a hydrochloric acid solu- tion of myosin. By adding, however, a solution of a metallic salt of such a nature that it does not react with ammonium chloride, to an ammonium chloride solution of myosin, a precipitate is pro- duced, which as our experiments show, is ordinarily a compound of myosin with the metal or metallic oxide. This is readily seen by adding either zinc sulphate or ferric chloride to such a solution of myosin and then washing the precipitates with water, until the washings give no reaction for chlorine or for sulphuric acid. On now warming the iron precipitate with dilute nitric acid, a solu- tion will be obtained, giving a distinct iron reaction but no reaction with silver nitrate for chlorine. Similarly on warming the zine pre- * Zeitschrift fir physiologische Chemie, v, p. 160, Compounds of Albumin and Myosin. 323 cipitate with hydrochloric acid, the solution gives no reaction for sulphuric acid with barium chloride. With cupric sulphate and cupric acetate the same is ordinarily true. It is possible, however, to prepare a myosin-copper compound in which cupric sulphate appears to unite directly with the myosin. (a.) Copper compounds. By adding either cupric sulphate or cupric acetate to a neutral ammonium chloride solution of myosin, a heavy greenish colored precipitate is obtained, which when freshly formed and after thor- ough washing with water, so that the washings are entirely free from chlorine and from copper, shows the following reactions. It is insoluble in moderately strong nitric, hydrochloric or sulphuric acid. The compound, however, is immediately broken up by the action of acids, the copper being completely removed, leaving the myosin as an insoluble residue having in the case of nitric acid a yellow color, and in the case of hydrochloric and sulphuric acids a white color. In acetic acid, the compound is more soluble, first, however, becoming semi- gelatinous. In ammonium hydroxide, the compound dissolves slowly or partially, taking on a blue color. In dilute sodium hydroxide, the compound swells up, takes on a purple color, but does not dissolve. In dilute sodium carbonate, the substance is likewise insoluble, but swells up and turns of a bluish color. Following are the results of the analyses of the various prepara- tions made. The compounds were in every case composed simply of the metallic oxide and myosin. Copper was determined, as in the case of the albumin compounds, by simple ignition and weighing as oxide. In order to ascertain how much ash was retained by the myosin compound, a few duplicate determinations of copper were made by dissolving the oxide after ignition, and precipitating the copper as sulphide and weighing as subsulphide, after ignition with a little pure sulphur in a current of hydrogen gas. Serigs I. With CuSO,. Amt. Sub, Per cent. Per cent. No. taken. Wt. CuO. Cu. Wt. Cuss. Cu. la 05426 gram. 0:0074 gram. 1-08 0:0056 gram. 0-81 b 0°7176 00097 1:07 00073 0-80 2a 0°6359 0:0087 1:08 00071 0°88 b 0:°5738 0:0078 1:08 0:0061 0°83 Chittenden and W hitehouse— Metallic Amt. Sub. taken. 075425 gram. 06241 0-577 04455 0°3527 0:4401 0°5708 Amt. Sub. taken. 0°7425 gram. 0°7463 0°7785 0°7767 0°5488 0°4836 0:9477 0°8150 0°8522 08466 0°6589 0-6572 0°8673 0°7120 Amt. Sub. taken. 0°7218 gram. 06584 0)-7564. 0°6817 0-7820 06218 0°7137 05863 0°7782 0°7407 0°7447 ()*7429 Per cent. Per cent. Wt. CuO. Cu. Wt. Cu,s. Cu. 0:0072 gram. 1:05 0-0051 gram. 0-73 0-0082 1:05 0-0063 0°80 0:0109 1°50 0-0081 1°48 0:0046 1:02 0:0062 alfesteal 00082 1°13 Series II. With CuSO. Wt. CuO. Percent. CuO. Per cent. Cu. 0-0059 gram. 0-79 0°68 0-0060 0-81 0°64 0-0057 0°73 0°57 0:0056 ; 0°72 0:58 0:0052 0-94 0°74 0-0041 0-94 0-73 00085 0:89 0-74 0:0074 0-91 0°73 With Cu(C,H,0,),. 00133 1:56 1-24 0-0188 1°62 1°29 0:0124 1°88 2 | 2°60 0:0123 1:87 1°50 0-0156 1:79 1°42 0:0129 1:81 1°44 Series III. With CuSO, Wt. CuO. Per cent. CuO. Per cent. Cu. 0-0171 gram. 2°37 1°88 0:0161 2°44 1°94 0:0168 2°22 gL 0°0156 2°29 1°88 0:0189 2-41 {Dias 0:0154 2°48 1:97 0-0144 2°01 - 1°61 0:0124 2°11 1°68 0:0164 2°12 1°68 0:0161 2°18 1°74 0-0188 2-45 1°96 00188 253° 2-01 Compounds of Albumin and Myosin. 325 With Cu(C,H,0,),. No. Amt. Sub. taken. Wt. CuO. Per cent. CuO. Per cent. Cu. Ta 0°7360 gram. 0°0161 gram. 2°18 1°73 b 0-7088 0°0154. Pill iba: 8a 07789 0°0174 2°24 1:78 b 0°8111 0°0181 2:23 uri 9a 0°7376 0:0169 2°29 1°81 b 0°6155 00188 2°24 1°78 10a 0°7815 00191 2°44 1:94 b 08774 00213 2°48 1:93 ila 0°8048 0:0170 2-11 1°67 b 0°8308 0:0174 2°10 1:67 12a 0:6780 0-0150 2°21 1°75 j b 0°7695 0:0168 2-718 1°74 Comparing these results with one another, there is to be seen a very noticeable lack of agreement in composition, and further it is to be seen that the myosin-copper compound has on an average a somewhat higher content of copper than the albumin-copper precipi- tate. The average composition of all the copper myosins shows about 1°42 per cent. of Cu, and deducting 0-25 per cent. of ash, the average content of Cu would be 1°17 per cent. Examining the individual results, it is apparent that the compounds made from the same myosin solution are approximately, at least, the same in composition and without doubt the difference in the composition of compounds made from different myosin solutions is due to variation in the concentra- tion of, and possibly also in the reaction of, the myosin-containing fluid. It would appear as if variations in the conditions of precipita- tion made a greater difference in the case of the myosin-copper com- pounds than in the compounds of copper with albumin. Several times, also, we have found that our myosin-copper precipitate con-_ tained sulphuric acid, even after thorough washing and when the wash-water was proved to be entirely free from any reaction with barium chloride. One such compound, after drying at 110° C., was analyzed with the following results: 0°7240 gram substance* gave 0:0343 gram BaSO, = 1°63 per cent. SOs. 0°8420 gram substance gave 0:0831 gram BaSO, = 1°73 per cent. SOs. 0-4705 gram substance} gave 0:0084 gram CuO = 1°78 per cent. CuO. 0°6011 gram substance gave 0:°0107 gram CuO —=—- =-_:1°78 per cent. CuO. * Roasted and then ignited with pure sodium carbonate, the residue dissolved in hot water acidified with hydrochloric acid and precipitated with barium chloride. + Ignited, the residue dissolved in nitric acid, precipitated with hydrogen sulphide, ete. 326 Chittenden and Whitehouse—Metallic The molecular weight of CuO and SO, being the same, it is evident that the two are present in just the proportion to form cupric sul- phate. b. Iron compounds. By adding a solution of ferric chloride to an ammonium chloride solution of myosin, a semi-gelatinous precipitate is formed of a red- dish yellow color, and consisting of a combination of myosin and oxide of iron. The compound when thoroughly washed contains no chlorine. When freshly precipitated, it is partially soluble in dilute ammonium hydroxide, as also in sodium hydroxide, the residue becoming gummy or gelatinous and brownish yellow in color. It swells up in sodium carbonate, but is insoluble. In nitric acid the compound turns yellow, but is wholly insoluble and does not swell up. In hydrochloric and also in sulphuric acid the compound is like-_ wise insoluble. In acetic acid, however, it is soluble completely, forming a semi-gelatinous fluid. In this, as in other metallic com- pounds of myosin, acids simply dissolve out the metal and then exert their usual action on the myosin. The various preparations, washed free from iron and chlorine, and dried at 110° C. were analyzed with the following results : Seriges I. No. Amt. Sub. taken. Wt. Fe.0s. Per cent. FegQs. Per cent. Fe. la 0°7046 gram. 0.0194 gram. 2°16 1°98 a .0°7021 0:0198 2°74 1:92 2a 0°3762 0°0128 3°40 2°38 b 0°36238 00121 3°33 2°33 38a 05837 0:0141 2°42 1°69 b 0°5618 0°0137 2°43 1°70 4a 054388 0:0177 3°25 2°26 b 06088 0°0197 3°24 2°26 5a 04761 00140 2°95 2-06 b 0°33881 j 0°0100 2°95 2°07 6a 05101 - 00173 3°40 2°37 b 05927 00194 3°28 2°29 Series II. i 0:4847 00188 3:87, 2-70 2 03200 00115 5 251 3 0-4118 00158 3°72 2:59 4 0.5205 0-0184 3:58 2-45 Compounds of Albumin and Myosin. 327 No. Amt. Sub. taken. Wt. Fe.0s. Per cent. Fe.03. | Per cent. Fe. 5a 0°4551 gram. 0-0167 gram. 3°68 2°57 b 0°3668 0-0137 3°73 2°61 6a 03783 0-0138 3°64 2°53 b 0°3675 0-0131 o5o7 2°50 Ta 04284 0-0148 3°34 2°33 b 04041 0-0136 D871 2°36 Serres III. With a large excess of ferric chloride. den 04385 0-0269 6-13 4-29 62 06939 00436 6°27 4°38 In none of these preparations was there any attempt made to add a definite amount of ferric chloride, but the iron salt was added until a good precipitate was obtained. Undoubtedly, the amount of iron salt added, modifies materially the composition of the compound. In series III it is seen that the content of iron is about double the aver- age amount contained in the other preparations. The average amount of iron (Fe) in the first two series of compounds is 2°29 per cent. ce. Line compounds. With zinc sulphate, myosin is thrown down from its ammonium chloride solution as a heavy gelatinous precipitate. Like the iron compound it is partially soluble in sodium and ammonium hydroxides, swelling up to a gelatinous mass. I[t is insoluble in nitric, hydro- chloric and sulphuric acids, but is partially soluble in acetic acid. In composition, it is seen to be very closely allied to the zine albu- minate. Following are the results obtained by analysis of the dried - compounds : ; SERIEs I. No. Amt. Sub. taken. Wt. ZnO. Per cent. ZnO. Per cent. Zn. la 0-6108 gram. 0-0049 gram. 0°81 0-64 b 0°6619 00055 0°83 0°66 2a 0°6611 0:0047 0-71 0-57 b 08006 00059 0:73 0°59 3a 04666 - 00046 0:99 0-79 b 04494 0:0044 0:99 0-79 4a 05936 0:0048 0-80 0°64 b 06926 0:0055 0-79 0°63 328 Chittenden and W hitehouse— Metallic Series II. No. Amt. Sub. taken. Wt. ZnO. Per cent. ZnO. Per cent. Zn. la 0°6258 gram. 0:0051 gram. 0°82 0°65 b 0°6618 0-0054 0°81 0°65 2a 0°5815 00064 1:21 0:97 b 0°5485 0-0065 1°18 0°94 3a 0°7925 00064 0°81 0°65 b 0°6913 00057 0°83 0°66 4a 0:4858 0:0062 1°27 1°02 b 0°53818 0:0066 1°24 0:99 5a - 0°66385 0:0056 0°85 0-68 b 06504 00050 0°76 0°61 6a 06268 00055 0:87 ee OD b 066389 0:0059 0:88 0-71 - The average content of zinc (Zn) is 0°72 per cent. Unlike the iron and copper compounds, there is here less variation in the composition of the various preparations. d. Nickel and cobalt compounds. The extremely low percentage of zine in the zinc-myosin com- pounds, as contrasted with the iron in the iron compounds, led us to make a nickel and cobalt preparation for the sake of comparison. The results, in both cases, accord more nearly with those of the iron compound, for although containing a higher percentage of metal than the latter, it was necessary to prepare them both under just such conditions as in the iron compound led to the highest percent- age of iron, viz: a large excess of the precipitant. Following are the analytical results obtained with both substances: With Ni (NO,),. No. Amt. Sub. taken. Wt. NiO,. Per cent. NiO,. Per cent, Ni. la 06165 gram. 0:0045 gram. 7°29 4-71 b 0°7016 0-0051 7°26 4°70 With CO (NO,),. la 06479 0-0064 9°95 ; 6°45 b 06696 0-0065 9°70 6°28 2a 0°6451 0°0056 8°75 5°67 b 0°6842 0-0060 8°84 5°72 Compounds of Albumin and Myosin. 329 e. Uranium compounds. The addition of uranyl nitrate to an ammonium chloride solution of myosin produces a heavy gelatinous precipitate of a uranyl-myosin compound, which in solubility resembles the other myosin prep- arations. Washed free from excess of uranyl nitrate and from ammonium chloride and then dried at 110° C., the various prepara- tions yielded on analysis the following results, the uranium being determined by simple ignition and weighing as uranoso-uranic oxide: Series I. No. - Amt, Sub. taken. Wt. U;Osz. Per cent. U30s. Per cent. U. la 0°6373 gram. 0:0489 gram. 767 6°51 b 0-6836 0:0526 769 6°53 2a 07081 0:0592 8°36 7-09 b 0:7689 0°0655 8°51 7°22 3a 06418 0:0586 9-13 yar 3) b 0-7809 0:0715 9-16 7-78 4a 0°6621 0:0541 8-17 6°93 b 0°7455 0:0608 8-15 6°91 5a 0°7525 0:0663 8°81 7-48 b 06964 0-0615 8°83 7-50 6a 0°7208 0-0590 8:18 6°94 b 0°7439 00607 8-15 6-91 Ta 0°6648 0°0527 7-92 6°72 b 0°7101 0°0565 7°96 6°76 Series II. la 08736 00837 958 8-13 b 0°8570 0:0820 9°57 8-12 2a 0°5857 0:0593 10°12 8:59 _ b 0°7088 0:0715 10-09 8:56 3a 0°5715 0°0568 9:94 8-44 b 0°6958 0:0688 9°89 8-40 4a 0°6929 0.0556 8:02 6°82 b 0°7667 0°0615 8:02 6°82 5a 0°8031 0°0874 10°88 9-23 b 0:8272 0-0901 10°89 9-24 6a 0°7034 0°0553 7°86 6°67 b 06810 0:0540 7-92 6°72 These results show a variation in the content of uranium, amount- ing to nearly 3 per cent. (6°51-9°24 per cent.) Further, a compari- TRANS. Conn. ACAD.. Vou. VII. 42 Nov., 1886. 7 330 Chittenden and W hitehouse—Metallic son of the two series shows plainly that there is something in the nature of the second myosin solution, which tends to raise the content of uranium in the uranyl compounds ; probably, the greater concen- tration or dilution of the solution. Evidently, then, the composition _ of the compound is, in part at least, determined by the conditions under which the uranyl salt and the myosin solution are brought together. The average amount of uranium contained in the prepa- rations is 7°49 per cent. J. Mercury compounds. By adding a solution of mercuric chloride to an ammonium chlo- ride solution of myosin, a heavy gelatinous precipitate is formed which soon changes to a flocculent one. Freed {rom the excess of mercury salt and ammonium chloride, the compound is found to be ~ entirely free from chlorine. The substance is somewhat soluble in sodium hydroxide, swelling up first and then gradually dissolving. Dried at 110° C. and then analyzed, the following results were ob- tained. The mercury was determined as already described under mercury albuminate. Series I, No. Amt. Sub. taken. Wt. of Hg. Per cent. Hg. la 07609 gram. 0-0166 gram. | 2°18 b 1:2911 0-0268 2°07 2a 12386 0-0270 2°17 b (09525 0-0198 2°07 3a 1:1856 00218 1°84 b 09261 0-0178 1°86 4a 173504 0:0257 1:90 b 0:9332 0-0178 1:90 Series II. 1 0-8917 0-0241 2°70 2 11196 —-0-0310 2°77 3a 0:9587 0-0271 2-84 b 08813 00259 2-93 4 0:9209 0:0270 2°93 5 08352 0-0241 2-89 08564 00217 2-538 Ta 1:0696 0-0344 3-22 b 0°8550 00264 3°09 Compounds of Albumin and Myosin. 331 The average content of mercury (Hg) is 2°43 percent. The results of each series show a fairly close agreement, but the two series do not compare with each other at all. Thus, the average amount of mer- cury in the compounds of the first series is 1°99 per cent., while in the second series the average amount rises to 2°87 per cent. This is another good illustration of the influence of the strength of the solu- tion on the composition of the precipitate, and as in this case the presence of any ash could not interfere with the ultimate result, since the mercury was separated by distillation, it follows that the appar- ently higher content of mercury in the second series must be due to combination of the myosin with a larger amount of the metal. Fur- ther, it has been claimed* that in the case of the silver albuminate, it is possible under certain circumstances for the albuminate, when formed in a concentrated solution, to inclose a variable amount of albumin mechanically, and thus the apparent percentage of silver in the albuminate be reduced. If such was true of the myosin-mercury compounds, a far greater variation would be expected in the per- centage of mercury in the different preparations of the same series. The following table of comparisons shows the average content of metal in the albuminates formed from the two kinds ot proteid matter. Egg-albumin. Myosin. Copper compound, 0:94 per cent. Cu 1:17 per cent. Cu tron x 0-95 Fe 2°29 Fe Zine oe 0-91 Zn 0°72 Zn Uranyl ss 4-60 U 7-49 U Mercury ‘“ 2°89 Hg 2°43, Hg Lead ean’ 2°56 Pb eee Sy Silver ef 4-09 Ag a sine Nickel #5 4-70 Ni Cobalt ce 6-03 Co < Apparently, the two forms of albuminous matter, the albumin and globulin, do not form corresponding compounds with the metallic salts experimented with. * See Loew, Pfliiger’s Archiy far Physiologie, Band xxxi, p. 393. XXI.—Ece-ALBumMIn anp Atpumoses. By R. H. Currrenpen . AND Percy R. Borron, Pu.B. Ever since the albumose bodies were first separated from the pro- duets of fibrin digestion* with pepsin-hydrochloric acid, it has been our intention, already expressed, to subject the various individual albuminst to the action of purified pepsin under like conditions, and thus ultimately to acquire a comparative knowledge of the albumose bodies obtainable from these different sources. Already the albumose bodies from fibrin and the globulosest have been subjected to a care- ful study and we present here the result of a study of the albumoses from egg-albumin. In doing this, we have to report at the same time, the results of a study of the composition of egg-albumin itself. For we have made it a rule, in the series of experiments shortly to be described, to analyze a sample of each lot ofalbumin prepared for digestion. In this manner we have obtained data for a direct comparison of composition between the original sample of albumin and the products formed by its digestion. This we have deemed of considerable importance, for the data so obtained may throw considerable light on the nature of the changes involved in the formation of the albumoses ;> particu- larly, as to whether they are hydrolytic in their nature. Four distinct samples of albumin were prepared, three of which were prepared in large quantities and served as material for the subse- quent digestions. . Albumin A. This was a preparation of coagulated egg-albumin, prepared espec- ially with the view of obtaining a product wholly free from globulin. The method employed was essentially that recommended by Ham- marsten.§ The whites of 120 eggs were freed from the yolks, then *W. Kiihne and R. H. Chittenden, Ueber Albumosen, Zeitschrift fiir Biologie, Band xx) pill. + W. Kiihne and R. H. Chittenden, Ueber die nachsten Spaltungsproducte der Wiweisskérper, Zeitschrift fiir Biologie, Band xix, p. 159. { W. Kiihne and R. H. Chittenden, Globulin und Globulosen, Zeitschrift fur Biologie, Band xxii, p. 409. See K. V. Starke, Beitrige zur Kenntniss des Serum- und Eialbumins, Jahres- bericht fiir Thierchemie, 1881, p. 18. rs. Chittenden and Bolton—Egg-Albumin and Albumoses. 333 finely divided by shaking with glass, the fluid mixed with an equal volume of water or more, then shaken vigorously with air and finally filtered through cloth. The solution so obtained, was then saturated with crystals of magnesium sulphate at 20° C., for the com- plete removal of the globulin. The mixture was filtered through paper and the clear filtrate saturated with sodium sulphate. The precipitated albumin was then filtered and washed with a saturated solution of sodium sulphate, after which it was dissolved in water and dialyzed in running water until the magnesium and sodium sulphates were entirely removed. The fluid was then again filtered and the albumin finally coagulated by being poured into eight litres of boil- ing water, slightly acidified with acetic acid. The great bulk of the coagulum so obtained was at once placed in four litres of 0-4 per cent. hydrochloric acid, while a small sample for analysis was washed with 95 per cent. alcohol, finally with absolute alcohol and then dried, first at 100° C., and finally at 106° C., in vaeuo, until of con- stant weight. The following table shows the results of the analysis of the product. The various determinations were made as described in the previous articles on these subjects, the sulphur being deter- mined by fusion with potassium hydroxide and potassium nitrate in a silver crucible, according to the method designated by Hammar- sten* as la. Albumin B. This albumin was prepared from the whites of 12G eggs by a some- what different method. The albumin solution, after dilution with water, was made very distinctly acid with acetic acid, and the heavy precipitate of globulin, after it had well settled, removed by filtration. The acid fluid was then made exactly neutral with sodium carbonate and again filtered ; it was then thymolized and dialyzed in running water for eight days. A little globulin, not precipitated by the acetic acid, was found in the bottom of the dialyzers when the salts had diffused out. This was filtered off and the perfectly clear fluid evap- orated at 35—45° C., to perfect dryness. A sample of this preparation was ground fine, dried at 106° C. in vacuo and analyzed. It is perhaps questionable, whether all of the globulin is removed by this method. The precipitate with acetic acid was quite heavy, and as H. Dillnert has recently shown that the amount of globulin in egg-albumin, as * See Zeitschrift fiir physiolog. Chemie, Band ix, p. 289. + Ueber die Globuline im Hihnereiweiss, Jahresbericht fiir Thierchemie, 1885, p. 31. ind Albumoses. uUmin ¢ Chittenden and Bolton—Egg-Alb 334 00-00T 60-86 — 2 os 7 aes a —— aad ae ae ©) 16-1 L6°T F6-1 16-1 le "a a “= a $ O8- ST hi co a on aad C8-CT =6ph-ST * §8-CT ie a. 96-9 = oN oo a rs eS 86-9 6-9 H Lé-6. paca a aie Ei ar ae 81-6 6-6 O ‘OSRIOAV ‘aun zsqns daatf-yso ay) fo Uoripsodwumod abpyUWad1ag | | | | LE-0 €600-0 ee Se, ta Boas — 3 a ee A es oe le 4 a — 9969-0 Be een ae 96-1 91600. «jee (eee ee a eee le Ce ie enh LIP9-0 aS ects 6-1 | SUCOIOMS = liar “gel ee eae ome : | een a a a we ik OOs im HOL0.0= “etme en es a ea ee ic Site a | TELE-0 a i SoS et eee i18-ST 6-992 8-9 9°90<|- Soa Se > ooh = - | 606-0 | a: i ay SN, oy ee a? 89-CT | C. Poh 8-9 8° Lie areas ome + Pet ae 6819-0 i er | 7 Se irks (84: | T-6hL 6:8 C08). ean i ee aa Or a ae 6&19-0 a2 el as ial aa, Fa rh Tike ~ * | 86TS | Lvee-T | 26-9 | LOOF-0 9079-0 | Sai a sae a ea ME eae Sale ss hy 3 | 20°89 | LISL-0 | 66-9 | ScPe-0 8&6E-0 ; “TUBS ‘mu +O)ine : aes . . Py hooked of “ONT + HOM % | emssarg 1 ay % Smead oe) “Wes mead ysV punoy g YqIA woIsny x 9 punoj i panoy pesn : ysVy roqye 'OSeq # a ae: [Oo OH souRysqug ‘Vy NINOGTY 4O SISA IVNY 330 Chittenden and Bolton—Egg-Albumin and Alhumoses. GL-T OTT 00-001 46-66 &8-T 68.97 86-9 8-69 ‘95k10A VW ¢°00-0 6F00-0 } 18-1 68-1 IL-1 06-1 CLT URL “ON + HOW UUM moIsny TOE 'OSPL PLT PL ST 04ST BLT T6-CT 48-ST 46-9 6&-6¢ ‘aoungsqns aatf-yso ayy fo worursoduod obn}UadLaq 9-CT “cu Oyen ALNSSOL rth *punoy N VIP 9-4 ‘0 °O -eece “meas “punoy O°H TO6P-0 O8PP-0 069-0 OFOS-0 P9FS-0 99T9-0 OF69-0 8808-0 66FF-0 0¢89-0 Cece. 0 “UBS ‘pesn eouRysqug 336 Chittenden and Bolton—Egg-Albumin and Albumoses. determined by the magnesium sulphate method, never reaches 1 per cent., but averages only 0°667 per cent., it seems probable that the greater portion is separated by the acetic acid. Further, Dil!ner has found that on the dialysis of a neutralized egg-albumin solution, the matter which separates out after a few days dialysis, is only in part globulin, but consists, in addition, of a somewhat insoluble body rich in sulphur. Hence, the substance which separated in our dialyzers, after precipitation with acetic acid and neutralization, may not have been composed wholly of globulin. The following table shows the composition of the uncoagulated albumin B. Albumin C. This preparation was much the same as albumin B, except that it was finally coagulated. Globulin was separated by acetic acid, the fil- - trate neutralized, again filtered and the fluid dialyzed in running water until all soluble salts were removed. The albumin was then coagu- lated by being poured into a large volume of boiling water acidified with acetic acid. A sample, after drying at 106° C. in vacuo, was found to have the composition shown in the accompanying table. Albumin D. This sample of albumin was prepared in exactly the same manner as albumin A; the globulin removed by magnesium sulphate, the albumin precipitated by sodium sulphate and after dialysis, coagu- lated as already described. Its composition is shown in the follow- ing table. Comparing now, the results of the analysis of these four samples of albumin, it is seen that the first three agree almost exactly in com- position, while the fourth shows a somewhat lower content of carbon, A B C D Average. C 52°21 52°33 52°46 51°74 52°18 H 6:96 6°98 7:00 6°81 6:93 N 15°80 15°89 15°88 15°68 15°81 Ss 1:94 1:88 1°69 2-02 1°87 O 23°09 22:97 22:97 23°75 23°21 Ash 0°37 aN kit O17 0°45 Further, the coagulated products (A and C) do not differ at all in composition from the non-coagulated albumin B. Schiitzenberger,* as a result of his work on proteid matter, ascribed * Bulletin de la Société Chimique de Paris, T, 23 et 24, 2 0 00-001 ra ao Ls 46:66 ied = Faget ears eo =re~eea li) a S 69-1 89-1 69-1 mbes San Pa Sgt) 5 S 88-91 ee ari 00-91 LL-ST. aay pS call a s 00-4 ae “ea 2 ge oar 86-9 w0-L = 91-69 (ae aie et = 6P-6G 6h-cS =O Ss ‘OBRIOA VW § ‘aounjsqns aatf-ysv ay} fo Uuor1prsodwods abnzUWaLagq § | | Ss LT-0 S100;07 ae 4 ee cae 7 Ra ae hos a, ed geo pea ae ee §S&L-0 TIA ge es Be ee eae ecient alma eee 2 erred oe See le ae | 68th-0 | IA S eae eT aon, oF eee ee |p ee |(Riee d | eee ak IP ee os ; | 69-1 9810-0 : CLES: 0 sAgs vive ware eR ater te eee pees pes eee ® SS esas a 86-CT | 8-F7oL | &-9T | 8-09 6PLE-0 Al f, = = 5 Key dle Stew 9 Ae 0) ots PLST | b-2Gh | bt | S6L | | geek Aer 2 ge6c-0 | UI ; 4 S = Sie ae Sh rN nthe mw vi ~— “ees “==> | =*"" 1@8-6¢ | S800-T | 46-9 | &868-0 8929-0 II = S : A ; is ae ee 9 ri ene ree ee “| = 1 eP-6e | SE9S-0 | 10-4 | OS8T-0 0866-0 f q x ye ! a : 3 uw (@) | ! av Be 3h, Se ede pe . ¥ “med, “wu WO~ || oe : : : : = a, vmeid of “On + HOM %. | easserg “1 bya) % | onead 4g “mBI8 ‘weIs Zi §$ punoy punoy punoy pesn ON 6 = UsV SV 8 YUAN worsng N STs wee H O*H | sourysqng ) SS , Toye OSeA |. . ‘punoy Nv ; a we sot Pian bs A ES a ‘O NIWOAATY AO SISATVYNY ‘ ttenden and Bolton—kgg-Albumin and Albumoses. / Ch 338 ‘ e 9F-0 CPO) sv 00-001 “OL-9 6L-1G “Weds “panoy °00 Gh 86 a4 60-0 £0-6 60-6 Ahir erad 89-91 roots oan L9-GT 69-¢T 18-9 Pas og =e “ae L1G ae oe oa arn ‘O.ORI0A WV ‘aounpsqns aatf-ysp ayy fo Wworpsoduod abnyua1agq POCO OF ae tees idl Ae 2 Sail Sole gu00-0 | ~~~" ei piel ae SE ee c nee 60-6 6860-0 fea 9 OL Soe ee oe 10-6 6LEL-0 =: : rr my a ieee! atte aaa mea a ax 09-ST | &-SOL Leh 0:96 res Se SE la iat al g9-0r | 999% | 0-9 | T-99 | ~--- ee = oe Boa ae Ce sae ROSES Caran’ mak tos, Sethe ae Se lpoe ee, POFaLe “WRI “UU 10) : MP | Kicltona+ wow) % |, emesod |ycmr | ° log panos ILM TOISN =e 9) at S l isny | N EAS JOIe "OSkA ‘punoy NI » H “mes “‘punoy O°H ‘d NINOATYV WO SISATVNY £L60-1 IITA L066-T TIA ¥999-0 IA GOF6-0 A 6C9L-0 | Al F60S-0 | itl POCP-0 Il 6009-0 | I | “TURLS *pasn oouRysqug ‘ON Chittenden and Bolton—Egg-Albumin and Albumoses. 339 >to albumin the formula C,,,H,,,N,,0,.S,, which requires a content of carbon not far different from the average of our results, but which on the other hand demands a content of nitrogen nearly 1 per cent. higher than we found. The well-known Lieberkiilin’s formula requires 53°59 per cent. of carbon, or 1 per cent. more than was found in our highest result. Harnack’s formula for albumin,* C,,,H,,,N.,O,,8,, with 204 66 29 a molecular weight of 4618, based on a study of the copper compounds of albumin, requires too high a content of carbon and altogether too low a percentage of sulphur. Lieberkiihn’s formula requires 1:98 per cent. of sulphur, while Harnack’s formula requires only 1°39 per cent. ; and as this was one of the main points on which Harnack based his formula, it is well to consider it. Our lowest result on sulphur is 1°69 per cent., and as the other three show a close agreement, it is proba- - ple that the former is somewhat too low. The average of our results, however, is but 0°04 per cent. higher than found by Lieberkiihn. O. Loewt has recently considered this question, and he found on deter- mining the sulphur in coagulated egg-albumin by a modification of Piria and Schiff’s method, 1°70 and 1°87 per cent. of sulphur respect- ively. O. Nasse,f{ likewise, found in coagulated albumin a content of 1-72 per cent. of sulphur, and lastly, Hammarsten§ found in non- coagulated albumin 1°93 per cent. of sulphur. There would seem to be plenty of confirmatory evidence, therefore, that the content of sulphur in egg-albumin is much larger than indicated by Harnack’s formula. ; The nitrogen, as determined in our preparations, is seen to be somewhat higher than found by Hammarsten, with whose results in other respects ours most closely correspond. Dumas, however, found nearly the same percentage of nitrogen as contained in our preparations. The accompanying table of analyses shows the aver- age of our results, compared with those of others. z Albumoses. Three distinct digestions of albumin (preparations A, B and C) were made with pepsin-hydrochloric acid, and the albumose bodies isolated. In this way it was possible to prepare the bodies under somewhat different conditions, and to notice the influence, if any, on the nature and composition of the products. The pepsin-hydrochloric acid used in two of the digestions was prepared with a special view * Zeitschrift fir Physiolog. Chemie, Band v, p. 207. + Pfliger’s Archiv fiir Physiologie, Band xxxi, p. 395. _ $ Jahresbericht fiir Thierchemie, 1873, p. 13. § Ibid, 1881, p. 19 16-12% CL. 19-91 gC. 8G Ll ‘61 “d ‘TEs ‘almoyosoryy, Inj yyoteqseiye sr ‘gop ‘d ‘ta (g) ‘shyg ‘woeyo ‘aay ‘ytd ‘egg ‘Wepeuay s,yropuessog 60G ‘d ‘ayx pueg ‘ermeyy ‘yyead inj jeaanoe ‘repynyy Aq pozATeue sy *] 68-16 86 I ¢9-CT. £6-9 6S-€¢ (FQ OF NEES TP HOG) BUI; Ss YoRUIBFT 0} Suppfiod9e uottsodmoo [Roye10eyT, “8 (8924092 9N B58 OFF) B[NUMIOJ $,1eSioqueznyoY 04 Sarp1ooow uonisodmo0d [eoyesooyy, *) "(F8OS 8 NEO) BpoMIOT SUYNYyIqory 0} Sutpcoooe uoitsoduroo [woye1o0ay,T, “9 ‘doyog pue uepusyiyg Aq sq[nsor ey] JO 9SvIOAY “G ‘uoysuvumvyy Aq pozdjear sy ‘P ‘snoye, pae seuing Aq pozdTeue sy “¢ ‘aynyseqory Aq pozATeue Sy “7 * LY ”n Ss S ~ . s > > ~ ~ ‘= > Ss > ns ) l => =) S = S ~ = 3 = ~~ ~ ~ =~ ~ ~ ‘= < i) 340 ‘ a ‘NIMAGTY-DOW TO SASXIVNY Chittenden and Bolton—Egg-Albumin and Albumoses. 341 v to removing all traces of albumose bodies, formed by the self-digestion of the mucous membrane, and was prepared as follows: 700 grams of mucous membrane from the cardiac portion of six pigs’ stomachs, freed from the muscularis, were finely divided and warmed at 40° C, for fourteen days, in two and a half litres of 0°5 per cent. hydro- chloric acid. At the end of this time, all albumose bodies presumably having been converted into peptone, the solution was filtered from the residue of nuclein, antialbumid, ete., and the filtrate saturated with ammonium sulphate. The precipitate, consisting mainly of pepsin, with perhaps some albumose, was filtered off, washed with a saturated solution of ammonium sulphate, and then dissolved in two litres of 0°2 per cent. hydrochloric acid. ‘The acid solution was then thymolized and dialyzed in running water, until the ammonium sul- phate was entirely removed. On opening the dialyzing tubes, quite a precipitate was found, which on being dissolved in 0:2 per cent. hydrochloric acid showed marked proteolytic action. The filtrate also, on being acidified, showed vigorous digestive power. These two solutions of purified pepsin were used in the digestion of two of the albumins, while with the third a pure glycerin extract of pepsin was employed. The general method of procedure, both in the digestions them- selves and in the separation of the various albumoses, was much the same as that previously employed by Kiihne and Chittenden. Digestion of Albumin A. The albumin, as previously described, was placed in four litres of 0-4 per cent. hydrochloric acid and the mixture raised to a tempera- ture of 45° C. Then 600 c¢. c. of the purified pepsin-hydrochloric acid solution were added and the mixture kept at a temperature of 45° C. for three hours, after which it was neutralized with sodium hydroxide and filtered. The pepsin solution, although quite active, did not act very vigorously on the coagulated albumin. The neu- tralization precipitate, therefore, together with the unaltered albumin, was again treated with a fresh quantity of the pepsin-hydrochloric acid, under like conditions as the preceding, for four hours. The two neutralized fluids were then united and treated together. The total volume was about six litres. The clear fluid was saturated in the cold with crystals of sodium chloride, by which a precipitate was obtained, which from analogy should consist of proto-, dys- and heteroalbumose, ! 342 Chittenden and Bolton—Kgg- Albumin and Albumoses. In making these separations of the albumose bodies, we intention- ally avoided raising the temperature of the fluid above 45° C., for fear that heat might induce some change in the character of the bodies; hence the first neutralized fluid was saturated directly with _ salt, in spite of its large volume, and the bodies were ultimately all separated without having been exposed to a temperature higher than that above-mentioned. The use, however, of such a large quantity of rock salt introduced into the solutions some calcium sulphate, which adhered very tenaciously to the albumose bodies and thus unavoidably raised the content of ash in the preparations. The precipitate produced by the addition of sodium chloride in substance was filtered, washed with a saturated solution of sodium chloride, then extracted successively with a ten per cent. solution of sodium chloride, a five per cent. solution of the same salt, and lastly with water. The residue remaining undissolved after these succes- sive treatments with dilute salt solutions and water, presumably con- sisted of dysalbumose, while the solutions contained a body precipi- table by acetic acid and soluble in excess, and also precipitable by potassium ferrocyanide; presumably protoalbumose together with heteroalbumose. The original salt-saturated filtrate contained all of the deuteroalbumose, together with considerable protoalbumose and some heteroalbumose. A. Protoalbumose. . The five and ten per cent. sodium chloride solutions of the first salt precipitate, together with the aqueous solution of the same, were united and then dialyzed in running water for removal of the hetero- albumose. The solution, partially freed from the latter, was concen- trated somewhat and the protoalbumose again precipitated by satu- rating the solution with sodium chloride. This precipitate was again dissolved in water, dialyzed until the greater portion of the salt was removed, the solution then concentrated and the albumose precipi- tated by aleohol. This precipitate was redissolved in water, dialyzed until no chlorine reaction could be obtained with silver nitrate, the solution concentrated on the water bath to a syrup and finally pre- cipitated with alcohol, washed with alcohol and ether and then dried at 106° C. in vacuo until of constant weight. In the last dialysis, there was no separation whatever of heteroalbumose, hence the pro- toalbumose is to be considered as quite pure. The composition of the substance is shown in the accompanying table. The ash gon- tained no sulphate. 3 S 00-001 s 19.46 oT = es: 5a = = O z 18-1 98: QL. ce ae og = S S LheGT 2 ee GL-GT 64-41 ee oe N 3 06-9 2% ass a rig 18-9 76-9 H = 86-09 5 a Sage = "s as G0-1¢ &6-08 a ~ “OSRAOA ; s aUDISgns aatf-ysp ayy fo worysodwmos abnzua.1eq S reas a 7 ae <= =e oe i. = eS : a ges ui Oe) Se aS alae 7 ee ee a alae SS ae 9¢99-0 ILA s 4 | . es ese oS eet 6810-0 ee a cel el ees ae gee ae ees as 9199-0 LA $ ele Sol Wed 6190-0 Bae SN agi WB eae eee We ep Se to 680-0 | A fe Sa S752 | eS eS Sho Oe | se ohoS Grviei rede em | po ikea seor-0 AT Pa mee ee ot at ee Gp Gel p GGe ||, GeO Tes eh fe saline lar a Sie isos eeer-0 TIT : S ’ : at eae eels ae ee eae a “"=- | =| Q1.6h| 8987-0 | OL9 | LO9T-0 c99e-0 TE as | | Sa ae Se cle ee oS) oe “777 | --"- | @9.67] 3908-0 | £49 | 6698-0 Serr-0 | I > — a = SS ee eS SS SS , Ss a ‘aed oa Dorel 9 UOTE Fo) ee 9h |e 2) -¥ S| tone ous) oe mee zg “ures % “ues eee eal ‘ Ss py | | 8) | wet oon | QO | ‘punoy #99 | HH | ‘puny O°H ; me ; ne ysy ¥ 7 souRysqug _ s 1oye "OSPA puno} N Bes. “ISOWAMIVOLOU “VY dO SISATVNY ae si deaielaaal < ’ . ‘ 2 . 5 . ; Bi eee Te. | . i ‘s ’ 344. Chittenden and Bolton—Egg- Albumin and Albumoses. The protoalbumose was readily soluble in water and, unlike the protoalbumose from fibrin, dissolved to a perfectly clear solution with a neutral or very faintly alkaline reaction. The aqueous solu- tion was rendered somewhat turbid by the addition of a little acetic — acid, the turbidity disappearing, however, on the addition of an excess of acid. The aqueous solution, strongly acidified with acetic acid, was precipitated by the addition of potassium ferrocyanide; the pre- cipitate, however, dissolved on heating the mixture, reappearing as the solution became cool. a An aqueous solution of the albumose, acidified with acetic acid to such an extent that the first turbidity was re-dissolved, was not ren- dered at all turbid by the addition of a little sodium chloride; the addition of more salt, however, gave a very strong turbidity which disappeared entirely on warming, reappearing on cooling. As with - the protoalbumose from fibrin, it is possible to add such a quantity of sodium chloride as to induce a very heavy precipitate, yet have it wholly disappear on boiling the mixture, separating out again, how- ever, as the solution becomes cool. Finally the addition of a larger amount of sodium chloride gave a precipitate in the acidified solution, which was not at all affected by even boiling. An aqueous solution of protoalbumose, when treated drop by drop with concentrated nitric acid, was rendered noticeably turbid at the point of contact, the turbidity disappearing as the mixture was shaken, On adding just the right proportion of nitric acid, a point was reached where the solution showed a permanent turbidity, which disappeared on the application of a little heat, returning as the solution cooled. A slight excess of nitric acid produced even in the cold, a very distinct reddish yellow coloration of the fluid, the turbidity disappear- ing. By adding crystals of salt to the acid solution, a precipitate was again formed, which disappeared on the application of heat, and reappeared as the solution cooled. By saturating an aqueous solution of protoalbumose with salt, a heavy precipitate was formed, but in the filtrate more albumose was always found on the addition of a little acetic acid. In fact, each time protoalbumose was precipitated by sodium chloride in substance there was always a loss; a certain proportion of the substance re- maining in the filtrate, precipitable only by the addition of a lit- tle acetic acid. Protoalbumose heated with acid, or treated in the cold with dilute alkalies was not apparently converted into acid albumin or alkali-albuminate-like bodies, for on neutralization, no precipitation whatever ocenrred. Heated with potassium hydroxide a Chittenden and Bolton—Lgg-Albumin and Albumoses. 345 and plumbic acetate, there was a decided blackening of the fluid. The protoalbumose likewise gave the characteristic reddish violet color with potassium hydroxide and cupric sulphate. Cupric sul- phate alone, added to an aqueous solution of protoalbumose, gave a heavy greenish colored precipitate, not very soluble in excess of the copper salt. Mercuric chloride and lead acetate also precipitated the albumose. In its reactions, therefore, the protoalbumose formed from egg- albumin does not differ, essentially at least, from fibrin protoal- bumose. A. Deuteroalbumose. This body was obtained from the first salt-saturated fluid, by the addition of a little acetic acid (sp. gr. 1042) also saturated with salt. As Kiihne and Chittenden have already pointed out, all of the proto- albumose is not precipitated by saturation of a neutral fluid with sodium chloride. Hence, it is to be expected that the deuteroalbu- mose solution would contain some protoalbumose, which latter would be likewise precipitated by the salt-saturated acetic acid. We en- deavored to make a separation, however, by rejecting altogether the first precipitate produced by the addition of a little acetic acid, and then to obtain the deuteroalbumose fairly free from the former, by the subsequent addition of more acetic acid. The final precipitate so obtained, was dissolved in a small amount of water and then dialyzed for several days. The solution, in which was noticed a small deposit of heteroalbumose, was concentrated and finally precipitated by alcohol. The precipitated deuteroalbumose was then redissolved in water, the solution made exactly neutral with sodium carbonate and dialyzed in running water for many days, after which the solution was concentrated to a syrup, the albumose precipitated with - alcohol and finally, after washing with ether, dried at 106° C. in vacuo until of constant weight. The composition of the product is shown in the accompanying table. The ash was composed mainly of ferric oxide and calcium phosphate ; it contained no sulphate. The pure white powder, after being dried at 106° C, was found readily soluble in water. The solution was not rendered at all turbid “by saturation with sodium chloride, but the substance was more or _ less completely precipitated by the addition of a little acetic acid to the salt-saturated fluid. Nitric acid added to an aqueous solution of the substance gave no precipitate whatever, but colored the solution - decidedly yellow even in the cold. A little sodium chloride added to ¢ Trans. Conn. Acap., Vou. VII. 44 Nov., 1886. ‘4 7 i D 4) nd Albumoses. Chittenden und Bolton—KEgg-Albumin a 346 Fe a ek a a eel ak ele elie ae 00.001 OT a « ae Sa Sf oe ae ee ee eS Se a eS O8-T L8-1 PLL cee ye Meise 3) eae oes GHGS. Ea Er, i 5 D, F9-GT &8-GT" it £69 ee PE ee ee eee ae 16-9 LOS a ei ere A oe —os 61-6¢ ‘Q0B10A ‘gounysqns aauf-ysp ayy fo worrsodmoa abppuaolag ea oe —=— es eS : : c=: 10-E | 6900-0 We 6b ee aS ce iret ae ee ae eS 29 OLE \s8200-02) So |< |e -s) = Se — ae | el me | ee ms Ss ae is ar a 8-1 €080-0 et Sas = eg eee el a | mii. * ns oe oe i IL-1 990-0 eke ea: ce oe ic a he ae ken ts ah et et la eee LPC | 9-192 | OFT | 8:08 | | ~ Si ee: Se ai ane oe he 99-7 | ¢-89h, | 8-87 | OT Rea | ra “ pe erase ale eae age re Bes er G9.TC | OTE8-0 06-9 | lige Sa a Biren S|. es Se i Se Rh ee one eh aes a oe “OF TS 9696 0 16-9 ; “mu 0 Ye RE “meds | ainsselg| “IL oe % ee g) sonst Hom foe= (= 2! y “ues 2 sv us S gta MOIsny | N | +) “punoy 7 eye) H BY’ rye "OS" | ‘punoy N | | | ‘ASOWACTIVONRLAAG ‘VY AO "umRIs ‘punoj OH CDZAZALO L08S-0) &PL9-0 196¢°0 Gocr-0) 6669-0 ‘mes ‘pasn aoURISGNG S A SISA TYNY Chittenden and Bolton—Egg-Albumin and Albumoses. 347 the nitric acid solution gave a decided turbidity, which disappeared on warming the solution and reappeared on cooling. The addition of acetic acid to an aqueous solution of the albumose gave no precipitate whatever, nor was any change to be observed on heating the fluid; neutralization, at least, caused no precipitation. The addition of a little sodium chloride solution to a solution of deuteroalbumose acidified slightly with acetic acid gave no precipi- tate whatever, but as with deuteroalbumose from fibrin, the applica- tion of a little heat induced a slight turbidity, which disappeared on raising, the temperature still higher. Again, on the further addition of sodium chloride, a heavier precipitate was produced which disap- peared completely on heating the solution and reappeared on cooling; and lastly, by adding more sodium chloride, a precipitate was ob- tained which was permanent even on heating the mixture to boiling. In these, as in nearly all other respects, the deuteroalbumose showed itself the same in nature as the deuteroalbumose from fibrin, and the reactions given for that body can well be applied here. In one reac- tion only was there any very noticeable difference; viz: in the reaction with cupric sulphate. Deuteroalbumose from egg-albumin gave only a slight precipitate with cupric sulphate, even on the addi- tion of a minimum amount of the copper salt.* With acetic acid and potassium ferrocyanide, the reaction was much the same as with pro- toalbumose. Boiling with sodium hydroxide and lead acetate gave a decided blackening of the fluid, from the presence of sulphur. A, Heteroalbumose. The greater portion of the heteroalbumose was obtained by the dialysis of the 5 and 10 per cent. sodium chloride solutions of the first salt precipitate, viz: in the purification of protoalbumose. Some, too, was also found in the dialysis of the precipitated deuteroal- bumose. In both cases, the albumose was left as a more or less gummy precipitate, closely adherent to the parchment of the dialyser, sepa- rating out as the sodium chloride left the solution. The product was purified by solution in 5 per cent. sodium chloride, re-precipitation by the addition of salt in substance, re-solution in 5 per cent. sodium chloride and separation by dialysis, continued until all chlorine was * This fact simply shows the greater purity of this preparation of deuteroalbumose or rather its freedom from protoalbumose, for as Dr. Neumeister has recently shown, perfectly pure deuteroalbumose gives no precipitate whatever with cupric sulphate. Later, we were able to prepare deuteroalbumose entirely free from protoalbumose. Chittenden and Bolton—Egg-Albumin and Albumoses, 348 98-1 “meis | “punoj sv 00-001 “Sr Og eS aay a ie | Set ake a ate (Sie. too 3! Ts ah le eg a oe aa oa tee le = = aL | 62Gou ates oe he ee loo cele ee ae aes eee wee, | ee So Poole el GOP G2GCk a (erate ee Sil onal at baie al ee al ena ah SS i a 5 fal aes ng era ~--- | =>" | @p-Fh | 8808-0 | 90-9 60LE-0 | | pees eae, A eS ee cates super este ee Seer sree 1@-9 0861-0 ee at Ear | aes 2 a ew ‘aounys | pie meis 5 Ce Oo PD) I “mRIs a “Ris % SONM+ HOM -qng jo 4 yse % punoy % |emmsselg| “L | % ‘punoy % -‘punoy S WM WOISNT | rey yo g| OU} WOME | “USV ere ee Ie — @ 500 H Of Joye TOSPE “| "OS*®a | ‘punoj N ‘aSOWAGIVOLOU ‘Gq AO SISA TVYNY pL9b-0 IA 8609-0 A 8609-0 A w1e-0 | AT FoLe-0 TT e960 TI oree-0 I Sota Ps} ‘pasn QoURISgNy } ‘ON Chittenden and Bolton—Egg-Albumin and Albumoses. 351 Digestion of albumin B. 340 grams of the dry albumin, non-coagulated, were soaked in 2 litres of 0-4 per cent. hydrochloric acid for 24 hours, then warmed up to 45° C. and 1 litre of the purified pepsin-hydrochloric acid added, also warmed at the same temperature. The mixture was kept at 45° C. for 16 hours, then neutralized and filtered. The filtrate, con- taining the albumose bodies formed from the non-coagulated albu- min, was then treated as already described under Albumin A. B. Protoalbumose. The protoalbumose isolated from this digestion, was purified in - much the same manner as A protoaibumose, and did not differ from it in its reactions, except that with water it did not dissolve to quite so clear a solution; in fact its solution in water resembled more closely the aqueous solutions of protoalbumose from fibrin. During its final purification, it was dialyzed in running water until no chlorine reaction could be obtained with silver nitrate. In spite of this fact, however, the preparation contained a large percentage of ash, con- sisting mainly of calcium sulphate, ferric oxide and a little calcium phosphate. The accompanying table shows the composition of the substance after drying at 106° C., in vacuo, until of constant weight. In the purification of this protoalbumose, the substance was repre- cipitated three times by saturating the aqueous solution of the pre- cipitate with sodium chloride. By this treatment, as already stated, there is considerable loss, inasmuch as the precipitation of proto- albumose with salt in this manner is never complete, considerable remaining each time in the salt-saturated fluid. By adding a very little acetic acid, however, the protoalbumose is completely precipi-- tated from the salt-saturated solution. The filtrates therefore, from the second and third precipitations of protoalbumose with salt alone were united, and the albumose remaining in them precipitated by the addition of a little acetic acid, saturated with sodium chloride. Our object was to see whether the protoalbumose which had at one time been precipitated by salt alone and then had finally become soluble in the salt-saturated fluid, differed at all in composition or in reaction from the protoalbumose still insoluble in the salt solution. The albumose separated in this manner was purified by being dis- solved in water, the solution made exactly neutral with sodium car- bonate and dialyzed for several days. The fluid was then con- ‘ysy JO§ Sunonpep 19yJeB g Chittenden and Bolton—Egg-Albumin and Albumoses. 352 00:00F 8648 86-1 16-S1. 68-9 T6-0¢ ‘OSVIOAW 68-9 16-06 OoHanoe ‘aoupngsqns aatf-ysp ayy fo uorprsoduoa abnquaosag 1600-0 2 — 18:6 “meds “yse % aT} WOIF) “YSy *osed Gru sees ai | eae be ae oe "> | 6P-GT | 8-992. | 0-6E | 8:0F | 77 | -7 ef ee a a “= | =""* | 9F.6P | 820-1 “TRIS ae 0 : 0°90 a "mes /-punoy fe OINSSOTg L ei: ; pi i ey “punoy NV } "00 | 69-9 of “meas ‘punoy O°H ‘ F80¢-0 AT ch6F-0 IIIT orer-0 TIT PITE-0 IT c0s¢-0 I wmei3 ‘pasn aouRysqnug ON ea: *. . 9L-% HO80<0: ~{) “=e Sie so G0 “med ? o..| “ONM+ HOM | ny qug jo % 8 ME Howwny ey 30 6 Joye 'oged | “HOOO-HO 4q ‘uornnos [OVEN woay poyeyidioarg “ASOWNETVOLOUG ‘q AO SISA TVNY Chittenden and Bolton—Egg-Albumin and Albumoses. 353 centrated to a syrup and the albumose precipitated by alcohol. This precipitate was again dissolved in water, the solution made exactly neutral and again dialyzed. After suitable concentration, the albumose was again precipitated by alcohol, washed with alcohol and ether and finally dried at 106° C. in vacuo. The results of the analysis are seen in the accompanying table. The ash in this preparation is seen to be much smaller than in the protoalbumose precipitated by salt alone. In other respects the two analyses are closely comparable, particularly the carbon and sulphur. The reactions were in almost every case the same as with the pre- ceding preparation, excepting perhaps a somewhat greater solu- bility. . B. Deuteroalbumose. This body was separated and purified in exactly the same manner as in the preceding digestion. The analysis of the product is shown in the accompanying table. The ash contained some calcium sul- phate and a little ferric oxide. The reactions of the body were the same as those of A deuteroalbumose. From this digestion, more or less heteroalbumose was separated but no analysis was made of the product, as the amount was rather small for the necessary purification. Digestion of Albumin C. In the digestion of this sample of coagulated albumin, a much “more vigorous pepsin-hydrochloric acid was employed than in the preceding digestions. The freshly coagulated albumin was placed in 3 litres of 0-4 per cent. hydrochloric acid and brought to a tem- perature of 45° C., then 400 c.c. of a pepsin solution, made from a~ pure glycerin extract of pepsin, were added and the mixture kept at 45° ©. for 24 hours. The fluid was then neutralized, filtered and the clear filtrate saturated with sodium chloride. The albumose bodies were then separated and purified according to the methods already described. : The several bodies showed the same reactions as observed in the preceding preparations. -Protoalbumose and deuteroalbumose were analyzed. The results are shown in the accompanying tables. The ash of the deutero- albumose contained no sulphate, but was composed almost entirely _ of ferric oxide. TRANS. Conn. AcAD., Vou. VII. 45 Nov., 1886. Chittenden and Bolton—Egg-Albumin und Albumoses. 354 ‘USV JOS Suyonpep Toye § | .g 00-001 ie : 80-46 =e ee aes said Boe roe - 808 20-8 20-8 ris Sage ae eer yh ale “Sy oager be Wer eed = ee 16-9 +555 egy Pose ae 26-9 96-9 6I-19 a as = a gT-1¢ 1@ Te ‘OBRIOA VW ‘aounysqns aat{-yso ay, fo woupsodmos abpzuardag (CEG Sie ea | Gee ced se es sek ore Praptlege. | erase“ seo ORO, ae ee | eas go ales ee SRO ea Cees cea a eae Sao 800°) "| GE00.0. eee boot ae Sop Ne coe eae cae ie Sie ae ae) eee Oo BALI ee | 2 eee ae oe aie ga aie ca Pigg mea" hoiGy | He0OR- | Ole laaetes eee ie Renae Mae pre gore ieee Heese segeugp | OD. | ORRID Me-RO: Ico ee Se ie ie ae Se Seliger | eer alleen sage ee. gaat Ep.g CE ae ag ae se a EO Geta oe eee ee ice 8 aati MR CYA “mes | "TRIS lest: ‘0. Ay) «|| ° . 3 y | . i . Psi “ONH+HOX |ang jo gf TV!) % |conos| % aioe bee a dees WA won TE Tog] mou | wv | ey | NX oa qaqye 'OSRA rose 4 ‘punoy N ‘ASONONATVOURLOANCG “G AO SISA TVNY oMano OSFT-0 6CE6-0 8LCE-0 666-0) Fore-() FEFE-0) €6EC-0 CP6F-0 L1l¥e-0 Tese"0 “wRAs “punoy "mes “pasn ‘ON OF eouRysqng } Chittenden and Bolton—Egg-Albumin and Albumoses. 355 Dysalbumose. This form of albumose was found in all three digestions, but the amount was much smaller than noticed in the fibrin digestions. The substance was obtained as an insoluble residue, after extracting the first sodium chloride precipitate successively with 10 and 5 per cent. salt solutions and with water. It was then dissolved in 0-2 per cent. hydrochloric acid, and after filtration precipitated by neutralization. After precipitation in this manner, a portion of the substance was found soluble in sodium chloride and on dialysis of the solution, sep- arated in much the same manner as heteroalbumose. The portion still insoluble in salt solution was then washed thoroughly with water and lastly with alcohol and ether. Not enough of the albu- mose was separated from any one digestion for analysis, but by unit- ing the products from all three, sufficient was obtained for the following analytical data: I. 0°2537 gram substance gave 0°1500 gram H,O=6°57 per cent. H and 0°4550 gram CO,=48'92 per cent. C. II. 0°3700 gram substance gave 46°3 c.c. N at 13°8° C. and 761°3™™ pressure= 14°96 per cent. N. III. 0°1354 gram substance gave 0:0069 gram ash=5:09 per cent. The ash-free substance therefore contained 51°52 per cent. C, 6°92 per cent. H, 15°79 per cent. N. The ash was composed wholly of ferric oxide. The peculiar behavior of dysalbumose after solution in either dilute acids or sodium carbonate and neutralization, shows plainly that the substance is simply heteroalbumose rendered insoluble by action of the sodium chloride. Dysalbumose, wholly insoluble in sodium chloride, is readily dissolved by sodium carbonate of 1 per cent., and on neutralization of the alkaline fluid is in great part pre- cipitated. The substance however, is now soluble in sodium chlor- ide and has evidently been reconverted into heteroalbumose. It is very apparent, however, from our results that heteroalbumose from egg-albumin is not so readily converted into dysalbumose by the action of sodium chloride, as heteroalbumose from fibrin. In all three of our experiments, the amount found was very small. Further, it would seem as if heteroalbumose from albumin was somewhat more resistant to the action of alcohol and ether than heteroalbumose from fibrin. Still the former did become quite rapidly insoluble in sodium chloride after standing under alcohol Chittenden and Bolton—Egg-Albumin and Albumoses, — : 356 00-001 86-66 he Fae 2 ata ae 00-6 00-6 oad ie ra ST-9F ri 8T-9T re bi me OF +k aes oS CO-L 3) 11-16 onl rae SPT 6-1 ‘QDLIOA VW “QOUDISQNS aauf-ysp ayy fo woryrsodmos abozudotaq cg. } | 18-& OMS sees jk eae seneg saci ob acne Sipe Mack — it = ae a a at (PSehe sk ae ee ee eee O > = @8-T ear €9-T 80-6 &0-6 08-1 00-8 = 86-1 10-3 TO(e cal. stant oe ee come ee Ss S ¢8-CT 61-1 cg. CT 86-ST LL-ST SL-CT ST-9L F6-G1 &T-91 WE) ies ee a ns eee N i . 86-9 66-9 C6-9 60-4 ¥6-9 16-9 OTL 68-9 60-4 06-9 SR ee Se races eet S &E-6E 6G. 1¢ 90-6¢ 09-1T¢ 61-1S L0-6¢ wets =| 16-0¢ F6-0¢ BOHOG Maine 5. tr Sci ctint aie pibeuee ean : a Ree c ‘ | fe Geen? (OMe ava ony 9 d v 0 | he wf es se hE as ep Vines ese: bee Ss rH - | ~ - -esoumnaye |‘esommn ; : * | iS UIUINg, y al alg asouINq[Bvo1EyNa(] : ASOUING[VO}OIg | SS S 3 lass o 360 Chittenden and Bolton—Egg-Albumin and Albumoses. ibrin products. | Kgg-albumin products, eat ai = | oe | | | on , o o merle a) o | $8| 88] 2¢ |\Sa3|/888| 281 83) we ) 28) so) £1208) ee8) 28 | she ) us| 82| 2 |e s| 2h5\ a2) gs | Me o os wl 3 oa Oe mre ae ne ee 50°77 50°65 | 52°68 | 50°89 | 51-04 | 51°07 51°62 | 52°33 fo ee eee | 6-78 | 6°83 | 6°83 | 6°81 | 6°89! 6°98 | 6°97 | 6°98 Nees seer al 17°14 | 17-17 | 16°91 | 15°98 | 15°79 | 16°00 15°82 | 15°85 Sa eee ae | 2°08.|. OBY [ LPO | ostcayleioe) Gb) ieee eee 6 NE, oe | 24°23 | 24°38 | 22°48 | -_-- ---- | 24:00 | 23°63 | 23-02 Deuteroalbumose is seen to contain 0°7 per cent. less carbon than egg-albumin, while protoalbumose contains fully 1°25 per cent. less. The nitrogen in the two compounds is in accord with the content of | carbon and the composition of the two products certainly suggests hydrolytic action. ‘The lower content of carbon in the albumose bodies has been ex- plained by some writers on the ground that the precipitants used, principally acetic acid, would tend to form an acid compound, and that even when dried and ready for analysis, the compound would still contain some acid; which fact would be sufficient to account for the low content of carbon found. It is to be noticed, however, in these results that the deuteroalbumose, not only on an average but in each individual case, contains more carbon than protoalbumose, which was precipitated by sodium chloride alone without the addi- tion of any acetic acid whatever. Further, comparing the two preparations of protoalbumose B, one of which was precipitated by salt alone and the other by acetic acid, it is seen that the. percentage of carbon is the same in both. Unquestionably proto- and deuteroalbumose both do combine with acids, but after neutral- ization and long continued dialysis the albumose body certainly exists in a free state, uncombined with either acid or alkali. The greater portion of this work was completed before we had any knowledge of Dr. Neumeister’s work on the complete separation of protoalbumose from deuteroalbumose.|| It was, therefore, too late for * See Kithne and Chittenden, Ueber die naichsten Spaltungsproducte der Hiweiss- korper. Zeitschrift fir Biologie, Band xix, p. 174. : + Average of all the products analyzed. Zeitschrift fir Biologie, Band xx, p. 40. ¢ According to Hammarsten. § Average of all products. | Zur Kenntniss der Albumosen. Zeitschrift fiir Biologie, Band xxiii, p, 381. Chittenden and Bolton—Egg-Albumin and Albumoses. 361 us to take advantage of his method of separation. Our preparations _ of deuteroalbumose unquestionably were not wholly free from proto- albumose, but that they did not contain much of this body is evi- _ denced by the small precipitate obtained with cupric sulphate; for, as Dr. Neumeister has recently shown, deuteroalbumose entirely free from protoalbumose gives no precipitate whatever with cupric sul- _ phate. ‘Trans. Cony. Acap., Vou. VII. 46 Noy., 1886. - ‘ fe c XXII.—CaseEin anv iTs Primary CireavacEe Propvucts. By . R. H. Currrenpen anp H. M. Painter, B.A., Pu.B. FoLiowineG out the general plan of procedure indicated some time ago with other albuminous bedies,* we have endeavored to prepare and study the primary products formed in the digestion of pure casein with pepsin-hydrochloric acid. Assuming that casein in its conver- sion into peptone by artificial gastric juice, passes through certain intermediate stages, in which bodies akin to the albumose bodies are formed, we have applied the methods of separation used so suc- cessfully in the past and have been able to isolate a class of bodies. bearing the same relationship to casein that the albumoses do to albumin. For this class of bodies we propose the name of caseoses. In studying these substances and particularly their composition, we deemed it essential to be certain of the purity and composition of the casein to be digested; particularly in view of the recent con- troversy between Hammarstent and A. Danilewsky as to the single nature of this albuminous body. Assuming, as claimed by Dan- ilewsky,{ that casein is a mixture of two albuminous bodies and that the numerous analyses recently made by Hammarsten of various preparations of casein are incorrect, particularly in the percentage of sulphur, made it incumbent on us to obtain some data on these points confirmatory of one or the other view, before advancing to a study of the products formed by the digestion of casein. Of the various methods used for the preparation of pure casein, that depending on repeated precipitation by acids and re-solution in alkalies has been the most in vogue; for although theoretical objections might be advanced as to the possibility of change in the nature of the substance under the influence of acids and alkalies, the results obtained have in some respects, at least, been very satisfac- tory. Alex. Schmidt, in conjunction with Kapeller,§ showed plainly that dialysis of milk and then precipitation of the casein by acetic acid, while it gave fairly good results, was not sufficient in itself to * See the preceding articles on globuJoses and albumoses. + See Zeitschrift fir physiologische Chemie, Band vii, p. 227. Zur Frage, ob das casein ein einheitlicher stoff sei, Also same volume, p. 427. + See Zeitschrift fiir physiologische chemie, Band vii, p. 433. + § Beitrag zur Kenntniss der Milch, Jahresbericht fiir Thierchemie, 1874, p. 154. << ’ af i “A Casein and its Primary Cleavage Products. 363 wholly remove the inorganic salts, and thus recourse was had to repeated precipitation by acids, after solution in dilute alkalies. By this method, Hammarsten* came to the conclusion that milk con- tains but two albuminous bodies, viz: casein and lacto-albumin, and the same investigator has repeatedly made use of this method for the preparation of pure casein. Lundbergt has plainly shown the notice- able resistance of casein to the action of acids, and Hammarsten has indicated the possibility of using acetic acid, if necessary, in place of the stronger hydrochloric acid. Millon and Commaille, however, have claimed that in the precipitation of casein with either acid, the precipitate does not consist of free casein, but is a compound of casein with the acid used. This erroneous view, Hammarsten shows depends simply on the great difficulty of washing the precipitated casein completely, and he suggests that it is perhaps impossible to prepare large quantities, of casein absolutely pure. For the preparation of the substance, however, Hammarsten recommends acetic acid in preference to hydrochloric acid and final drying of the compound at a temperature of 110°C. Danilewsky and Radenhausen,} however, prefer to use hydrochloric acid in the preparation of pure casein, and for this purpose they use skimmed milk, diluted with 4-5 volumes of water, to which the dilute acid is added little by little, until a good precipitate is obtained, After filtration and repeated washing with distilled water, the casein is rubbed fine, then dissolved in water to which a little ammonia has been added, the fluid filtered and the clear filtrate again precipi- tated by the addition of a little dilute hydrochloric acid. Casein, so prepared, after being washed with distilled water, reacts acid to test papers and shows the usual reactions of this body; but if when freshly precipitated, the substance is boiled with perfectly neutral- 50 per cent. alcohol and filtered hot, according to Danilewsky and Radenhausen, the casein is separated into two bodies, one of which is partially soluble in hot alcohol and separates out on cooling, while the other is insoluble. The soluble portion is termed caseoprotalbin, the insoluble portion caseoalbumin. The former, it is stated, gives * Zur Kenntniss des Caseins und der Wirkung des Labfermentes, Jahresbericht fiir Thierchemie, 1877, p. 158. + Kleinere Beitrage zur Kenntniss des Caseins, Jahresbericht fiir Thierchemie, 1876, p. 11. + Untersuchungen iiber die Hiweisstoffe der Milch, Jahresbericht fir Thierchemie, 1880, p. 186, 364 Chittenden and Painter— Casein and its no sulphur reaction when boiled with 2 per cent. sodium hydroxide, but contains 1°13 per cent. of sulphur, while caseoalbumin is stated to contain 1°23 per cent. of sulphur. Hence, according to these investi- gators, casein is a mixture of two bodies, one of which is rich in sul- phur, while the other contains a somewhat smaller amount. The objection which these investigators make to the use of acetic acid in the preparation of casein, is that from a sodium acetate solution, casein itself is not precipitated, or only in part, but that the precipi- tate consists mainly of the protalbin body. Further, Danilewsky and Radenhausen claim that caseoalbumin dissolved. in 1 per cent. sodium hydroxide and allowed to stand for 24 hours at the tempera- ture of the room, is changed almost completely into caseoprotalbin, with loss of sulphur and calcium phosphate. In a similar manner, protalbin dissolved in lime water, with addition of alcohol and phos- - phorie acid, can be changed into caseoalbumin. Hammarsten,* however, takes exception to these views and points out that the peculiar behavior of Danilewsky’s casein towards boiling 50 per cent. alcohol, depends in part upon its content of calcium phos- phate, the presence of which impurity depends upon the use of hydro- chloric acid in the precipitation of the casein, which acid does not favor the removal of the salt as well as acetic acid. Using acetic acid as the precipitant and then employing a casein three times so precipitated, and which analysis showed to be almost entirely free from calcium phosphate, Hammarsten, by treatment with boiling alcohol, was unable to obtain anything more than a trace of sub- stance corresponding to caseoalbumin. Further, casein is unques- tionably changed by boiling with alcohol, as Hammarsten clearly shows; in fact it is well known that heating an albuminous body in water is liable to change its nature, at least its solubility, and there is no reason why treatment with 50 per cent. alcohol should not lead to a like result. Again, Hammarsten points out clearly another inconsistency in the reasoning of Danilewsky and Radenhausen in connection with the so-called conversion of caseoprotalbin into caseo- albumin. ‘The former body is stated to be poorer in sulphur than the latter, and yet we are told that the protalbin body can be con- verted into caseoalbumin by simple solution in lime water and addi- tion of phosphoric acid, with or without alcohol. Yet how it is possible by this method of treatment to convert a body with a small * Zur Frage, ob das Casein ein einheitlicher Stoff sei. Zeitschrift fiir physiologische chemie, Band vii, p. 227. ay ad sing 5 eS ds TEA “¥ 7 nw) 05 ‘ ‘= ‘ 4 Primary Cleavage Products. 365 content of sulphur into a body richer in sulphur, is hard to see. Much more plausible is it, as suggested by Hammarsten, that in these two bodies we have to deal with the same substance, in the one case united with calcium phosphate, and in the other uncom- bined with this salt; or in other words that the so-called protalbin body in the absence of calcium phosphate is soluble in boiling 50 per cent. alcohol, while in the presence of that salt it is insoluble. This view being correct, and Hammarsten’s observations would tend to show that it is, it is obvious that the caseoprotalbin of Dan- ilewsky is simply a portion of the casein, which, owing to lack of a sufficient amount of calcium phosphate, passes into solution on being boiled with dilute alcohol; while caseoalbumin, on the other hand, is likewise a portion of the casein, insoluble on account of the presence of calcium phosphate; changed, however, more or less by action of the boiling alcohol. Further, the reason why casein precipitated several times by acetic acid does not contain as much calcium phosphate as when precipitated by hydrochloric acid, and thus reacts differently with alcohol, depends on the far greater insol- ubility of freshly precipitated casein in excess of acetic acid than in hydrochloric acid. In the precipitation of casein with hydrochloric acid only the slightest excess of acid can be added, on account of the ready solubility of the precipitate in this dilute acid. With acetic acid, however, a moderate excess can be added without solution of the precipitate, and thus in the latter case, a larger proportion of mineral salts are removed at each re-precipitation. Danilewsky and Radenhausen have further called attention to the fact that casein precipitated with hydrochloric acid yields a larger amount of alkaline sulphide than when precipitated by acetic acid. This statement, Hammarsten has several times been able to verify, but the latter investigator seeks an explanation for this fact in the- occasional presence of a second albuminous body, richer in sulphur, presumably serum-globulin, precipitable like casein by acids. Serum- globulin too, is readily soluble in excess of acid, even more so than casein, and hence by the acetic acid method of precipitation, which allows a far greater excess of acid, the casein would be much less liable to contamination by this hypothetical globulin than by the hydrochloric acid method. In this connection it may be well to notice that Musso and Menozzi* claim the presence in milk of a peculiar albuminous body containing 53°74 per cent. C, 15°52 per * Studien iiber das Eiweiss der Milch. Jahresbericht fiir Thierchemie, 1878, p. 139. 366 Chittenden and Painter— Casein and its cent. N and 1:55 per cent. S, for which they claim a position midway between serum-albumin and casein. It can be partially pre- cipitated from fresh milk at ordinary temperatures by the addition of acetic acid. Further, Lebelien* has proved the presence in milk of a. globulin-like body, lacto-globulin, which can be precipitated by satu- rating the fluid remaining after removal of the casein with sodium chloride, with magnesium sulphate. The substance appears to be identical with paraglobulin, and thus this fact, just discovered, would seem to confirm Hammarsten’s theory as to the cause of the greater content of sulphur sometimes noticed in casein precipitated by hydro- chloric acid. Accepting then, Hammarsten’s views as correct, it is obvious that casein precipitated by acetic acid, if not a single body, must be com- posed of two bodies, more or less alike and both precipitable by — dilute acids. In attempting to settle this point definitely, Hammars- ten has sought by analysis of a large number of preparations made under different conditions, to obtain data as to the exact composition of casein variously prepared. Naturally in this connection, consider- able attention was paid to the content of sulphur, since, Danilewsky and Radenhausen’s views being correct, variations in the content of sulphur would naturally be expected. If casein is a mixture of equal parts of caseoprotalbin and caseoalbumin with 1°13 per cent. and 1:23 per cent. of sulphur, respectively, then casein itself would nat- urally contain 1:18 per cent. of sulphur; an amount somewhat higher than has been found heretofore. | Recently, Dainlewskyt+ has modified his views somewhat, and now considers casein, as before, to be a mixture of two distinct bodies, but of nucleoalbumin with nucleoprotalbin instead of caseoalbumin and caseoprotalbin. As the reactions of these two bodies are appar- ently much the same as those given as characteristic of the caseo- bodies, this change of view appears to be mainly a change of name. Danilewsky still claims the correctness of the high content of sul- phur in casein and assumes that the variation in the results obtained by different workers is due simply to difference in the methods of determination, and that unquestionably pure casein contains over 1:0 per cent. of sulphur. The content of sulphur in casein as determined 30-40 years ago by * Beitrag zur Kenntniss der Hiweisskérper der Kuhmilch. Jahresbericht fir Thier- chemie, 1885, p. 184. ; +See Zeitschrift fir physiologische chemie, Band vii, p. 433. Primary Cleavage Products. 367 Lehmann, Riihling, Viélckel and others,* varies from 0°85 per cent. to 1:10 per cent. Ritthausen,t from several analyses of the copper compound of casein, found a content of sulphur equivalent to 0°80- 1°12 per cent. in the free casein. Schwarzenbach,{ by a study of the platinum cyanide compound of casein, ascribed to casein itself a con- tent of 0°19-1°10 per cent. of sulphur. Hammarsten, however, found by analysis of eight distinct preparations of casein, some of which had been reprecipitated even ten times with acetic acid, a content of sulphur ranging from 0-619 per cent. to 0°775 per cent.§ Later, Hammarsten|| analyzed four other preparations of casein and each by six distinct methods. Omitting two or three results, which were altogether too low on account of inaccuracies in the method, Ham- marsten found in these different preparations of casein, as a result of twenty-nine distinct determinations by five different methods, 0-798 per cent. as maximum, 0°726 per cent. as minimum, or 0°758 per cent. as the average, content of sulphur. Taking, however, the results obtained by what Hammarsten considers as the more correct methods the average content of sulphur is raised to 0°77-0°78 per cent. In no case did Hammarsten obtain results in any way con- firmatory of Danilewsky’s views. Hammarsten further made a large number of phosphorus determinations, and these as well as the results obtained for carbon and nitrogen showed too little variation to war- rant the idea of a mixture of two bodies of unlike composition. While therefore, Hammarsten’s results would seem to point conclu- sively to the unit-like nature of casein, we have, however, made quite a number of different preparations of the substance, both from fresh milk and from skimmed milk, with the idea of obtaining confirmatory data, with which to make direct comparisons between the composi- tion of casein and its primary cleavage products. Preparation and composition of Casein. The casein was precipitated in some cases by acetic and in others by hydrochloric acid. In both cases the acid used was very dilute, *See Gmelin-Krauts’ Handbuch der Organische Chemie, Band iv, Abtheilung, iii, 1870, p. 2254, + H. Ritthausen und R. Pott, Untersuchungen iiber Verbindungen der Kiweisskorper mit Kupferoxyd. Journal fiir prakt. Chemie, 1873, Band vii, p. 361. ¢ Annalen der Chem, u. Pharmacie, Band exxxiii, p. 185. . § Zeitschrift fiir physiologische Chemie, Pand vii, p. 259. || Ueber den Gehalt des caseins an Schwefel und iiber die Bestimmung des Schwefels in Proteinsubstanzen. Zeitschrift fiir physiologische chemie, Band ix, p. 273. 368 Chittenden and Painter— Casein and its the hydrochloric acid being 0:2 per cent. In dissolving the casein for re-precipitation, a very dilute solution of ammonium hydrox- ide was employed; in fact so dilute as to consist of hardly more than water with a trace of ammonia. We used ammonia, in preference to sodium or potassium hydroxide, as this alkali would seem less liable to induce any alteration in the content of sulphur. Further, in dis- solving the casein in ammonia, the solution at no time became more than very faintly, if at all alkaline; usually being hardly more than neutral to test papers. The general method of procedure was to dilute fresh cow’s milk with about four volumes of water (skimmed milk diluted considerably less) and then to precipitate the casein with either hydrochloric or acetic acids, adding the precipitant cautiously, until complete precipi- tation was obtained. The precipitate was then washed as completely as possible with large quantities of water, both by decantation, tritura- tion with water ina mortar and on a cloth filter. The casein was then dissolved in the ammonia water, filtered through paper and reprecipitated, each time being thoroughly washed with water. In the portion used for analysis, the final precipitate was further washed with alcohol and ether and lastly soaked in a mixture of alcohol and ether for the more complete removal of any fat. The preparations were then dried in the air and lastly on a water bath ‘ata gentle heat. When dry, they were powdered and extracted with boiling ether in a fat extractor for several hours, to insure complete, freedom from fat. Ultimately, the products for analysis were dried at 105° C. in vacuo until of constant weight. In all, seven preparations of casein were made for analysis, as follows: No. I. From fresh milk, precipitated twice with hydrochloric acid. «* TI. From fresh milk, precipitated twice with acetic acid. «* JII, From skimmed milk, precipitated three times with acetic acid. «* TV. A portion of No. III, precipitated a fourth time with acetic acid. ‘© VV, From skimmed milk, precipitated three times with hydrochloric acid. « VI, | rgee-0| ar Bae ee ie at |e oF ae ee BGT | B-G9L | 8-08] 9-99] ~-- | > | | o> | OS8F-0 | THT Serre eae ae | Oct Cue ee sz | sree |oo=- | =2> | gua | gon9-0 | go-e | 188-0 | 6678-0 | TI calle hae ea aR 5 ee ee tall eke a ie < moe ees yom | BLE | S86L-0 | GO-2 C098-0 | FOLP-0 I Fe “TUBIS . mRIs “cur Welles : | : | mes x |.mes | ¢ |ona+Hou| % |"onut+Hon | % |lemssug) “Zt | °°] 4 Soe | 2 ae } spasn | gg ‘ysy | os | g TIM WoIsNy g yg uoisny N fe) H a0uR}s . UsV O°H Joye “O*g*3jq 1o}Ze 'OSRT ‘punoy J *99 -qng ‘T ‘(ON NIGSVO dO SISATVNY 00.00F 68-0 88-0 371 98-66 88-0 94-0 08-ST T0-4 61-&¢ ‘ODBIOA WV 82-0 eee PL-0 GL ST 98-CT sae 90-2 LoS OHAnwumo ‘aounjsqns aatf-yso ayy fo uoyprsodwmos abpjUa1aq Primary Cleavage Products. 9¢-0) 89-0 2B ‘qsV 0&00-0 f&00-0 “punoy ‘meas d ysy ot TUBS SONN+HOW | 2 YA worsny Joye +O% gS Lb §L-0 6180-0 oe es te LUST "7 Sella eo 99-CT. UVtEN ta} SONM+HOH| 2 S | QI uolsng N 1aye "OSLg GLP 6:6P G-9Sh 6-8Eh, 9-06 6-16 cerca eimsselg | ‘ZL “‘puuoy N ‘II ‘ON NIBSVD dO SISATVNY PEES-0 9866-0 20PL-0 9868-0 S0PL-0 9869-0 BPSE-0 8698-0 SE6P-0 £898-0 "meas ‘pesu eourys “Qugs Chittenden and Painter— Casein and its 372 GPT ré-T 1900-0 £900-0 88:0 00.001 78-1é 68-0 68-0 66-9T 60-4, 08 8G ‘OSBIOAY “wuUBIs “ONN+HO™ IM WOIsny Joyye “OF q Sq 68-0 88-0 * aS” (tony i nC ne 7 . OY a ee ae ars eons > aif O Roy Sink: TF : 7h Vie d 68-0 SF sie! me Ve ces S$ ath F0-9L C6-CT rie * a N Se icy 4 ae P0-2L, FI-h H Bode: feo et 0&§-¢ T§-6¢ @) ‘aoUDysgns aatf-Yysy ay? fo WO1psO0dumod aboyUad/aT ae ae Pipe a |, ee cea (a So 8. eee 06c7-0 IITA Danian cages Leys ea eg Cee |e te a ea CésP-0 | ITA ae ae ay Peas eae te eS |b ees | co 99F¢-0 | TA 0¢80-0 yess eal Fedcontl|Rcostemman lack 2 er || noes ett A donee! 9979-0 | A Sa eS l= Jereteyee WN OTE Ih [ESI] Sm Sees = Sel ae 9896-0 AT eer a VL-SL | FLEL | 0-16 | §-87 | ~~ BS at Pee, hee 99°E-0 | III ese : ees ree ~~" | "=" | 28-68 | S82-0 ¥6-9 CLES-0 | SO8E-0 | II 4 Gey yee Urs “~~~ | ~~~ | 8&-6G | SSPL-0 , PO-L | L&Ps-0 | $988-0 | I arden es} “CU TOs alle ‘ ; | Beene esi | “ON + HOM % jeinsseig| “f a2: p Rane 2 ane ‘pesn | ‘ON YA WOIsNy N 9 H gry | 900848 Joye 'OQLg Oo oe “ans "punoy N ‘TIT ‘ON NIGSVOD HO SISATVNY wee Dill seis. < 00-001 ; 373 66.16 aes 48:0 88-0 48-0 a Cale eae 68-0 ao is 8-0 64:0 ie 48>9T fake :™’. aes a oe 86-ST LL-Gt ape 90-4 sal i ie. ou lat et.) 60-2 TT-h 68-89 eae eats ae os wae) 0&-&¢ 67-&S "98RI10A ¥ ‘gounysqns aatf-ysp ay) fo uoryrsodwmos abnzuadag OMAMMO 92:0 | 600-0-| -7"~ oes aioe | peice eRe rane oom eae, Alki) 48-0 | #8000) ----} poral egw seacoa) epcheal lanect | eaten Pageccen cme espe isc) Poke Ee ce nati Maro ices a. ea ek ila ne oe (ee lo vare Esere araeeee sensi gue) | TTT. se-yibeece eral netoue Alen Se Pee ck [acid |e? ena [ree Gera Mociee Fem Pinta = aur barn encase FON RbGOT Wes ue ees eee shee oll oo ee em OOO aoe La ens ee eee Sin Mees nee ale soca CA oceans (ape Nicaea! cite? Rte (pees ore (oh ay LN Bree |teece = [ene= eo se ee se ===: anon L8-OE | -OsF02 79:08 | TPG:)) => | 8) BOOP OAT ee ee oe Ser SSS 00ST). OPOk 1-706 | SBP) oe le Cr Be 03) ALE Sse) seem 4 | Seine hee = | =a.) bees seen | ossse [mess |---| $8.8¢ | 0818-0 | 86-9 | 2082-0 | OOLF-0 | IT : Pearl ete oben een seas 1-2 alia cere | cme | osos 4 "= | 80.8¢ | LL89-01 0-2 | 0802-0 | 9428-0 |. 1 t Primary Cleavage Products. “6 ube % | bale rae Pesos % pay % ee ON + HOM d| ‘L mes ‘pesn | , ON e0uR}s N : % a ue mon |. @ | Uy uoleny | Np |2——— = |g H eal soe +08g°ST rye *OSkT ‘punoy N “00 O'H | -qng | 374 00-001 86-66 78-0 GL-0 GL-GT TT+& ne AGEG ‘OSRIOAY ia 80-4 FI-8g CE-E¢ a0UDISQns datf-ysp ay? fo UOYsOdwmod abn WAdILAT Chittenden and Painter— Casein and its 98-0 8-0 “UsV 9700-0 CP00-0 "meds “panos qS¥V &8-0 FL-0 CTL TS-€¢ “URIs “ONM+ HOM TL WOIsny oye +O*g*syw “me13 “ON + HOW YIM UOIsNy raqje 'OQeg 2B N -6T P81 anti ‘0. ainsselg | “ “‘punoy N ‘A ‘ON NIQSVO WO SISATVYNY 096S-0 | 60-2 ! i 0h) 2) GLGT-T | 60-4 9TS-0 6L6¢.0 0fé8-0 OfE8-0 8688-0 699§-0 F80E-0 c&19-0 Of6T-0 E188-0 | OL6E-0 URIS “punoy | *00 fd xe ‘mes "meIs “pesn “punoy | l Otn | souls “qus ‘ON 00-001 16-16 oe ac ; 48-0 ats 375 Primary Cleavage Products. 60-1 60-1 | 9700-0 | 700-0 | 48-0 80-91 GO GG-EG ‘OSRIOA VY 66-0 18-0 OT-9T 40-91 ‘20UDISQns aatf-ysp ayz fo UoYpsOdwod abnyUddLaq C8-0 Bee (e820 C610-0 "mes “ON + HOM ali [gla uoIsny lye +O q°SW | 00-2 66-¢ OMAnwHto “mRIs “ONM + HOM WIM woisny Jayye "OSeg ‘TA ‘ON NIGSVO dO SISAIVNY N “wUr aInssolg “‘punoj N 4-67 8-LP ¥-06 9-61 te aie iL 69-6¢ | OFOL-0 9L-6G | cO&L-0 “punoj 4, “TUBIS 9 | %9 F6c0-0 LESs-0 URLS *‘punojy O°H 8667-0 | X 9EF-0 | XI 0269-0 6169-0 0ce9-0 | TA 6109-0 | A OF9E-0 | AT 0egs-0 | TIL Sv9s-0 IL PLLE-0 | I “meIs ‘posn 9ourys -qug ‘ON Chittenden and Painter— Casein and its 376 00-001 Sere tiie want 7 E |) aR A Pot est See er ey O 88-0 ce MR File i as atanceactics ed Mea ga cans et OP ce 2 d SosOF? 2 Means 88:0 Phe eee eee ir aa cieire, Dm aeees Bae 8 Geen n Maser iee Oe aa L0-91 0 a As De ORI N OD ok Speirs, Ueet. ats eva ae PLL LOL H GEG TERT gas aM Bay Ome Sioa eed pets 6F-8S 89-8¢ ©) , ‘OSUIOA VW ‘aounjsqns daauf-ysp ay? fo uorrsodmos abpzuaosag | | g8-1/2900-0)""= | <7 7-7 pause |) ee ae as wees, Be pei dS URS Salle eo eae cea SIIF-0 TILA 18-1/9900-0|""={° 77 >>-> ere || ee ee Peaches cs th pea | a eal a ets Gaon habia 6S0F-0 ILA seni es a9 Oia cOLCOsOuiehla | ee aes Oe Maha ie Sinker ira © |e pe ate nr Saw |i oar 8829-0 |LA OIE ant en gs 98-0 62F0-0 om Uwe t eo caate olise tal as eu ee ee ee ae @8L9-0 1A “SCRCC Fe Re arias PPE a Ree 28-91 | 9-09% | G6L | @@g |---| a> ray pe a 0868-0 |AT Feo SS Sno ie oem EE es GiGi) eeeaun alk Os0Gh lakers Wanner es a= oer ceLy-0 TH eee ae glo nt ee ce Sak sell Capes "7" | -7"" | gg.ge] oLgt-0 | 80-4 | 68¢¢-0 | e90F-0 [II AS SR i la aa rere CO a “--- | === | 9g.g¢ | 0926-0 | 86-9 | T9Te-0 | Te0¢-0 {IT ‘ URIS “TRIS Beoneet Hayes ; re A 2 ; g |e |g ltona+Hou) % |“oNy+Hou| % Jomssag] “| °°] ee oe Hees i aS ‘ysy| Soe | ad | ya uomny | g | wim uosny | N passe | bye Baa eae | 3 Hina (eaeenace has ey JOYR +O* G23 Joye 'OSeg “punoy N 00 O°H ke 498 TIA ‘ON NIGSVO HO SISATIVNY Primary Cleavage Products. 377 phorus, the alkaline fluid was acidified with nitric acid, evaporated to dryness, the residue dissolved in a little water acidified with nitric acid, filtered, and the phosphoric acid precipitated in the usual man- ner with ammonium molybdate. After standing 24 hours at 40° C, this precipitate was filtered off, dissolved in ammonium hydroxide and the phosphoric acid re-precipitated as ammonio magnesium phos- phate and ultimately weighed as magnesium pyrophosphate. The accompanying tables show the results of the analyses of the different samples of casein, Comparing now the average composition of these different prepa- rations of casein, it is to be seen that they all show a very close _ agreement throughout. Thus the percentage of phosphorus in the seven preparations varies only from 0°84 to 0°89, sulphur from 0°75 to 0°89, nitrogen from 15°75 to 16°08, hydrogen from 7:01 to 7:11 and carbon from 53°19 to 53°53 ; or leaving out one preparation which for some reason showed a high content of carbon, from 53°19 to 53°39 per cent. The results therefore show a constancy in composition fully as marked as observed by Hammarsten and thus tend to confirm the latter in the view that casein is a single body of definite com- position. | Comparing our results collectively, with those obtained by Ham- marsten (see table showing average composition), we find a fairly close agreement throughout, although minor differences are to be observed. First, all of our preparations show a content of carbon somewhat higher than found by Hammarsten. The latter investiga- tor found the carbon in his preparations to vary from 52°78 to 53:09 per cent., while in all of our preparations, the content of carbon cal- culated to the ash-free substance is above 53 per cent. The possi- bility of our preparations still containing some fat was rendered improbable by the thorough treatment with ether which they had received, and further by the fact that the nitrogen in our preparations was also somewhat higher than found by Hammarsten. One of the preparations, however, with a high content of carbon, was extracted _again for several hours with boiling ether, but on analysis the con- tent of carbon was found unchanged. The content of phosphorus agrees exactly with Hammarsten’s results, while the sulphur is, on an average, 0°] per cent. higher. There is nothing in the content of sul- phur, therefore, to even suggest confirmation of Danilewsky’s views. ~The amount of ash in our preparations was somewhat larger than found by Hammarsten and further, there is no especial connection to be seen between the content of ash and the precipitant used; the Trans. Conn. Acap.. Vou. VII. 48 Nov., 1886. Chittenden and Painter— Casein and its 378 49s neyyyny 04 Sup 1000 V 86-0 BL-2B | 80-82 c8.0 18-0 6L-0 68-0 9.e] 16-1 CO.) LO-2, 96-8S 08-88 peyased S|. st aes Coes SON -1B UU FY OsBIOAY JO ADRIOAV TOH Wis poyeqyidioaid Soul} F ITA ON TOH WI poyeyidro -o1d sow Pp TA ON ‘gz ‘d ‘eng ‘ormayosarqy, MJ JYoWogseyep oeg = *S}[NseI oaIy} JO osB1OAv ‘UIAsBO Jo punoduos toddoo oy jo stshyeue mousy uasneyyyy Aq poyenopen + "69g ‘d ‘Ha pueg ‘ormayo oypstsoorsAyd any 4yliqos}leZ 90g » OH YIM poyeyidro -e1d saumly ¢ “A ON ‘HOOO"HO payeqidyo yy -aid sow) 7 “AT ‘ON ‘HOOD *HO WLM poreqidto -o1d samy ¢ ‘TIT “ON ‘SNIGSVO AHL dO NOWMISOdWOD ADVURAY AHL YNIMOHS CM ES A 6-0 ‘HOOO-*HO (DIM pele} -1diooud = 90TMy TI ON OH TIM poqey -1dideid 901.4 ‘TON ©) Primary Cleavage Products. 379 amount in the acetic acid precipitate being fully as large as in the casein precipitated by hydrochloric acid. Compared with Ritthausen’s results (see table showing average composition), obtained by analysis of the copper compound of casein, the percentage of carbon comes very much too high. It is question- able, however, how close a comparison should be drawn between indirect results obtained by analysis of a metallic compound of casein and those obtained by analysis of casein itself. _ In conclusion then, we must affirm that our results accord closely with those of Hammarsten’s, while the two together make it very improbable that in casein we have to do with a substance composed of two bodies of unlike composition. Digestion of Casein and Formation of Caseoses. In the digestion of casein with pepsin-hydrochloric acid, the casein was prepared by precipitation and reprecipitation with acetic acid, and rendered as pure as possible by thorough washing with water. While still moist it was placed in 0°4 per cent. hydrochloric acid, as preliminary to its treatment with pepsin. Pure pepsin solution, free from peptone and albumose, was prepared from the mucous mem- brane of pig’s stomachs by the method already described.* Digestion Pe = 4a-G GA-0 GL-0 FL-0 oe “2 = a) ; 84°91 pe | =. BL-ST 89-CT Ae oan eh 86-9 mye << aos abe 00-2 %9 H 65-1 aa =? ies Aer 6G- TS 99-1¢ O “Q5B10A V ‘aounysqns aatf-ysp ay, fo worpsodwuos abnpUalag 96-01 8960-0 | - = i, me de See lo Whe RCP! Sg Seis oe oe = 6876-0 ITA as el bate ee 89-0 6660-0 -— | 2S aoe ee ok Satria See ae 0609-0 IA fe on | aoe 49:0 ¥660-0 Se al Nee tree < ee Oe fa hae oo F609-0 A Aa —s rae pa ak LI-PL | 6-992 #80 ebay A). es a Se ea? eS OScE-0 AI Se ce ee yar eecn 80-60 PSer ah ch cine eee ah eee. pe eS See ee oe = x =e ““"" | “""" | F8.9F'| 2829-0 | 46-9 | 2966-0 cOOF-0 II | ae Sle a < ape fa ae oe "7" |.7""" | 88.97 | 089-0 | 66-9 | 1986-0 LOOF-0 I uURIs - nmwas d boa ) 2 a) aden “TURIs TURLD U > pane] font a Ne e ae ih is pauoy ze ‘punoy | — ‘pasn ‘ON ca ‘ raft rosea : “puno} N *09 Hs. OH L aoUBISGUS ‘VW aSOUSVOOUR LAAT AO SISATIVNY Primary Cleavage Products. 387 acetic acid and potassium ferrocyanide, but later on, these reagents gave a heavy precipitate, showing plainly that the substance was dissolving. The entire precipitate was thereupon dissolved in very dilute sodium carbonate, the solution made exactly neutral with hydrochloric acid and then dialyzed. After remaining in the dial- yzers for nearly a week the fluid was removed, filtered from some heterocaseose which had separated, evaporated to a syrup on the water-bath and precipitated with alcohol. This precipitate was re-dissolved in water, the solution made exactly neutral to test papers and again dialyzed. From this solution, the caseose was finally precipitated by alcohol, after suitable concentration of the fiuid, washed with alcohol and ether and dried at 105° C, in vacuo. The final solution, prior to precipitation by alcohol, was perfectly neutral and quite clear, showing no evidence of the presence of any heterocaseose. The composition of the substance is shown in the accompanying table. Digestion B. In this digestion, 750 grams of freshly prepared casein were mixed with 4 litres of 0-4 per cent. hydrochloric acid, the mixture warmed at 45° C., and then 800 c.c. of pure pepsin solution added. The mix- ture was warmed at the above temperature for one hour and a half, then neutralized and filtered, and the clear filtrate saturated with sodium chloride. This precipitate, as in the preceding digestion, was washed thoroughly with saturated salt solution, then succes- sively extracted with 10 and 5 per cent. salt solution and finally with water, leaving a small residue of dyscaseose soluble only in 0-2 per cent. hydrochloric acid. The united filtrates, containing proto and heterocaseose, were again precipitated with salt and then _ treated as described under protocaseose B. B. Protocaseose. The protocaseose formed in this digestion and twice precipitated by salt, was dissolved again in water, filtered through paper and then dialyzed until no chlorine reaction could be obtained with ‘silver nitrate. Quite a little heterocaseose separated from the solu- tion during dialysis, which was removed by filtration. The fluid was then concentrated, the substance precipitated by alcohol, again dissolved in water and dialyzed. This time, as no heterocaseose ‘separated from the fluid, the solution was concentrated, precipitated Chittenden and Painter— Casein and its 388 00-001 87-86 es ee ee oes pe Ever O 06-0 06-0 ra aed “Pee tel S) : G9-ST cs ta 69-ST T9-ST ae Lor% N 90-4 iia 7 at i. a4 FOL 60-2 H 16-29 redge Sees a 88:6¢ 6-66 a) € ‘ODBIOA VY ‘goupysqns aaif-yso ay, fo worpysodmos abnzualod ; - ae | | | LL-0 6S: 1 OBB0 Oca | = ee See ener meade hes: ~ 308s See cee ae eo Sota eh Se | emai | Bette aera | ~gc9-0 | IA | i | iS ae ean we a cL-0 | 0080-0 ae i a | eae tte Sea tal eerie =a le Oy 6696-0 | GA nen Sei me We yey ey me Sees Ie 99-2 ASLED OF a a eS > See | Pavol orale ao“ e a ee | 6698-0 A Ee ie et Seas Soony Alt ae Sol 7S Oey Is] SOGu: leGe et Gx0dal en la” e Sian (a ae eR epee! fee ee & PRES ccut ay! eer Ss arn ve STi es SL ors ia, SB: 09Le is O-60 a d-89 ie ae se eee ee | 8cL7-0 | TI Lie Se " | 88-8F | Cy9L-0 | TS-9 FOLE-0 | g9¢F-0 | TT aie oe es. 3 eating | earn =n] SME te gl eicoee Spe | 68.6 || 2689-0 [299-9 | 8966-0 | FP8s-0 | I | | | : oa es | -- b ‘aes eobae ues j POE A emai 30 a) | GP, ; - Agel 4 wes vy (eaNsSalg Th a med Pata | wes wes Soe as ON nae -qng jo % eG a os “punoy 4 & “punos A | “punoj ‘pasn ‘ON Meee Gey Ogee yl Hey 509 O*H eauRysqng Ta}Fe § Jaqye *OS¥a rosed punoy N | ‘l q ASOUSVOOLOUd JO SISATVNY 389 Primary Cleavage Products. 18:6 UsV 00-001 At.86 Kah GeS ae oie awe Se O 06-0 68-0 16-0 Pepe eS =e) a 8 67-91 a pee 16-91 LT-9T . Weal N T0-£ or eos pe asc 00-4 Ok H 81-69 = oo. ee ea 9F-69 The ) ‘OSRIOAV ‘aounysqns aatf-ysp ayz fo Uoipsodmos abnyuaovag GRO Ora lae ee Oe a. Dene, ts ke Fy eel Eoemeeaeen see rae i Sa eae OF6E-0 TIA eer era 080-0 Oe Nee ie aes: ae ae yl ee ce ae eae 0109-0 | TA weeeee €8-0 1980-0 mie ilies ae Skee ees eee keane geet a ete ante Fe00-0. FA ~- 220: recs Saar (LGD SPL | GOES | F-Oly | e001 wire ene ee lee lee te aes OT0S-0 | AT ee agek are 89-FT | &-89L | 9-6 | 0-99 | ~~~” eo ie Sa Tesy-0 | TIT E eae wee Ec eae So Sey lear ee sige = ae eS ay ea 0819-0 18-9 1006-0 0688-0 II ae oar ssteee Se Soe las 2 ee | eee) 68-9 1686-0 P80G-0 it mUeIs J shou Oe f , ee | % |Yonm+wom | % femssoza| zt | °° | x | eis eae es pee ea qs downy | NN 0, | Puno} *0O | H | PUNO} OTH | gonenaqn 3 ey qaqye 'OQeg \ *punoy N asqns ‘6 G UOASVOOLOUd AO SISATVNY 390 Chittenden and Painter— Casein and its with alcohol and the substance finally dried at 105° C. in vacuo (Protocaseose B 1). Its composition is shown in the accompanying table. Such portion of the protocaseose as was not precipitated the second time by salt alone, was precipitated by a little acetic acid. At first, the precipitated substance was insoluble in water (no reac- tion with acetic acid and potassium ferrocyanide), but after being washed with salt solution until the acid reaction had nearly disap- peared, it then dissolved quite appreciably in water, as evidenced by the reaction with acetic acid and potassium ferrocyanide. The bulk of the precipitate, after being washed, was dissolved in a little very dilute sodium carbonate, the solution made exactly neutral with hydrochloric acid and then dialyzed until all chlorine was removed from the solution. The caseose, after concentration of the solution, © was precipitated by alcohol, washed with alcohol and ether and finally dried at 105° C. iz vacuo. The composition of the substance (Protocaseose B 2) is shown in the accompanying table. B. Deuterocaseose. Deuterocaseose was separated from the filtrate from the first sodium chloride precipitate, in the same manner as in the preceding digestion. Like A. deuterocaseose, this precipitate was at first in- soluble in salt solution and in water, but after being washed for some time with saturated salt solution, it was found to be gradually dissolved, as shown by the rapid disappearance of the precipitate and the pronounced reaction with acetic acid and potassium ferrocy- anide in the wash-fluid. Evidently the acid is easily removed from the compound by simple washing and when that has been effected, the substance becomes soluble, or else the acid compound is more insoluble in water containing a little free acid, than in water or salt solution alone. Hydrochloric acid and acetic acid seem to act alike. The substance is readily soluble in dilute sodium car- bonate and is not precipitated by neutralization, but is quickly thrown down by a slight excess of hydrochloric or acetic acid. This compound was not analyzed, but was used in studying the reactions to be described later. Digestion C. In both of the preceding digestions, the products formed resulted from the action of an exceedingly vigorous pepsin mixture. In the - — i aal Primary Cleavage Products. 391 present case, the pepsin solution employed was much weaker and the pepsin-casein mixture was warmed at 45° C. for several days instead of hours. 750 grams of casein were employed and 4 litres of 0°4 per cent. hydrochloric acid, to which was added a reasonable amount of pure pepsin solution. About an hour after the addition of the latter the mass began to gelatinize, and at the end of 18 hours the whole mix- ture was a perfectly stiff jelly. Thereupon, 2 litres more of 0:4 per cent. acid were added together with a little pepsin. The mixture was then kept at 40-45° C. for three days longer. Quite a large residue of undigested matter remained, semi-gelatinous and soluble only in alkalies. The mixture was then neutralized and filtered. The filtrate contained considerable caseose, as evidenced by the heavy precipitate obtained on saturating a portion with sodium chloride. In the preceding digestions, the caseoses were separated directly from the dilute solutions, without previous concentration, thereby avoiding any possible change due to the action of heat. In the pre- sent case, however, the perfectly neutral solution was evaporated to a small volume and then all of the caseoses were directly precipitated by saturating the fluid with ammonium sulphate. The precipitate produced was exceedingly gummy, but was washed as thoroughly as possible by trituration with a saturated solution of ammonium sulphate. The caseoses were then dissolved in water, the solution filtered from the small residue of insoluble matter and saturated with sodium chloride. The protocaseose so separated was freed from heterocaseose, etc. by repeated precipitation and dialysis. Carbon and hydrogen were determined in a portion of the dried ‘substance (Protocaseose © 1) with the following results : I. 05107 gram substance gave 0°3101 gram H,O=6°74 per cent. H and 0-9386 gram CO,=50°'11 per cent C. II. 0°4088 gram gave 0:0194 gram ash = 4°80 per cent. The ash-free substance would, therefore, contain 52°64 per cent. of carbon and 7:08 per cent: of hydrogen. In the filtrate from the first salt precipitate, the protocaseose re- maining was precipitated by the addition of a little salt-saturated “acetic acid. The precipitate after being washed was dissolved in a little dilute sodium carbonate, the solution neutralized and dialyzed. ' As no heterocaseose separated from the solution, it was concen- trated and then precipitated by alcohol. After purification by re- solution, dialysis, etc., it was dried at 105° C. tz vacuo and analyzed with the results shown in the accompanying table (Protocaseose C 2), Chittenden and Painter— Casein and its 392 00-001 st.te ae i sa ie aa ) 4 64-0 6L-0 ss as ea, ah >) $891 i 66-ST LL ST = rs N GT +k 23 4- ae a Co 8T-h H GhTG Fa ae ome a Be _ 18-Tg @) "ODR10A V ‘gounjsqns aatf-ysp ay, fo Uuorprsodumos abozUdasag oe | 6600-0. (9-5 | eS a: = ae pea ee ee aes es Re aa: PIEP-0 ITA peas | S200: 03 a | ae an eel ee es ee See | eas Baal 8907-0 IA ae rad Bt) CTg0-0 =. aaa ee we ace 2 oie eee i Ps 8009-0 A lo ae ae a Se OF-FT| 8-994 | 1-08 | GER | | lee < Meg 08cé-0 | AI eels gS aig ay O8-FL) F6SL | 66T Bee |e iS Pee he C6EE-0 il ~sjs | Poa Sa ae ell Sa ea et ee] ee ae 8°6L-0 cP-9 8oFs-0 CECF-0 Il lie ee liga 4) Ea ale | We ete oe | LO OP 80L-0 1¢-9 8696-0 O80F-0 I 3 | “Wd 06 | *2), “a . “CRIS ° ag NS 2 ers ie domencenen|) 8 S| et Sel 2 ‘wes 2 ‘wes asin fas qsV ra 4S Ygta GoIsny N | .O | ‘punoy*o9 | H | ‘punoy O*H te hee ele aay rae ‘ose ‘punoj N palm ‘60 ASOUSVOOLOUd HO SISAIVNY Primary Cleavage Products. 393 Deuterocaseose was then separated from the filtrate from the acetic acid precipitate, by saturation of the fluid with ammonium sulphate, ‘as recommended by Neumeister* for deuteroalbumose. It was then purified by dialysis, etc., and its reactions carefully studied. So far as we could see, it differed from deuterocaseose A and B in two respects only, but these points showed so marked a difference it was plainly evident that the deuterocaseose separated by ammo- nium sulphate was quite different from deuterocaseose A, separated by acetic acid. Thus deutero C was not precipitated at all in an aqueous solution by acetic acid, nor by acetic acid and potassium _ferrocyanide, neither was its aqueous solution precipitated by cupric sulphate. The significance of these points of difference will be dis- cussed later on. After studying the reactions, there was not enough _ substance remaining for analysis. 5 Digestion D. In this digestion, 2 kilos of freshly prepared casein were used, together with 6 litres of 0:4 per cent. hydrochloric acid and an appro- priate quantity of strong pepsin solution. The mixture was warmed at 45° C. for five hours, then neutralized and filtered from the semi- gelatinous residue. D. Protocaseose. The neutral fluid was concentrated to about 14 litres and then filtered from the slight flocculent precipitate which had formed. Saturation of the fluid with sodium chloride gave an exceedingly heavy precipitate, somewhat more gummy than usual. The entire ~ fluid, however, was only partially saturated with salt, with a view to see whether the precipitate produced in this manner would agree wholly with the precipitate produced on complete saturation. Thus a fractional precipitation was made, in which the first fraction represents that portion of the caseose precipitated by about two- thirds saturation of the fluid with salt. This precipitate was there- fore filtered off, washed as usual, dissolved in water and re-precipi- tated with salt. As dyscaseose was generally found only in traces, ‘the precipitate, after being washed, was dissolved at once in water and dialyzed until all chlorine was removed from the solution. On opening the dialyzing tubes, quite a large quantity of heterocaseose was found adherent to the sides of the paper. The clear solution of * See Zeitschrift fiir Biologie, Band xxiii, p. 381. » Trans. Conn. Acap., VoL. VII. 50 Noy., 1886. 394 00-00T 00-66 0 Bt hag 88-0 GO.9T ADek &6-€9 Pa ‘aSB10A VW oLk 60-S ‘aounjpsqns aatf-ysp ayy fo worpsodwuod abhnpuasiag Te-L ¥8-6S oHAznnmoe > ae. 0 \89-0 LPTO-0 ee Boas Bal ee |) Seteshs liner lee eae SPR Men oelce: |e 4 aol Po cae. kes.er AP Ome ene 10-0 69-0 6710-0 siete ra aly ~ |b i leat al ie Poa Soe lige | | Cae ie et met eae hae (P109-0 aks eee eae = = 4==> OM Or | AG00-Oel 2 ota la ey ae Soya Aake els Wer yee lene el ae =~ | -""" (6707-0) XI ee eee ---- ---> |TL-8/89T0-0) ~~ | pe Poh SA PSmeaie colic (ere ey ama det ae Teer-0 TMA oe foo ---- -"2> S\F:812910-0|-7- | ey eo eapaian (AS = a) | ie Pome! ees SF On ae, ISL ee Ola EA eS Se oer he gaa esse 10 F180-0 Seer hector bec let eo eek en (Os a il pee ea Nerod|(e 20 ea < -=-= Seen ay erase ME) SFk0-0 Soe es ee tie eal | eee! 1 ee PO ee peeeeieee |) ==> ---- See leslie |= tks OG-CT8-G9L'G-8T|\S-8F] | | -- | --*- l¢x98-0] AT 5 ee a Sep Ve ghee conc) ere ee ee OF-GTIS-COLP-STIE6S| “~7= | ~~" | > | 777 18eSP-O TIT So jroo) a=5-2 ---- ane eel S's tl io Rees ---- |----_| ~~ | “> 1@6.T@ GPLL-0 98-9'8086-0 G90F-0 IT See ER | = 22-2 Sil eee eel ae aa ae “== |==-= | => | =" 198.76 0686-0 96-9 8928-0 1128-0 | Bb | “mes ‘gourys, ‘ured | | “me a oe | ips g “wes ‘st 10 leony + HOM Hans 30 nye | |e | “oNN+HON | % ens fee go ae |g eam ae d Our) = | wr uoisng | % ‘yse | oy Moly |YSV | panos! g | yyrm aoisny | N eid @) eee al eee aounys ON -youpep) TY layye 'OQe | ‘00 | O°H | -qug oye di Joye ‘O° g*3W | Jod /Ord*3i4 | ‘puno) N | | ‘TQ GSOUSVOOLOUg AO SISATVNY 395 Primary Cleavage Products. 00.001 9-83 40.1 98-81 OT +k VATAS ‘OSBIOAV OT 66-0 C9 &e-9 Usv “cIBIS ‘panos Ysy FO-T 66-0 % “ON + HOW g “TRIS TBIM WOISHy ey ace Shae eed g F8-C1 68-G1 eer ay N ane reall OTL Olek H alae ts 88-6G 61-6 @) ‘a0UDISQNS aatf-ysy ayz? fo UOLpsOdUod dbp] WAdAT Bees si| ses ---- | ---- | ------ eae | ie ak 98TF-0 IIA a ei We IY NAN i Pe ee Fae | ec OGGP-0 TIA eee ye rises |b Sar 9 |e core een cl (ere a 0°89-0 IA Fame aae scene | erie Rae a= ee a 6619-0 A U2) ait lik GX SI 2) i fre al Se ae Ate ecm 969E-0 AI JASE) Pa SEY AS FSC) a faa tga | SR aa =r Re ee 68LP-0 Iii SNe ee ew OS Gr | ENSLEO0) 3 S005) SOTeO TTS8-0 Il BEE ae “-- | "<7" | Sper] L09L-0 | 29-9 | TTSs-0 | 96TF-0 I “au ie) i 0°0 “UeIS “TIBI “med einssoig | ‘ZL % ‘punoj % “punoy “posn ‘ON : 0 cop. «|| BH | otH | coumsqng “‘puno} N Joye "OSA °8 @ BSOUSVO \y OLONT AO SISATVNY 396 Chittenden and Painter— Casein and its protocaseose was concentrated, precipitated with alcohol, the precipi- tate dissolved in water, again precipitated with salt in substance, the precipitate dialyzed, this time without showing any heterocaseose, and finally precipitated with alcohol, washed with alcohol and ether and dried at 105° ©. in vacuo (Protocaseose D 1). The product was analyzed with the results shown in the preceding table. On adding more sodium chloride to the first filtrate from the above precipitate, thereby completely saturating the solution, a second precipitate of protocaseose was obtained, which was purified in the same manner as the preceding preparation. The only difference noticed while purifying the substance was that, on dialysis, nothing corresponding to heterocaseose separated from the fluid. After final washing with alcohol and ether, the substance was analyzed with the results shown in the accompanying table (Protocaseose D 2), The difference in the results will be discussed later on. The original salt-saturated filtrate was precipitated with a little acetic acid, and the protocaseose so precipitated dissolved in dilute sodium carbonate. The solution was then neutralized and dialyzed in running water until all chlorine was removed. |The substance was then separated by precipitation with alcohol, and ultimately purified as described previously (Protocaseose D 3). The addition of a little — more acetic acid to the acetic acid and salt-saturated filtrate from the above, gave a still further precipitate of caseose ; presumably proto- — caseose with perhaps a trace of deuterocaseose, which was filtered off, washed with saturated salt solution and then freed from acid and purified in the same manner as the preceding preparation (Proto- caseose D 4). The two last products, after being dried at 105° C, in vacuo, were analyzed with the results shown in the following tables. Comparing these two tables, it is seen that the difference in per-_ centage composition of the two ash-free substances is not very great, — and taking into consideration the large percentage of ash, it is proba- ble that the two latter precipitates have approximately the same composition. D. Deuterocaseose. The filtrate, from which certainly all protocaseose had been re- moved by acetic acid, and in fact nearly everything precipitable by acid from the salt-saturated fluid, was treated with ammonium sulphate in substance. A gummy precipitate resulted, which natu- rally enclosed considerable salt, and which for purification was dis- — 397 Primary Cleavage Products. 96-0 00-1 8 “meas SONI + HOM YIM WOIsny 1aqye 'OSed 00-001 90.T F0-1 80-1 Go oe Tha Ea $ 6T-9F Pr Toe 8T-9L 10-91 oui woe N 81-4 a Be 4 vw sare 9T-h Ihd H GO-6G a sere tk i is & OL-6S 10-6¢ @) *OSVIOAY ‘aoungsqns aatf-ysp ayy fo uorpsoduoa abnzUaLag pe Sen zm ‘aero ag a ea DIC py FRc ee F868:0 IIA ant ape Wal Can Pe egies to ee — eae es PLOP-0 IA §8-2 | 9660-0 | ~~ i. ere ite Oa pat Lae ay 980F-0 A Wa Soo | TOFGT |S SSOk | TSE | BGR lee os aa tee Wee 9cSE-0 AI ced ===> | 68:7E | O90L) | WSE | eRe ol y= os eae = ke G9CE-0 IIl oe ae Ea 5 Fabs TR Lele maa a teictey 7 LOLL-O ¥9-9 6696-0 F8EP-0 II ee TA dee Lan ie dL ‘9 saul EGLey 6611-0 66-9 9TFs-0 SLOF-0 1 é “uu Oo vit : - : % pe % \einsserg| ‘L he % TURIS % “UIeAS ties te nig ey NE anions 9 | ‘puno¢*00 | H | PUN OTH | goueysqng a ; pon ke a a EE ee ‘@ ( ASOUSVOOLON AO SISATVNV Chittenden and Painter— Casein and its 398 TL 6L-¢ UsVv 00:00F 91-91 © LO-L 18-6¢ ‘OUDISQNS aatf-YsD ayy fo uorvyrsodiwuods abnyualag 46-06 Seah 86-0 86-0 §1-9T Sate) LO-k i. 88-69 oa 958104 V £6-0 GLP0-0 ¥6-0 SLP0-0 oHano a med *ONM + HOM qj aoisny qoye TOSe 08-ST 6-992 9-81 PE-T 1-894 9-81 “Wau Do einsselg | *y fe N “‘panoj N TE-0¢ C6-0¢ vA o 8078-0 918¢-0 “mes “punojy 00) TL-9 TL-9 fa Ss “TUR.LS *punoj O°H S0L¥-0 LECP-0 LP09-0 £609-0 L696-0 8I8s-0 CLSP-0 9CTE-0 "med ‘pesn eouRysqug IIA Iil Il ‘ON ‘$ ( ASOUSVOOLOUG AO SISATVNY 399 Primary Cleavage Products. 00-001 66-86 0 ALT 00-91 GO-k 6GkTG “OOBIOA YW 61-1 ST-T 60-91 L6-ST F0-4 vit ‘aounpsqns aauf-yso ay) fo woyrsodumoos abnzualaq 60-0 60-0 Yse jo d 5u1 -gonpep loye d OL-0 69-0 B d TST0-0 a OfT0-0 ‘read *ONM+ HOM YIM WoIsny Joye “OF G3 “900848 -qus Jo “TRIS “yse % YSe | oy} uLody Jo d |*0*a° 3X “mes “ON +HO YA WOTSNy] raqje "OS" LO-k ¥8-1¢ OMTA2nxwO 08-CT|6-992L 86-CT|F-994 0-81 1-67 G-81/8-6¢ *‘punof N ‘d ASOMSVOOUELAAG AO SISATVNY 1699-0 Lv06-0 “punoy £09 0066-0 0808-0 0609-0 ¥§09-0 GGOF-0. GGO0F-0 €80P-0 0609-0 PE09-0 0988-0 oITP-0 FP9E-0 x XI VITA TILA ITA 666P-0 400 Chittenden and Painter— Casein and its solved in water and dialyzed until the greater portion of the salt was removed. The solution was then concentrated and the substance reprecipitated by saturating the neutral solution with ammonium sulphate. This precipitate, after solution in water, was then dialyzed until all of the ammonium sulphate was removed, after which the solution was concentrated and precipitated with alcohol. When dry, the substance gave by analysis the results shown in the accom- pavying table, This body differs from all of the preceding preparations, in that it is not precipitated from an aqueous solution by acetic acid. Neither is it precipitated at all by the addition of salt in substance; but the addition of a little acetic acid to the salt-saturated fluid gives a heavy precipitate which, however, does not represent all of the deutero- caseose, since the filtrate gives an additional precipitate with. ammonium sulphate. Apparently about one-half of the substance is precipitated by acetic acid. Further, the acetic acid precipitate in this case differs from the protocaseose precipitate with acid, in that it is readily and completely soluble in water. This body, therefore, which certainly must represent pure deuterocaseose, shows a close resemblance to the pure deuteroalbumose separated by Neumeister. Like the latter, it does not give any precipitate whatever with cupric sulphate nor with ferric chloride and only the faintest turbidity with acetic acid and potassium ferrocyanide. D. Heterocaseose. In each digestion, evidence was obtained at various points in the _ process of separation, noticeably on dialysis of the first protoalbumose precipitate, of the presence of a body insoluble in water but soluble in dilute sodium chloride solution. The quantity of the sub- stance, however, was in most cases exceedingly small, so much so that nothing more than a few reactions could be tried with it. In the present digestion, however, the amount was somewhat larger, and sufficed for a partial analysis. The substance was obtained as a more or less gummy residue, on dialysis of protoalbumose 1. It was purified by solution in 10 per cent. sodium chloride and separation by dialysis. Like heteroalbumose, the whole of the substance was not now soluble in salt solution, for a portion had apparently been converted into a body resembling dysalbumose, insoluble in salt solution but soluble in 0°2 per cent. hydrochloric acid, a ‘ scarey Reema * Wee ini RT POE, 4 Primary Cleavage Products. 401 Analysis of the dried substance gave the following results : I. 0:-4011 gram substance gave 0°2463 gram H,O=6°82 per cent. H and 0°7434 gram CO.,=50°'54 per cent. C. II. 04287 gram gave 53°6 c.c. N at 18°6° C. and 760°1 mm. pressure =14-70 per cent. N. III. 0:4118 gram gave 0:0256 gram ash =6:21 per cent. The ash-free substance would therefore contain 53°88 per cent. C, 7°27 per cent. H, 15°67 per cent. N. Reactions of the caseoses. Under this head little need be said. The reactions characteristic of the albumose bodies in general will apply here. Certain differ- ences, however, have already appeared in our description of the processes incident to separation of the caseoses. Protocascose, unlike protoalbumose, is precipitated from an aqueous solution by acetic acid. The precipitation, however, is not complete; satura- tion of the acid filtrate with sodium chloride, invariably gives an additional precipitate which is the heavier of the two. Further, long-continued washing of the acid precipitate with water or salt solution appears to partially remove the acid from the caseose body. Protocaseose is likewise precipitated from an aqueous solution by hydrochloric, nitric and sulphuric acids; the precipitate, however, is far less soluble in excess of sulphuric or nitric acid than in excess of the other two acids. In very dilute acids, protocaseose is soluble and is partially precipitated by addition of stronger acid of the same kind; thus the substance is readily soluble in 0°4 per cent. hydrochloric acid, from which solution it is precipitated by the addi- tion of a little concentrated acid, this precipitate dissolving on the addition of more acid. Evidently then, protocaseose as fast as formed by the action of pepsin-hydrochloric acid, would dissolve in the acid gastric juice and not be mixed with the jelly-like insoluble residue. Boiled with dilute or strong acid, protocaseose is appar- ently not changed; at least no precipitate is obtained on neutraliza- tion of the acid fluid. The acetic acid solution of protocaseose gives avery heavy precipitate with potassium ferrocyanide. In an aque- ‘ous solution of the substance, cupric sulphate gives a heavy curd-like precipitate, while ferric chloride gives a similar precipitate read- ily soluble in excess of the precipitant. Like protoalbumose, proto- caseose is precipitated by saturation of its aqueous solution with sodium chloride, but never completely ; there always remains in the Trans. Conn. Acav., Vou. VII. 51 Marou, 1887. 402 Chittenden and Painter— Casein and its filtrate, a portion of the substance precipitable only on addition of acetic acid. Of the several preparations of deuterocaseose, those precipitated by ammonium sulphate are evidently the only ones perfectly pure. Further, it is evident that this substance can be obtained pure only by complete removal of all protocaseose from the solution, which implies precipitation of a large portion of the deuterocaseose also, and then precipitation of the small amount of dentero remaining, by saturation of the fluid with ammonium sulphate. D deutero, pre- pared in this manner, shows several very marked points of differ- ence from protocaseose. In the first place, it is not precipitated in an aqueous solution by acetic acid. Further, the addition of potas- sium ferrocyanide to a solution acidified with acetic acid gives no precipitate whatever. Cupric sulphate and ferric chloride both fail — to produce any precipitate in an aqueous solution. Pure deutero- caseose, as already mentioned, is not precipitated by saturation of its aqueous solution with sodium chloride; addition of acetic acid, however, to the salt-saturated solution gives a heavy precipitate, which represents perhaps half of the deutero, the remainder of which is precipitated only by saturation of the fluid with ammonium sulphate. It is thus evident that in the precipitation of protocaseose from a salt-saturated solution by acetic acid, more or less deuteroca- seose will be likewise precipitated, the amount depending probably on the concentration of the solution and other minor circumstances, Hence, A deutero is unquestionably contaminated with some proto- caseose, and on the other hand protocaseose D 3 and 4, and perhaps protocaseose C 2, without doubt contain some deuterocaseose. That A deutero contains some protocaseose, is evident from the fact that it gives a precipitate with cupric sulphate, and further its aqueous solution is rendered decidedly turbid by acetic acid. Moreover, the protocaseose precipitated by acetic acid, and which may contain some deutero, appears to differ in one or two respects from either proto or deuterocaseose. Thus, an aqueous solution of the purified substance is precipitated like pure protocaseose by acetic acid, but the precipitate is only partially soluble in excess of the acid and even that requires a large excess. With nitric acid, pure deuterocaseose gives no precipitate, but on warming the solution the xanthoprotein reaction comes out strongly. Composition of the caseoses and their relation to casein. In studying the composition of the various caseoses we have been hampered by the large percentage of ash invariably present in all of Primary Cleavage Products. 408 TABLE SHOWING RELATIVE COMPOSITION OF THE CASEOSES. Protocaseose. Cel EE ai) N Ss O pele Nal yprecipitate: -_-.-:2- 22.2222. 5.-2 52°50 | 7:15 | 15°73 | 0°96 | 23-66 ano. * 1° (GM heen eae ne 53°85 | 7°21 | 15°84 | 0:98 | 22-12 | Ao ACehG acid precipitate. -_..-.----....- 52°59 | 7°17 | 15°70 | 0°90 | 23°64 Eee acel precipitate. 22222. 2-- 2222-2 2+. 52-91 | 7-06 | 15°65 | 0:90 | 23-48 B 2 Acetic acid precipitate. ..........---- 52°43) 7:01 | 16°19 | 0°90 | 23-47 Sie wah precipitate: .-.222./.-..2.---..-- De OA TOS oo ar | te aie C 2 Acetic acid precipitate__...-..:.....- 51°73 | 7°15 | 15°85 | 0°79 | 24-48 Pile Nanen precipitate. 2.9.2... 2.-..-222-2 53°93 | 7:17 | 16:05 | 0°85 | 22-00 De « 2 Sah eee 5284) 7-10 15°86 1-04 23-16 D 3 Acetic acid precipitate__-_-.------- ----| 52°05, | 7°18 | 16°12 | 1:06 | 23°64 Bo. « Paid Pe), 52°88) 7-07 1613) 0-98 22-94 Deuterocaseose. A. Acetic acid precipitate...__..__...-- 51:59 | 6-98 | 15°73 0-75 25-03 © D. (NH,).SO, es paces ft tt .----| 51°79 | 7:05 | 16°00 | 1:17 | 28°99 Heterocaseose. Lee 2 ire 58°88 | 7-27 | 15°67) _... | 2 Casein. | Beaverage of Nos. I-VIT___-___-_.:..__--2- 53°30 | 7:07 | 15°91 | 0°82 | 22:03 404 Chittenden and Painter— Casein and its the preparations. We have already commented on the difficulty, in fact, impossibility, of removing certain inorganic salts after they have once been brought in contact with a caseose body. Repeated precipitation appears to affect the percentage of ash but little. The reason for the large percentage of ash lies in the precipitation of the caseoses from such large volumes of fluid. We thought it unwise at first, to expose the bodies to the long continued evaporation neces- sary for precipitation with a small amount of salt. To avoid the possible danger of change, therefore, the large volumes of fluid resulting from the several digestions were saturated directly with salt, and as this involved the use of large quantities, calcium salts and some iron as impurities in the sodium chloride, were unavoid- ably introduced. These, the caseoses seemed at once to catch hold of and retain, in spite of oft-repeated purification. In the digestion | D, in which the fluid was concentrated somewhat before precipita tion, the percentage of ash is seen to be somewhat smaller than in preparations from the other digestions. | In comparing the composition of the individual protocaseoses (see the accompanying table) it is seen that two of the bodies show a content of carbon somewhat higher than casein itself, while the average of all the others, with one exception, shows a content of carbon a little lower than casein. Leaving out the acetic acid pre- cipitate C 2, the average of the remaining ten preparations of proto- caseose shows the following composition for this substance ; Ch iT N S O Y Protocase0se 4244: - oe 52°89 7-10 15°94 0°95 23°12 @aseines. tote ern 53°30 7-07 15°91 0°82 22°03 Plainly, the average of our results would indicate that protocaseose does not differ essentially in composition, from the casein from which it is formed. A slightly smaller content of carbon is the only notice- able difference. To be sure the individual results show noticeable variation in the percentage of carbon, but bearing in mind the large amount of ash present in the preparations, it is evident that the aver- age result is of more value than the results obtained in any one case. As to the lower content of carbon in so-called protocaseose C 2, it is probable that this body is composed mainly of deuterocaseose. The two caseoses being precipitated together in this digestion by ammo- nium sulphate and then separated afterwards from a fairly concen- trated solution by saturation with salt and addition of acetic acid, renders it probable that the protocaseose was more completely precip- Primary Cleavage Products. 405 itated than usual by salt alone; and further, it is probable that on addition of acetic acid to the concentrated and salt-saturated fluid, a much larger proportion of deuterocaseose was precip- itated. In confirmation of this view it was noticed that the amount of deuterocaseose obtained by the later precipitation with ammonium sulphate was quite small; far smaller proportionally than obtained in D. That the body contained some protocaseose, was evident from its reaction with cupric sulphate and with acetic acid, Pure deuterocaseose evidently contains a smaller content of car- bon than protocaseose. It is equally evident that it is a body further removed from casein than protocaseose. Its general reac- tions show a closer relationship to peptone than to casein or the proto-body. Heterocaseose, on the other hand, judging from analysis of a single preparation, contains fully as much if not more carbon than casein itself. Nearly all of the caseoses show a somewhat higher percentage of sulphur than casein, but probably the increase (0°1 per cent.) is due mainly to a trace of sulphate in the ash, not accounted for. Owing to the large amount of phosphate in the ash of the different prepara- tions, phosphorus was sought for only twice. In both of these, how- ever (protocaseose D 1 and deuterocaseose D), the phosphorus in the ash was the exact equivalent of the total phosphorus found after fusion with potassium hydroxide and nitrate. This might indicate that in the cleavage of casein with pepsin-hydrochloric acid, the phosphorus of the casein is removed in the form of a phosphorized body, leaving the thus non-phosphorized matter to break down into the caseoses. With this thought in mind, we propose to study later the nature and composition of the insoluble, semi-gelatinous body separated in the first stage of digestion. We also hope to extend our work by a study of Weyl’s commercial ‘ casein-peptone,” pre- - liminary examination of which has shown us the presence in large quantities of caseoses. In this way and by a somewhat different method of isolating the individual caseoses, we hope to verify our present work and at the same time obtain products comparatively free from ash, with which to establish beyond question the composi- tion of the caseoses. We are also occupied in a study of pure casein- peptone, purified according to the method made use of by Kiihne and Chittenden in the study of fibrin-peptone. XXII1.—Isrivence or Some OrcGanic anp InorGanic Sus-. sTANCES ON Gas Merasposism. By R. H. CuirreEnpDEN AND G. W. Cummins, Pu.B. Wuiter much time has been spent during the past few years in studying the influence of various substances on proteid metabolism, far less attention has been paid to the effects of these substances on the consumption of oxygen and the elimination of carbonie acid. Naturally in studying the influence of any substance on the nutrition of the body, we need to know not only its action on the excretion of nitrogen but also its influence on the production of carbonic acid. In. this way only can we arrive at a true understanding of the influence of the substance on total metabolism, and obtain the necessary data from which to draw conclusions as to its influence on the consump- tion of either nitrogenous or non-nitrogenous matter. The difficulties, however, in the way of carrying on consecutive determinations of the relative amount of carbonic acid eliminated by the lungs are consid- erable, and in the absence of the necessary respiration apparatus, the difficulties are greatly increased, We have, however, endeavored to carry on some experiments in this direction, and although lacking the ordinary apparatus we have still been able with the meas at our disposal to obtain some interesting results, a portion of which are simply confirmatory of previous work, while others are wholly new. The apparatus employed in measuring the amount of carbonic acid eliminated is shown in the accompanying illustration (see Plate). The chamber in which the animal was placed during the experiment, was a bell jar of 32 litres capacity, with ground edge fitting closely upon a smooth glass plate. This when coated with grease made a perfectly tight joint, but in order to avoid any possibility of error, the jar and plate were placed in a shallow pan of galvanized iron, and water poured in to the depth of 2-8 inches, thus insuring a perfectly air-tight joint. In the top of the bell jar was an opening, closed with a doubly perfor- ated rubber stopper, through which passed two tubes; one bringing air into the chamber, the other carrying it to the absorption apparatus. The inlet tube (to the left of the figure) was prolonged so as to admit the air vearly at the bottom of the jar, while the outlet tube came just through the stopper, thus insuring a perfect circulation of air, Air was drawn through the chamber by means of three aspirators, PLP OR AD IE Bt LM them o5 ee oe Se lp RE tts Chittenden and Cummins— Gas Metabolism. 407 two of which had a capacity of 15 litres and one of 74 litres. The three aspirators working together would therefore draw through the chamber 374 litres of air at every filliug, and the flow was so regulated that 30 minutes were required to draw that amount of air through the apparatus. The flow of water from the aspirators was quite regu- lar, since the inlet tubes went to the bottom and the air had to bubble up through the water, as the latter ran out, on the principle of Mariotte’s bottle. The rate of flow was regulated by carefully changing the difference in height between the inlet tube (for air) of the aspirator and the outlet tube (for water). This of course, at the outset, was a tedious operation, but when once perfected and the apparatus permanently set up, the three aspirators ran exactly to- gether, with a maximum variation of 15 seconds for the half-hour, which variation, however, was seldom observed. In addition, each aspirator was marked off into eight divisions, the last one of which was equal to only one-half of the others. In the two large aspirators these divisions indicated exactly the same volume, while in the small aspirator the divisions represented half the capacity of the former ; but the flow of water in the latter was regulated to consume the same amount of time as in the former. Hence four minutes were required for the water to flow by each of the first seven divisions, and two minutes for the last, making a total of thirty minutes for the entire volume of water to flow from each aspirator. The tube drawing the respired air from the chamber in which the animal was enclosed, was divided a short distance from the chamber, as seen in the figure, and two-fifths of the mixed air was drawn successively through three absorption tubes filled with a standard solution of barium hydroxide for absorption of the carbonic acid. The absorption tubes were about two-thirds of a metre long and the lower tube (@) contained 100 ¢. c. of a standard baryta solution, the middle tube (4) also 100 c. c. of the solution, and the upper tube (ec) 50 ¢c.c. The amount of carbonic acid absorbed was, at the end of the experiment, determined by titration with a standard solution of oxalic acid, using phenol-thalein as an indicator. Two titrations were made, one of the contents of tube @ and one of the contents of the two tubes b and ¢. By using the three tubes, absorption of the carbonic ‘acid was quite complete. In order to aid absorption, the air was broken into small bubbles by being forced through a small tube dip- ping beneath the barium hydroxide. Frequent blank experiments showed that all of the connections were perfectly tight, and further, all of the tubes being in the same position, that the flow of water 408 Chittenden and Cummins—Influence of Some Organic from each aspirator was perfectly uniform, and that the aspirators could be relied upon to draw the given volume of air through the apparatus in the time designated without any appreciable variation. In addition, the two-fifths drawn through the absorption tubes for determination of the carbonic acid was always exactly two-fifths of the aspirated air; since the aspirators, as already remarked, worked with perfect uniformity. Any tendency to variation, either in the time, or in the action of the individual aspirators, was noticed at the very outset of the experiment, as the water reached the level of the different marks on the aspirators, and could be at once checked or controlled by moving slightly the water outlet tube so as to either increase or diminish the difference in height between the latter and the inlet tube for air, Theoretically, variations in the temperature of the water in the aspirators might affect somewhat the volume of air analyzed, but a constant determination of the temperature of the water showed such slight variations that they did not seem to justify us in making any corrections for possible change in the amount of air aspirated. Naturally, all of the supports for the three absorption tubes were permanently placed, so that there could be no change of position ; the tubes themselves placed in the same position in the holders; the volumes of baryta solution invariably the same, so as not to increase or decrease the pressure to be overcome; and lastly the aspirator tubes and stoppers fastened so as not to admit of any change. With these precautions, the results obtained, both as to the volume aspirated and the time consumed, were quite satisfactory. ; As already mentioned, the total capacity of the three aspirators was 374 litres or 5$ litres more than the capacity of the bell jar. This amount of air drawn through the chamber in 30 minutes, was more than enough to supply the largest rabbit experimented on, with the necessary amount of oxygen. But there must have been a slight accumulation of carbonic acid in the air of the chamber; this, how- ever, was a constant factor throughout the experiments. Further, the results obtained, expressed in milligrams of CO,, do not represent the total amount of carbonic acid eliminated by the rabbit during the thirty minutes of the experiment, but simply the amount of CO, contained in the 374 litres of air aspirated during that time. Such a result, however, ought certainly to show just as plainly any influence on the elimination of carbonic acid, as a determination of absolute quantity and thus be equally valuable as an indication of influence or lack of influence on the gas metabolism of the body. Further, the results thus obtained ought to express equally as well, the comparative ‘action of the various substances experimented with. . and Inorganic Substances on Gas Metabolism. 409 In every experiment, the time at which the animal was introduced into the bell jar was exactly noted and then two minutes were allowed before starting the aspirators, to make all of the connections properly. The rabbit was therefore under the bell jar, in each determination of earbonic acid, for exactly thirty-two minutes. The aspirators were started simultaneously and their progress carefully watched, in order to check any slight irregularity that might show itself. The animals experimented with were wholly rabbits, and prelimi- nary trials showed us plainly that it was very necessary to have them in a condition of hunger during the experiment, in order to avoid the irregularities incident to change in digestion. Further, we soon found that this was best accomplished by depriving the animal of food for three days, after which the experiment was commenced and allowed, as a rule, to extend through three con- secutive days, the animal being deprived of food during the entire period. On the first of the three days, eight determinations of carbonic acid were made and the results obtained were used as a control, with which to compare the results obtained on the two fol- lowing days, when the animal was being dosed with the substance experimented with. This, as a rule, we found to be the most satis- factory method of procedure, since small differences could not be relied upon as expressing anything of importance; for the varying restlessness of the confined animal, involving more or less muscular activity, would many times lead to variations in the amount of car- bonic acid excreted, as may be noticed in the control experiments on those days when the animals were not dosed. Hence, the average of several consecutive results must necessarily express more correctly the average elimination of carbonic acid than any single result. Further, we deemed it better to allow the experiments to extend, as a rule, over several days and thus study the action of small, repeated _ doses of the various substances rather than to observe the effects of a single large dose, where violent action might naturally be expected. The following table of results illustrates the way in which our experiments have been conducted, and at the same time shows the extent of variation, in the amount of carbonic acid, to be expected under normal circumstances from day to day. In this experiment, the rabbit had been deprived of food for three days, and the results show the amount of carbonic acid in the 37°5 litres of aspirated air for four distinct periods, during the fourth and fifth days. As already stated, the total amount of baryta solution employed in the three absorption tubes was 250 c. ¢., of which 100 ¢. c. were used in the first Trans. Conn. AcaD., Vou. VII. 52 MARCH, 1887. 410 Chittenden and Cummins—Influence of some Organic or lower tube (1), while the remainder was used in the two other tubes b and c. Several solutions of oxalic acid were employed, the average strength of which was such that 1 ¢, ¢. equaled about 20 milligrams of carbonic acid. Oxalic acid to Bo; ier: 2» B. . ¢ neutralize ba-| © = 4 SEITE RS a o 0 5 ryta solution.| og |So°| 9 5 Sie Ss Time. So saa og 2 10 55) | R. |see| ge | @ oe a mS Dae ilien$s' Oa BOes- Hrs [hao eT: eo |geo| 32 ess) os | se | se | ae ee Jose aa 3) Sg March 18. | 9:48 to 10:18 | 10-4 | 25°0 | 35:4 | 45-1 9°7 180°2 - 450°5 | 38°8 11:51 to 12:21 | 10°6 | 25°2 | 35:8 | 40-1 93 | 1727 | 432°0 | 38-1 2:59 to 8:29 | 10°5 | 25°3 | 35°8 | 40-1 9:3 172°7 | 432°0 | 38°2 | 5:03 to 5:33. | 10-4 | 24:9 | 35°3 | 45-1 9°8 182°0 | 4551 | 38:3 March 19. | ; 8:59 to 9:29 | 10°2 | 25-1 | 35:3; 45:1 9-8 | 182:0 | 455:2 | 38:3 10:53 to 11:23 | 10-9 | 25°3 | 36-2 | 45-1 ee) 165°3.| 413°4 | 37°7 2:53 to 3:23 | 10°2 | 24:2 | 34:4) 451 { 10-7 198-7 496°8 | 38-7 | 4:51 to 5:21 10-6 24-9 | 35°5 | 45-1 96 | 1783 | 445°9 | 38-9 35-5 | 45-1) 9:6 | 179-0 | 447°6 | 38-4 Average, 10:5 | 25:0 Action of uranyl nitrate. As stated in a preceding article,* the physiological action of ura- nium salts has been little studied. Experiments are now in progress to show the influence of uranium on proteid metabolism, and our present results show the influence of this substance on the excre- tion of carbonic acid. The rabbit first experimented with was de- prived of food for three days, and on the fourth day the experi- ment was commenced, extending through three entire days, during which time the animal was without food. The accompanying tables show the results obtained. The body temperature was ascertained - by inserting a sell-registering thermometer into the rectum. * Chittenden and Hutchinson, this volume. and Inorganic Substances on Gas Metabolism. 411 A study of the first results shows plainly a decided action on the part of the uranium salt. The influence of the salt, however, mani- fests itself somewhat slowly, and it is not until the third day that its action becomes very pronounced, when the increased excretion of car- bonie acid becomes very noticeable, accompanied with a slight rise in temperature. The first action of the uranium appears to cause a diminution in body temperature and in the amount of carbonic acid eliminated. The total amount of uranium salt given was quite large (1:175 grams in divided doses), and although no especial toxic symp- toms showed themselves, the animal died on the day following the conclusion of the experiment. A second series of experiments was tried, using smaller amounts of uranium nitrate and extending through four days, the results of which are also shown in the accompanying tables. The rabbit was deprived of food for four days prior to commencing the experiment. The amount of uranium nitrate given was considerably smaller than the quantity employed in the first series of experiments, and the animal did not’ suffer any permanent ill effects from its use. The following table shows the average daily result, expressed in milli- grams of CO, contained in the 37°5 litres of aspirated air, together with the average body temperature. May 3. - 38°9° C. 574°3 milligrams CO, cor A 39°0 540°8 a ge Os 39°9 581°2 & ‘ Cae Bip 38°'5 716°3 6 as The uranium nitrate was introduced by hypodermic injection in the following quantities : May 3. ‘0 ee) A 5:18 p. m. 0-080 gram of the salt. i TP RaP 8:40 a. m. 0:00 0 race a Fen: 10:20 a. m. Oa00res: ae eS 3 12:40 p. m. O° 1a ii cf Ds 3:25 p. m. O50 ye os a Bay 5:15 p. m. 0-200 “ $s ae Als 0 0-770 In this second series of experiments it is to be noticed that the first two days are given up wholly to determining the normal excretion of carbonic acid, and the results show fully how close an agreement may be expected under normal circumstances. Taking the results 412 Chittenden and Cummins—Influence of some Organic First SERIES OF EXPERIMENTS WITH URANIUM. Normal period, without uranium nitrate. Oxalie acid to 4 ; Sgn ttt S ‘ a ; neutralize ba-| 1S aan bee) Bs % E Date. ryta solution.| © Bs ao = re a 8 is ae) ae gc 2 wien a April 19. | a Ne oeel rers 4 © FA 3 = Nes: Rees o® =e q 2 3, mits A — wo Tears || eo “= 69 = > O BS eh oll seco ll REEL A Cees “5 ae hy eat CAE a 8 8 1a A.M, ; 9:47 to 10:17 65 | 24°8 | 31:3) 46-3 | 15-0 2789 | 697°3 | 38°7 10:45 to 11:15 7-8 | 25:8 | 33°6 | 46:3 | 12:7 235°3 | 5883 | 387 11:46 to 12:16 79 | 25:8 | 33°7 | 46°3 | 12-6 233°4 | 583°6 | 38°6 . M. P 2:04 to 2:34 | 8-4 | 26:3] 34:7] 46-3} 11°6 | 2149 | 537-3 | 38-4 2:57 to 3:27 | 8-4 | 96-1] 34:5 | 46-3} 11°8 | 218°6 | 546°6 | 38-7 3:58 to 4:28 | 8-2 | 261 | 343 463 12:0 | 221-4 553'5 | 386 4:52 to 5:22 | 85 | 26-2 | 34:7 | 46-3 | 11°6 | 2149 | 537°3 | 38-7 Average, 7-9 | 25:9 | 33°8 | 46:3 | 12°5 231-0 577°7 | 389 April 20. With uranium nitrate. 9:04 to 9:34 | 7-3 | 25-9) 382) 463 184 | 242-7 | 606:8 | 38-4 9:57 to 10:27 | 8-3 | 26-8| a5-f| 463 | 11:2 | 207-5 | 5x88 | 38-4 10:59 to 1129 | 9-8 | 266) 36-4 | 463 9-9 | 188-4 | 458-6 | 38-2 11:53 to 12:23 | 8-7 | 263} 35:0 463 | 11:3 | 2093 | 523°5 | 38-1 P M. 1:59 to 2:29 8°8 | 26°3 | 35:1 | 46:3 11°2 207°5 5188 | 384 2:01 to 3:21 88 | 265 | 35:3 | 46°3 11:0 204°8 512°0 | 38°8 3:46 to 4:16 8°4 | 2671 | 34:5 | 46°3 11°8 | 218°6 446°6 | 39-1 46°3 14:3 | 264:9 662°4 | 389 4:41 to 5:11 6°8 | 25°2 | 32:0 Average, 8:4 | 26:2 | 34:6 | 46:3 11°7 217°3 535°9 | 385 and Inorganic Substances on Gas Metabolism. 413 With uranium nitrate—continued. Oxalic acid to Se ee ee hd neutralize ba-| <3 Pe rl) oes bz 2 to = Tate: ryta solution. | & .; Be 7 | ts a Se 3 go lena) goo | 2 tn Pe April 21. : es oe S 2e oS - 58 5 Sor iinet Dee tle Ws ees @° |oC°| S2lscs| 85 ier efiast mile = iS] ete eo (ac nar = = * eNa one S) 15 =) = So A oO Oo fea) A. M. | 9:02 to 9:32 5:0 | 22°4 9:58 to 10:28 6:0 | 24:9 | 380°9 | 46°3 15°4 285°1 712°8 | 38:9 10:55 to 11:25 6:0 | 24:7 | 30:7 | 46:3 15°6 289-0 722°6 | 39-0 11°47 to 12:17 57 | 24:8 ras) 3 eS a SE oo — C C [o 2) A A s co oO S pee oo ~I un on ey) Je} ras) 2:00 to 2:30 | 7-3 |257 | 380! 463| 133 | 246-4 | 6x62 | 39-1 2:55 to 3:25 | 6-0 | 25-0 | 31-0 | 463] 15:3 | 288-4 | 7087 | 39-1 3:47 to 4:17 | 84 126-2 | 346 | 463] 11-7 | 216-7 | sqres | 39°2 4:42 to 5:12 | 93 | 266 | 35:9 | 46-3] 10-4 | 192°6 | 48z°7 | 39-4 Average, 6°7 | 25:0 | 31°7 | 46°3 14:6 | 269°5 673°9 | 391 The following figures give the average daily result in body tem- perature and in the amount of carbonic acid contained in 37°5 litres of aspirated air: April 19, 38'9° C. 577°7 milligrams CO, 6é 20, 88:5 6c 585°9 “é 6c “ec 21, 39-1 ‘é 673°9 “e “é The uranium nitrate was introduced by hypodermic injection in - the following quantities: April 19, 5:40 p. m. 0:050 gram of the salt. of a 20; 8:55 a. m. 0-100 ae ee PANDO: 10°35 a. m. 0-100 es ; 20; 12°40 p. m. 0150 «e ef Us 1:35 p. m. 0-150 ee 8s oma!) 60:30 P.M. 0°300 oS oe Sek, 8°55 a. m. 0-200 ee i mel, 2°45 p. m. 07125 ae ap ja AY i) Oa OLS AS | iris toa Fes oD S34 a4 a Be OMe Ore a sees ee Oy Bcs aa| = Big =r) sau fe) im © (eo) ° =) = a o B 1) i) fa 9:00 to 9:30 9-4 | 26-1 | 35:5 | 45°5 | 10-0 | 2021 | sos4 | 39:0 9:52 to 10:22 | 10-4 | 265 | 369 | 45:5) 8-6 | 172-8 | g3ar | 38-9 10:41 to 11:11 | 9-1 | 261 | 35-2 | 45°53} 10:3 | 208-2 | s20r5 | 391 11:36 to 12:06 | 9°6 | 263) 35:9 | 455 | 9-6 | 194-0 | 485-2 | 39:0 PM: 2115 to 2:45 | 9:6 | 26-5) 36-1 | 45-5 | 9-4 | 1900 | 4751 | 39°2 3:08 to 3:38 | 9:6 | 265 | 36:1 | 45-5 | 9:4 | 190-0 | 475°1 | 38-9 8:57 to 4:27 | 10-3 | 26:5 | 36-8 | 45-5 ‘~ | 195-8 | 43977 | 39:2 4:47 to 5:17 | 10-0 | 26:5 | 86-5 | 45-5 | 9-0 | 181-9 | 454°8 | 39-2 Average, 9-7 | 26-4 | 86-1 | 455] 9-4 | 1893 | 473°5 | 391 Average daily excretion of carbonic acid expressed in milligrams of CO, per 37°5 litres of aspirated air, and average temperature is as follows : June 7 389° C. 586°6 milligrams CO, cea 38°9 480°4 ae rf io 39°1 473°5 as is The arsenious oxide was introduced by way of the mouth in small gelatin capsules, in the following doses : June 7 5:25 p. m. 0:005 gram =As.,O; aoe) 8:47 a. m. O:005 ses ss pie) 12:12 p. m. 0-005." * a ceo 5:25 p. m. 0-005 ** 9 8:50 a. m. 0-005 ** os .* “ 9 1212 p.m. 0-010 « ‘ 0:0385 422 Chittenden and Cummins—Influence of some Organic FIRST SERIES OF EXPERIMENTS WITH ANTIMONY. Normal period, without tartar emetic. Oxalic acid to yA a Bs ' neutralize ba-| © = . of i ts z E Date. ryta solution. | & 5 |S5°| © = is S aia ao [sa s| og 2 i 5: 2, March 31. Hla se] ge es ms & 5 os. pleas cepa = Ole Boss a as mee . Diet = cot = Be 4 - 3% \285| 82 £25) 8s ok SE | Be res |= = S) A o ‘= aa) A. M. 9:08 to 9:33. | 13°9 | 30°9 | 44°8 | 52°6 78 144:5 | 361°3 | 38°6 10:01 to 10:31 | 138 31:0 | 44-8 | 526] 78 | 144.5 | 361-3 | 388 10:56 to 11:26 | 15-0) 31:0) 46-0 52:6) 66 | 1222 | 305-7 | 38-9 11:52 to 12:22 13:7 | 30°8 | 44:5 | 52°6 8-1 150°0 | 375°2 | 39°2 P.M. | 1:55 to 2:25 12°5 | 30°0 | 42°5 | 52°6 | 10-1 187-0 «| s467°7 ies 2:47 to 3:17 15°0— 31:1 | 46:1 | 52°6 6°5 120°4 | 3o0rx | 39-2 3:41 to 4:11 | 14:4) 380-4 | 44:8 | 526) 78 | 1445 | 361-3 | 89°2 4:36 to 5:06 | 18-7, 30°8| 445 | 526| 81 | 150-0 | 375:2 | 39°3 Average, | 14-0! 30-7! 447! 526] 7.85 | 145-4 | 3636 |:39-0 April 1. With tartar emetic. A. M. 3 8:57 to 9:27 | 14-1 | 30°8 | 44-9] 526] 77 | 141-7 | 354-4 | 88-1 9:54 to 10:24 | 145 | 31-0 | 45-5 | 52-6 | 7-1 131°5 328'9 | 36-9 10:50 to 11:20 | 15-4 | 31:0 | 46-4] 526] 62 | 1148 | 287-2 | 86-4 11:44 to 12:24 | 15:4 | 31-0 | 46-4 | 526] 62 | 1148 | 2872 | 35-7 P. M. 2:01 to 2:31 17°0 | 31:2 | 48:2 | 52°6 4:4 81°55 2038 | 34°6 Average, 15°3 | 31:0 | 463 | 526| 63 116-9 292°3 | 36°3 The antimony was given in the form of tartar emetic and was introduced by hypodermic injection as follows: March 31. 5:20 p. m. 0:012 gram tartar emetic. April 1. 8:45 a. m. 0-085 es ye ee ils 12:43 p. m. 0-035 s “¢ 0-082 Rabbit died at 3:30 p. m., April 1. and Inorganic Substances on Gas Metabolism. 423 SECOND SERIES OF EXPERIMENTS WITH ANTIMONY. Normal period, without tartar emetic. Oxalic acid to ah Sigs A ; = 3 neutralize ba-| = ae ee @ 5 Date. rytasolution. | 25 |S5 ° 5 5 = ¢ PO cee le rj. Ml 2 ee | 8, April 5 s |Sem| 83 AR ete = aa Per ess cos | | ae |e. Pees a2 ieee) 2a) 28 | Tek | eS Sips |S. | 3s Sg seo ies A. M. 9:14 to 9:44 | 14-4 | 30:9| 45:3 52°6| 73 | 185-2 | 338% | 38-4 10:09 to 10:39 | 13-0 | 30°3 | 43:3 | 526) 93 | 1723 | 430°8 | 385 11:04 to 11:34 | 13-1 | 305 | 43°6 | 526) 9:0 | 166-7 | 4169 | 38-4 11:57 to 12:27 14°5 | 30°8 | 45°3 | 52°6 7:3 135°2 | 338°1 | 38°6 P.M. 2:05 to 2:35 | 13-8 | 30-7 | 445 | 526) 81 | 150-0 | 375-2 | 38-9 2:58 to 3:28 13-1 | 30-5 | 43°6 | 52°6 9°0 166°7 | 4169 | 38°8 3:52 to 4:22 | 12°7 | 30:5 | 43-2 526 | 9-4 | 1741 | 435-4 | 38°9 4:45 to 5:15 12:9 | 30°7 | 486 | 52°6 9-0 166°7 | 416°9 | 38°7 Average, 13-4 | 30°6 | 44:0 © 52°6 8°5 158°4 | 396°0 | 38°6 April 6 | With tartar emetic. A. M. | 8:59 to 9:29 | 14:5 | 30°8 | 45:3 52°6 73 135°2 | 338°1 | 37-4 9:57 to 10:27 | 15°2 | 31:1 | 46°3 | 52°6 6°3 116°7 | 291°8 | 36°7 45°1 | 52°6 75 138'9 | 347°4 | 36°6 10:52 to 11:22 14:5 30°6 11:46 to 12:16 14°5 | 31:0 | 45°5 | 52°6 Til 131°5 | 3289 | 37°3 1:58 to 2:28 153 | 30-9 | 46-2 | 52:6 | 6:35 | 117°6 | 2g4°r | 36-2 2:50 to 3:20 | 145 | 30-9 | 45-4 | 52:6 | 72 | 1838-4 | 333°5 | 36:2 B42 to 4:12 | 15-4 | 31-2| 46-6 526) 5:95 | 110-2 | 275°6 | 85-4 4:38 t0 5:08 15-7) 31:2 | 46-9 526 | 5-7 | 115°6 | 289°0 | 35:7 124:9 | 3122 | 36°7 Average, 149 |. 31:0 | 45-9 | 52°6 | 6°68 424 Chittenden and Cummins—Influence of some Organic With tartar emetic—continued. | | | Oxalie acid to a el ae | ‘ B ; neutralize ba-| cg poe id = | a ob E . ° rene . csc | | ~ Date. ryta solution. | @ 5 | F's g a ae s =| io | [aS ey i Aes © F a a) oe aa ow aa owt = ex = a April 7. 4 So ee e a 5 ro = cS) Ss Oo ye) a e f=heen =3 2 5 oS |Sus\ su (Sos o2 2 | 7a ele 2s 125s] o° [eno Be Wo. oa 5 £ a) eo) = iS) oS = A. M. 9:00 to 9:30 | 17°6 | 31:2 | 48°8 | 52-6 3°8 70'4 | 1760 | 30-0 9:59 to 10:29 | 18°5 | 31°3 | 49°8 | 52°6 28 | 61'S | x20 jeee0 10:53 to 11:23 | 18°9 | 31:4 | 50°3 | 52°6 2°3 42°6 | 106°5 | 27-0 Average, | 183 | 31:3 | 496 | 526|) 2-97 | 54:9 | 137-4 | 283 i Average daily excretion of carbonic acid expressed in milligrams of CO, per 37°5 litres of aspirated air, together with average temper- ature is as follows : April 5. 38°6° C. 396°0 milligrams CO, 6. 36°7 312-0 fe Be te 28°3 137°4 % et The following amounts of antimony were injected : April 5. 5:30 p. m. 0-015 gram tartar emetic. 6. 8:45 a, m. 0-015 iF es 6. 12:39 p. m. 0015 se c— 6. 5:24 p. m. 0-010 eA te 0-055 Rabbit died at 12 m., April 7. and Inorganic Substances on Gas Metabolism, 425 Action of morphine sulphate. Boeck and Bauer* have already made a careful study of the action of morphine on the elimination of carbonic acid and the absorption of oxygen. By experiments on a cat and on a dog they found that the action of morphine on metabolism was mainly an indirect one, affecting especially the consumption of non-nitrogenous matter. Further, that its action hinged mainly on its power of affecting mus- cular activity ; thus in the case of a cat the first action of morphine was to increase the elimination of carbonic acid and the consumption of oxygen, due to the increased muscular activity induced by the poison, while in the case of a dog, where narcosis was half induced, there was a diminution in the amount of carbonic acid eliminated ‘amounting in one case to 27 per cent. This diminished excretion was due almost wholly to the quieting action of the morphine and was followed by an after period in which there was increased produc- tion of carbonic acid, due to the increased activity of the muscle tissue. In these experiments the dose of morphine was 0:05 gram, in the form of chloride, and was introduced by subcutaneous injection. The injection of the poison was followed soon after by convulsions, ete., indicating vigorous toxic action. In our first series of experiments we endeavored to have the toxic action less pronounced, and for this reason the morphine was introduced by way of the mouth in repeated doses, the experiment extending through three days ‘and into the fourth. The rabbit was deprived of food through the entire period and had also been kept without food for three days prior to the experiment. The data are to be found in the accompanying tables. The results do not show any very marked action, either on the excretion of carbonic acid or on the body temperature. At no time was there any noticeable indication of increased muscular activity, the rabbit remaining fairly quiet in the chamber and showing no symptoms of tetanic convulsions. On the other hand there was no very profound narcotism. © | 5) r P neutralize ba- = eee 2 a = : peeeaes| oe 3 a: = Date. ryta solution. | ©, |35°| ° |; «a Ko = Se Pear Pero eet re es Pe 5) reo I CS ome | eure walt t= March 26. ie ee Fa) eo a See |e aes Ee ers OP O'S, aie 25 = oS logs! eau |2Bs| ow: eae ans Zoe ion] aa) = as co —_ 2; (285/57 |s- =i ene an % Ze 2 = 4 5S Ss 2 5 ial e) = >) S) = | t | 9:24 to 9:54 | 176! 35°8| 58-4| 605) 71 | 131-5 328'9 | 388 10:19 to 10:49 | 16-4 | 356 | 520 605) 85 | 157-4 | 393°7 | 38-8 11:14 to 1144 | 165 | 35-7| 522| 60:5) 83 | 153-7 | 384-5 | 38-6 12:09 to 12:39 | 16:5 | 35:7 53-2 / 605) 83 | 153-7 | 3845 | 38-9 1:58 to 2:28 17-4 | 35°8 | 53°2 | 60°5 7:3 135°2 | 3381 | 38°9 2:55 to 3:25 | 17:0 | 35-7 | 52:7 | 60°5 S| AASD ol 363-3 38°7 3:49 to 4:19 | 16:1 | 35°5 | 51°6 | 60°5 8-9 164°9 | 412°3 | 38-9 4:48 to 5:18 16°0 | 35°3 | 51:3) 60°5 O2) al Ti0ra Cazes eS Average, 16-7 85-6 “52:3 “605! 82 | 151-3 | 378-4. 38°8 March 27. A. M. 8:33 to 9:03 16°2 | 35°6 | 51:8 | 60°5 8:7 1611 | 403°0 | 38-9 9:30 to 10:00 | 15:7 | 35°5 | 51°2 | 60°5 9°3 172°3 | 430°8 | 38°9 Following is the average daily excretion of carbonic acid, expressed in milligrams per 37°5 litres of aspirated air, together with the aver- age body temperature : March 24. 38°0° C. 381°0 milligrams CO, sone oOs 38°4 363°7 ae $6 Soe mes 38°8 378-4 x aia hs 389 416°9 ss £S The morphine was introduced into the stomach in solution in the following amounts: March 24. 6:00 p. m. 0-075 gram morphine sulphate. Soc TaD. 8:35 a. m. 0:075 oh ee ie geco: 12:35 p. m. 0-075 ee Ke Son ens 5:50 p. m. 0-100 ce oC Sos 9:15 a. m. 0-100 e¢ xe er aU: 12:50 p. m. 0-100 ae ts 0-525 428 Chittenden and Cummins—Influence of some Organic jarge dose of morphine sulphate was given, and given by hypo- dermic injection. In this case, as before, there was no increased muscular activity, but there was a sudden and rapid fall both in tem- perature and in the amount of carbonic acid excreted, the latter of which was stili quite pronounced on the following day, although the temperature has gone back to normal. This diminution in carbonic acid was accompanied by a profound narcotism, the animal lying almost motionless and with a respiration ranging from 8 to 20 per minute. ‘Che results, which are to a certain extent corroborative of Boeck and Bauer’s, are shown in the accompanying table: SECOND SERIES OF EXPERIMENTS WITH MORPHINE. Normal period without morphine sulphate. i 4 ' 2 ° eS (o) = 4 3 0 : Oxalie acid to SD es z os fm an S neutralize ba- = ‘Sus ‘ = ee | = ryta solution.) «5 |3S 2 a = S 5 . * oO Time. Sis Sal ees 2 ee = = ee all Le Son one > os g KH. | as Bc ee 30) 3) : Ket 9 os oO eS) o fs Pa, a0 ay fas} o be ill aS) “= op 6S) = Se ie AS rie ae Se O +5 = BO BO EO) ree 1 Gea eels ak oF Ze ee, |eg 5a C See iS oOo; 8 Ss (=) oS oOo jea) March 12. A. M. 9:12 to 9:42 15.4 | 34:1 | 49°5 | 59°25 9-75 180°6 465°0 | 39-1 >. 10:09 A. M., injected subcutaneously 0-100 gram morphine sulphate. A. M. | | 10:23 to 10:53 16°9 | 34:5 | 51:4 | 59°25 7°85 145°4 363°5 | 36°6 11:29 to 11:59 | 17°83 | 34:5 | 51-8 | 59-25) 7-45; 1880 | gq25 | 84-4 12:32 to 1:02 | 163) 34:1 | 50-4 | 59:25) 8-85 | 1640 | gro-o | 33:3 P. M. 2:48 to 3:18 17-0 | 34:2 | 51:2 | 59°25) 8:05 149-2 37370 | 34°4 3:49 to 4:19 17°8 | 84°6 | 52-4 | 59-25 | 6°85 | 126°9 317°3, [-onU 1 | 4:47 to 5:17 | 17-1 | 84:3 | 51:4 | 59-251 7-85 | 145-4 | 363°5 | 863 March 13. 10:08 to 10:38 16:9 | 34:3 | 51:2 50:25 8:05 | 149:2 373°0 | 39:2 Action of quinine sulphate. The importance of quinine as a therapeutic agent and particularly its value as a febrifuge has led to a thorough study of its physiolog- and Inorganic Substances on Gas Metabolism. 429 ical action. In this connection, the action of quinine on proteid metabolism has been very thoroughly investigated, but as to its exact influence on the decomposition of non-nitrogenous matter, as shown by its effects on the elimination of carbonic acid, there is less una- nimity of opinion. This is naturally a point of considerable import- ance for if, as is generally supposed, the alkaloid has the power of diminishing body temperature, it would presumably be due to its in- fluence on the combustion of non-nitrogenous matter in the body. Ranke, Kerner, von Boeck and others have plainly shown the power of quinine to diminish proteid metabolism, but Strassburg, by an elaborate series of experiments* found that the alkaloid had no very decided effect upon the elimination of carbonic acid, either in healthy or fevered rabbits. Boeck and Bauer,t however, from experiments on cats, claim that quinine in the first stage of its action diminishes somewhat the pro- duction of carbonic acid, owing to its inhibitory action on the tissue cells; but when large doses of quinine are given, so that con- vulsions appear, then there is an increased production of carbonic acid, owing to the greater decomposition of non-nitrogenous matter incident to increased muscular activity. With small doses of the alkaloid, it is to be presumed that the slight diminution in carbonic acid noticed by Boeck and Bauer comes simply from diminished proteid metabolism. In our experiments, rabbits only were used and these in a con- dition of hunger, having been deprived of food for three days prior to the experiment. In the first series of experiments, the total amount of quinine given was quite large, so that at last the animal finally died from its effects. No decided action on the production of carbonic acid was noticed until just before the animal’s death, when both the body temperature and the amount of carbonic acid fell quite noticeably. On the second day of the experiment, when the quinine was first being given, the body temperature, as taken per rectum, fell quite gradually until it finally reached a point 1°5 a: below the average of the normal period. The results of the experi- ment are to be seen in the accompanying tables. The quinine given was in the form of hard, gelatin-coated pills and possibly was not as rapidly absorbed as might otherwise have been. At no time was the rabbit in convulsions. * Quoted from Dr. H. C. Wood, Therapeutics, p. 75. + Zeitschrift fiir Biologie, Band x, p. 350. 430 Chittenden and Cummins—Influence of some Organic First SERIES OF EXPERIMENTS WITH QUININE. Normal period, without quinine sulphate. | “ ' ; x ie ie om Oxalic acid to) Wy hopes £ 3 & en neutralize ba- = Fa) NTS i os rh . | ¢€ = ; =I 3 Date. | ryta solution.| 5 |FSs| ° 2 = | ee oe > hie = 1D 5: = oO | = oS - =|"s Som oS-8 : ae April 12. ] Bal nice | Bore Ped s cies : Als Ors "ey 5S) Dre ee 20 ES D aa |jSee| os “~* oo ee RES a HERS 5? pe ig am os oF ae = 0 sO z° |3 ex |o A <) s) A.M. 9:11 to 9:41- 9:5 | 24:2 | 38:7 | 41°5 78 144°5 361'°5 10:05 to 10:35 | 10°8 | 24-4 | 35:2 | 41:5 | 6:3 116-7 | 291°8 10:58 to 11:28 | 10-8 | 24:2 | 35:0 | 41°5 | 6:5 120-4 | 30r°r 11:54 to 12:24 | 10°7 | 24:2 | 34°9 | 41°5 | 66 | 122°2 | 305-7 P. M. | 1:58 to 2:28 | 10-0 | 24:0] 34:0 | 415] 75 | 1389 | 34774 2:51 to 3:21 110-0) |) 24-2) 934-27) e415 UPBY alate 3381 3:49 to 4:19 | 105 | 24:3 | 848] 41:5] 67 | 1244 | 320-4 4:41 to 5:11 | 11:0 | 24:3 | 35-3] 41:5 | 62 | 1148 | 2847-2 415 68 171 | 3178 Average, 10-4 | 24:2 | 346 | | April 13. With quinine sulphate. A. M. 9:14 to 9:44 9°3 | 23°9 | 38:2 | 41°55 | 8-3 | 10:10 to 10:40 11:3 | 23°9 | 35°2 | 41°5 Br] 1164 291°8 11:05 to 11:85 11:0 | 24:4 | 35°4 | 41°55 | 6-1 | 11:58 to 12:28 10°3 | 24:2 | 845 | 41:5 | 7-0 | 129-7 314°3 P. M. 1:55 to 2:25 9-4 | 24:0 | 88:4 | 41°5 81° | 150:0 375'2 2:46 to 3:16 | 10-1 | 24:2 | 343] 41:5 | 72 | 188-4 | 3333 8:40 to 4:10 | 98} 23:8] 88:1] 41:5) 8-4 | 168-7 4:32 to 5:02 | 10°3| 24:1] 34:4] 41:5! 71 | 1485 | 282°6 Average, 10-1) 241 | 842) 415) 73 | 1386 | 325°6 | , Body temperature, co os a | and Inorganic Substances on Gas Metabolism. 431 With quinine sulphate—continued. r ' = . ey fo} fxg : ao. : f Oxahe acid to we hee tee S eae 3 neutralize ba-| = (3° .; 2 eS ox & EI ryta solution.) sco |Se-| ¢ a isle = Date. ay ee |o.m | oS re Ei c= Rou ae ee 2 12 = : S So om S-s Z -"S a} April 14. | Mo. jae) Eg os reas 5B A 25 | ore. ) po Fe el cs d® = inv} A n 54 Shen i= os 7 OG =) bBo oo joCO; Sa 1S 2m) gs s 4 a 3 So 25 22s) © | 4 ret ers One =) Oe oO QA eS) :) faa) —— -—_ — ——_ | | eS — A. M. | 8:54 to 9:24 1070 | 24:3 | 34:3 | 41°5 72 133°4 3335 | 38°6 | 72 9:50 to 10:20 10-2 24-1 34:3 | 41-5 10:43 to 11:18 | 9-8 | 24:0 | 33-8 | 41:5 11:38 to 12:08 | 10-9 | 24:3 35:2 | 415 —_ (Jt) ow te Ww Ww Sa un ou) oe © P.M. | | 1:57 to 2:27 | 11°3 | 24:5 | 35°8 | 41-5 | By 105°6 264°0 | 34°7 Average, 10:4 | 24:2 | 34-6 | 41.5 | 6:9 126:3 | 37579 | 37-5 | | | | The following amounts of quinine were given by way of the mouth: April 12 5:20 p. m. 0:130 gram quinine sulphate. raeetile 9:10 a. m. 0-260 <“ ms a pete t00'p.-m. 07390 << as os CO re aks} 5:15 p. m. 0:520 = <* ee ss «14 8:45 a. m. 0°520 “* oe a eas I 12:00 m. 0-780 s¢° e oe 2-600 Rabbit died at 3 p. m. April 14. Following are the average daily results in temperature and in the amount of carbonic acid excreted per 37°5 litres of aspirated air. April 12 - 38°6° C. 317-8 milligrams CO,. Sai 3: aat,'** 325°6 oe oa SOB ee! ano 315°9 ae 432 Chittenden and Cummins—Influence of some Organic SECOND SERIES OF EXPERIMENTS WITH QUININE. Normal period, without quinine sulphate. | . Oxalic .acid to) po.) [een : feet Ps ‘ neutralize ba-| = a) < = pe | a 2 E . Date. ryta solution.) «5 |3'S 3 8 A hoe 2 le™ al og | 2 ey | & May 17 Cae B ,|eet| a § os 2 e a 5 Fo Oks Allee een | ori oo Seo) 28 faee| 22 | SF | Se | SP eS /Bes/ 2 [6 Pie 5 Des A.M. | 8:40 to 9:10 53 | 22°2 | 27°5 | 41°8 | 14:3 289-0 722°7 | 38°8 9:33 to 10:03 | 6:1] 22°8 | 28:9 | 41:8} 12:9 260°7 651°9 | 38°8 10:29 to 10:59 6°2 | 22°6 | 28°8 | 41°8 13°0 262°7 657°0 | 38°8 11:24 to 11:54 | 4:9 | 21-9 | 266) 41°38) 15:0 | 303-2 | 7582 | 80-4 - a P. M. 1:55 to 2:25 BO.) 220 Sed |e 4al-Bol Ss TA ood 742°9 | 39:0 2:48 to 3:18 | 4:5) 22:0) 265) 41:8 15-3 3092 | 773:2 | 38-9 8:43 to 413 | 67 | 21] 29-8) 418 12:0 | 2425 | 6o6rs | 38:8 4:35 to 5:05 | 6-7 | 28-0 | 29-7] 41:8) 124 | 244-5 | 6x27 | 38-9 Average, 57 | 22°4 |] 28-1 | 41°8 | 13:7 | 2763 | 6g0°5 | 386 May 18. With quinine sulphate. A.M. l . ‘7 | 41-8 | 18-1 | 2648 | 662°0 | 38°6 5 | 418 | 11:8 | 928-4 | s7xx | 88-4 22-1 | 27-7 | 41-8 | 14-1 | 285:0 | 7126 | 383 22:5 | 28:5 41-8 133 | 2688 672'2 | 388 9:05 to 9:35 6°2 | 22°5 | 2 LO to) 14:21 5'6 11:46 to 12:16 6:0 P.M. 2:14 to 2:44 6°5 10:00 to 11:30 75 | 28:0 | 3 | 4:58 to 5:28 | 6°0 | 22-1 | 28-1 | 41°8 | 18-7 276° 22:8 | 293 | 41-8) 125 | 252-6 | 63177 | 389 3:07 to 3:37 | 6-5 | 22-9! 29-4 41-8 | 19-4 | 249-6 | 62gcr | 39-0 4:01 to 4:31 | 7-0! 28-4] 304) 41-8] 11-7 | 2365 | sox-3 | 39-0 6 Average, | 6-4 22°6 | 29°0 | 418 | 128 | 257-8 | 644-7 | 387 | | a ee i eS ee ees and Inorganic Substances on Gas Metabolism. 433 With quinine sulphate—continued. Oxalic acid to Nae Rae by S | B. 3 neutralize ba-| © re gard Poke cs) Sogn ee Date. ryta solution.| «5 |3'S © a =| = | Seley S\ gat pe ee | 2. May 19. Md. jee | 29 a «© | 8 Boe Wu es (SSS Bo Ae SO GF a eS |$o6/ 82 eS) 83 | ce | oe | se gd |ees/4 |S Span as Ss |8 A.M. | ae Poel eal ed 9:02 to 9:32 f-3 |. 200) }-30°3' | 41°8 | 11-5 | 239-4 | s8x-2 | 389 Sage | | 9:57 to 10:27 6) 23°2\| 80-8") 41-8 | 11-0 | 222°8 | Ss55°o | 38-4 10:50 to 11:20 72 | 2371 | 30°3 | 41°38 | 11°5 | 282-4 | 58r°2 | 38-7 | 11438 to 12:13 | 6-9 | 23-0 | 29°9 | 41-8 | 11°9 | 240°5 | Gor-4 | 38°6 | ; P. M. 2:03 to 2:33 75 | 23°2 | 30°7 | 41°8 | 11°71 | 223°3 | 558:4 | 38:9 2:54 to 3:24 8-2 | 23°6 | 31°8 | 41:8 | 10°0 | 202-1 | 505°4 | 385 3:47 to 4:17 | 7-4 | 23:3 | 380-7 | 41°8| 11:1 | 2243 | s6r-o | 38°8 4:40 to 5:10 7A |: 23-1 | 80-2 | ‘41-8 |’ 11°6 | 284°5 | 586-3 | 38-8 Average, | 74 | 28:2 | 30°6 | 41:8 11°2 226°4 56674 | 38°7 The quinine was given by way of the mouth in gelatin capsules, in the following quantities : 1 May17 5:30 p. m. 0:250 gram quinine sulphate. ete Ufo, 8:50 a. m. 0:250 ‘ ee fs S18, 12:30 p. m. 0-250 “* a fs aA Us: 5:35 p. m. 0-250 ‘ fe re aalo 8:50 a. m. 0-325 ‘ SS F eer a 1:55 p. m. 0:250 * ie 1:575 Following are the average daily excretions of carbonic acid ex- pressed in milligrams of CO, per 37°5 litres of aspirated air, together with the average body tempeyature : * May 17. 38°6° C. 690°5 milligrams CO, q “e 18 38°7 66 644:7 ee ee ce 19 38°7 “é 566°4 oe “é Trans. Conn. AcaD., Vou. VII. 55 MARcH, 1887. 434 Ohittenden and Cummins—Influence of some Organic THIRD SERIES OF EXPERIMENTS WITH QUININE. Normal period, without quinine sulphate. ; : \ 4 a Oxalic acid to ie sis © 3 fn bin 2 ; neutralize ba- a |Bes ° Zz Sc dees Time. ryta solution.| © 5 |3o 9 a 4 & oO Sone ei r = 10 et a) a ra ee os ee | = oS a" x ~ March 8. ae Palau 8 Ss a = = os ~” = leet a ro] SD lone LON =| oo eae od |\2a5|\/ aa S23 a ae er BO 2. |28.| 85 (fe) o's oF oF Zo FEIN (he ac he i a= as) a) ea) A. M. 9:50 to 10:20 | 15:5 | 33:9 | 49:4 |,59°25 9°85 182°5 456°3 | 39°2 10:58 A. M. injected subcutaneously 0:088 gram quinine sulphate. 11:15 to 11:45 16°9 | 34:5 | 51:4 | 59°25 | 7:85 1454 | 363°5 | 384 12:25 to 12:55 15:3 | 33:9 | 49:2 | 59-25 | 10-05 186°2 | 465°5 | 39°9 P. M. 3:00 to 3:30 15°6 | 34:0 | 49°6 | 59°25 9°65 178°8 | 447°0 | 39°8 4:06 to 4:36 15°8. | 88:7 | 49-5 | 59°25 | 9°75 | 180°7 | 451°8 | 39°9 SS |. |W | | | Mm _ — | _ Average, ita. | 15:9. (3840 499 59°25 9°35 | 1728 432°0 | 39°5 FOURTH SERIES OF EXPERIMENTS WITH QUININE. Normal period, without quinine sulphate. . . 1 “4 . et : 2 Oxalic acid to = SS gS ses = PS} a a 2 neutralize ba- 3 sis S = ae = Time. ryta solution. = Ome = ) iE 4 B moO |BA 8] gid 2 rar a March 10. ae Hale ee| 38 wf oo 8 5 ees f SCS lope Do fS)) ao beara 3 |\2od| 82 |SaS| oO WP eae ce fe SS. |Sa.| se = |S3h| ga a8 Gaol tee 3 0 5 8 0 | = 5 = ) =) 1S) oO ia) A. M. } | 9:11 to 9:41 15:0 | 33°8 | 48°8 | 59°25 | 10°45 193°6 | 484°0 | 38°8 10:15 A. M. injected subcutaneously 0°15 gram quinine sulphate. 10:38 to 11:08 | 16°5 | 34°6 | 51:1 | 59:25] 8-15 151°0 | 377°5 | 38°4 11:39 to 12:09 15:4 | 33°9 | 49°3 | 59:25 | 9°95 1844 461'0 38:9 12:38 to 1:08 | 15°3 | 84:1] 49-4 | 59-25] 9:85 | 1825 | 4563 | 38-4 P. M. } 2:54 to 3:24 16:0 | 34°3 | 50°3 | 59°25) 8°95 165°8 | 414°5 39°1 4:07 to 4:37 15°9 | 34:1 | 50:0 | 59:25] 9:25 171°4 428°5 | 38°8 _ Average, 15°8 | 34:2 | 50-0 | 59°25] 9:25 | 171°0 | 427°6 | 38-7 and Inorganic Substances on Gas Metabolism. 435 EXPERIMENT WITH CINCHONIDINE. Normal period, without cinchonidine sulphate. Oxalie acid to ee 3 f: B : By neutralize ba-| BBs.) ae ao | & Date. ryta solution.) S95 | > apes 2 aS Aaa be Sh (ee ail as 2 | 1 5! 2 June 24. S |Soenr| Ss = | 5's =) Fe ais od eaoN anes a Bete al eg Se oe ee ieso sass) fe P| we | ee Be Wars wR hp eh) Sol he A. M. | 9:00 to 9:30 10°6 | 28°8 | 39:4 | 50:2 10°8 218°3 545°8 | 364 9:56 to 10:26 |- 11:2 | 29:1 | 40°3 |) 50:2) 9-9 200°1 500°3 | 37°4 10:47 to 1117 | 120 | 29-0) 41-0 502) 9:2 | 1859 465°0 | 381 11:38 to 12:08 10° 28-7 393 50-2 109 | 2203 5509 | 36-3 9:08 to 8:88 | 103 285 | 38-8 502 | 11-4 | 280-4 | s76-r | 86-9 2:51 to 3:21 9-4 | 28:2 | 87-6 502) 12°6 | 2549 | 636°8 | 37-1 3:42 to 4:12 9:7 | 28°5.| 38:2 | 50-2 | 12:0 | 2425 | 6065 | 37-0 4:30 to 5:00 | 9-7 | 285 | 38-2 | 50-2 | 12°0 | 242°5 | 606°5 | 36-4 Average, 10°5 | 28°6 391 DOAN aed 224°3 | 561°0 | 37-0 June 25. With cinchonidine sulphate. A. M. | 9:08 to 9:33 | 10°38 | 28:6 39-4 | 50-2) 108 | 2183 | 5458 38-6 9:58 to 10:28 | 114] 28:9 40:3) 50:2 9-9 | 200-1 | 500-3 38°6 10:58 to 11:28 | 11:1 | 98-7 | 39-8 | 50-2) 10-4 | 210-2 | 525°6 38-6 11:48 to 12:18 | 11-6 | 28-7 | 40°3| 50°2| 9:9 | 200-1 | s00°3 | 38-7 P. M. . 2:04 to 2:34 11:1 | 28°8 | 89°9 | 50°2 10°3 | 208°2 | 520°5 | 38°9 2:59 to 3:29 = 115 | 29:0 40°5 ee 97 | 196-0 | 4go°2 | 39:0. 0 3:56 to 4:26 | 11°6 | 29:0 | 40°6| 50:2 96 | 1940 | 485'2 | 39-0 4:50 to 5:20 | 12:1 | 29:1 | 41:2 | 50-2 | 9:0 | 181-9 | 454°8 | 89-1 Average, 11-4 | 28:8 | 40-2 | 50:2 | 10-0 201-1 502°8 | 38°8 Following are the average daily amounts of carbonic acid excreted, expressed in milligrams per 37°5 litres of aspirated air, together with the average body temperature. June 24, 37°0° C., 561:0 milligrams CO,:. oa e0; 38°8 502°8 - “e Following are the doses of cinchonidine : June 24, - §:12 P. M., 1:000 gram cinchonidine sulphate. eos Ob. 10:35 A. M., 0250/0 ss Le ee 20s 12:35 P. M., 0-325 «SS “ % 1°575 Rabbit died at 5:40 p. M., June 25, in convulsions. ‘436 Chittenden and Cummins—Influence of some Organic In the second series of experiments, smaller doses of quinine were employed, so that no special toxic action was observed. In this case there seemed to be a gradual falling off in the amount of carbonic acid produced; such a decrease as might be assumed would naturally result from diminished proteid metabolism, The results are shown in the accompanying tables. The body temperature, as determined per rectum did not show any change whatever under the influence of this quantity of quinine (total dose 1575 gram of quinine sulphate). There does not appear to be any proof that moderate doses of quinine lower the body temperature of healthy animals or even man, and Kerner found in his experiments that a full dose of quinine given to a healthy man would prevent the usual rise of temperature resulting from vigorous exercise, but did not affect the temperature under ordinary circum- stances. Liebermeister* has also reported that the alkaloid has no constant depressing action on the bodily heat in health. Two other short series of experiments were tried with quinine, also with rabbits in a condition of hunger. In both of these cases the quinine was introduced by sub-cutaneous injection in the form of . sulphate. In both cases there was a slight fall in temperature, accompanied with a noticeable decrease in the amount of carbonic acid eliminated, directly after injection of the quinine. This effect, however, was only temporary, for the temperature quickly rose to the normal, and even somewhat above the normal point, while the carbonic acid in the third series came quite back to the normal | and in the fourth series remained only a little way below. It would appear, therefore, from our experiments, that in a healthy, hungry rabbit moderate doses of quinine sulphate exercise at the most only a very slight depressing influence on body temperature, and have but a minimum effect on the production of carbonic acid. Action of cinchonidine sulphate. Previous experiments ¢ on man have shown that cinchonidine has the power of lessening materially the elimination of nitrogen, pre- sumably through its inhibitory action on proteid metabolism. Cinchonidine is supposed to have much the same physiological action as quinine and cinchonine, only weaker. Our present experi- * Deutsch. Archiv fiir Klinische Medicin, Band iii. + See Chittenden and Whitehouse, Studies from the Laboratory of Physiological Chemistry, vol. i, p. 164. and Inorganic Substances on Gas Metabolism. 437 ments with the alkaloid, using quite large amounts of the sulphate, show a somewhat different action from quinine. Using a large rab- bit, without food for three days, and giving it by way of the mouth, in gelatin capsules, large doses of the alkaloid, there was a very noticeable and constant rise in temperature up to the very time of death, accompanied by a slight but gradual diminution in the amount of carbonic acid given off. In all, 1°575 grams of cinchonidine sul- phate were given; an amount exactly equal to the quinine sulphate given in the second series of experiments with quinine. With cin- chonidine, however, the rabbit was much prostrated, showed symp- toms of tetanic convulsions, and finally died in a vigorous tetanic spasm at the end of the second day. The results are shown in the preceding table. Action of Antipyrine. ' Antipyrine or dimethyloxychinicine has of late been much experi- mented with. Among the many statements which we have seen recently concerning its action are the following, which are of interest in this connection. Arduin* found that 3 grams given to a rabbit produced cataleptic stiffness, diminished reflexes, etc., followed by violent convulsions. There was also a very marked fall of bodily temperature. Anseroff,t by experiments on animals, found that the alkaloid caused an increase of blood pressure and a decrease of inter- nal temperature, as shown by a thermometer in the rectum, but a considerable rise in the external temperature, sometimes as much as 12°C, Pavlinoff{ has reported that autipyrine produces a very con- siderable quickening of the respiration, while Dr. Walter, of St. Petersburg,§ is reported as having found that the alkaloid while reducing febrile temperature, also reduces nitrogenous tissue changes ; and further, that the assimilation of proteids is materially favored by the drug. F. Miiller|| has also found that in fever antipyrine dimin- _ ishes the excretion of nitrogen. Coppola, however, states that in the case of a dog, 0°3-0°4 gram of antipyrine was wholly without action on its excretion of nitrogen. Coppola has further found that the * Abstract in Therapeutic Gazette, 3d series, vol. i, p. 677. + Abstract in Therapeutic Gazette, 3d series, vol. ii, p. 315. + Abstract in Therapeutic Gazette, 3d series, vol. ii, p. 339. § Abstract in Therapeutic Gazette, 3d series, vol. ii, p. 53. || Jahresbericht fiir Thierchemie, xiv, 242. 4 Jahresbericht fiir Thierchemie, xv, 97. 438 Chittenden and Cummins—Influence of some Organic FIRST SERIES OF EXPERIMENTS WITH ANTIPYRINE. Normal period, without antipyrine. Date. May 24, iNET pease 8:54 to 9:24 9:52 to 10:22 10:47 to 11:17 P. M. 1:55 to 2:25 2:47 to 3:17 3:40 to 4:10 4:35 to 5:05 Average, May 25. A. M. 8:56 to 9:26 9:58 to 10:23 10:49 to 11:19 11:47 to 12:17 P. M. 2:15 to 2:45 3:15 to 3:45 4:15 to 4:45 5:09 to 5:39 Average, May 26. A. M. 9:02 to 9:32 9:59 to 10:29 Oxalie acid to Bs 6 neutralize ba-| © 5 cab ei de ryta solution.| & cules ees 25 |SA al go s Som! oo owen RS | or rom As dee, Ae = eS Dig, 2° |o35| 38 feee| 5 oe ssn Cus = Bas Mess iets 5 =) BH (=) =) | 8-6 | 26-4 | 35-0 | 46-1 | 11:1 8:8 | 26°5 | 35°3 | 46-1 | 10°8 9:5 | 26-4 | 35-9 | 46-1 | 10-2 9:1 | 26:3 | 35-4 | 46-1 | 10°7 9:5 | 26-4 | 35°9 | 46-1 | 10:2 9:1 | 26°5 | 35°6 | 46-1 | 10°5 8°8 | 26-3 | 35-1 | 46-1 | 11-0 9:0 | 26-4 | 35:4 | 46-1 | 10-7 With antipyrine. 9-6 | 26-4 | 36-0 | 46-1 | 10-1 9:6 | 26-4 | 36-0 | 461 | 107 7-5 | 25°6 | 38-1 | 46-1 | 13-0 8:4 | 25:9 | 34:3 | 46-1 | 11°8 79 | 26:0 | 33-9 | 46-1 | 12-2 87 | 25:8 | 34:5 | 46-1 | 11°6 8:4 | 26°0 | 84:4 | 46-1 | 11-7 7-4 | 25:6 | 33:0 | 46-1 | 18-1 8:5 | 25°9 | 84-4 | 46-1 | 11°7 | | | 11°3 | 26°8 | 88-1 | 46-1 8-0 | 9:4 26-0 (85-4 | 461 | 10-7 mg. | CO, in a, b and e. e. ; F ® o ea) 561°0 | 384 545°8 38-4 513°0 | 38-4 540°8 38°3 513°0 | 38°6 530°6 | 38-4 555°9 | 384" 535°7 | 38-4 | 510°4 38°2 510°4 | 3881 657°0 | 38:0 596°3 | 381 616°6 | 381 586°3 | 38:3 591°3 | 38-0 662°0 | 881 591°3 38-1 404°3 | 36°2 540°8 | 35°83 4 ee > and Inorganic Substances on Gas Metabolism. 439 alkaloid not only has a noticeable antipyretic action in conditions of fever, but also reduces the temperature in healthy organisms. The extent of reduction, however, is not great, ranging only from 0°1 to 0°6 of a degree. Further, the diminution in temperature is to be ascribed, according to Coppola, not to diminished metabolic activity, but to increased giving up of heat, due to dilatation of the blood- vessels by the antipyrine. Jacubowitsch * also claims for antipyrine an inhibitory action on the excretion of uric acid. So far as our knowledge extends, however, no experiments have been tried as to _ the influence of this therapeutic agent on the production of carbonic acid. Our experiments have been confined wholly to rabbits, and those in a condition of hunger. In the first series of experiments, during the antipyrine period, the alkaloid was given in large and oft-repeated doses, in the form of powder, in gelatin capsules, at follows: May 25, 8:33 A. M., 0-2 gram antipyrine. eo: 9:35. °* OrDit sc8 a i 20, 10230. = Q-22) fe ws “S, 25; AOE 1S 0-205 Cray 12:27 P. M., 0:6, Globulose Bodies, 212. Glucose, Dehydration of, 252-259. Glycogen, 184, 185. Heteroalbumose, 347. Heterocaseose, 400. Heteroglobulose, 215. nium Salts on the Amylolytic Action of Saliva, the Proteolytic Action on Pepsin and Trypsin, 261-273. Hyptiotes, 454, 455, 456. Americanus, 444. cavatus, 444, 456. Influence of Antimonious Oxide on Met- abolism. R. H. Chittenden and Jo- seph A. Blake, 293-300. Bile, Bile Salts and Bile Acids on | Amylolytic and Proteolytic Action. | R. H. Chittenden and Geo, W. Cum- mins, 134-148. Certain Therapeutic and Toxic Agents on the Amylolytic Action of Saliva. R. H. Chittenden and H. M. Painter, 60-83. Cinchonidine Sulphate on Metabol- ism. R. H. Chittenden and Henry H. Whitehouse, 166-178. Potassium and Ammonium Bro- mides on Metabolism. R. H. Chit- tenden and W. L. Culbert, 153-165. Some Organic and Inorganic Sub- stances on Gas Metabolism. R. H. Chittenden and G. W. Cummins, 407-442. Temperature on the Relative Amy- lolytic Action of Saliva and the Dias- tase of Malt. R.H. Chittenden and W. E. Martin, 125-133. Uranium Salts on the Amylolytic Action of Saliva and the Proteolytic Action on Pepsin and Trypsin. R. H. Chittenden and M. T. Hutchinson, 261-273. Various Inorganic and Alkaloid Salts on the Proteolytic Action of Pepsin-hydrochloric Acid. R. H. Chittenden and 8. E. Allen, 84-107. | | 461 Influence of Various Therapeutic and Toxic Substances on the Proteolytic Action of the Panereatic Ferment. R. H. Chittenden and Geo W. Cum- mins, 108-124. Iodide, Mereuric, 62, 88, 110. Potassium, 71, 104, 119. Tron compounds, 314, 326. Juice, gastric, 223. pancreatic, 230. Knots, with a Census for Order Ten, C. N. Little, 27-43. Kiihne, W., Globulin Bodies, 206-219. Peptones, 220-251. and Globulose Lambert, Alexander, Post-mortem For- mation of Sugar in the Liver, in the presence of Peptones, 179-206. | ? . aS) . > Hutchinson, M. T., Influence of Ura- | ba¥ Poaeror ny Tarepie Shooting: (Eh L. DeForest, 1-8. Lead acetate, 64, 86, 111, 313. compounds, 312. Little, C. N., On Knots, with a Census for Order Ten, 27-43. | Malt, Diastase of, 44-57, 125-133. Magnesium sulphate, 69, 94, 114. Manganous chloride, 91, 114. Martin, W. E., Influence of Tempera- ture on the Relative Amylolytic Ac- tion of Saliva and the Diastase of Malt, 125-123. Mercuric bromide, 62, 88, 110. chloride, 62, 79, 86, 109. cyanide, 62, 88, 110. iodide, 62, 88, 110. Mercury compounds, 317, 330. Metabolism, Influence of Bromides on, 153-165. Influence of Antimonious Oxide on, 293-300. Influence of Cinchonidine Sulphate on, 166-178. Metallic Compounds of Albumin and Myosin. R. H. Chittenden and Henry H. Whitehouse, 301-331. Metallic Salts, 78. Mithras, 456. Moore, Jr., Eliakim Hastings, Extension of certain Theorems of Clifford and of Cayley in the Geometry of x Dimen- . sions, 9-26. Morphine sulphate, 72, 120, 425. Myosin, Metallic compounds of, 301-331. Narcotine sulphate, 121. Neutral peptone, 48. solutions, 143. New England Spiders of the Family Ciniflonidee, 443-458. 462 Nickel compounds, 328. Nitrate, Potassium, 70, 96, 117. Uranyl, 262, 267, 410. Organic and Inorganic Substances, in- fluence of, on Gas Metabolism, 407— 442, Oxalate, Ammonium, 98. Oxide, Antimonious, 288, 293-300. Arsenious, 64, 89, 111, 418. Oxychloride, uranic, 264, 269, 272. Painter, H. M., Casein and its Primary Cleavage Products, 362-405. Influence of certain Therapeutic and Toxic Agents on the Amylolytic | Action of Saliva, 60-83. Pancreatic Ferment, Proteolytic Action of, 108-124. juice, 230. Pepsine, 140, 224, 261-273. Pepsin-hydrochlorie acid, Influence on, 265. Proteolytic Action of, 84-107. Peptone, Acid, 54. Gland, 235, 237. 239. Neutral, 48. Peptones, 179-206. W. Kiuhne and R. H. Chittenden, 220-251. Permanganate, Potassium, 68, 94, 115. Phillyra, 454. ripaira, 454, Phosphotungstic acid, 244. Post-mortem Formation of Sugar in the Liver, in the presence of Peptones. R. H. Chittenden and Alexander Lam- bert, 179-206. Potassio uranic oxychloride, 264, 269, 22. Potassium antimony tartrate, 66, 113, 419. bromide, 71, 104, 119, 153-165. chlorate, 70, 96, 1177. chloride, 101, 118. cyanide, 69, 95, 115. dichromate, 95, 115. ferricvanide, 70, 116. ferrocyanide, 69, 95, 116. iodide, 71, 104, 119. nitrate, 70, 96, 117. permanganate, 68, 94, 115. Proteids, Acid, 51, Proteolytic action, Influence of Bile on, | 134-148. of Pancreatic Ferment, Influence of various Substances on, 108-124. of Pepsin, Influence of Salts on, 261-273. of Pepsin-hydrochlorie Acid, Influ- ence of various Salts on, 84-107. Protoalbumose, 342, 351. Protocaseose, 381, 384, 387, 393. Protoglobulose, 212. INDEX. Quinine sulphate, 72, 122, 428. | Relative Distribution of Antimony in the organs and tissues of the body under varying conditions. R. H. Chitten- den and Joseph A, Blake, 275-292. Saliva, Amylolytic Action of, 60-83, 125-133, 261-273. Salts, Alkaloid, 106, 120. | Influence of various, 84-107. Nature of the action of, 78. — Uranium, Influence of, on Ferment Action, 261-273. Silver compounds, 318. Smith, Herbert E , Absorption of Arsenic by the Brain, 149--152. Sodio uranic sulphate, 264, 268, 272. Sodium carbonate, 45, 47, 49.- chloride, 71, 100, 118. sulphate, 117. tetraborate, 71, 97, 116. Solutions, Acid, 143. Alkaline 143. Neutral, 143. Stannous chloride, 67, 89, 111. Steatoda borealis, 453. Strychnine sulphate, 75, 121. Sugar in the Liver, Post-mortem For- mation of, 179-206. Sulphate, Atropine, 74, 121. Brucine, 75, 121. Cinchonidine, 73, 122, 166-178, 436. Cinchonine, 73, 122. Cupric, 63, 80, 85, 111, 418. Ferrous, 68, 92, 113. Magnesium, 69, 94, 114. Morphine, 72, 120, 425. Narcotine, 121. Quinine, 72, 122, 428. Sodium, 117. Strychnine, 75,121. 4 Uranie, 264, 268, 272. Uranous, 263, 268, 272. Uranyl], 268, 271. Zine, 67, 80, 91, 114. Target-shooting, Law of Error in, 8. L. DeForest, 1-8. Tartar emetic, 281, 282, 284, 288. Tartrate, Potassium antimony, 66, 113, 419. Tegenaria, 450. Tetraborate, Sodium, 71, 97; 116. Tetragnatha, 454. Theorems of Clifford and Cayley, 9-26. | Therapeutic and Toxie Agents, Influence of, on Saliva, 60-83. Substances, Influence of, on Pan- creatic Ferment, 108-124. Therididve, 443, Theridion, 444. foliaceum, 444, 450, morologum, 444.” INDEX. Theridion, sublatum, 444. Titanceca, 453. americana, 453, 454. brunnea, 453. quadriguttata, 453. Trypsin, 119, 120, 143, 147. Influence of Salts on, 261-273. Uloboridze, 443. Uloborine, 443, 454. Uloborus, 454, 456, 457. plumipes, 454. walckenzerii, 456. Uranic citrate, Ammonio, 264, 268, 272. oxychloride, Potassio, 264, 269, 272. sulphate, Sodio, 264, 268, 272. Uranium compounds, 316, 329. Salts, Influence of, on Ferment Ac- tion, 261-273. 463 Uranous sulphate, Ammonio, 263, 268, 272, Uranyl acetate, 263, 267. nitrate, 262, 267, 410. sulphate, 268, 271. Veleda, 454. Whitehouse, Henry H., Influence of Cinchonidine Sulphate on Metabolism, 166-178. On Some Metallic Compounds of Albumin and Myosin, 301-331. Zine compounds, 316, 327. sulphate, 67, 80, 91, 114. =e éor fe Ret \ : Oye ed etnias’ mae Peal : | 49 7 Py 7 A Rat a * 7 . th ne i ’ . ’ a , \ ; ~ 4 ' "(oie ey 7. ; As - . . rete | : ave phere 1" (4 (ae ; , A os x . 7 « ‘ ace Mb Phi) i ‘ ; ¥ - fe ' 5 i . — F > 7 ’ Pe (eile ; i ‘ dl 7 j ; i 7 ’ * ‘ : Fr F & ‘ : \ : iY : = + i ' * rr ; ¥ _ ue j | A ‘ ¢ ~ ’ F " * : ‘ 7 7 é- > i iw s La. Lbs hay A , 7 ; " i i : bd Z eo = +? . t : 2 ny - men i 1 uae: lid 2 had, mie, ¥ 7 “NT . - tz l A ~~ i A t an ; -\ 7 rat: y ee * Ae nd if A, , { i ; aie - fi i P i NA 7 ' . ‘ 4 : is any, : ne} an a Ne BINDING SECT. SUN 9 1971 Q Connecticut Academy of Arts 1: and Sciences, New Haven ve7 Physical & Applied Sa. PLEASE DO NOT REMOVE CARDS OR SLIPS FROM THIS POCKET —————— UNIVERSITY OF TORONTO LIBRARY STORAGE ta 4 ari "hey oy ar ° esi enor serge ee pis Tbr peter i! 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