COLUMBIA LIBRARIES OFFSITE HLAI IH Si II Nl I s M ANDAMI) HX64141250 QP514.H151 Aiext-bookol( RECAP m CHEMICAL PHYSIOLOGY AND PATHOLOGY PRINTED BT SrOTTISWOODE AND CO., NEW-STREET PQirARE LONDON A TEXT-l'.dOTs' CHEMICAL PHYSIOLOGY PATHOLOGY BY W. D. HALLIBUETON, M.D., B.Sc, M.E.C.P. PROFESSOl; or PHYSIOLOOT at king's college, LONDON' LKCTURER ON PHYSIOLOGY AT THE LONDON' SCHOOL OP MEDICIXE FOR WOMEN' I.AIE ASSISTANT PROFESSOR OF PHYSIOLOGY AT UNIVERSFfY COLLEGE, LONDON WITH 104 ILLUSTRATIONS LONDON LONGMANS, GEEEN, AND CO. AND NEW YORK : 15 EAST 16"> STREET 1891 All righls reserved Digitized by the Internet Archive in 2010 with funding from Open Knowledge Commons (for the Medical Heritage Library project) http://www.archive.org/details/textbookofchemicOOhall PEEFACE It is some years since a complete textbook of Cliemical Physiology has appeared in the English language, and the rapid strides that have been made in this department of science appear to me to justify the produc- tion of a new work on the subject. It is with the object of filling this gap in our literature that I have written this book. I have selected the title Chemical Physiology in preference to that of Physiological Chemistry, as the subjects are treated rather from the point of view of their function in the body, than fn)m that of their chemical relationships to one another ; hence the book deals with a department of Physiology more than with a department of Chemistry. I have added the words ' and Pathology ' to the title, as I have endeavoured to include the chief facts in relation to the blood, urine, and tissues which have a chemico-pathological bearing. I am in hopes that the book may be not only useful to students of physiology, and those pursuing original investigations in chemical physiology, but also to the student of practical medicine and the medical practitioner. In the preparation of this volume I have received much help from a large number of friends, among whom I would especially mention Prof. ScHAFER, to whom I owe a number of valuable .suggestions and references ; Dr. Sidney Martin, who read my manuscript of the chapters on proteids, foods, diet, and pathological urines, and made both corrections and suggestions ; Dr. MacMunn, who kindly read the proofs of the sections relating to the pigments of the bile and urine, and Dr. R. N. Wolfenden, who generously placed in my hands the manuscript of a number of valuable tables relating to the urine ; these have formed the basis of the tables given on pp. 712 to 713, 716 to 719, 745 to 746, and 784 to 785. The books and original monographs that have been consulted have been very numerous. T believe it will be found that in all cases I have vi PHlsFACE given my authority for any statements T liave made, and I take this o})portunity of thanking all those who have unwittingly, by their researches and writings, thus aided me in the production of this volume. The illustiations are also culled from various sources, and I have to tliank the following authors and publishe)-s for the loan of blocks, or for permission to use certain illustrations : Dr. Dupr^, Prof. M. Foster, Mr. A. W. Gerhard, Mr. C. E. Groves, Prof. McKendrick, Prof. Schafer, Dr. Schunck, Mr. F. Suttox ; the Council of the Chemical Society ; Messrs. Churchill, Messrs. EN(iEL.MAN\ of Leipzig, Messrs. Macmillan, Messrs. Viewe(; and Sox of P>i-unswick, and Mr. Hawksley. The duty of reading the proof sheets has been greatly lightened for me by my friend Mr. C. J. Martix, B.Sc, Demonstrator of Physiology in King's College, who read the tinal revises, and I have to thank him for many valuable suggestions and alterations. W. D. HALLIBURTON, King's College : Octoher I, 1890. CONTENTS PART I METHODS OF RESEARCH AND ANALYSIS CHAPTER I APPARATUS, REAGENTS, WEIGHTS AND MEASURES , . 3 CHAPTER II ANALYTICAL METHODS Gravimetric, and volumetric analysis . . . . . . . .7 Filtration 10 Washing precipitates . . . . . . . . . . .10 Drying 11 Cooling after drying ........... 11 Incineration ............. 12 Evaporation . 12 Boiling 12 Di-stillation i;^ Dialj'sis .............. IH Determination of specific gravity . . . 15 Determination of reaction. Alkalimetry, acidimetry ..... 16 The centrifugal machine . . . 17 Determination of relation of solids and water in any substance , . .18 CHAPTER III ULTIMATE ANALYSIS OF ORGANIC COMPOUNDS Introductory 19 Tests for nitrogen ........... 19 Tests for sulphur ............ 19 Tests for phosphorus ........... 20 Quantitative analysis of substances consisting of carbon and hydrogen, or of carbon, hydrogen, and oxygen ........ 20 Determination of the carbon and hydrogen in nitrogenous substances . 21 Determination of the nitrogen in organic compounds ..... 22 vm CONTENTS Pik.UK Analysis of organic compounds containing sulphur 24 Estimation of phospliorus in organic substances 25 Determination of iron in organic substances 25 To deduce empirical formula; from percentage compositions . . . 26 CHAPTER IV GAS ANALYSIS Methods of collecting material intended for ga-; analysis .... 28 The extraction of the gases from the materials under investigation . . 30 Analysis of the gases 32 CHAPTER V OPTICAL IXSTKUMKXTS USED IN CHEMICO-PHYSIOLOGICAL INVESTIGATIONS The microscope . 36 Polarisation of light, and polarisers 3 Relation between circular polarisation and chemical constitution . . 44 The spectroscope 45 The spectrojihotometer 50 The spectropolarimeter 53 PAET II THE CHEMICAL CONSTITUENTS OF THE ORGANISM CHAPTER VI INTRODUCTORY 57 CHAPTER VII INORGANIC COMPOUNDS Water . . . 5S Hydrogen peroxide, sulphuretted hydrogen, ammonia ..... 59 Acids 60 Salts 60 GHAPTER VIII THE SIMPLER ORGA^^C PROXIMATE PRINCIPLES The paraffins and their derivatives (alcohols, acids, fats, &c.) ... 64 Aromatic oompounds ........... 74 Nitrogenous organic compounds (amido-acids, bile-acid>, uric acid group. Sec.) '80 CONTENTS Intioductoiy . Dextrose Levulose Galactose Inosite . . Cane sugar Lactose . Maltose . Starch Dextrin . Glycogen Cellulose Gums and other car Glucosides Glvcuronic acid CHAPTER IX THK CARBOHYDRATES )ohv lrat( I'ACE 92 '.H !)9 100 100 101 102 103 104 105 106 107 108 109 109 CHAPTER X THE PROTEIDS Introductory ...... Composition and constitution of the proteids Tests Quantitative estimation . Classification . . . Proteids as poisons Tables illustrating methods of testing for proteids 111 112 117 126 127 137 139 CHAPTER XI THK ALBUMINOIDS. FERMENTS, AND PIGMENTS The albuminoids The ferments The pigments 143 146 146 CHAPTER XII FERMENTATION Introductory ............. 151 Unorganised ferments . . .158 Organised ferments . . .161 CHAPTER XIII PTOM.UNES AND LEUCOMAINES Introductorj' Methods of separation of ptomaines General properties of the animal alkaloids Enumeration of the animal alkaloids 169 175 177 177 X CONTENTS PART III THE TISSUES AND ORGANS OF THE BODY CHAPTER XIV THE C'KLL PAGE Introductory ............. 183 Protoplasmic movement .......... 186 Physical and chemical properties of protoplasm ...... 190 The nucleus at rest and during division ....... 194 Functions of cells 205 Functions of the nucleus . 207 Comparison of animal with vegetable cells ... ... 208 Appendix^chlorophyll 211 CHAPTEE XV THE BLOOD Introductory ............. 217 Coagulation of the blood 221 The plasma ............. 227 The serum ............. 230 Fibrin 231 Fibrinogen ............. 284 Serum-globulin ............ 236 Fibrin-ferment 239 Historical account of the theories of coagulation 242 iSerum-albumin ............ 245 Extractives of the plasma and serum 251 Inorganic constituents of the plasma and serum 254 White blood-corpuscles or leucocytes 257 Blood-tablets 261 Red blood-corpuscles 262 Haemoglobin ............ 267 Compounds of hfemoglobin 274 Estimation of hemoglobin 282 Composition of hfemoglobin ........ . . 286 Tests for blood 295 CHAPTEK XVI THE BLOOD IN DISEASE Introductory ............. 297 The blood in anaemia 298 Conditions in which the coagulation is abnormal 305 The blood in inflammation . . . . . . . . • • 306 Parasites in the blood ........... 308 The blood in diseases of various organs . . . . . • .311 Haemoglobin crystals in septic diseases . ■ . 315 CONTENTS XI THE BLOOD OF INV AFTER XVII EKTKBKATK ANIMALS Intniiliii'litry Tlif hloixi of echinoderms The blood of worms Hiuinot'vaiun 'J'h«> hlood of molluscs Tho hlood of Crustacea The hlood of arachnida The hlood of insects PAGE 816 H18 :ny 321 823 324 328 328 CHAPTER XVIII LYMPH AND ALLIED FLUIDS Introductory . Lymph .... Chyli' .... Lymph in serous cavities in healtl Dropsical fluids Peritoneal tiuid Pleural fluid Pericardial tiuid Hydrocele tiuid The tiuid of subcutaneous cedema The aqueous humour Perilymph and endolymph Synovia .... The fluid in ovarian cysts The fluid in hydronephrosis . The fluid in hydatid cysts The amniotic fluid Cerebro-spinal fluid Pus 331 333 335 338 339 342 346 347 348 349 350 351 351 352 353 354 354 355 361 CHAPTER XIX RESPIRATION Introductory .... . . The gases of respiration .... The gases of the blood . . . • Tissue respiration .... The gases of lymph, chyle, and similar fluid: Cutaneous respiration .... FtEtal respiration .... Respiration in flshes .... 365 366 378 390 392 394 394 395 Xll CONTENTS CHAPTEE XX MUSCLE Introductory ....... Microscopic .study of niuscular lihres Chemical composition of muscle .... Muscle-plasma and muscle-serum .... Pigments of muscle ...... Extractives of muscle (creatine, glycogen, lactic acid, &c.) Inorganic constituents of muscle .... Gases of muscle ....... Contraction of muscle (summary) .... Effects of muscular contraction on the urine Ayjpendix — Electrical organs .... I'AGK 398 399 405 40(> 417 418 427 428 431 436 440 CHAPTEK XXI KPITHKLIUM Introductory ............. 441 Pavement ejjithelium ........... 442 Columnar epithelhim .I.43 Ciliated epithelium ........... 443 Mucus .............. 444 Secreting epithelium ........... 448 Compound e])itlielia 45I Keratin .............. 459 Melanin .............. 453 Turacin 454 Skeletins (chitin,=conchiolin, &c.) 454 Tunicin 456 The retina 456 CHAPTEH XXII THE CONNECTIVE TISSUES Introductory 4fi6 The cells of connective-tissue . 47O The white fibres ; collagen and gelatiti 470 The elastic fibres ; elastin 473 The ground substance ; mucin . 475 Cartilage ; chondrin . . . . 481 Cartilage of invertebrate animals . 485 Hyalins and hyalogens 486 Adipose tissue (fat) 487 Bone 492 Dentine, enamel, and other calcareous and skeletal stnictures . . . 495 The fat of marrow ■ . 496 CUNTKN rs CHArTEll XXI 11 THF, t'ONNECTIVK TISSUES I.N DISKASK PACK Introductory 497 Pigments of melanotic .sarcoraata ......... 4i)9 Myxcfidema ............. 501 Gout 508 Rickets 510 Mollities ossium, or osteomalacia ......... 512 Brittle bones 513 Caries and necrosis . oKS CHAPTER XXIV THE NERVOUS SYSTEM Introductory ............. 514 General composition of nervous structures . . . . . . .516 The proteids of nervous tissue ......... 528 The phosphorised constituents of nervous tissue (lecithin, protason) . . 524 Cholesterin ............. 531 Cerebrins 533 CHAPTER XXV THE ORGANS OF THE BODY Introductory 535 The liver (composition, proteids, glycogen, &c.) ...... 536 The spleen 553 The lymphatic glands . 555 The thymus 556 The thyroid and suprarenal body 557 The pancreas and salivary glands 558 The kidneys 559 The lungs 560 The testis 561 The ovary 564 The eye 564 The ear, the skin 566 CONTENTS PART IV ALIMENTATION CHAPTER XXVI FOOD PAGE [iitroductory 569 The proximate principles of food 569 Milk 572 Eggs 592 Meat 596 Vegetable foods 597 Accessories to food (alcohol, tea, coffee, kc.) COO CHAPTER XXVII DIET (502 CHAPTER XXVIII THE DIGESTIVE JUICES AND THEIR ACTION 012 CHAPTER XXIX Introductory The physiology of salivary secretion The structure of the cells that secrete salivii The composition of saliva The action of saliva .... 616 616 619 622 626 CHAPTER XXX 0.4STRIC JUK.'E Introductory • • The physiology of the secretion of gastric juice .... The .structure of and changes in the cells tliat secrete gastric juice The composition of gastric juice . . . . . The action of gastric juice (;30 631 6n8 637 648 CHAPTER XXXI DIGESTION IN THE INTESTINES G52 fn NIK NTS xv CHAITEIJ XXX I [ THK SKCRKTION OK TKi: l'AN( KKAS 1'Ai;K Jiitro(lucti)ry ............. 6.54 Composition of pancreatic juice ......... OM't The action of pancreatic juice on foods ....... 05!) CHAPTER XXXIII SUCCUS ENTKRICUS 004 CHAPTER XXXIV riiLK Introrlucton- 668 The secretion of bile ........... 668 The characters of bile and its constituents . 674 The uses and fate of bile.in the intfestine . . . . . . . 686 Abnormal and pathological conditions in bile-formation .... 688 The secretion of the gall-bladder ......... 689 The invertebrate liver 690 CHAPTER XXXY PUTREFACTIVE PROCESSES IN THE INTESTINE . . 091 CHAPTER XXXVI THE F.ECES ...... G!);") CHAPTER XXXVII ABSORPTION ..... 700 PART Y EXCBETION CHAPTER XXXVIII THE URINE The kidney 709 General characters of urine 711 CHAPTER XXXIX UREA, URIC ACID, AND ALLIED SUBSTANCES Introductory 720 Urea 721 XVI CONTEXT^ Uric acition spectrum of myohaematin Goblet cells ..... Alveoli of serous gland . Pigmented epithelium of retina A rod and a cone .... Absorjition spectra of retinal pigments Fat-cells ..... Calcium carbonate ciystals from an otolith Corpora amylacea .... Cholesterin crj'stals Hepatic cells with glycogenic deposit Cellular constituents of colostrum Alveoli of serous gland . Mucous cells ..... A simple cardiac glaud . Alveoli of jjancreas Diagram of intestinal fistula . Origin of bile canal iculi within hver-cells Absorption spectra of bile-pigments Klein . Langley Max Schultze Max Schultze Schdfer Frey Frey Frey Heidenhain Heldenhain . Langley . Langley . Langley Kiihne and I^ea Kupffer PA'.E no 94 101 102 119 195 196 199 200 201 202 215 221 2.54 263 264 270 271 276 277 283 291 293 303 367 368 888 889 401 401 418 445 450 458 460 463 488 496 496 531 540 574 620 621 634 655 664 670 685 XX LIST OF ILT.rsTliATIONS Kri. 89. Villus of rat killed during fat absorption 90. Mucous membrane of frog's intestine durint 91. Crystals of urea ..... 92. Crystals of nitrate and oxalate of urea . 93. Crystals of uric acid .... 94. Crystals of acid sodium urate 95. Crystals of acid ammonium urate . 96. Crj'stals of hippuric acid 97. Absoi-ption specti-a of urinary pigments 98. Crystals of calcium oxalate . 99. Crystals of triple phosphate , 100. Dupre's urea apparatus .... 101. Garrard's urea apparatus 102. Garrard's percentage glj'cosomet er . 103. „ „ ,. „ . . ]0i. Dulongs Calorimeter .... fat absorption r.VGK . Schiifer 704 . Schiifer 704 Frey 721 Freij 722 Frey 728 Frey 731 Frey 731 Frey 739 748 Frey 755 Frey 762 Diqyrc 812 . Gerrard 813 . Gerrard 815 ,, 815 McKtndrick 850 Errata On p. 86, the formula for Cystin is wrongly given. It should be C-JT^NSO,. Its formula and constitution are correctly given on pp. 768, 769. On p. 303, the formula for Spermine (Charcot's crystals) is incorrectly given. It should be C.jHjX. This is given correctl}- on p. 563. PART I METHODS OF RESEARCH AND ANALYSIS CHAPTI'K I APJ'.iinrrs. jii:a(h:.\ts, wkights axjj mkascjujs ' Tin: iiit'tliods (if analysis of, and modes of exannnini;- the sul)stances of Mliicli tlic l)(idy is roniposcd vaiy a good deal from those 2 grain 1 centigramme = 0-01 „ = 0-1 54:523 „ 1 decigramme = 0"1 „ = 1-543235 „ 1 gramme = 15'43235 grains 1 decagramme = 10 grammes = 154'3235 „ 1 hectagramme=100 „ = 1 543-235 „ 1 kilogramme = 1000 „ = 15432-35 = 21bs. 3oz. ll!}-8 „ Measures of Length. (English system.) 1 inch = 25-4 millimetres 1 foot =12 inches = 304-8 millimetres (Metric system.) The standard of length is a metre ; subdivisions and multiples of which, with the prefixes milli-, centi-, and deci- on the one hand, and deca-, hecta-, and kilo- on the other, have the same relation to the metre as the subdivisions and multi- ples of the gramme, in the table just given, have to the gramme ; thus : 1 millimetre =0-001 metre = 0'03i)37 inch 1 centimetre = 0-01 „ = 0-3937 „ 1 decimetre =0-1 „ = 3-93707 inches 1 metre = 39-37079 „ Measures of Cajiacittj. (English system.) 1 minim = 0059 cubic centimetre 1 fluid drachm = 60 minims = 3-549 cubic centimetres 1 fluid ounce = 8 fluid drachms = 28-396 „ 1 pint = 20 fluid omices =567-936 „ 1 gallon = 8 pints = 4-54837 litres (Metric system.) In the metric system the measures of capacity are intimately connected with the measures of length ; we thus have cubic millimetres, cubic centimetres, and so forth. The standard of capacity is the litre, which is equal to 1,000 cubic centimetres ; and each cubic centimetre is the volume of 1 gramme of distilled water at 4° C 1 cubic centimetre (generally written c.c.) = 16-931 minims 1 litre = 1,000 cubic centimetres = 1 pint 15 oz. 2 drs. 1 1 min. = 35-2154 fluid ounces 1 cubic inch = 16-386 c.c. ^ 4° C. is the temperature at which water has the greatest density. For practical purposes, measures are more often constructed so that a cubic centimetre holds a gramme of water at 16° C, the iisual temperature of a room. The true c.c. contains only O'OOt) trramme at 16° C. Al'l'AKATlS. KKACKNTS, WKl(iHTS AND .MEASURES THEKMOMETKIC SCALES Tlio scale most frequently used in this country is tlie Fahroulieit scale ; in which the freezing point of water is ii'I", and tlio boiling p<»int 212°. On the Continent the Reaumur scale is largely employed ; in which the freezing point is 0^ C, the boiling point SO'. In scien- tific work the centigrade scale has almost completely taken the ])lace of these ; in this system the freezing point is 0°, and the boiling point 100^ To convert degrees Fahrenheit into degrees centigrade, subtract 32 and multiply by §, or C = (F— 32)^. Conversely, degrees centigrade may be converted intt) degrees Fahrenheit by the following formula : F=gC+32. TENSION OF AQUEOUS VAPOUR IN MILLIMETRES OF MERCURY FROM 10° TO 25° C. 10°. 9 12G U=. 11-882 18°. 15-351 22='. 19-G75 11°. !i-751 15°. 12-677 19°. 16-345 23°. 20-909 12°. 10-421 16°. 13-519 20°. 17396 24°. 22-211 13°. 11130 17°. 14-409 21°. 18-505 25°. 23-582 These numbers are used in correcting measurements of wet gases — for instance, of the nitrogen obtained from urea by tlie hj-pobromite method. TABLE OF THE DENSITY OF WATER AT TEMPERATURES BETWEEN 0° AND 30° C. 0°. 0-99988 1°. 0-99993 2°. 0-99997 3°. 0-99999 4°. 1-00000 5°. 0-99999 6°. 0-99997 7°. 0-99994 8°. 0-9998S 9°. 0-99982 10°. 0-99974 11°. 0-99965 12°. 0-99955 13°. 0-99943 14°. 0-99930 15°. 0-99915 16°. 0-99900 17°. 0-99884 18°. 0-99866 19°. 0-99847 20°. 0-99827 21°. 0-99806 22°. 0-99785 23°. 0-99762 24°. 0-99738 25°. 0-99714 26°. 0-99689 27°. 0-99662 28°. 0-99635 29°. 0-99607 30°, 0-99579 SYMBOLS AND COMBINING WEIGHTS OF THE PRINCIPAL ELEMENTS Aluminium Al 27-3 Fluoj'ine Fl 191 Fhospliorus - P 30-96 Antimony Sb 122 0 Gold Au 19G-2 Platinum Pt 196-7 Arsenic As 749 Hydrogen H 1-0 Potassium K 39-04 Barium Ba 136-8 Iodine I 126-53 Silver Ag 107-66 Bismutli Bi 210-0 Iron Fe 55-9 Silicon Si 28-0 Boron B 11-0 Lead Pb 206-4 Sodium Xa 22-99 Bromine Br 79-75 Magnesium Mg 23-94 Strontium Sr 87-2 Cadmium Cd 111-6 Manganese Mn 54-8 Sulphur S 31-98 Calcium Ca 39-9 Mercury Hg 199-8 Tin Sn 117-8 Carbon C 11-97 Nickel Ni 58-6 Tungsten W 1840 Chlorine CI 35-37 Nitrogen N 14-01 Zinc Zu 64-9 Copper Cu 63-0 Oxygen 0 15-96 t) .ArETlIODS OF RESEAKCH AND ANALYSIS REAGENTS AND APPARATUS The reayentfi chiefly employed are distilled water, physiological saline solution (0-6 per cent. NaCl), alcohol, ether, glycerine, the mineral acids, acetic, oxalic and tannic acids, potash, soda, ammonia, lime water, baryta water, silver nitrate, barium chloride, lead acetate, copper sidphate, mercuric cldoi-ide, sodium chloi-ide, carbonate and sul- phate, magnesium sulphate, the carbonate, chloride, molylxlate, oxalate, sulphide and sulphate of nmmonium, ikc. itc. Normcd Solutions used in analysis are of such a strength that 1 litre at 15° C. contains the hydi'Ogen equivalent of the reagent in grammes. A normal solution of hydrochloric acid, for instance, will contain (H = l + Cl=35-5 = 36*5) 36'5 grammes of the acid in a litre of water. Or in the case of caustic soda, NaHO (Na = 23 -1-11 = 1 +0=16), 40 grammes must be present in the litre. In the case of a bivalent substance the equivalent is half the atomic or molecular weight ; thus a normal solution of oxalic acid which is dibasic (CgHgOj +H,0 = 126) contains 63 instead of 126 grammes dissolved in the litre. Decinormal solutions are 3^^, and centinormal solutions y^^ of the strength of normal solutions. Empirical Standard Solutions are generally constructed so that 1 c.c. corresponds to 0-01 gramme (1 centigramme) of the substance to be estimated. The Apparatus necessary consists of the ordinary appliances of the chemical laboratory : test-tubes, beakers, flasks, funnels, hlters, dishes and crucil)les, stirring rods, pestle and mortar, wash-bottles, retorts, pipettes, blow-pipe, balance, air and water-baths, exsiccators, mea- sures, tfec, and in addition certain forms of apparatus which are moi-e fully described in subsequent chapters, such as microscope, spectroscope, polarimeter, dialyser, apparatus for gas analysis and for combustions, specific gravity bottles, urinometers, cfec. itc. In addition, apparatus for the manufacture of carbonic acid, suljiliui-etted hydrogen, and other gases, is often needed. CHAPTER II . 1 X. 1 /. ] TIC. I L METHODS GRAVIMETRIC AND VOLUMETRIC ANALYSIS Gravimkthic analysis, or quantitative analysis by weight, consists in separating out the constituents of any compdunrl in a pure state, or in the form of some new compound of known composition, and accurately weighing the products. V^olumetric processes are, as a rule, more quickly performed, and consist in submitting the substance to be estimated to certain chaiacter- istic reactions, employing for such reactions solutions of known strength, and from the volume of solution necessary for the pnxluction of such reaction deteiTnining the weight of the substance to be estimated. Volumetric analysis consequently depends on the following conditions for its successful prac- tice : — 1. A solution of the reagent, the chemical power of which is accurately known, called the ' standard solution.' 2. A graduated vessel from which portions of it may be accurately delivered, called the burette (fig. 1). 3. The reaction produced by the test solu- tion ^vith any given substance must either by itself, or by an indicator, be such that its termination is unmistakable to the eye, and thei*eby the quantity of the sub- stance with which it is combined accu- rately determined. The great advantage of volumetric processes is that the substance to be estimated need not be isolated in a pure condition, but the reaction chosen is generally one which is not interfered with by the presence of other substances.' Suppose, for instance, one requires to know the amount of phos- 1 The above introductory sentences are taken almost verbatim from Sutton's Volumetric Analysis. 8 METHODS OF RESEARCH AND ANALYSIS phoric acid in the urine ; a measured amount of urine is rendered acid and boiled, and to it is added from a burette, a solution of known strength of uranium acetate (which for accuracy has been previously- titrated • with a standard solution of sodium phosphate) ; the result of this is the formation of a compound of the uranium with the phosj)horic acid, and this compound, called uranium phosphate, is insoluble in hot acid urine, and so a jDrecipitate occurs. The precipitate, which is yellowish-white in colour, continues to form until all phosphoiic acid is combined. When the precij^itate ceases to form, one knows that there is no more phosphoric acid in solution, and if one adds more uranium acetate it will ])e left uncombined and free. One's object then is to add just sufticient of the standard solution to precipitate all the phosphoric acid. The volume of urine oi'iginally taken is known, the strength of the standard solution is known, and the amount of the standard solution that has been used can be ascertained by reading the burette.^ It is, however, difficult to determine by the eye when pre- cipitation is finished ; an indicator is therefoi'e used to detect any excess of the uranium salt ; this is done by testing a drop of the mixture with a drop of potassium ferrocyanide on a white porcelain plate or testing-slab ; this gives a reddish-brown precipitate with any uranium salt not combined with phosphoric acid. The appearance of such a brown precipitate indicates the end of the reaction. As an example, suppose the amount of urine taken was 50 c.c, and the amount of standard solution required for the ajjpearance of the terminal reaction 24 c.c. The standard uranium solution used is of such a strength that 1 c.c. will exactly precipitate 0*006 gramme of phosphoric acid. 24 c.c. will precipitate 24 times 0"005=0-l 2 grammes. This is the amount of phosphoric acid in 50 c.c. of urine : the amount in 100 c.c. of urine will therefore be twice as great — c.c. 0'24 per cent. This is an operation which can be completed in a few minutes, whereas if it had been necessary to separate out the phosphoric acid in a pure condition and weigh it, the processes would have extended over several days. There are, however, certain substances to which a volumetric method cannot be applied, and it is then necessary to use the gravi- 1 A titrated solution is one of which the strength has been accurately found by experiment. When a solution is directed to be titrated, the meaning is that it is to be quantitatively tested for the amount of pure substance it contains by the help of standard or previously titrated solutions. ^ It is usual to note the position of the lowest point of the curved surface (meniscus) of the fluid in a burette. Sometimes accuracy is obtained by using a glass float which rises and falls with the fluid in the burette without wavering ; this has a horizontal line drawn on it, and the coincidence of this line with the graduation mark on the burette is accepted as the true reading. ANALYTICAL .M H'l lloDS metric luetluxL The precautious to bo used iu such a method may he best illustrated by taking- an example, and we may again select ouf example from the urine : in certain forms of morlnd urine, proteid matter is present ; it is often desirable to estimate it, and the Ijest method for doing so accurately, is to pi-ecipitate it, and weigh the l)re- Fiu. 2.— S|a-uu;;Lro Uliur pump. Water enters by tube tr, and escapes by d, ilrawiiig air witli it_ from the rest of the ai>paratus, so producing a partial vacuum in the flask i, into whicli filtration is performed. The gauge / indicates the pressure, and the clips a and b regttlate the rate of flow of water. (Gschleidleu.) cipitate. Among the many precipitants of proteids, alcohol is on the whole the most convenient. A known volume of urine is evaporated to a small bulk, and if alkaline is rendered faintly acid ; about ten times the amount of alcohol is added, and the mixture boiled. The precipitate is collected on a previously dried and weighed filter. The filter used must either contain no ash, or the amount of ash (i.e. mineral constituents) must be known. 10 MK'J'HODS OF KESEAKCH AND ANALYSIS The precipitate is then thoroughly washed with alcohol and ether to remove all the other constituents of the urine ; the filter, Avith the precipitate on it, is (hied at 110° C, cooled in an exsiccator, and again weighed. The increase in weight is the amount of albuminous substance in the volume of urine originally taken. Proteid, however, carries down with it a certain amount of ash ; this is estimated as follows : A crucible is dried and weighed ; in this the filter and precipitate are carefully burnt, until ash only remains, allowance being made for the ash of the filter, this amount of ash must be deducted from that of the proteid previously found. FILTRATION The filter should be smaller than the funnel into which it is inserted, and generally should be moistened with water before being used. Filtration may be hastened by the use of ribbed filters ; hot liquids also filter more quickly than cold. Filtration under pressure may be accomplished by using one' of the many forms of filfer pump of which one is here figured (fig. 2).' In order to keep a liquid ]uA during filtration, the ordinary glass funnel is en- closed in a hollow copper funnel filled with hot water (fig. .3). In the case of fine precipitates, a small portion of the precipitate may at first pass through the filter ; soon, however, the larger pores of the paper become plugged, and the portions, which first passed through, must be returned to the filter. With voluminous, dense, or gelatinous deposits the separation of the greater part of the precipitate may be accomplished by filtering through muslin or linen. Precipitates are now often collected on asbestos filters ; these are readily made by perforating the bottom of a platinum crucible with fine holes and filling up the bottom of the crucible with finely divided asbestos. They are advantageous, as they can be easih^ dried and weighed, are permanent, and where incineration is necessary are free from ash. Fi(i. 3. — Sectional view of Iiot water funnel. (Gsuhleidleu.) WASHING PEECIPITATES Precipitates may be washed on the filter ; it is best to use a wash-bottle, and care must be taken that the wash-liquid penetrates to every part of the precipi- tate, which may be gently disturbed for the purpose with a glass rod. Washing by decantation is only applicable to precipitates that are heavy and subside readily ; the precipitate is well mixed with the wash-liquid and allowed to settle ; the wash-liquid is then poured, syphoned, or piiJctted off, more liquid added, and the process repeated as often as necessary. * Simple forms of filter pump can now be purchased for a few shillings, and can be attached to any ordinary water-tap. ANALYTIC. \I, .MF.TIIODS 11 DllYlNG A Nv.itt r-owii ;it tlie temperature of 100°, or, better still, .-in air-bath 10^ or 20° hij^'lar, ami tlie temperature kept constant by a gas regulator, ma}' be used for (Irvirg filter-jyapers and organic substances generally. Crucibles may be Fig. 4. — Hot-air oven with gas regulator (g). (Gsclileidleii.j readily and quickly dried by holding them with tongs for a few seconds in a Bunsen flame. A flask is dried by warming it and then sucking the air from the interior with a long glass lube dipping into it. COOLING AFTEE DEYIXG Substances must not be weighed hot, otherwise air currents are set np which disturb a delicate balance. They must not be allowed to dry in the air, or they (especially if hygroscopic) become moist again. They are generally cooled in an exsiccator — a closed glass vessel containing a tray of sulphuric acid. See figure 5. A filter is usually allowed to cool and is weighed between two watch- glasses clipped together, or in a thin wide-mouthed glass bottle. The bottle or watch-glasses, however, must be dry, and cooled before weighing, in an exsiccator. After weighing any substance that has been dried, it is again heated to 110° for some hours, cooled and weighed as before ; the process being repeated until two Tig. 5.— An exsiccator. (,G.sciaeidlen.) successive weighings give the same result — that is, till there is no more loss of 12 METHODS OF RESEARCH AND ANALYSIS weight from evaporation of water. Tliis is called weighing to constant weight. Exsiccators may be used for drying in vacuo at a low temperature such sub- stances as would be injured by the application of heat. In such cases the top of the bell jar of the exsiccator is connected by a tube to an air-pump. The tube should be fitted with a stop-cock. The air is thus exhausted either by an ordinary air-pump or by a water air-pump constructed on the same principle as that already described (fig. 2). When the vacuum is as complete as possible the stop-cock just mentioned should be closed and tlie pump can be detached. Moisture rapidly passes off from the substance wliicli is to be dried, and is absorbed by the sul])huric acid. INCINERATION The substance to be incinerated must be dry and must not touch the side of the crucible more than is absolutely necessary. A crucible of known weight is placed ux3on a triangle over a Bunsen fiame and at first heated very cautiously, or the contents are apt to froth and be partially lost. The heat is gradually increased, and ultimately the flame is allowed to surround the crucible, which should be tilted. The process, which is a long one with porcelain crucibles, is allowed to continue till the ash is white, when it may be cooled and weighed. Rose's method is better than the preceding, and is as follows :— The dry substance is carefully carbonised in a crucible over a Bunsen flame. After cooling the contents are heated with distilled water again and again to dissolve all soluble salts. The hot aqueous extracts are mixed and filtered through a small filter of known ash. ' The insoluble matters together with this small filter are dried at 1 10° and ignited at a red heat ; when the residue is white the crucible is cooled and weighed ; this gives, subtracting the weights of the crucible and filter ash, the weight of the insoluble salts. Either in the same crucible or in a separate one the aqueous extract is evaporated to dryness, dried at 110°, and ignited at a red heat ; the crucible is then cooled and weighed; the increase in weight is the amount of soluble salts. EVAPORATION The usual temperature employed is 10U°, which is most easily obtained with a water-bath ; for lower temperatures, a water-bath is kept at a constant tem- perature by a gas regulator. Ethereal or alcoholic solutions must never be evaporated over a naked flame, but over a water-bath. In heating glass vessels it is also advisaVjle to interpose a flat iron plate, or a piece of wire gauze, or asbestos cardboard, or a sand-bath between the glass and the flame. BOILING The boiling point of a liquid is that temperature at which the liquid becomes no hotter, but at which any further heat is used up in converting the liquid into vapour. If the liquid, however, be enclosed in a vessel so that no vapour can escape, its temperature will continue to rise. Hence it is possible to heat aqueous solutions above 100° C. bj- enclosing them in sealed tubes or a Papin's digester, and placing tliese in a liquid such as oil, which boils at a higher tem- perature than water. 1 The ash of a filter may be ascertained by incinerating, say, 12 similar filters, weighing the ash, and obtaining the average by dividing by 12. ANALYTICAL 31ETHUD.S 13 Sometimes one requires to heat a liquid for a long time without its losing much of its bulk ; a long glass tube or a contlenser is then attached by a cork with a hole in it to the neck of the flask, the vaiiour condenses in the tube and runs back into the flask. DISTILLATION Some substances are much more volatile — that is. boil at a lower temperature — than others. Advantage is taken of this to separate such substances by a process of distillation. A distillation apparatus consists essentially of a boiler and a condenser. The boiler may be a flask or a retort closed with a cork through which a tube passes : the tube leads to the condenser, which in the form commonly used (Liebig's) consists of a long tube suiTounded by an outer tube ; cold water is made to circulate between the two tubes, and thus the vapour which passes along the inner tube is condensed, and the fluid so formed is collected at its far end. A thermometer is fixed into the retort through the cork at its summit : fractional distillation consists in collecting the substances in separate vessels that distil over at different temperatures. DIALYSIS If a solution of albuminous, gelatinous, or mucilaginous substances, mixed with saline and crystalline substances be placed in a dialyser, in distilled water, it will be found that the crystalline substances pass through the parchment mem- brane into the water, while the proteid or gelatinous substances remain in the dialyser. The substances which pass through membranes in this wav are Pig. 6.— Liialy^er. T;../ ^.^ver -i*i;:i:^ ..: :;.- !..-:: iar ^u^- peuilol ill water is tightly covered %ritli iiarc-liment XKiper. TLe fluid to be dialysed is placeil wtliiu this vessel, the crj-stalloids pass out into the distilled water outside, through the parchmeut paper. li. 7. —In tlii* form of dialyser the substance to be dialysed is 7>lace a scale attache I to C divided into millimetres. (Gschleidlen.) Sodium chloride . 4-0 Sulphuric acid Sodium sulphate . 11-0 Caustic potash Potassium sulphate 120 Alcohol Magnesium sulpliate . 11-5 Sugar . Copper sulphate . 9-5 Thus 4 grammes of water would pass through the membrane into the endosmo- meter for 1 gramme of sodium chloride, 11 for 1 gramme of sodium sulphate, and so on. Sometimes negative osmosis occurs, i.e. more of the suV^stance i:)asses out of the osmometer than water passes in ; this is the case with acids. The rate of osmosis increases with the concentration of the substance with the temperature of the liquids used. There is also no doubt that the nature of the membrane affects osmotic action ; different varieties of dead membrane affect the rate of osmosis ; the osmosis that . occurs in living membranes is also no doubt very different again, but is a difficult subject to investigate experimentally. A ANALVTilAL METHODS 15 living nipniliraiie is iR)t fixed or stable, but is constantly undurgoing processes of building up and breaking down.' Thus the discussion whether the formation of lymph is due to liltratiou or diffusion of the blood plasma through the vessel walls has not yet received a satisfactory answer.- The question is still further complicated in the living body by tlie fact that the fluids on the two sides of any membrane are almost invariably at different pressures; and in addition it is possible that there may be some kind of attractive influence exerted by the tissues themselves, analo- gous to the selective activity of secreting cells. DETERMINATION OF SPECIFIC GRAVITY The specific gravity of liquids is usually ascer- tained by a hydrometer ; and these instruments adapted for the range of specific gravities in urine and milk, are termed uriuometers and lactometers respectively. But when the specific gravity must be deter- mined with greater accuracy, a small light flask of known weiglit, called a pycnometer or specific gravity bottle, is employed. This is fitted with a stopper, through which a capillary canal passes. Fig. 9. — UriuometL-r fliKitiny in urine in a testing glass. FlO. 10. — Geissler's specific gravity bottle, a is a light flask, 6 an accurate thermometer ; c is a tube connected with a, through which fluid escapes when the tliermo- meter is inserted in the bottle ; il a cap to fit ou to the top of c. 1 In a recent paper, Prof. Waymoutli Reid [Brit. Med.Journ. vol. i. 1.S90, p. 165) brings out very clearly the difference as regards diffusion between dead and living membranes. The membrane he experimented witli was the skin of the frog. As he points out, we have doubtless in a living membrane to deal with an absorptive force dependent on protoplasmic activity, and comparable to the excretive force of a gland cell. This is excited especially to make osmosis take place more readily in one direction than in the other ; in the case of the frog's skin from without in. * A discussion on this subject will be found in Foster's Physiology, vol. ii. p. 303, 5th edit., 1889. 16 .^IKTHODS OF KESEARC'ir AND ANALYSIS and contains when filled a known weight of water (25-30 grammes) at 15° C, Some pycnometers are fitted with thermometers, used at the temperature of the air, whatever it happens to be, and then the weight of water in it calculated from the table on p. 5. The bottle is filled with the liquid the specific gi-avity of which is to be deter- mined ; it is weighed, and the specific gravity or density obtained by the formula : sp. gr. = - ; w = the weight of the liquid, w' = the weight of the water. The result is then obtained in comparison with water, which is taken as unity. In medical work it is often found more convenient to take water at 1000 ; a urine of specific gravity 1020 means one which is 1-02 times heavier than water bulk for bulk. DETERMINATION OF REACTION. ALKALIMETRY. ACIDIMETRY Litmus papers, or a neutral litmus solution, are usually employed to determine whether a substance is neutral, acid, or alkaline. Neutral substances have no effect on either red or blue litmus, but in presence of organic materials a neutral solution will often turn delicate glazed blue litmus papers faintly red, and red ones faintly blue. Acid substances turn blue litmus red. In the case of volatile acid, the red colour passes off as the acid evaporates. Alkaline substances turn red litmus blue. At night it is best to examine the transition in colour by monochromatic (sodium light); the red colour appears coloixrless, the blue is blackish. In measuring the amount of acidity or alkahnity of a solution it is titrated with a standard solution of acid or alkali respectively ; the indicator of the end of the process being the change in colour produced in the litmus. It is, in fact, a simple example of the volumetric method. Recently, however, more delicate indicators than htmus have been employed, and the follow- ing is a list of the principal ones : — 1. Methyl orange, 1 gramme dissolved in a litre of water. This is only applicable to titration with mineral acids : it is not affected by carbonic or sulphydric acids in the cold. It is an admirable indicator for ammonia and its salts. The colour given is i)ink with acid, yellow with alkali. 2. Phenacetolin, 2 grammes dissolved in 1 litre of alcohol. The solution is dark brown ; it gives a scarcely perceptible yellow with caustic soda or potash ; with ammonia and the normal alkaline carbonates a dark pink ; with the bicarbonates a brighter i)ink ; and with the mineral acids a golden yellow. 3. Phenolphthalein, 1 gramme in 1 litre of 50 per cent, alcohol. A few drops of the indicator show no colour in the ordinary volumes of neutral or acid liquids, the faintest excess of caustic alkalis gives a sudden change to purple-red. It possesses the advantage of great delicacy, but the disadvantage of being useless for the titration of free ammonia or its compounds. 4. Rosolic acid, 2 grammes in 1 litre of 50 per cent, alcohol. Its colour is pale yellow unaffected by acids, but turning to violet-red with alkalis. It is not reliable for organic acids. 5. Lacmoid. This is prepai-ed from resorcin, and behaves like litmus. Lacmoid paper is also prepared. Lacmoid, rosolic acid, phenacetolin and ANAL^'I'ICAI. MI"riln|)^ 17 phonolplithaloin air capable of showing change of colour with i of the quantity of acid or alkali necessary in the case of methyl orange or litmus.' The normal solutions most frequently used in estimating acidity or alkalinity are those of sodium carbonate (r)3 grnis. NaX'Oj per litre), i)otassium carbonate (t)9 grms. K..CO3 per litre), sulphuric acid (49 grins. HoSO, )K;r litre), oxalic acid (().■? grms. of C._,H.p,.2II.A>, or l'> grms. of C.U.O^ per litnO, hydrochloric acid (i?!)-."?? grms. HCl per litre), caustic soda or potash (10 grms. NaHO, or 50 grms. KHO per litre), and semi-normal ammonia- (8-5 grms. NH^ per litre). 100 c.c. of any of the acid solutions exactly neutralise the .-^anie volume of any of the alkaline solutions. excei)t in the case of semi-normal ammonia, which rccjuires onl}' half tie ([uantity of at'id. THE CENTRIFUGAL MACHINE The separation of jirecipitates too line to filter off, of corpuscles from serum, of cream from milk. &c. kc, may be facilitated by subjecting the fluid to the action of a centrifugal machine (see figure). The liquid is placed in tubes at the margin of a horizontal rotating disc, worked at a high rate of speed by machinery (1000 Vm. 11. — Centrifugal machine as made by Fr. Runne of Basel. Glass vessels containing the sub- stances to be centrifugalisecl are placed wilhui the six metallic tubes which hang vertically wiiile the disc is at rest : when the machinery is set going they fly out into the horizontal position. A water motor is a very convenient motive power for these instruments. revolutions per minute). The tubes fly into the horizontal direction, and the heavy particles settle to the far end of the tube ; the upper fluid can then be decanted or pipetted off. The time that this takes varies with the relative densi- ties of the substances to be separated. Serum and blood corpuscles are usually -eparable by this means after about 30 to 60 minutes' whirling. • For full particulars see Thompson, Chem. News, vol. 47, pp. 12S, 185, vol. 48, pp. 32, 119. - It is unsafe to use normal ammonia. 18 MI-rninDS ()]• 1{KSKA1J('H AND ANALYSIS HEAT COAGULATION AND SATURATION AVITH SALTS are processes used especially in connect ion with the proteids undi'i- which tlicy are fnlly described. DETERMINATION OF RELATION OF SOLIDS AND WATER IN ANY SUBSTANCE If the substance is liejuid. a weighed quantity is evaporated t(i n' a mixture of nitric acid and jxjtassium clilorate, at tii-st in the cold, and finally with the application of heat ; the solution is evaporated neai'ly to dryness, diluted, filtered, if necessary, and then tested for sulphates. 3. The following test serves to detect sulphur in organic compounds in the unoxidised state only. The substance is boiled with a strong solution of potassium hydrate, and evapctr- ated nearly to drjTiess. The residue is dissolved in a little water, poured into a small flask A, which is then loosely corked. Through the cork a funnel tube c passes, which is allowed to dip into the fluid at the Vjottom. A slip of paper b, moistened with lead acetate and then with a few drops of ammonium carlx)nate, is allowed to hang down the neck of the flask ; dilute sulphuric acid is poured down the funnel ; if stdphur is present the slip of paper is turned brown from the action of the sulphuretted hydrogen which is evolved ; or the sulphide of potas- sium may be detected by a solution of lead oxide in soda, which is turned black or brown. TESTS FOE PHOSPHORUS The methods 1 and 2 just described in testing for sulphur may also be employed for phosphorus. The solution obtained is examined for phosphates, either by a mixture of magnesium sulphate, ammonium chloride and ammonia, which gives a white precipitate, or preferably by the yellow ciystalline precipitate given by a nitric acid solution of ammonivuu molybdate. If method 2 is used the greater part of the nitric acid must be first removed by evaporation. QUANTITATIVE ANALYSIS OF SUBSTANCES CONSISTING OF CARBON AND HYDROGEN, OR OF CARBON. HYDROGEN. AND OXYGEN The principle first proposed by Liebig for the analysis of these compounds was as follows. The substance is burnt and carbonic acid and water are formed ; these products are separated fi'om one another and weighed ; the carbon is calculated from the weight of carbonic acid, the hydrogen from the amount of water. If the sum of the carbon and hydrogen is equal to that of the original substance, that substance contains no oxygen ; if it is less than the weight of the substance, the difference expresses the amount of oxygen present. Methods have been proposed for the direct estimation of oxygen, but the oxygen is generally obtained bv difference. ri;i-l.MATK ANALYSIS ol' OUGANIC t'OMPOUNDS 21 Tlie coiiihustioii is ofiif'cted eitluT by ii^'nitiiii; the oi-ojanic substance witli oxyjijeiiated substances that part I'eadily witli tlieir oxygen (copper oxide, U'ad chroniate, itc), orin the case of ditiicultly eomljustible bodies, fi-ee oxygen is used also. Volatile substances reciuire special precautions described in detail in works on organic analysis.' The usual method adopted for solid, readily combustible, non- volatile substances such as sugar or starch is that of combustion with oxide of coj)per. The substance to be analysed is finely pulverised, dried, and weighed. A combustion tube is carefully dried and half filled with warm oxi a trough containing mercuiy. About 6 cm. of the far end of the tube is then heated, the rest being protected by a sci-een ; the bicar- bonate is decomposed and the carbonic acid propels the air before it, expelling it from the tube. After some time the end of the delivery tube is dipped under the mercury and a test-tube tilled with potash solution inverted over it. If the gas bubbles are completely absorbed by the potash, all air must have been expelled from the tube ; if not, the evolution of gas is continued till this desired point is obtained. The actual combustion is then commenced. A graduated cylinder half filled with mercury and half with potash is inverted over the delivery tube. The combustion tube is heated, commencing with the copper foil, and gradually all the burners are lighted till those under the second half of the bicarbonate are all in full blaze. The whole of the gases evolved are driven on by the carbonic acid so evolved, the oxides of nitrogen are reduced by the metallic copper, and nitrogen in a moist state alone collects in the cylinder, the carbonic acid being absorbed by the potash. The weight of nitrogen is calculated from its volume, correc- tions to normal pressure and temperature being made, due regard being paid also to the tension of aqueous A-apour (see further Gas Analysis). 2. Method of Varrentrapp and Will.^This method is founded »)n the same principle as the test for nitrogen already described (p. 19, test 2). The substance to be analysed is reduced to the finest powder, dried and weighed. It is mixed with soda-lime. A combustion tube is filled so that the mixture lies between two portions of pure soda-lime, and is then jDlugged loosely with asbestos, and finally with a cork which is ri/riMAI'K ANALYSIS OF oliCANIC Ci i.M |>( XNDS 23 perfonitod, allow iiii; the tube and a Inilb aj)i)aiatu.s coiitaiiiiii,i( hydro- chloric acid to he j>ut in connection with one anothei". The tube is ^nidually heated, ('(ininiencini,' at the fore part and progressing slowly towards the closed end. The nitrogen present is all thus convei'ted into anunonia which is absorbed by the hydi'ochloric acid in the bull)S. The anunonia present is estimated by adding platinic chloride and weighing the aiunioniuni platinum chloride which is thus pi-ecipitated. 3. Kjeldahl's Method. The difficult and lengthy operations invoh ed in the two methods just described, and present also in the many modifications of them which chemists have proposed from time to time, are howe\ tT now unnecessai-y, as the simple and accurate method oi Kjeldahl' has very largely replaced them. I take the following account of the method with the modifications proposed by Warington^ from Sutton's Volumetric Analysis.^ I have myself fre(iuently used the method, but it hardly needs now my testimony to its usefulness, as it has been so widely praised and so much adopted by others. From O'l to 1 gramme of the dry powdered substance is put into a boiling flask holding about 100-120 c.c. The acid used for the destruc- tion of the (trganic material is made Ijy mixing 200 c.c. pure oil of \ itriol, 50 c.c. Nordhavisen oil of vitriol, and 2 grammes of phosphoric acid in sticks ; all these must of course be free from ammonia. 10-20 c.c. of this mixture is poured over the substance in the flask and hetited on wire gauze over a small Bunsen flame. The temperature must be kept below boiling ; with prolonged heating the organic matter is gradually desti'oyed, and the liquid becomes clear and .quiet. The nitrogen originally present is thus converted into ammonia, and this may be hastened by adding to the liquid very minute pinches of pure potassium permanganate. A violent commotion takes place with every addition, but there is no fear of any ammonia being lost. The operation is ended when the mixture becomes permanently greenish, and moderate heat is continued for a few minutes more. The flask is cooled, some water added, and the contents washed out into a large flask of 700 c.c. capacity with as little water as possiljle. It is then made alkaline with excess of either pure caustic soda or potash solution (sp. gr. 1"3). A little metallic zinc is added to prevent bumping during the subsequent distil- lation. The flask is then closed with a perforated caoutchouc stopper, thi'ough which passes an upright tube with two bulbs about an inch in diameter blown upon it ; these arrest and cai-ry back any spray of soda from the liquid. The tube above the bulbs is bent over and connected to a condenser, and the delivery end of the condenser leads into a flask 1 Zeit. Anal. Chem. xxii. p. 36G. - Chem. News, lii. p. KJ'i. '' pp. 6a-70. 24 :\iETH(ti)s OF j{EsE.\];('ii and analysis containing a measured excess of standard acid.' The mixture in the flask is then distilled, the ammonia passes over into the acid. The amount of acidity is then determined in the distillate by titration with standard potash or soda, methyl orange being used as the indicator of the end of the reaction. Example. — Suppose 0'15 gramme of a nitrogenous substance is taken, treated with acid, neutralised and the ammonia distilled over and received by 100 e.c. of a decinurmal solution of hydrochloric acid ( = 10c.c. normal acid). The distillate is then titrated with decinormal soda and it is found that the neutral point is reached when 60 c.c. of the decinormal soda have been added. The other 40 e.c. must therefore have Ijeen neutralised by the ammonia derived from the nitrogenous substance under investigation. This 40 c.c. of decinormal acid =4 c.c. of normal acid =4 c.c. of normal ammonia =4 x 0017 =0"0G8 gramme of ammonia. 0"15 gramme of the substance therefore yields 0'068 gramme of ammonia, and this amount contains 0'056 gramme oi nitrogen ; 100 grammes of the substance will therefore contain 100x0-056 .,_ ., — =.3/ -.3 grammes or nitrogen. ANALYSIS OF ORGANIC COMPOUNDS CONTAINING SULPHUR The method we have described for determining the carbon in sulphur free substances would give too high a result with substances containing sulphur, as the sulphurous acid formed on combustion would be absorbed in the potash bulbs. Carius recommends that the svibstance containing the sulphur should be burnt with lead chromate in a combustion tube 60-80 cm. long, care being taken that the anterior 10-20 cm. which contains pure lead chromate is never heated above low redness. The pure lead salt retains the sulphurous acid produced Ijy combustion, and thus never contaminates the potasli in the bulbs beyond. Hydrogen and nitrogen are determined by any of the methods described. As regards the estimation of the sulphur itself, that element is weighed in the form of barium sulphate, into which it may be converted by many methods, the principle of which has been already given under tests for sulphur (1 and 2, pp. 19, 20). If the substance contains oxygen this is estimated by difference. * One can guess approximately how much ammonia is expected ; in a proteid, for instance, there is speaking roughly 15 per cent, of nitrogen ; suppose O'o gramme of this is taken, we should obtain in round numbers 0'08 gramme of nitrogen from this, which would be converted into O'l gramme of ammonia. 1 c.c. of normal acid corresponds to 0'017 of ammonia ; therefore 10 e.c. of normal acid or 100 e.c. of decinormal acid, which is more often employed, would neutralise 0'87 gramme of ammonia, and therefore be quite a safe quantity to take. Decinormal hydrochloric acid is the acid I myself use. • ri/riMATK .\N.\I,YSIS (»K ORCANIC CO.MrorNDS 25 ESTIMATION OF I'HOSPHOEUS IN OlRiANIC SUBSTANCES The estimation of i)lnisjili(iius is effected in a nianncr similar to that of sulj)hur ; i.e. the suhstance is oxidised either in tlie dry or wet way, and the phosphoric acid produced is determined generally by weighing the precipitate caused by a mixture <>f magnesium sulphate, ammonium ddoride, and ammonia. Phospliorus cannot be estimated in an organic substance by incin- erating and determining the phosphoric acid in the ash, as all the phos- phorus is not converted by this means into phosphoric acid. In analysing animal tissues, one has to deal with substances, such as proteids, to which small quantities of mineral bodies obstinately adhere, and are not removable by any known means before analysis. After analysis one has therefore to incinerate, weigh the ash, and allow for this in subsequent calculations. This method can, however, only be regarded as approximate ; first, because certain salts like sodium chloride are \olatile to some extent and pass ofi" ; and, secondly, because if sulphur or phosphorus, or both (as in nervous tissues), are present, a certain amount of sulphuric or phosphoric acids respectively are formed during ignition ; these are weighed with the ash, though they liave really been derived from the organic substance itself. If a substance contains phosphates as well as phosphorus in organic combination, a separate portion is Ijoiled witli dilute hydrochloric acid, filtered, and the phosphorus present as phosphoric acid estimated in the solution ; this amount is deducted from the total quantity of phosphorus. DETERMINATION OF IRON IN ORGANIC SUBSTANCES In some few substances in the body, e.(/. h;emoglobin, iron is present in addition to the other elements we have mentioned. A weighed amount of the material is incinerated ; the ash dissolved in hydrochloric acid, and the amount of ferric chloride so formed ascertained by one of the many volumetric processes now in use. The following is Oude- mann's method.' To the dilute ferric solution, which should not con- tain more than 0-1 to 02 gramme Fe in 100 c.c, nor much free HCl, 3 c.c. of c. 1 per cent, solution of cupric sulphate are added, 2 c.c. of concentrated HCl, and 1 c.c. of a 1 per cent, solution of potassium sulphocyanide. The mixture is slightly warmed, and a standard solu- tion of sodium thiosulphate (1 c.c. of a decinormal solution of which corresponds to 0-0056 Fe) is run in from a burette, until the pre^dously red mixture becomes as perfectly colourless as water. • Zeit. Anal. Clicm. vi. 12!), and ix. 342. 20 :METir()i)s of kesearck axd analysis The amount of iron in lueuK^globin is 0'4 per cent. Kno\vin<,' this, hiemoglubin may be estimated quantitatively from the amount of iron present in the ash of an unknown amount of the hiemoglobin. TO DEDUCE EMPIRICAL FORMULAE FROM PERCENTAGE COMPOSITIONS From the percentage composition the empirical formula can be calculated, provided that the combining weights of the elements are known. The actual size of the molecule and its constitutional formula are obtained by other methods. The wa}' in which an empirical formula is deduced may be most readily described by giving examples. Example 1. Suppose starch has been subjected to elementary analy.^is and it was found to contain 4:4-44 carbon, 6-17 hydrogen, and 4939 oxygen per cent. Knov\-ing that C=12, H = l. and 0 = 16, what is the empirical formula for starch 7 Divide the percentage numbers by the combining weights of the elements, and we obtain Cg-YOS H|j-i7 Og-OR which gi\ es us a rough guide to the formula ; from this we can see that the hydrogen atoms are twice as numerous as the oxygen atoms, and the carbon atoms are also rather more numerous than the oxygen atoms. We must next find some common factor which will convert the above numbers into whole numbers; it is, however, generally impossible to do this exactly, and it will be found in the present instance that the number 1-62 is the smallest number which will give us approximately whole numbers, viz. : — C.jtKJO Hyyy O^-gj The nearest whole numbers to these being taken, the simplest empirical formula for starch is CbH,oO-. Example 2. When we are dealing with a substance containing more than three elements, the arithmetical processes become more comijlicated. The example I wiU choose is that of mucin obtained from tendons. Loebisch found that the percentage composition of this material was C, 48-3 : H, 6-44; N, 11-75 ; S, OSl ; 0, 32'7. Divide each of these numbers by the combining weight of their respective element, and we obtain a guiding formula, viz. :— C402.5 Hfl-44 No.h;s Soo-25 t)-2a,-, It will be found that the lowest common factor which will convert these numbers most approximately into whole numbers is 39 75 ; this will give us — Cl59'99 H.233-g9 ^3.2-1);) So<)9 Oho-4,S or approximately, C,«o H.^ N33 SOs,. The numbers do not correspond with equal exactness throughout ; this is especially noticeable with regard to the oxygen. It must, however, be remembered that there are certain unavoidable small errors of analysis which have always to be allowed for. Moreover, in this particular instance the correspondence between the percentage calculated from the formula and that obtained by analysis is closer than in many other cases. Such methods give us only an empirical formula ; the true molecular weight ri/I'lMA'I'K ANAIA'SIS OF OROANIC CO.MI'f )rNI)S 27 may be « times as great, and in substances in which the molecular weight can be ascertained by determination of vapour density, &c., the calculation is simplified. But with re^^ard to the albuminous and starchy substances we have to deal with in animal chemistry, these methods arc not available. The constitutional formula of any substance, i.e. the wa}' in which the atoms are united to one another, must be determined by other methods also. Here, again, the substances we have chietiy to deal with in animal chemistry are those, in regard to the constitution of which, we arc almost entirely in the dark at present. 28 METHODS DF RESE-IECH AND ANALYSIS CHAPTER IV GAS AXAL YSIS The gases with which the physiologist has to deal are those of the atmosphere, and those concerned in respiration, those present in the blood and other fluids, as well as those obtainable from the solid tissues of the body. In the greater proportion of cases, a physiologist has to investigate three gases or mixtures of these : viz. : — oxygen, nitrogen, and carbonic acid. Small quantities of carbonic oxide are also pro- duced in the body, and in the alimentary canal, fermentation processes iii^y gi^e rise to hydrogen, marsh gas, and sulphuretted hydrogen. The reader is, however, referred to larger treatises dealing more especi- ally with gas analysis, for the methods of investigating these more rarely occurring gases. In examining the gases obtainable from either fluid or solid animal tissues, the methods adopted rlivide themselves naturally into three parts : — 1. The collection oi the blood or other tissue in a suitable manner. 2. The extraction of the gases from this material. 3. The analysis (jf the gases so obtained. 1. METHODS OF COLLECTING MATEKIAL INTENDED FOR GAS ANALYSIS o. Collection of blood. In some cases it is possible to lead the bloofl direct from the blood-vessel of the animal, by a tube into the vacuum chamber of an air-pump. The gases can be then pumped from it forth- with. The vacuum chamber can be weighed before and after the entrance of blood into it ; the increase of weight giving the weight of blood used. In other cases it is advisable to collect and measure the blood m a separate vessel before introducing it into the air-pump. It must then be collected over mercury, and the following apparatus, as describe. 5). Th6n, V'= Vx(r, -T) 7(50 X (1 +0()o:){j(;.5o If the gas is drv, then y. _ V X B 7(JOx(l + 0U0H(i(i.')O The number OOOiJGG.'i is the coefficient of expansion of gases. The number 7fi0 x (1 +00036650 is obtained from tables, and tlie calculations arc much simi)lificd by the use of logarithms : thus, log V = log V + log (B -T)- log [760 x (1 + 0003665O], or, for dry gases, log V' = logV + log B-log [760x(l + 0-00.S665O].' ' Mr. F. Sutton, Norwich, will forward a cox^y of these tables, printed separately for labniatoiy use, to any one desiring them, ou receipt of the uecessarj- address. 36 :metii()J)s of research and analysis CHAPTER V OPTICAL IXSTUUMEXrS USED IX CFIEMICO-PHYSIOLOGICAL IX VESTIGA TIOXS THE MICROSCOPE This instrument is of value in observations on crystals which are too small to be seen, much less measured, by the naked eye. In performing chemical reactions with small quantities of material, it is sometimes convenient to do so on a microscope slide, and observe the result with the microscope. Such operations are designated micro- chemical. A familiar instance of a micro-chemical reaction is the test for blood, which consists in the formation of hremin crystals. POLARISATION OF LIGHT If an ol)ject, such as a black dot on a piece of white paj^er, be looked at through a crystal of Iceland spar, two black dots will Ije seen ; and if the crystal be rotated, one black dot will move round the other, which remains stationary. That is, rays of light entering such a ci'ystal are split into two rays, which travel through the crystal with different velocities, and consequently one is more refracted than the other. One ray travels just as it would through glass ; this is the ordinary ray, the ray which gives the stationary image ; the other ray gives the inovable image when the crystal is I'otated ; the oixHnary laws of refraction do not apply to it, and it is called the extraordinary ray. Both rays are of equal brilliancy. In one direction, however, that of the optic axis of the crystal, a ray of light is transmitted without double refraction. Ordinary light, according to the wave theory, is due to vibrations occurring in all planes transversely to the direction of the propagation of the wave. Light is said to be j^lane polarised when the vibrations take place all in one plane. The two rays i^roduced l:)y double refraction are both polarised, one in one plane, the other in a plane at right angles to this one. Doubly refi-acting bodies are called anisotropous ; singly refracting bodies, isotropous. The effect of -polarisation may be very roughly illustrated by a model. If a string be stretched as in the figure, and then touched with the finger, it can be made to vibrate, and the \ibrations will be free to OPTICAL 1NST1{IMKNTS 37 occur from above (lown, or from side to side, or in any intermediate position. If, liowever, a disc with a vertical slit be placed on the course of the string, the viljrations will be all obliged to take place in a vertical plane, any side to side movement being stopped by the edges of the slit.' Light can be polarised not oidy by the action of crystals, but by roriection from a surface at an ani,de which varies for ditt'erent substances (glass 54° 35', water 52° 45', diamond 68°, quartz 57° 32', lire). It is also found that certain non-crystalline substances, like muscle, cilia, t^-c, are doubly refracting. The Nicol's Prism is the polarlser usually employed in polariscopes; it consists of a rhombohedx'on of Iceland spar divided into two by a section through its obtuse angles. The cut surfaces are polished and cemented together in their former position with Canada balsam. By this ineans the ordinary ray is totally reflected through the Canada balsam ; the extraordinary ray passes on and emerges in a direction parallel to the entering ray. In this polai'ised ray there is nothing to render its peculiar condition visible to the naked eye ; but if the eye is aided by a second nicol's prism, wdiich is called the analyser, it is possible to detect the fact that it is polarised. This may be again illustrated by reference to our model (fig. 18). Fig. w. Suppose that the string is made to \-ibrate, and that the waves tra^"el in the direction of the arrow. From the fixed point c to the ' Such a model is, of course, imperfect ; it does not, for instance, represent the splitting of the ray into two ; and moreover the polarisation takes place on each side of the slit ; whereas in regard to light, it is onlj- the raj-s on one side of a polariser, viz. those that have passed through it, which are polarised. 38 :\IETIIOI)S OF RESEARCH AND ANALYSIS disc a, the string is theoretically free to vibrate in any plane ;' but fifter passing through the verti-cal slit in a, the vibrations must all be vertical also ; if a second similar disc h be placed further on, the vibrations will also pass on freely to the other extremity of the string f?, if as in the figure (fig. 18) the slit in h be also placed verti- cally. If, however, b Ije so placed that its slit is horizontal (fig. 19), the vibrations will be extinguished on reaching h, and the string between h and d will be motionless. c here represents a source of light, and the vibrations of the string the undulations which by the nicol's prism a are polarised so as to occur in one plane only ; if the second nicol or the analyser b is jmrallel to the first, the vibrations will pass on to the eye, which is represented by d ; but if the planes of the two nicols are at right angles, the vibrations allowed to pass through the first are extinguished Ijy the second, and so no light reaches the eye. In intemnediate positions, h will allow only some of the light to pass through it. It must be clearly understood that a nicol's prism contains no actual slits, but the arrangement of its molecules is such, that their action on the particles of tether may be compared to the action of slits in a diaphragm to vibrations of more tangible materials than aether. The Polarising Microscope consists of an ordinary microscope with certain additions ; below the stage is the polarising nicol ; in the eye-piece is the analysing nicol ; the eye-piece is so arranged that it can be rotated ; thus the directions of the two nicols can be made parallel and then the field is bright ; or crossed, and then the field is dark. The stage of the microscope is arranged, so that it also can be rotated. The polarising microscope is used to detect doubly refracting substances. Let the two nicols be crossed, so that the field is dark ; interpose between the two, that is, place upon the stage of the microscope, a doubly refracting plate of which the principal plane is parallel to the first prism or polariser ; the ray from the first prism is unaftected by the plate, but will be extinguislied by the second ; the 1 The imperfection of the model has been ah-eady explained. ( >ITIC.\L INSTKl'MENTS tield therefdio still remains dark. If the plate is parallel to the second nicol the tield is also dark; but in any intermediate position, the li.Ljht will he transmitted by the second nicol. In other words, if between two crossed nicols, which consequently appear dark, a substance be interposed which in cei'tain positions causes the darkness to give place to illumination, that substance is doubly refractive. How this takes place may be explained as follows : — Let N|N| (tig. 20) represent the direction of the principal plane of the fii-st nicol, and NoNj that of the second. They are at right angles, and so the ray transmitted by -po- the tirst, will be extinguished by the second. Let PP represent the principal plane of the interposed doubly refractive plate. The extraordinary ray transmitted by X , N , ^-ibrates in the plane X j X , , ^ y^ — ^g and falls obliquely on the plate PP : it is by this plate itself split into two rays, an ordinary and an extraordinary one, at right angles to one another, one vibrating in the plane PP, the other in the plane P ' P ' . These two rays meet the second nicol, which can only ti-ansmit vibrations in the plane XaXg. The vibrations m PP can be resolved into a \-ibration in X,X, and a vibration in X2X2, the former is extinguished, the latter transmitted. Similarly the ^-ibration in P^P' can be resolved into two sub-rays in X,X, and XoX^ respectively, the latter only being transmitted. The illumination is thus due to two sub-rays, one of the vibrations in PP, the other of those in P'P' which have been made to vibrate in X.2X.2. Xow, although these two sub-rays ^-ibrate in the same plane, they are of diflerent velocities : hence the phases of the A-ibrations do not coincide, and thus the phenomena of interference are obtained. If we have two sets of vibrations fused, the crest (jf one wave may coincide with the crest of the other, in this case the wave will be higher ; or the crest of one may coincide with the hollow of the other; that is, the undulation would be extinguished ; in other intermediate cases, the movement would be interfered A^-ith, eithei- helped or hindered more or less. Interference in the case of many kinds of doubly refracting substances (Iceland spar is in this an exception) shows itself in the extinction of certain rays of the white light, and the light seen through the second nicol is white light minus the extinguished rays ; Pig. 20. 40 METHODS OF RESEARCH AND ANALYSIS those extinguished and those transmitted will together form white light, and are thus complementary. Moreover, the rays extinguished in one position of the plate will be transmitted in one at right angles and vice versa ; thus a crystal showing these phenomena of 2)leochromatis77i as it is termed, will transmit one colour in one position, and the complementary colour in a position at right angles to the first; blue and yellow, and red and green, are the pairs of colours most frequently seen in this way. The subject of double refraction and polarisation of light will be discussed in ^certain special aspects in connection with hemoglobin crystals and muscle. Rotation of the plane of Polarisation.— Certain crystals such as those of quartz, and certain tiuids .-^uch as the essence of turpentine, aniseed, ic, and solutions of certain substances like sugar, and albumin, have the power of rotating the plane of polarised light to the right or left. The polarisation of light that is produced by a quartz crystal is different from that produced by a rhombohedron of Iceland spar. The light that passes through the latter is plane polarised ; the light that passes through the former (quartz) is circularly polarised ; i.e. the two sub-rays are made up of vibrations which occur not in a plane, but are curved. The two rays are circularly polarised in opposite directions, one describing circles to the left, the other to the right ; these unite on issuing from the quartz plate ; and the net result is a plane polarised ray with the plane rotated to right or left according as the right circularly polarised ray or the left proceeded through the quartz with the greater velocity. There are two kinds of quartz, one which rotates the plane to the right (dextrorotatory), the other to the left (Isevorotatory). Gordon' explains this by the following mechanical illustration. Ordinary' light may be represented by a wheel travelling in the direction of its axle, and the A-ibrations composing it executed along any or all of its spokes («). If the a ibrations all take place in the .^. £X a. l> h' !>' h'" Fig. 21. same direction, i.e. along one spoke, and the .spoke opposite to it (i), the light is said to be plane polarised. The two spokes as they travel 1 Physical Treatise on Electricity and Magnetism. OPTICAL INSllM.MKNIS 41 ■lUmii in tlio direction of the arrow will trace out a plane (nt^f figure 21) between /> and b'. If this pohirised beam be made to travel now through a solution of sugar, the net result is that the i)laiie so traced out is twisted or rotated ; tlie two spokes, as in bb', tlo not trace out a plane, but we must consider that they i-otate as they tra\'el along, as though guided by a spiral or screw thivad cut on the axis, so that after a certain distance the vibrations take place as in i'' ; later in b'", and so on. This effect on polarised light is due to the molecules in solution, and the amount of rotation will depend on the strength of the solution, and on the length of the colunui of the solution through which the light passes ; or in the case of a quartz plate on its thickness. Tf a plate of quartz be interposed between two nicols, the light will not be extinguished in any position of the prisms, but will pass through various colours as rotation is continued. The rotation produced for ilifterent kinds of light being different, white light is split into its various constituent colours ; and the angle of rotation that causes each colour to disappear is constant for a given thickness of quartz plate, being least for the red and greatest for the violet. These facts are made use of in the construction of polarimeters. Polarimeters are instruments for determining the strength of solutions of sugar, albumin, &c., In' the direction and amount of rotation they produce on the plane of polarised light. They are often called saccharimeters, as they are specially useful in the estimation of sugar. Soleil's Saccharimeter.— This instrument (see fig. 22) consists of a nicol's jn'ism r/, called the polariser ; the polarised ray passes next Flu. 22.— Soleil's Saccharimeter. through a quartz plate (b) 3-75 m.m. thick, one half (d in fig. 23) of which is made of dextrorotatory, the other half {g in fig. 23) of lievo- rotatory quartz. The light then passes through the tube containing the solution placed in the position of the dotted line in fig. 22, then through a quartz plate cut perpendicularly to its axis (g in fig. 23), then through an arrange- ment called a compensator {r in fig. 23), then through a second nicol (a) called the analyser, and lastly through a telescope (L in fig. 23). 4-2 3IETH0DS OF KESEARCH AND AX.U.YSIS The compeiisatoi' consists of two quartz prisms (RR, fig. 23) cut perpendicularly to the axis, but of contrary rotation to the plate just in front of them. These are wedge-shaped and slide over one another, the sharp end of one being over the blunt end of the other. By a screw the wedges may be moved from one another, and this diminishes the thickness of quartz interposed ; if moved towards one another the amount of quartz interposed is inc reased . The effect of the quartz plate (d, g) next to the polai-iser {c in fig. 23) is to give the polarised light a violet tint when the two nicols are -ioqIq' a® fe B' Fig. i3. — Diagram of optical arraugemento in Soleil'.s Saccharlmeter. parallel to one another. But if the nicols are not parallel, or if the plane of the polarised light has been rotated by a solution in the tube, one half the field will change in colour to the red end, the other to the violet end of the .spectrum, because the two hahes of the quaitz act in the opposite way. The instrument is first adjusted with the compensator at zero, and the nicols parallel, so that the whole field is of one colour. The tube containing the solution is then interposed ; and if the solution is optically inactive the field is still uniformly ^dolet. But if the solution is dextrorotatory the two halves will have different tints, a certain thickness of the compensating quartz plate which is la?vorotatorv must be interposed to make the tint of two halves of the field equal again ; the thickness so interposed can be read off in amounts corresponding to degrees of a circle by means of a vernier and scale (E in fig. 23) worked by the screw which moves the compen.satoi". If the solution is la^vo- rotatory, the screw must be turned in the opposite direction. Zeiss's Polarimeter is in principle much the same as Soleil's ; the chief difference is that the rotation produced by the solution is corrected not by a quartz compensator, but by actually rotating the analyser in the same direction, the amount of rotation being directly read off in degrees of a circle. Laurent's Polarimeter is a more valuable instrument. Instead of using daylight, or the light of a lamp, monochromatic light (generally the sodium flame produced by volatilising common salt in a coloui'less OITICAT- IN'STH r:\IKNTS 43 o-MS tliinu') is (Mn})l(>yo(l ; tlie aiiittuiit of i'ot;itiou v;iries for diflerent colours ; and now ;dl observiitions are recorded as having been taken with hght coi-responding to the D or sodium Une of the spectrum. The essentials of the instrument are as before, a polariser, a tube for the solution, and an analyser. The polarised light before passing into the solution traverses a quartz plate, which how^ever covers only half Fiu. 24.— Laurent's Polarimeter. the field, and retards the part of the ray passing through it by half a wave-length. In the 0° position the two halves of the field appear equally illuminated ; in any other position, or if rotation has been produced by the solution when the nicols have been set at zero, the two halves appear unequally illuminated. This is corrected by means of a rotation of the analyser, that can be measured in degrees by a scale attached to it. Specific Rotatory power of any substance is the amount of rotation in degrees of a circle of the plane of polarised light produced by 44 METHODS OF EESEAKCII AND ANALYSIS 1 gramme of the substance dissolved in 1 c c. of li(|uid examined in a column 1 decimetre long. If «== rotation observed. t(;=weight in gi'ammes of the substance per cubic centimetre. Z=length of the tube in decimetres. (n)p=specific rotation for light with wave-length corresponding to the D line (sodium tlame) then ((,)^=±^ 1. irl In this formula + indicates that the substance is dextrorotatory ; — that it is laevorotatory. If on the other hand [u)-^ is known, and we wisli to lind the value of ?/• ; then (")i,X/ The specific rotatory powers of a few of the more important optically active substances found in the body are as follows : — Dextrorotatonj Lcevon Siicrose . . („)j,= -|- 73-8° Levulose . Dextrose . . = -f 56-0° Egg albumin. Lactose . . . =+ 59-3° Serum-albumin Dextrine. . . = + 138-8= Gelatin otatory (r,)„=-106° . =- 33-5= . =- 56-0= . =-130° Gh'cocholic acid . =+ 29-0" Cholalic acid . . =+ 35-0° Sodium taurocholate =: + 24 -5° Chondrin (alkaline solution). = -213-5=^ EELATIOX BETWEEN CIECULAR POLARISATION AND CHEMICAL CONSTITUTION The first work in this direction was performed by Pasteur,- and it was his publications on this subject that brought him into prominence. He found that racemic acid, which is optically inactive, can be decomposed into two isomerides, one of which is common tartaric acid which is dextrorotatory, and the other tartaric acid differing from the common variety in being Iffivorotatory. The salts of tartaric acid usually exhibit hemihedral faces, while those of racemic acid are holohedral. Pasteur found that, although all the tartrate crystals were hemi- hedral, the hemihedral faces were situated on some crystals to the right, and on others to the left hand of the observer, so that one formed, as it were, the reflected image of the other. These crystals were separated, purified by recrystallisation, and 1 As solution may cause condensation of volume, the density {d) or specific .gravity of the solution should be also taken ; then (0)0= ± -" and w=± — u Id (a)D X la- 2 Ann. Chim. PJnjs. (2) xxiv. 442; xxviii. 56. CoinjJtes rend, xxxvi. 26; xxxvii. 162. Poggendorff's A)nialen, Ixxx. 127 ; xc. 498, 504. OPTICAL IXS'l'IMMKNTS 45 those wliich oxhibitt'd (lestiM-hcinila'dry possL'sscd dextrorotatory jiower, whilst the others were huvorotatory. Pasteur' further showed that if the moiMjH'mc'illiniu f/laiu'iim. be grown in a sohitioii of raceuiic acid, destro-tartaric acid first dis- appears, and the la;vo-acid alone remains. The subject remained in this condition for many years ; it was, however, conjectured that probably there is some mole- cular condition corresponding to the naked eye crystalline appearances which produces the opposite optical effects of various substances. What this molecular structure was, was pointed out independently by two observers — Le Bel- in Paris, and Van't Hoff* in Holland — who published their researches within a fewdaj's of each other. They pointed out that all optically active bodies contain one or more assymmetric carbon atoms, i.e. one or more atoms of carbon connected with four dissimilar groups of atoms, as in the following examples :— cir, co.oH r I U—(c)-CE, H— @— OH I I CH,.OH CH.,— CO.OH Amyl alcoliol Malic acid The question, however, remained — do all substances containing such atoms rotate the plane of polarised light ? It was found that they do not ; this is explained by Le Bel by supposing that these, like racemic acid, are compounds of two molecules — one dextro-, the other l;^vo-rotatory ; that this was the case was demonstrated by growing moulds, the fermenting action of which fs to separate the two molecules in question. Then the other question— how is it that two isomerides, which in chemical characteristics, in graphic as well as empirical formula, are precisely alike, differ in optical proi3erties .' — is explained ingeniously by Van 't Hoff. He compares the carbon atom to a tetrahedron with its four dis- similar groups. A, B, C, T), at the four corners. The two tetrahedra represented in tig. 25 appear at first sight precisely alike ; but if one be super-imposed on the other, C in one and D in the other could never be made to coincide. This differ- ence cannot be represented in any other graphic manner, and probably represents the difference in the way the atoms are grouped in the molecule of right- and left-handed substances respectively. THE SPECTROSCOPE When a ray of light passes from one medium such as air into another such as water or glass, it is bent out of its original course, or as it is termed refracted. The ratio of the sine of the angle of incidence to that of the angle of refraction is called the refracti^'e index. When white light is passed through a glass prism or a triangular bottle con- taining a substance like carbon bisulphide with a high refractive ' Cviiqit. rend. li. 158. - Bull. Soc. Chim. (2) xxii. 337. ^ La chitnic dans Vesjjacc. 46 :method.s of research am» analysis index, it undergoes two bendings, one at each surface of the prism ; the whole i-av is however not equally bent, but it is split into its constituent coloui-s, which may be allowed to fall on a screen ; the red rays will be found at one end of a continuous band of colour, the A-iolet i-avs at the other ; orange, yellow, green, blue and indigo Toeing the colours which are intennediate in the oi-rler named. This band of colours is termed a sjiectrum. The i-ainbow is an instance of a spec- trum produced in nature by the suns rays passing at a certain angle through drops of water. A spectroscope is an instrument pr(j\ided wiuh a prism or prisms to enable us to obtain a spectnim artilicially. In the spectnim the red rays are the least, the violet rays the most, refmnoible. If the spectnim produced by one piism be immefliately passed thi-ough a second prism like the first, but invei-ted, the coloured rays are reunited, and build up a white ray emerging from the second prism. The different colours are due to vibi-ations of jether of different rates of rapidity ; the wave-length of red light is greater than that of yellow, that of yeUow than that of gi-een, and so on, violet having the shortest wave-length. The wave-length of the ray changes in different media, and thus the velocity of propagation varies : the \-iolet is the most, the red the least, retarded ; the ^-iolet is thus bent the most, the red the least ; lience, arises the dispersion which results in tiie foi-ma- tion of the spectrum. In addition to the A-isible rays, other rays at either end are present which can be detected by their effects, though not by the eye. These i-avs are called respectively the ultra-red and the ultra-violet. The ultra-red rays are those in which heating effects preponderate, the ultra-violet are rays of chemical activity, i.e. produce such chemical changes as those on which the art of photography depends ; the visible ravs have heating, and chemical effects also, but in a subordinate degi*ee. The spectnim of sunlight is interrupted by numerous dark lines crossin"^ it vertically, called Fraunhofer's lines. They are perfectly constant in position, and serve as landmarks in the spectrum : the more prominent are lettertd ; A, B, and C are in the red, D in the yellow, E and F in the green, G in the indigo, and H in the \-iolet. The a (in the i-ed) and h lines (in the gieen) are also well marked. These lines are due to the presence of certain substances volatilised in the solar atmosphere. If the light from burning sodium or its com- pounds be examined spectroscopically, it will be found to give a -l^right yellow line, or rather two bright yellow lines, very close together ; it is in fact a tnie monochromatic light. Potassium gives two bright OITICAL INSTKIMKNTS 47 red lines, ;uilioiograph taken by Dr. MacMunn, frmi McKendrick's ' Physio'ogy.') rupted by a number of dark bands corresponding to the light absorbed by the coloured medium. Thus oxyluemoghjbin gives two perfectly characteristic bands between the D and E lines, hsemoglobin gi^-ing only one ; and other red soluti(jns, though to the naked eye similar to rsxyhjemoglobin, will give characteristic bands in other positions. Chlorophyll again gives four well marked bands, especially one in the recL The study of the absorption spectra of animal and Aegetable pigments is full of interest, and has been followe. c mmences by causing the D or sodium line to coincide exactly with that part of the scale which expresses its wave-length, that is to say, with the division 589 of the scale (this expresses the fact that the wave-length usually denoted by the Greek letter X is 589 millionths of a millimetre). Having done this, the scale is set accurately for all other points. The usual method of determining wave-lengths, namely Ijy interpo- lation curves, is thus described by MacMunn : — ' A piece of paper ruled into square inches and tenths, obtainable from Letts & Co., has a scale of wave-lengths ruled off along the right -band edge, and the upper edge at right angles to this has a scale corresponding to the scale of the instrument marked on it. The value of the Fraunhofer lines on the scale of the spectroscope is observed, and by a reference to Angstrom's numbers, their value in wave-lengths ; - they are then marked in their proper places on the scale with dots. A curve is then drawn through these marks as uniformly as possible. When a band or bright line has to be mapped out, all that is necessary is to take its reading on the scale ; then knowing between what lines it is placed, we find its position on the ctirve opposite which its wave-lengtli is printed on the right-hand edge. THE SPECTEOPHOTOMETEK The spectrophotometric method for estimating the concentration of coloured solutions was originally proposed by Bunsen and Roscoe,' in 1857. In 1873 Vierordt* invented a spectrophotometer, but it is Hiifner^ who definitely intro- duced the instrument into physiological methods. 1 The Sjyectroscope in Medicine, p. 32. 2 Angstrom's calculations of the wave-lengths of the principal Fraunhofer lines are as follows in millionths of a millimetre : A, 7(50-4 ; a, 71«-5 ; B, 686-7 ; C, 6.56-2 ; D, 5«i)-2 ; E, 526-9 ; b, 517-2 ; F, 486-0 ; G-, 430-7 ; Hj, 306-8 ; Ho, 393-3. {Recherches sur le spectre solaire. Upsala, 1865.) •' I'oggendorfi's Annalen, \o\. ci. ]i.-2'd5. * Vierordt, Die Anwenclung des Spectralapparates sur Pliotometrie der Absorptions- spectrum und zur quant, chem. Analyse, Tiibingen 1873, and a later pamphlet in 1876. ■'' Hiifner, Jonrn. f. jnakt. Chemie, xvi. (1877). Zcit. phijsiol. Chem. vol. i. ii. vi. &c. (^PTICAI. INSTRUMENTS 51 A very excellent general account of tlie spectrophotometer and its applica- tions in physiological chemistrj- is given by Lambling ' in a paper, which the reader is advised to consult. The method consists essentially in measuring the diminution in intensity which a beam of light undergoes in its passage through a coloured solution, and in deducing the concentration of the solution from such a measurement. Given two rays of equal initial intensity, one of which is per- ceived directly by the observer, and the other after its passage through a coloured solution, what one has to do is to measure the relative intensity of the two rays. But such observations must be made not with white light, which is mixed light, but with homogeneous light ; in other words, the rays from a particular part of the spectrum ; hence the term spectrophotometry. The amount of absorption varies for the same substance and the same region of the spectrum with the concentration and thickness of the layer of liquid examined. A double layer of the liquid would produce the same effect in absorb- ing light as a single layer of a liquid twice as concentrated. The quantity of light absorbed, however, does not increase directly with the thickness or concentration of the coloured liquid. Suppose a luminous ray of intensity equal to 1 passing through a layer of coloured liquid of one unit's thickness, its intensitv is reduced to - ; when, however, this ray of diminished n intensity passes through another similar laj-er, its intensity is diminished by - X _ = — , and after passing through m similar lavers to — . » n n- ' «"* T/ie co-efficient of extinction (Bunsen) e of a coloured solution is the inverse of a number expressing the thickness of the layer of that solution which is necessary to reduce the intensity of the light to one tenth of its initial intensity. We have already seen if I' = final intensity, 1 = initial intensity, and m = thickness of layer, that From which we see that '"-™ — r7 (Equation 1). Converting these into logarithms : — m log 11= — lo' Therefore log«=--^'?— (Equation!'). But by the definition of the co-efficient of extinction : — I' = — and m = -. 10 e Therefore «'n = /i* and -r,~ If we put these values of ti"' and ^, in equation 1 we get «"=10 Which in terms of logarithms is : — ^ log « = log 10 = 1 e 1 Arch, de Physiologie, 18b8, j). 1. 52 METHODS OF EESEARCH AND ANALYSIS Therefore log « ^ e Putting this value of log ii into equation 2, we get : _ _ log I' 111 And if 7« = unity, €= —log I'. In other words, the co-efficient of extinction is obtained by taking the nega- tive logarithm of the fraction which represents the final intensity of the light. Suppose, for instance, that a solution of oxyhaemoglobtn observed in a layer 1 centimetre thick reduced the luminous intensity in the region of the /3 band to 0-225 of the original, then e= -log 0-225 0-64782 Suppose that C, C, C" . . . represent the respective concentrations of a series of solutions, and e, e', e" . . . the corresponding co-efficients of C C C" extinction, then : — =-7 = -n • • • =^- 6 r € This constant A can be easily measured in a solution of known strength : it is called the absorptive power : A=5 Therefore C = Ae. In other words, the concentration of the solution (number of "Tarns in 1 c.c. of solution) can be ascertained by multiphing the co-efficient of extinction by the constant A. This method can also be applied to mixtures of two colouring matters in solu- tion— e.g. haemoglobin and oxyhjemoglobin — provided that the constant A is known for both substances in two regions of the spectrum. The co-efficient of extinction in the same two regions is then determined by observation. The formula is some- what comphcated, and the memoir already referred to must be consulted for this matter, as well as for other interesting suggestions relating to the examination of other animal pigments, as of the bile, urine, &;c.. by means of the spectrophoto- metric method. The forms of spectrophotometer that have been invented for the determina- tion of co-efficients of extinction are very numerous. I select for description one invented by Glazebrook, and described by Dr. Sheridan Lea in the ' Journal of Phvsiology.'' Li principle it is the same as Hiifner's, but differs from it, in that the liectrum is eclipsed. Then set the pointer in some intermediate position in which the two spectra are of equal brightness. Now let the solution of the pigment of unknown 1 Vol. V. p. 239. OPTICA I, INSTRUMENTS 53 concentration C be introduced on the path of the liglit wliich forms one of the spectra. In order to produce equality of the spectra the pointer of the eye-piece must be rotated into a fresh position. Now if 6 be the angle through which the eye-piece was rotated from 0° in order to produce the original equality of the spectra, and 6' be the angle of rotation required to produce equality when the absorbing substance is interposed, our formula C = A€ becomes C = 2Alog-^ -■ tan « Suppose, now, that the same be done with a solution of known concentration, C, and that 6" be the angle of rotation required to produce equality when this solution is interposed in the path of the light from one source, then C = 2 A log tan e C log tan 6 — log tan 6' ^ • r. i i . Hence — = - -, „,, from whicli equation Lean be calcu- tan e" -fi^"*-*^' c' log tan 0- log tan 0" latcd. THE SPECTRO-POLAEIMETEE ' This instrument is one, in which a spectroscope and polarising apparatus are combined for the purpose of determining the concentration of solutions of sub- stances which rotate the plane of polarised light. It was invented by E. v. Fleischl, for the estimation of diabetic sugar in urine. Its chief adv^antage is, that no difficulty arises of forming a judgment, as to the identity of two coloured surfaces, as in Soleil's saccharimeter, or of two shades of the same colour, as in Laurent's instrument. The light enters at the right-hand end of the instrument, Fro. 30.— Spectropolarimeter of von Fleischl. is polarised by the nicol's prism b, and then passes through two quartz plates, cc, placed horizontally over one another. One of these plates is dextro-, the other Ifevorotatory, and they are of such a thickness (7-7o mm.) that the green rays between the E and b lines of the spectrum are circularly polarised through an angle of 90°, the one set passing otf through the upper quartz to the left, the 1 The following account of this instrument is taken from Dr. McKendrick's Phijsiology, vol. i. p. 154. 54 METHODS OF RESEAECH AND ANALYSIS other through the lower to the right. The light then continues through a long tube,_/f", which contains 15 c.c. of the solution under examination. It then passes through an analysing nicol, d, and finally through a direct vision spectroscope, e. On looking through the instrument, the tube _^' being empty, or filled with water or some other opticallj- inert substance, two spectra are seen, one over the other, but each shows a dark band between E and h owing to the extinction of these ra.vs by the circular polarisation, produced by the quartz. The analyser can be rotated : a vernier, (j, is attached to, and moves with it, round a circular disc (seen in section at K) graduated in degrees. The two bands in the spectra coincide when the zeros of vernier and scale correspond. If now the tube / is filled with an optically active substance like sugar, the bands are shifted, one to the right, the other to the left, according to the direction of rotation of the substance in/. The rotation is corrected by rotating the analyser into such a position that the two bands exactly coincide once more as to vertical position. The number of degrees through which it is thus necessary to move the analyser, measures the amount of rotation produced by the substance in/, and is a measure of the con- centration of the solution. The degrees marked on the circular scale are not degrees of a circle, but an arbitrarj' degree of such a length that each corresponds to 1 per cent, of sugar in the given length of the column of fluid in ^ (177-2 mm.). PART II THE CHEMICAL CONSTITUENTS OF THE ORGANISM CHAPTER A^I IXTROJDUCTOliY The chemical constituents of the animal Ijody are exceedingly numerous ; they consist of chemical elements, of inorganic compounds, and lastly of organic compounds. The organic compounds are the most numerous ; some of them have a simple structure, but the greater number are very complicated. The elements found in the body are carbon, hydrogen, nitrogen, oxygen, sulphur, pliosphorus, fluorine, chlorine, silicon, sodium, potas- sium, calcium, magnesium, lithium, iron, and occasionally manganese, copper, and lead. Of these very few occur in the free state ; oxygen (to a small extent) and nitrogen are found dissolved in the blood. Hydrogen is found in the alimentary canal. Particles of carbon bi^eathed in with the air may be found in the tissues of the lung. With some few exceptions such as these, the elements enumerated above are found combined with one another to form what are called compounds. The compounds, or as they are sometimes termed in physiology, the proximate principles, found in the body are divided into (1) mineral or inorganic compounds, and (2) organic compounds or compounds of carbon. The inorganic compounds present are water, peroxide of hydrogen, sulphuretted hydrogen, ammonia, various acids, and numerous salts (sodium chloride, calcium phosphate, etc.). The organic compounds present are the various groups of alcohols and organic acids, and their comj^ounds such as the fats ; various de- rivatives of ammonia, for instance amides, amines, itc. ; the aromatic bodies, and lastly the proteids, albuminoids, pigments, ferments, carbo- hydrates, and glucosides. 58 THE CHEMICAL C0XSTITUEXT8 OF THE ORGAXISM CHAPTER YII IXOItGANIC COMPOUNDS WATEE (H,0) Water forms about 58-5 per cent, of the weight of the body; it occurs in different propox'tions at difierent ages, the proportion becoming- smaller as life advances. In an infant the amount present is 6G-4 per cent. (BischoiF). The following table from Beaunis ' gives the proportion of water in various solids and fluids of the body, in parts per 1000. Enamel Dentme Bone . Fat . Elastic tissue Cartilage . Liver Spinal cord White matter of brain Skin Brain Muscle Spleen Thj'mus Connective tissue Kidneys . 2 100 486 299 496 550 693 697 700 720 750 757 758 770 796 827 Grey matter of brain . 858 Vitreous humour . 987 Blood . 791 Bile .... . 864 Milk . 891 Liquor sanguinis . 901 Chyle . 928 Lymph . 958 Serum . 959 Gastric juice . 973 Intestinal juice . 975 Tears . 982 Aqueous humour . 986 Cerebro-spinal fluid . . 988 Saliva . 995 Sweat . 995 An adult takes into the body in the form of food (solid and liquid) about 2,500 c.c. of water per diem. A small quantity is formed in the body from the combination of hydrogen and oxygen, and thus a larger quantity is excreted than is actually taken in. On the average, the daily excretions contain about 2,600 c.c. of water. A diminution of the quantity of water leads to the sensation kncjwn as thirst ; in frogs, when they have lost 30 per cent, of their weight of water, death ensues. A great increase in the quantity of water is harmful, as it increases tissue waste, and carries off a large amount of the solids, especially the saline solids of the body, in solution. 1 Plujsiologie humaine. INORGANIC CoMI'orXDS 59 Injection of water into the circulation in large quantities causes death, as it dissolves the luenioglobin from the corpuscles and so interferes with respiratcny functions (Picot).' In starving animals (pigeons) the relation of water to solids only- shows important changes when the total body weight is diminished by 34 per cent, and the animal has taken no solid or liquid food for 133 hours. The relation in some organs (heart, kidneys, thoracic muscles, alimentary tract, l^lood, brain, and lungs) even then undergoes little or no change ; in others (thigh muscles, and bones) the water is increased ; and in a third category (spleen, pancreas, liver) the water is diminished (Lukjanow).- PEROXIDE OF HYDROGEN (H.A) C. Wurster ^ uses paper soaked with a solution of tetramethylpara- phenylenediamine as a delicate test for active oxygen, a- blue-violet colour being formed. The development of this colour by means of certain tissues, and jSuids of the body (skin, sweat, &c.), is believed to be due to the evolution of active oxygen from peroxide of hydrogen, present in those parts. Peroxide of hydrogen coagulates albumin, and Wurster considers it possible that its presence may explain such phenomena as the coagulation of the blood and of muscle ; and by its action on the haemoglobin of the blood various other pigments, such as those of the skin and hair, may be produced. In want of further proofs of these and other functions assigned to peroxide of hydrogen, we must accept all such conclusions with the greatest possible reserve. SULPHURETTED HYDROGEN (H,S) This gas occurs free in the alimentary canal, being formed by putrefactive processes which occur there. AMMONIA This also is formed in small quantities during these putrefactive processes. It, however, soon enters into combinations to form salts or organic compounds. Ammonia also occurs in urine, especially if it has been allowed to undergo putrefaction within or without the body. The mineral salts and organic compounds in which ammonia occurs will be described under other headings. ' Comptes rend. 1874, p. 62. - Zeit. jjhysiol. Chem. xiii. 339. 3 Berichte deutsch. Chem. Gesellsch. six. 3195 ; xx. 263, 1033. 60 THE CHEMICAL CONSTITUENTS OF THE ORCtANL^M ACIDS Pree hydrochloric acid occurs in the gastric juice. Free sulphuric acid in the so-called saliva of certain gasteropods (Doliitm galea, ttc.).' The acids found in the body are, as a rule, not free, but combined with bases to form salts (chlorides, sulphates, &c.). Free carbonic acid is found in small quantities dissolved in the fluid and solid tissues of the body. SALTS The chief salts found, are the chlorides of sodium and potassium, the sulphates of the same metals, phosphates of sodium, potassium, calcium, and magnesium, and the carbonates of sodium and calcium. Bone and similar tissues like dentine and enamel are chiefly rich in calcium salts, especially the phosphate. Other solid tissues, except the lungs, are especially rich in potassium salts. In fluids (milk excei^ted) the most abundant salt is sodium chloride. Enumerations of the various saline constituents will be given when we consider the fluids, tissues, and organs themselves. The following general tables, however, compiled by Beaunis - may appropriately be quoted here. The figui'es give percentage quantities of mineral matters in the ash. 1 Boedeker, Pogg. Ann. vol. xciii. p. 614. Journ. pra'kt. Chem. vol. Ixiii. p. 170. Panceri and de Luca, Compt. rend. vol. Ixv. pp. .577, 712. These tliree observers found from 2o to 3'5 per cent, of free sulphuric acid in this remarkable secretion. - I am indebted for the reference to these tables to Dr. McKendrick's Textbook on Plnjsiology. The subsequent remarks on the individual salts are very largely a resume from the same work. iNDiiuANic coMrurxDs 61 1 Fluid Blooil Suruiu Bidod- eh.t Lviiipli Urine Milk Bile Excre- ments Analyst . Vcrdeil Weber \Vel)ei- Duliii- I'orter vViM.T- Rose Porter 1 1 72-88 1736 liiir.lt 67-26 stein 4-33 Sodium chloride 58-81 74-48 10-73 27-70 Potassium „ — — 29-87 — — 26-33 Soda 4-15 12-93 3-55 10 35 1-33 — 36-73 5-07 Potash . 11-!I7 2-95 22-3() 3-2o 13-64 2144 4-80 6-10 Lime 1-7(3 2-28 2-58 0-55 Bile '>■?,.', 02S Milk 0-S7 213 Urine 11 00 4-50 Other Sodium and Potassium Salts.— Bunge found that the soda salts are more abundant in embryonic and early life than in adult life. This is illustrated bv the followini:- table : — Na.,0 K.,0 Na.,0 K..0 Rabbit's embrvo 2-183 2-605 Cat, 29 days old . 2-292 2-684 Rabbit, 14 days old 1-630 2-967 Dog, 4 „ „ . 2-5S9 2-667 Kitten, 1 day ,, 2-666 2-691 Adult mouse . 1-700 3 -2X0 Cat, 19 days 2-285 2-790 62 THE CHE3riC.\L CONSTITUENTS OF THE ORGANISM This fact is probably due to the larger amount of cartilage (rich in soda salts), and the smaller amount of muscle (rich in potash salts) in early life, as compared with the adult condition.' Soflium phosphate (XagPO^), acid so^lium phosphate (Xa^HPO^), and acid potassium phosphate (KoHPO^) are found in the urine, the latter salts causing the acidity of that secretion. Phosphates of sodium and potassium also occur in the bld and tissues. Sodium carbonate (NaoCOs) and bicarbonate (XaHCOg) occur in the food, but are chiefly formed from the salts of vegetable acids (tartaric, citric. «tc.). They occur in the blood, and carry the carbonic acid from the tissues to the lungs. Sodium sulphate (Xa^SO^) and potassium sulphate (K.^SO^) exist in small quantities in the body ; they are partly introduced with the food, but cliiefly formed by the oxidation of proteids and other organic substances containing sulphur. Anunoniuin Salts. — Minute traces of ammonium chloride are found in the urine ; ammonium carbonate is formed from urea in decom- posing urine. The urine of reptiles, and birds is largely composed of ammonium urate. Small quantities of this salt, and also of ammonio- magnesic phosphate, are found in human urine. Calcium Salts. — About three-quarters of the total mineral solids in the body consist of calcium phosphate, Ca3(P04).2; this is because of the gi-eat preponderance of this salt in bone. Other calcium salts occurring in bone, dentine, and enamel are the carbonate, sulphate and fluoride. Calcium phosphate, urate and oxalate are found in the urine. Most tissues contain small quantifies of the phosphate and carbonate. Egg .shells, the shells of Crustacea, coral, and otoliths consist chietiy of carbonate of lime. MagnesiTun Salts. — Magnesium phosphate (Mg:3(PO^).2) occurs in the tissues, along with the calcium phosphates (Ca3(P04)2 and CaH4(PO,)2), but in smaller amount. It occurs also in the urine : ammonio-magnesium or triple phosphate (NH^Mg.jPO^ + GH.^O) is also often found in decomposing urine. Magnesium palmitate and stearate are found in the ftfces. Iron is an important con.stituent of the blood-pigment. The blood of an adult contains 3 grainmes of iron. Small quantities are found in other Uquids of the Ijody (chyle, lymph, bile, milk, urine, gastric juice): it is also contained in the black pigment of the skin and hair, and of melanotic sarcomata. A small quantity of ferric sulphide is found in the faeces, and small quantities of iron are found in both liver and .spleen. ' Bunge, Zeit. physiol. Chem. xiii. 399. jn()U(;anic co.Mi'drxDs 03 Other Metals. — Copper is found in two proximate principles, hieinocyanin, the blue pigment of the blood of many invertebrates (crustacea, cuttle fishes, scorpions, tire), and in the pigment turacin of birds' feathers. Small quantities of this metal, and also of aluminium, manganese and lead, may occur accidentally in other parts, being taken in Avith the food, and not excreted at once with the faeces, but deposited in some tissue or organ. Drugs and poisons (mercury, arsenic, ii:c.) may be similarly deposited. Silicon. — A minute quantity of silica exists in the blood, urine, liones, hair, and other parts. Phosphates. — The amount of phosphoric acid given in analyses of the ash of animal structures is not always correct, since a certain quantity is obtained during the process of incineration from the decomposition of organic compounds, which like lecithin contain phosphorus. The phosphoric acid which occurs in mineral compounds in the liody is derived partly from the food, and partly from the metabolism of lecithin and nuclein. It unites with soda, potash, lime and magnesia to form the various phosphates already alluded to. An adult man eliminates by the kidneys 2 -5-3 -5 grammes of phf)sphoric acid daily. Carnivora eliminate phosphates chiefly by the kidneys, herbivora chiefly with the f feces. Carbonates. — The presence of carbonates in the ash of animal matters is partly deri^•ed from the decomposition of organic compounds. Alkaline carbonates and bicarbonates are however found in blood, urine, lymph, saliva, ifcc. Sulphates. — These also may be partly formed during the process of incineration from j^roteids, and other organic compounds containing sulphur. The sulphuric acid in the urine is partly combined as ordinary sulphates, partly as ethereal sulphates. It is derived to a small extent from the food, but chiefly from the metabolism of proteids, the amount of sulphuric acid and urea in the urine running parallel with one another. 64: THE CHEMIC.IL CONSTITUENTS «1F THE oRG.lNLSil CHAPTER VIII TI{£ SIMPLER ORGANIC PROXIMATE PRIXCIPLES It was at one time supposed that the organic compounds differed from the inorganic, in the fact that it was not possible for the chemist to make them artificially from their elements. Many of the organic constituents of the body have however been produced in the laboratory since that time.' Organic compounds are now regarded as the compounds of carbon ; this definition would however include carbonates, which we have already considered with inorganic substances. Schorlemmer describes organic chemistry, as the chemistry of the hydrocarbons and their derivatives. Carbon is a tetrad element: its atomic weight is 12 (11"97). THE PAPtAFFINS AND THEIR DERIYATIVES The simplest hydrocarbon known is marsh gas or methane ; its- formula is CH4. This is the first raeml^er of the series known as the paraflins. The paraffins differ from one another by CH^: the second member of the series, etlian". has the formula CoHg ; the thu"d, CjHg, and so on. The tyjdcal fozTuula for the series is therefore C„H.^^.2 The lowest members of the series are gaseous, the next fluid, and the higher members form the solid or hard paraffins. By replacing an H of the hydrocarbon by hydroxy 1 (OH), a com- pound of the nature of a hydrate is formed. In the case of methane, the formula for this hydrate will be CH3.OH : in the case of ethane C.^Hj.OH : and so on throughout the series. These hydrates differ from the metallic hydrates, like potassium liydrate (K.OH) or sodium hydrate (XaOH), by the fact that the hydroxyl is combined, not with a metal, but with a group of atoms called a radicle : in the case of methane with CH3 which is called methyl : in the case of ethane with C.,H.5 which is called ethyl, and so throughout the series. These organic hydrates are called ahohoh, the first methylic, the second ethylic or common alcohol, and so on. There are, as we shall see, other series of alcohols in addition to those derived from this parafiin series. This tiret and simplest family • The synthesis of urea by Wchler ia 1828 was the first step in_this direction. THE SIMPLER ORGANIC PROXIMATE rRIXCIPLES 65 of alcohols are known as the inotintotnir dlcohoh ; tliat is, they contain only one hydroxyl group, and the radicle (methyl, ethyl, itc.) is therefore monovalent, like the metals hydrogen, sodium, potassium, itc. Another group of suljstances calle ^^^^ ^^^^ lowest term of these series is the simplest hydrocarljon known, namely, methane or marsh gas. There are in addition to these parathns, how- ever, other series of non-saturated hydrocarbons, each f)f which may be the basis of a group of chemical substances. The general formulae for these .series of paraffins are C„H,,„ C„H.:„_o, C,Ho„_j, C„Ho„_gj ^,ii^ii,~x and so forth. The series of paraffins starting with methane (CH^) forms the basis of the ordinary monatomic alcohols, aud the corresponding fatty acids. The series of paraffins with the general formula C„H2„ starts with THE snri'l.KlJ OIMANIC I'HOXl.MATE PRINCIPLES 67 €thene, ethylene or oletiant gas C2H4, and forms the basis of a series of alcohols, which are called glycols. These, like the first series of alcohols, aie hydrates, i.e. compounds of hydroxyl with organic ladicles, > C,,H^O^ nialonic CjH^lJ^ succinic CsHjOj P.vrotartaric 1^ or gliitaric )' C„H,oO< a) Tlie Oxalic acid series. — Oxcdic acid occurs in the urine as 72 THE CHEMICAL CONSTITrENTS oF THE ORGANISM oxalate of lime, where it is deposited as octahedral or dumb-bell crystals. Its relations to urea and uric acid will be discussed under urine. Succinic acid has been detected in the urine, after food containing malic acid or asparagine has been taken. Small quantities have been discovered in spleen, thymus, thyroid, hydrocele fluid, Arc. Traces of this acid (and also of glycerine) are formed during the alcoholic fermentation of dextrose by means of yeast. Succinic acid (CjHgO^) is closely related in composition to three acids contained in many vegetable foods, viz. malic acid (C^.H^O^), tartai'ic acid (C^HgOg), and citric acid (C,;H^^O-). Acids of the Acrylic Series. — Acrylic acid itself (C3H4O2) is obtained by the oxidation of acrolein (C3H4O), the aldehyde of allyl alcohol. Acrolein is also produced by the removal of two molecules of water from glycerine (C3HHO3-2H2O-C3H4O). Crotonic acid occurs in croton oil. Angelic acid occurs in croton oil, and angelica root. Its aldehyde occurs in essential oil of chamomile. Erucic acid (C.,2H4 202), a high term of the series, is found in rape- seed oil. Oleic acid (C18H34O2) is more important to the physiologist, as it occurs not only in vegetable oils (almond oil, olive oil, etc.) but also in the glyceride olein, an important constituent of the fat of adipose tissue, and of milk. Aiuido-acids. — These are acids derived from the fatty acids, by replacing one or more hydrogen atoms by the radicle amidogen (NH,). This important group of substances, which includes leucine, tyrosine, glycocine, taurine, creatine, ifec, will be more conveniently dealt with in connection with the nitrogenous proximate principles of the body. The Fats. — The fat of adipose tissue is a mixture of the glyceric ethers, or glycerides of palmitic, stearic, and oleic acids variously mixed together. In cream there ai-e in addition small quantities of glycerides of fatty acids lower in the sei'ies. Their chemical characteristics will be more fully described in con- nection with adipose tissue and milk. Small quantities of fat are, however, found in other parts. The table on the next page from Gorup-Besanez gives the percentage of fat in the organs and fluids of the human body. Moleschott found that in a man 30 years of age, weighing. 64 kilo- grammes, about 2-5 per cent, of the body weight was composed of fat. Burdach gives an average of 5 per cent. It need hardly be said that it varies immensely. TlIF SIMPLKK OKGAXir TR'^XIMAT?: PRINCIPLES 73 Sweat Vitreous luiiiiou Saliva Lymph Synovia Liquor aninii Chylo Mucus Blood Bile . Milk . 0-001 Cartilage 0-002 Bone . 0-02 Cryst.allinc lens 0-05 Liver . 0-06 Muscles 0-06 Hair . 0-2 Brain . 0-3 Egg . 0-4 Nerves 1--1 Adipose tissue 4-3 Marrow i-;5 1-4 2-0 2-4 3-8 4-2 8-0 11-G 22-1 82-7 96-0 Tripah)iitiu C3H5(O.C|qH3iO)3 and tristearin C;jH5(O.CigH350)3 are the solid fats of the body, they are held in solution at the body temperatui'e by ti'iolein C3Hr,(O.C,gH330)3. Trimargarin is a mixture of the two first-named fats. Tributyrin 03115(0.041170)3 is found in butter. Trivalerin 03H-,(O.O.r,H90)3 exists in the oil of marine animals like the seal. Tricaproin 03H5(O.OqH,iO)3, tricaprylin 03115(0.0^11150)3, and tricaprin 03Hg(O.CioH,90)3 are found in milk and butter. In the decomposition of fat, we may find propionic, acetic, and formic acid, which are absent from the fat in tlie fresh condition. This occurs when a fat becomes rancid, and is doubtless produced also by the action of putrefactive organisms in the alimentary canal. This is really a process of oxidation : the way in which a lower term of the series is tlius produced may be illustrated by the following equations : — OaH^Oo + 03=O.H,Oo + 00, + H,0 [propionic acid] [iicetic aciil] O.H.O, + 03=OH202 + OO2 + H9O [acetic acid] [formic acid] 0H2O2 + O=0O2 + HoO [formic acid] Lecii/dn. — This substance (042H84NPOy) is a wax-like material, which can be separated from the nervous tissues and blood cor- puscles. According to some observers it occurs in nervous tissues in combination with a nitrogenous glucoside called cerebi-in to form protagon. When boiled with an acid or alkali, lecithin yields glycero-phos- phoric acid, stearic acid, and an alkaloid called neurine or choline. Lecithin is abundantly found also in the seeds of many plants.' ^ Schulze and Steiger, Zeit. phijsiul. Cliem. xiii. 365. 74 THE CHEMICAL C(^NSTITUENT,S OE THE ORGANISM AROMATIC COMPOUNDS "We have already seen how several different substances may exist, having the same formula. Isomerism, as this is termed, is due to differences in the atomic constitution of the molecules. Take the substance known as ethyl chloride : this has the formula CoHjCl, and can be represented graphically only under one possible form : — H H I I , H— C — C— CI I H H But in the case of a substance having the formula CoH^O, there are two possible arrangements of atoms : — H H H H H— C— C— OH and H— C— O— C— H H H H H [etliyl axijho'] [methyl ether] and as a matter of fact the two substances, ethyl alcohol and methyl ether, actually exist. Or take the case of propyl chloride : again we have two, and only two, possible arrangements of atoms, and actually the two isomerides have been found to exist. H H H H H H ! I I i I H— C— C— C— Cl and H— C— C— C-H H H H H Cl H [orthripropyl chloride] [isopropyl chloriile] In the case of compounds containing more than three atoms of cai-bon, the isomerides possible are more numerous, and those that actually exist are also more numerous. It does not, however, neces- sai'ily follow that actual compounds exist, corresponding to all the possible combinations, and a laio of limitation is still wanting (Dittmar).i One of the most striking of these instances is that of benzene ; this substance has the formula CgHg ; there are more than thirty possible arrangements in which the atoms might be strung together, and yet 1 McKendrick's Phijsiologij, vol. i. p. 51. TIIK Sl.MI'LKIi ORGANIC PROXIMATE PRINCIPLES 75 only one benzene exists. Kekule' represents the constitution of benzene thus : — . H 1 C /\ H-C C— H H— C C-H c H that is, the six atoms of carbon do not form an open chain as in the substances of which the graphic formula have just been given, but a closed ring (the benzene ring). In this ring every two neighbouring C's are united alternately by a single and a double bond, and the fourth combining power of each C is satisfied by the H. This substance benzene is the fuundation of the aromatic group, which contains very numerous members. These can be derived by substituting one or more atoms of hydrogen, by more or less compli- cated radicles. If for instance one atom of hydrogen be replaced by one of chlorine, we obtain chlor-benzene, a su.bstance of great stability. If one atom of hydrogen be replaced by hydroxyl (HO), an alcohol-like substance, plienol is obtained ; but it is distinguished from the alcohols in the same way that chlor-benzene is distinguished from the alcoholic chlorides : viz. the OH is more strongly attached than it is in the alcohols. One of the hydrogen atoms is also replaceable by NOo (the radicle of nitric acid), to form nitro-benzene (C(;HgNOo), or by amidogen NHg (to form amido-benzene or aniline, CyHgNHg), and thus nitrogenous aromatic compounds are obtained. Or, again, hydrogen may be replaced by radicles containing carbon, and so substances containing more than 6 atoms of carbon are added to the group ; for instance 1, 2, 3 or more atoms of the hydrogen are replaceable by methyl, and we get the following series : — Benzene ..... C^Hg Methyl-benzene (Toluene) . . C6H.5(CH3) Di-methyl-benzene . . . CgH4(CH3)2 Tri-methyl-benzene . . . CgH3(CH3)3 Tetra-methyl-benzene . . . C5H^(CH3)4 1 Liebig's Annalen, vol. cxxxvii. p. 160. 76 THE CHEMICAL CONSTITUENTS OF THE OROANISM Again i\ similar series is obtainable with ethyl, and all the other alcohol radicles ; still further complication is produced by i-eplacing some H's with one, and some with another kind, of radicle; and even more complicated compounds than these can be reached, Ijecause the atoms of hydrogen in the methyl, ethyl. Arc, are replaceable by other elements, or other radicles. In addition to all this, isomerism has to be reckoned with. If we repi"esent the molecule of benzene by a hexagon, at the corners of which the carbon atoms are placed, it is seen that three isomeric di-methyl-benzenes can exist; in which the two methyl groups have the positions indicated by the figures : a. 1 and 2. Ortho-di-methyl-benzene. (j/\'> h. 1 and 3. Meta-di-methyl-benzene. | | c. 1 and 4. Para-di-methyl-l^enzene. "\/ No other cases of isomerism are here possible ; as 1 and 6 is identical with 1 and 2 ; and 1 and 5 with 1 and 3. One more example of this kind of isomerism may be given : ( OTT pyrocatechin or catechol has the formula CgH^ - ,-vtt ; i.e. two atoms of hydrogen in benzene are replaced by hydroxyl. This substance stands to phenol, as ethene glycol does to ethyl-alcohol ; it is a diatomic phenol. But the hydroxy Is may have different positions, as the methyls in the example just given, and thus we have two isomerides of pyro-catechin, which are known as resorcin and hydroquinone respectively. We may now take seriatim the members of this impoi'tant family, which are interesting to the physiologist, either because they occur in the body, or are useful as drugs or reagents. Phenol or Carbolic acid CgHg.OH is a white crystalline substance, fusing at 42°C., boiling at 184°, and forming the chief constituent of the heavy coal oils. Perchloride of iron gives with it a deep violet colour. A chip of fir or deal moistened with phenol and then with dilute hydrochloric acid and exposed to the light turns a deep greenish blue. Phenol reduces silver nitrate. When heated with nitric acid, tri- nitrophenol C6H2(N02)30H, commonly called picric acid, is formed. An aqueous solution of phenol gives with bromine water a yellow- ish crystalline precipitate of tri-bromo-phenol (CgHaBrg.OH). This reaction may be used for the quantitative determination of phenol. Phenol gives in addition the following colour reactions : a. A blue or greenish colour, on adding a quarter of its volume of ammonia and a few drops of potassium chloride. TTiE sT:\rrT,F,rv organic prvr)XT:\rATE PinN'OiPLEs 77 b. A blue colour in presence of a little aniline and an alkaline solu- tion of sodium hypochlorite. f. An intense red colour with Millon's reagent. d. A brown colour changing to green and blue on adding fuming niti'ic acid, or a 6 per cent, solution of potassium niti'ite in strong sulphuric acid. Phenol occurs normally in the urine, sweat, and ffeces in small quantities, but especially after medical or surgical treatment with carbolic acid or other drugs containing a benzene nucleus. It is seldom present in the free state, but usually as phenol sulphate of potassium (CgH.^O.SOaK). The dark colour of the urine in carboluria is due to one or both of the two isomerides, pyrocatechin and hydro- chinon. On exposure to the air (oxidation) in an alkaline urine these substances turn dark brown. Phenol is formed by the activity of the pancreatic ferment and putrefactive organisms on proteids in the intes- tine. That normally in the urine is absoi-bed from the intestine. Pyrocatechin or Catechol Q,^j^{Qi]A^).^. — This occurs in small quantities as a conjugated-sulphate in the urine. It is a crystalline substance, which turns brown on oxidation in alkaline solutions, and green on admixture with ferric chloride. It was called alcapton by Bodeker, when he found it in abnormally large quantities in certain urines. It must be carefully distinguished from sugar, as it reduces alkaline solutions of copper salts like Fehling's solution. It occurs in the cerebro-spinal fluid. It is one of the products of the decom- position of proteids. Cresol C7lI-(H0). — This is a derivative of toluene or methyl- benzene (C-Hg). It is contained in crude carbolic acid. It boils at 200°C. It also is a product of the decomposition of proteids, and so is found in the faeces, and a small quantity passes into the urine as cresol sulphate of potassium. Benzyl alcohol has the formula CfiH^CH,) ^ ., ,, , , ■^ '^ " jj^tO; its aldehyde is CgHyCOH, i.e. the alcohol minus H, ; and an acid is formed by replac- ing the H., by O. The aldehyde is known as oil of bitter almonds, and is the result of the decomposition of the amygdalin contained in the almond. The acid is called benzoic acid CyH^Oo. This occurs in the urine, especially of herbivora, combined with glycocine or amido-acetic acid (C.HjOo.NH.j) to form hipfmric acid (C9H9NO3). The radicle of benzoic acid is called benzoyl (C^H^O). Salicylic or Oxybenzyl group. — The members of this group are closely connected with the benzoyl group ; they are benzoyl compounds in which an atom of hydrogen is replaced by hydroxy! (HO). CoH^j, 78 THE CHEMICAL CONSTITrENTS OF THE ORGANISM The formula foi' salicylic acid is 0711^03. If two atoms of hydrogen in this be replaced by hydroxyl, a substance is obtained with the formula CjHgOg, which is called gallic acid. This acid is generally obtained from nut-galls. On heating it splits up into COo and pyrogallic acid (ti'i-hydroxyl-benzene C6H3(OH)3). Tri-methyl-benzene CgH3(CH3)3 is the starting-point of the aromatic compounds containing nine atoms of carbon. The most important members of the group are anethol, the chief constituent of anise oil; anisamic acid, which exists in the balsams of Tolu and Peru ; cumarin, found in Tonka-bean, and sweet scented grasses : tyrosine f OH C H /'"\'TT \rO TT' ^ pi'oduct of the decomposition of albuminous substances, hair, horn, &c.; it is found also in the cochineal insect. Being an amido-acid, it will be more fully descriljed with that group. Thymol, an important antiseptic, contained in oil of thyme, is a derivative of tetra-methyl-benzene. Aromatic oxy-acids. — Two of these, hydroparacumaric acid and paraoxyphenyl acetic acid, are found in the urine in combination with potassium in small quantities. They apparently are derived from the decomposition that takes place in proteids in the intestine ; tyrosine is probably an intermediate product (Baumann).' C9Hll^03 + H2= CgHjoOg +NH3 [tyrosine] [liyilroparacumaric acid] C,H,o03 = CsH.oO +C0, [hyilropai-acumaric aciil] [paraetliyl jilieuol] C8H,oO + 03 = CgHgOg -fH-^O [paraetliyl phenol] [paraosyplienyl acetic acid] The Indigo Group. — Substances belonging to this group are found not only in the vegetable kingdom, but also in animals. The pure colouring matter obtained from the crude commercial pi'oduct is called indigotin or indigo-blue CgHgNO. This by the action of reducing agents becomes indigogen or indigo-icliitp, CgHr.ISrO. On oxidation a body called isatin is formed, C^H.^NOo. When indigo-white is heated with zinc and water it yields indole Cj^H^N; and when isatin is acted on by potash, it yields aniline (CsH,NOo-f 4KOH = C6H-N + 2K,C0;, I TT \ [isatin] [aniline] This last reaction shows that the indigo group of substances contains the benzene group of atoms. The parent of all these substances in plants is a colourless 1 Zeit. pliysiol. Chem. vol. x. i>. 123. THE STMrT,EK Ol^aANTC I'TJOXIMATE rHTNCI I'EKS 79 substance called indican C.,,;H3,N0,-. Iiidioau is a o-lucoside ; when boiled with acids, it splits into indij,'()-blue, and a sugar-like substance called indiKlucin. C,oH,,NO,7 + 2H20=C,H,NO + 3(C,;H,oO,). [iii(iicaii] [iiicligo-blue] [imliu-luciii] The starting-point of the indigo series from a chemical point of view is indole. We pass from indole to indigo-blue by successive oxidations, and from indigt)-blue to indole by successive reductions. A full list of the various intermediate products with their formula' is as follows :^ Indole C.H^N I Isatin CgH^NOo Ox-indole C,H-NO ' Indigo-white C,H,,NO Diox-indole C^H-NOa Indigo-blue CgHsNO Isatyde C^H^NOo ' The formula for indole is thus represented graphically by Baeyer and Ennnerling : — C,H / >CH Of this group, two members are found in the body, viz. indole (from which indigo is obtainable) and skatole, a derivative of indole. IndoU CgHyN is an oily fluid, which crystallises when mixed with water ; the ciystals melt at 52°C. It has a fa?cal odour and is readily soluble in alcohol and ether. It gives a red precipitate with dilute fuming nitric acid. This precipitate is soluble in alcohol, and the alcoholic solution, mixed with hydrochloric acid, colours tir-wood cherry red, changing after a while to dirty brown red (Baeyer). It is a i^rodvict of the decomposition of proteids, and is formed from these substances dui'ing their stay in the intestine. It passes away partly with the faeces ; part is absorbed, and finally excreted with the urine as an ethei'eal sulphate. Indigo. — If the urine (especially in diseases where nuich putre- faction occurs in the alimentary canal, or after the administration of certain drugs — creosote, benzoic aldehyde, tui-pentine, etc.) be boiled with a lai'ge quantity of strong hydrochloric and a few drops of nitric acid, a violet-red colour appears, due to the formation of indigo-blue and indigo-red. There are also other methods employed for demon- strating the fact that, on oxidation, indigo may be obtained from the urine. The parent substance in the urine is called indican, but this must not be confounded with the indican of vegetables. Vegetal)le indican is a glucoside. The indican of urine is indoxyl-sulphate of potassium, and is derived fnmi the indole of the intestine. Its formula is CgHfiNS04K (i.e. the radicle of potassium sulphate, KSO3, plus 80 THE CHEMICAL CONSTITUENTS OF THE ORGANISM indoxyl, CgHgNO). This substance does not apparently occur in the sweat, or only in traces ; its presence has, however, been stated to have been demonstrated in cases of clirornidrosin, or coloured sweat (Bizio, Hofmann). Skatole CgHgN is methyl indole, CgHg(CH3)N, i.e. indole in which an H is replaced by methyl, CHg. When pure it occurs in dentate shining plates, having a fecal odour, and melting at 94°C. Fuming nitric acid gives with its solutions a white, cloudy precipitate, thus distinguishing it from indole. From its hydrochloric acid solution, it is thrown down, on the addition of picric acid, in the form of red needles. When present in the urine, it gives a violet-red colour with strong hydrochloric acid and chloride of lime. Skatole like indole is formed in the alimentary canal from proteids ;' most is excreted pe?' rectum ; a small quantity is absorbed, and finally excreted in the urine and sweat. Absorption of a large quantity of indole, skatole, &c., produces disturbances of the nervous system ; and many of the unpleasant symptoms of constipation may arise from this cause. In the urine skatole is found, like indole, in the form of an ethereal sulphate. The name of this compound is skatoxyl sulphate of potassium (C9HgNS04K). It has been surmised that this substance may, like the corresponding indoxyl compound, give rise to a pigment. Mester^ finds, however, that the amount of the so-called skatole- pigment is not proportional to the amount of the skatoxyl sulphuric acid • and he suggests that the chromogen of the pigment is a combination of skatoxyl with glycuronic acid. Skatoxyl-potassium-sulphate is also present in the sweat (Kast).^ NITEOGENOUS OKGANIC COMPOUNDS A few nitrogenous organic compounds have already been described ; we have to deal now more especially with the organic derivatives of ammonia. Amines. — An amine is a compound ammonia, which can be obtained by replacing one or more atoms of the hydrogen in ammonia (NH^) by alcohol radicles. Of these only one has been described in the body — trimethylamitie N(CH3)3, which occurs normally in human urine, and is found in guano, decomposing fish, and decomposing proteids generally. It is the substance to which the characteristic smell of fish is due. It 1 Fiist four.d ihere by Brieger {Ber. d. dcntsch. chein. Gcs. vol. viii. p. 722), and Ner.cki (Ccntralll. Med. Wiis. 1B78, No. 47J. - Ziit. phijsiol. Cliem. xii. ISO. ■' Ihid. xi. 501. TlIK SJ.MI'l.KU ORGANIC I'KOXIMATE PiaXCIPLES 81 is an oily fluid, strongly ;iIk;iliiio, soliihlo in ijcoliol, ctlicf, and water. It boils at 9° C. IfajyhthyJ amine (Ci„HgN) is a pi-odnct of the oxidation of proteids, and naphthalene (C|,)Hj^) has been detected by Hoppe-Seyler in the urine. Amides are derivatives of acids which have exchanged the hydroxyl (HO) of the acid for amidogen (NH,,). Urea (CONoH^) is a tyjjical member of the group. By some it is regarded as the diamide of carbonic acid. Hydrogen carbonate has the formula C0(0H)2 ; replace the hydroxyls by amidogen, and we get CO(NH2)2. From another point of view it may be regarded as being built in the type of two molecules of ammonia, in which two hydrogen atoms are replaced by the dyad radicle CO : — H ^ CO=CON2H4 H It is thus carbamide. Urea is isomeric with ammonium cyanate (NH4)CN0, from which it was first prepared synthetically by Wohler (1828). When am- monium cyanate is heated to 100°C., the atoms rearrange themselves to form urea. It may also be prepared by the action of ammonia on carbonyl chloride (COCl2 + 4NH3=CON2H4 + 2NH4Cl). By uniting with water, urea forms ammonium carbonate. This it does under the influence of a specific organised ferment {micrococcus ure(e) in decomposing urine (CON2H4 + 2HoO = (NH4)2C03). Urea is met with in nearly all the solids and fluids of the body, but chiefly in the urine ; about 30 grammes (.500 grains) are on the average excreted by the kidneys of an adult daily. Urea is the chief end product of the metabolism of the nitrogenous constituents of the body. Oxaluric acid is urea in which one atom of hydrogen is replaced by the radicle of oxalic acid (i.e. oxalic acid minus HO). The Amido-Acids are compounds which show partly the character of an acid, and partly that of a weak base. They may be considered as ammonias, in which one or more atoms of hydrogen are replaced by radicles of an acid, thus resembling alkaloids ; or, on the other hand, as acids in which one or more hydrogen atoms of the acid radicle are replaced by amidogen (NH2). The principal are as follows : — (rt) Glycocine. — This is also known as glycine, glycocoll, and amido- acetic acid, the last-mentioned name expressing its constitution. The formula for acetic acid is C2H,,02 ; if one atom of hydrogen be re- G 82 THE CHEMICAL CONSTITUENTS oF THE ORGANISM placed by NH.,, we obtain C2H3(XHo)Oo=C2H-XOo, which is the formula for glycocine or amido-acetic acid. It has been obtained syntheticaUv bv the action of monochloracetic acid on ammonia (CoHgClO. + XH3=HC1 + CoH3(XH.)02). In a pure state glycocine crystallises in rhom- bohedric prisms, soluble in water, but not in ether or alcohol. The aqueous solution is acid. When heated on platinum foil, it leaves a colourless residue, which, on warming with a drop of caustic soda solution, forms an oily drop which runs about without touching the surface of the platinum. (Scherer's test.) When heated in a glass tube open at both ends, it sublimes, and a smell of amylamine Fig. 31.— Gl;. cocine crystal.-;. -g (^iven off. Glycocine is found in combination in glycocholic acid (one of the bile acids), and in hippuric acid (in the urine). It is a product of decomposition of proteids, and also of gelatin, mucin, and other albuminoids. It occurs free in small quantities in the intestine as a result of the decomposition of the bile that occurs there. It is probably largely reabsorbed as such. Part may be transformed into ui'ea. (b) Leucine (CgHi3N0,) is amido-caproic acid, i.e. caproic acid (CgHigOo) in which an H is replaced by XH, : or, according to some, oxy-caproic or leucic acid (Ct^H^oO^), in which hydroxyl (HO) is re- placed by yji.. Leucine forms yellowish -brown spheres consisting of masses of needle-.shaped crystals, soluble in water and shghtly .soluble in alcohol, but insoluble in ether. By heat it is decomposed into carbonic acid and amylamine (CeH,3XO,=C0.2-fC.:;H,,.XH2) ; by hydriodic acid into caproic acid and ammonia (C6H,3X0.2 + H.2=CgH,202-f NH3) ; with sulphuric acid it yields ammonia and valerianic acid ; with potassium permanganate, oxalic acid, carbonic acid, valerianic acid and ammonia. Probably similar decompositions occur in the body. Probably also leucine is one of the intermediate products in the formation of urea. Leucine is most important as one of the chief decomposition products of proteids, and is formed when proteids are decomposed with strong acids or alkalis, or undergo putrefaction, and within the body by the activity of certain ferments, e.specially of one secreted by the pancreas called trypsin. It is found in small quantities as a constituent of many organs and tissues, particularly of the pancreas. In certain cases, however (spleen, thymus, Arc), it appears to be formed by post-mortem changes, and not to be a constituent of the healthv li\iug tissue. It THE SIMI'IJ:1; oUCWTr proximate PRIXf'TPLES 83 32. — Leucine crvstals. may be separated from an organ by making a watery extract ; this is boiled, acidified and filtered to separate any proteid; to the filtrate basic lead acetate is added, and again it is filtered ; excess of lead is removed from the filtrate by a stream of sul- phuretted hydrogen. The lead sulphide is filtered off, the filtrate is evaporated to a syrup, and extracted with hot alcohol. The alcoholic extract is evaporated, and the residue is leucine. (c) Tyrosine CyHnNOyis amido - oxy - phenyl - propionic acid. Propionic acid has the formula C^Uffi.,- Amido-propionic acid 0311^(^112)02 is also called alanine. Oxyphenyl-propionic acid is pro- pionic acid in which one H is replaced by oxyphenyl (CgH^.OH), i.e. C■^li.X^(^Ii.^.0H)0.2 ; if another H in this be replaced by NHo we get C3H^(Vh2)(CcH4.'0H)02=C9Hi,N03, which is amido-oxyphenyl-pro- pionic acid or tyrosine. To separate tyrosine from an albuminous fluid, the fluid must be boiled and the precipi- tated proteid filtered off. The filtrate is evaporated to a third of its volume on the water- bath, precipitated with lead acetate and filtered, a stream of sulphuretted hydrogen is passed through the filtrate, and the lead sulphide so precipitated, filtered off ; the filtrate is evaporated, and crystals of tyrosine sepa- rate out. Tyrosine crystallises in slender needles, often in groups, slightly soluble in water, but insoluble ^ig. 33.-TvT05me ci-ystau. in alcohol and ether. On oxidation it yields benzoic aldehyde, hydro- cyanic, benzoic, acetic, formic, and carbonic acids. G 2 84 THE CHE:\nC-\L CONSTITUENTS OF THE ORGANISM Tests, i. Heat in a watch-glass Avith concentrated sulphuric acid ; cool ; add water and a few pieces of chalk ; there will be an efferves- cence ; filter ; evaporate to a small bulk ; add a few drops of a neutral solution of ferric chloride ; a violet colour is produced (Piria).' ii. Millon's reagent gives a red solution, the tint of which deepens on heating (Hoffmann).^ Tyrosine is generally found along with leucine, and like it results from the decomposition of proteids. {d) Amido-valerianic acid (C.jHuXOo) is a product of decomposi- tion of proteids, but it only occurs in small quantities in comparison with leucine and tyrosme. (e) Sarcosine CgH^XO., is methyl-glycocine, i.e. amidoacetic acid in which one H is replaced by methyl C2Ho(CH3)(XH2)02. It is not found in the body, but is a product of decomposition of creatine. (/) Creatine C^HgX^Oo. Sarcosine (C3H7NO2) united to cyanamide CX.NHo X'IPLES 89 Pure uv'w acid crystallises in Cdloui'lcss rlioiniiic plates oi- prisms. When obtained from urine it is more or less tinged with pigment, and it assumes many crystalline forms (dumb-bells, whetstones, etc.). It is Avithout taste or smell. It is insoluble in alcohol and in ether ; it requires for its solution 15,000 parts of cold and 1,900 of hot water. Its solutions give only a feeble acid reaction. It is not found in the free condition in the urine, excei^t in cases of disease (gravel, calculus) ; but it is combined with bases to form urates.' The acid is dibasic. The amount excreted per diem by an adult averages 0*5 to 1 gramme, but its amount is raised by much animal food, and by want of exercise. Urates occur also in the blood, and as chalky deposits in and around the cartilages of gouty persons. The solid urine of birds and reptiles consists almost entirely of urates. Traces of uric acid have been separated from various tissues, kidneys, spleen, lungs, brain and muscle. Urates of sodium. — The neutral salt CgH2N403Na2 forms nodular masses, and the acid salt C5H3N403Na is usually amorphous in urine ; they form the deposit in urine commonly called lithates. The chalky deposits in gout are chiefly composed of the acid salt, which is then crystalline. Urates of jiotassiiim corresponding to these seldom occur. Acid ammonium urate Q^^H^ ^0-j^{^}i^) (neutral salt unknown) forms globu.lar collections of crystals ; it is found in the deposit in alkaline urine, and is the chief component of the excrement of I'eptiles and birds. Acid calcium urate (C^-^^ ^0-^.,Q?k occurs in the form of fine needles in urinary sediments, calculi, and in gouty deposits. Acid lithium urate C_5H3N403Li is the most soluble salt of uric acid, hence the use of lithia as a drug in cases of gouty diathesis. Murexide test. — Evaporate to dryness with nitric acid ; the residue is reddish yellow, and becomes reddish pui-ple on the addition of ammonia, and bluish violet with soda or potash. (h) Xanthine CgHjIST^Oo differs from uric acid by containing one atom of oxygen less. It is a pale yellow, amorphous powder, insoluble in alcohol or ether, soluble in cold water. When evajaorated to dryness with nitric acid, a yellowish residue remains which turns red with caustic potash, and reddish violet on being heated. Xanthine occurs normally in minute quantities in the urine, and has been obtained from many organs such as pancreas, spleen, liver, brain, and thymus. 1 The question of quaclurates will be disciissed under ' Urine.' 90 THE CHEMICAL CONSTITl-ENTS C)F THE OEfiANISil Urinary calculi, consisting of xanthine, varying in size from a pea to a pigeon's e^g, occasionally form. (c) Hypoxanthine C5H4N4O differs from xanthine by containing one atom of oxygen less. It generally occurs with xanthine. It has been described in the spleen, pancreas, muscles, liver, marrow, blofxl, and urine. In leucocythaemia its quantity in the Vjlood and urine is in- creased. In acute yellow atrophy, the amount in the liver rises. (d) Adenine C5H5X.5 can be obtained from the nuclei of cells. On heating it with sulphuric acid XH is replaced by O, and hypoxanthine thus formed : C5H5N".5 + H.20=C5H,N40 + XH3. Both substances [a'lenine] [hypoxanthine] contain a radicle C5H4N4 called adenyl (Kossel '). Adenine is a crystalline substance ; the crystals contain three molecules of water of crystallisation. («) Guanine C^H^X^O has been found in the liver and pancrea.s,. in guano, in the excrement of spiders, and in the skin of many reptiles and fishes. It bears the same relation to xanthine that adenine does to hypoxanthine (C5H5X.,0 + H.^O=:C5H4X402 + NH3). It Ls amorphous, [guanine] [xanthine] insoluble in water, alcohol, and ether, but readily soluble in acids and alkalis. The ci*ystals of chlorate of guanine are characteristic. {/) Allantoin CJ3.^JS> ^O^ is found in the amniotic and allantoic fluids and in the urine of new- born animals. It crystallises in colourless prisms which are solu- ble in water. By the action of dilute nitric acid it takes up water and splits into allanturic acid (CVHioNgOfJ and ux'ea. (y) Gamine 07113X403 which has been .separated from muscle, is a crystalline substance, the crystals containing one molecule of water of crystallisation. It is soluble in warm water, insoluble in alcohol and ether. It may be considered as a compound of hypoxanthine and acetic acid : C5H4X4O -f- C2H40,=C7H,N403 [hypoxanthine] [acetic [carnine] acid] Other Nitrogenous Acids, — («) Inosinic or Inosic acid C ^^y'il■[^S ^0 ^y. Fig. 40. — Allantoin cry.«tals. 1 Zcit. xjhijdol. Chein. xiii. 29-2. Hjpoxanthine is adenyl oxide; adenine is adenyl imide. Compounds of the radicle NH are called imides. 'nil'; siMiMj;i; oiiiiANic ri;nxi.M.\TK PTJiNriPLEs 01 An uiicrystiillisaljle substance uf doubtful nature, which lias been described as a constituent of muscle juice. (b) C ryptophanic acid C|oH,yN20,o an aiiiorphous acid said to exist in small quantities in human urine (Thudichum).' (f) Sulplio-cyanic acid CNHS united to potassium or sodium to form a sulpho-cyanide (CNKS) is found in saliva, and occasionally also in urine, milk, and l)lood. It gives a i-ed colour with ferric chk)ride, due to the formation of sulpho-cyanide of iron. ((/) Cynurcnic acid CaoHijNaOg is found in dog's urine.^ It is a decomposition product of j)roteids, but is apparently not derived fi'om the putrefaction, which occurs in those sul>stances in the alimentary canal (Baumann).-'' On heating its crystals, which contain two molecules of water of crystallisation, to 250° a base called cynurin Cj8H,4N.202 is obtained.^ By means of certain reagents Kretschy '^^ obtained chinolin CgHyN from it. (e) Urocanic acid C6HgXaOo + 2H20 was found in the urine of a dog in which the urea was diminished. At 212° C. it breaks up into carbonic acid, water, and a base urocanin CjiHidN^O (JafFe).'' 1 Journ. Chem. Soc. (2) viii. 132. 2 Hofmeister, Zeit. i)lnjsiol. Chem. v. 67. ^ Zeif. phijsiol. Chem. x. 123. * Scluniedeburg and Schultzeii, Ann. Chem. Pharm. clxiv. p. 155. * Ber. d. deutsch. chem. Gesellsch. xii. 1673. '^ Hid. viii. p. Sll. 92 THE CHEMICAL CONSTITUENTS OF THE ORGANISM CHAPTER IX THE CARBOHYDRATES The carbohydrates fonn a most important group of organic sub- stances. They are found chiei3y in vegetable tissues ; a fe^^' are found in the animal organism ; many of the vegetable carbohydrates are used as food for animals, and so they are of importance in a consideration of animal physiology. . The carbohydrates may be conveniently defined as compounds of carbon, hydrogen, and oxygen, the two last named elements being in the proportion in which they occur in water. They may be for the greater part arranged into three groups, according to their empirical formiilse. The names and formulae of these groups, and the most important members of each, are as follows : — 1. Glucoses 2. Sucroses or Saccharoses 3. Amyloses (CeH,,Oe) (C,^H,„0„) «(CeH,„05) + Dextrose -hCane sugar + Starch — Levulose + Lactose -f Glycogen + Galactose -7- Maltose -f Dextrin Inosite + Melitose Cellulose — Sorbin + Melizitose Gums — Eucalin 4- Mvcose Tuniciu — Synanthrose — The + and — sign in the above list indicate that the substances to which they are prefixed are dextro- and Isevo-rotatory respectively, as regards polarised light. The formulae given above are merely empirical ; and there is no doubt that the quantity n in the starch group is variable and often large. Investigations relating to the mole- cular weights of the different carbohydrates have yielded very unsatis- factory results.' The most recent work in this direction is that of Brown and Morris.- The method these observers adopted was devised by Raoult,^ and is the outcome of his elaborate investigation into the laws governing the freezing-point of very dilute solutions. Briefly 1 Musculus and Meyer {Bull Soc. Chun. (2) xxxv. 370) attempted to determine the relative size of the molecules by obser^-ing their rate of diffusion. 2 Trans. Chem. Soc. 1888, p. 610. 5 Ann. Chem. Phi/s. 1883, 1881, 1885, 1886. Comj^tes rend. 94, 1517 ; 101, 1056 ; 102, 1307. THE CARBOHYDRATES 93 stated, the law is as follows : — AVhen certain quantities of the same substance are successively dissolved in a solvent, on which it has no chemical action, there is a progressive lowering of the freezing-point of the solution, which is proportional to the weight of the substance dis- solved in a constant weight of the solvent. It is unnecessary here to describe the actual methods employed, and will be sufficient to quote the principal results obtained by Brown and Morris. Substance Formula of Molecule Molecular Weight Dextrose' C«H,A 180 Cane sugar before inversion . C,,H.,,0,. 342 Cane sugar after inversion C„H,A 180 Maltose- . C,.,H..,0„ 842 Arabinose ^5H7oO, 1.50 Kaffinose C„H3.,0,„,..oH,0 594 Galactose f"«H,A 180 Inulin ^(C3«H,A,) 1980 Dextrin . (C,,H,„0,„)« 1800 Soluble starch Starch . 5(C\,H,„0.A 9000 It was found impossible to apply the method satisfactorily to starch ; a num- ber of fairly concordant results, how- ever, pointed to a molecular weight of 20,000 to 30,000. i The carbohydrates are not however simple compounds of carbon with water ; their reactions and derivation products show that their molecular structure is more complicated : they are in fact derivatives of the hexatomic alcohol, mannite. The glucoses may be regarded as the aldehydes of mannite ; they contain in their rational formula the characteristic aldehyde group COH. Thus : — CH2.OH CH2.OH \ i ' (CH.0H)4 =C6Hh06 (CH.OH), -=CeH,20e CH2.OH Mannite COH Glucose The sucroses are condensed glucoses, i.e. they are formed by the combination of two molecules of a glucose with the loss of one mole- cule of water (C6Hi20g + C6Hi20o- H20 = C,2Ho.20h). Theamyloses may be regarded as the anhydrides of the glucoses (CgHioOy — H9O =CeH,oO,). 1 This confirms KiUani's observation that dextrose and levulose yield hydroxy-^vcids containing seven atoms of carbon. ^ Cane sugar and maltose are thus isomeric, not polymeric. The difference in their properties must therefore be due to difference of the arrangement of the atoms in their molecules. 94 THE CHEMICAL CONSTITl'ENTS <)F THE DRGANL^M By oxidation of the sugai-s by means of nitric acid, an acid is obtained ; that is, the Ho removed from the alcohol (mannite) to form the aldehyde (glucose) is replaced by O. The formula of the acid so obtained is therefore C^HijO^ : this is monobasic, and called mannitic acid. On repeating the process, that is, replacing another H^ by O, we obtain an acid with the formula CgH,QO^ ; this is dibasic ; of this there ai'e two isomerides, one called rnucic acid, which is slightly soluble in water, the other saccharic acid, which is readily soluble in water. On oxidation some sugars yield one, some the other, acid ; by fvirther oxidation, tartaric acid, then oxalic acid, and finally carbonic acid and water, are obtained. The most impoilant cai-bohydrates may now be described one by one. DEXTROSE OR GRAPE SUGAR This carbohydrate exists in fruits, honey, and in small quantities in the blood, and in numerous tissues, organs, and fluids of the body. It is the form of sugar found in the urine in the disease known as dia- betes. It is formed by the hydration of members of the amylose and saccharose gi'oup : such as is brought alx)ut by boiling with dilute sulphuric acid. Dextrose is soluble in hot and cold water, and in alcohol. It is not so sweet as cane sugar. It crystallises from an aqueous solution in white spheroidal masses, and from alcohol in transparent anhydi'oiis prisms. Its solutions rotate the ray of polarised light to the right ; (a)D= + 56" (Hoppe-Seyler).' Heated with alkali, dextrose gives a brown or yellow colour due to the forma- tion of glucic and melassic acids. ^ Xitric acid oxidises dextrose to sac- charic acid. In alkaline solutions, dextrose reduces Fia. 41.— Dextrose crvita-.s. salts of silver, bismuth, mercury and copper ; in the case of the first three, the metal is precipitated : cupric are reduced to cuprous compoimds, with the sepai-ation of cuprous oxide. In the presence of ammonia, dexti*ose is precipitated by neutral or basic lead acetate. 1 Tollens gives (0)0=^ +53"1^. Fresh watery solntions may indicate 10i°. * These acids are of doubtful composition. In Watisi' Dictionary the formula for glucic acid is given as C'.24Hi80ii, and for melassic acid a< CjaHjoOs. In Beihtein's IHc- tionary the acids formed are stated to be glycic acid ^Ci-z^^iOii) and sacchammic acid (CuHxaOji). TlIK CAiniOlIVDKATKS 95 Dextrose fi)mis conipuunds with certain acids and bases (e.g. potash, lime) : tliese are called gluco.sates. Under (he influence of yeast, dextrose is converted into alcohol and carhonic acid (C,;H|20,;:=2C,H^O + 2C().J.' Jt may also umlei-go the lactic acid fermentation, under the influence of certain bacterial growths. AVhen sugar is heated to 200°, a brown substance called caramel is formed. This has been separated into three bodies of complex formula' and doubtful nature {see ' Watts' Diet.'). Sugar is also turned brown by the action of suljohuric or hydro- chloric acid. This is partly due to charring. A number of other substances, called humous substances by Hoppe-Seyler, have also been separated. These brown products are similar to many produced in vegetable growths naturally, in peat, &c., and are of complex nature and doubtful composition. They have received various names (humin, ulmin, ulmic acid, phlobaphene, tannin-red, hymatomelanic acid, kc). Among the decomposition products of these substances are formic acid, pyrocatechin, and protocatechuic acid.^ The origin, role, and destination of dextrose in the body, and other physiological problems connected with its presence, will be more con- veniently described with the various tissues and fluids in which it occurs (.sv'^ liver, muscle, blood, urine, diabetes, food, digestion). Tests for Dextrose. — 1. Trommers test? — Add a few drops of dilute cupric sulphate solution to a solution of dextrose and caustic potash or soda in excess. The result is a deep blue solution ; the precipitate of cupric hydrate which is formed being soluble in the presence of dextrose. Heat the solution ; a little below the boiling-point a red precipitate of cuprous oxide, or a yellow precipitate of cuprous hydrate, is formed. This reduction is due to the formation of glucic and melassic acids which, having a strong affinity for oxygen, take it from the cupric compound. 2. Fehling's test. — The principle of this test is the same as that of the pi-eceding. Fehling's solution is thus prepared : — Solution A. Dissolve 36'64 grammes of copper sulphate in 500 c.c. of water. Solu- tion B. Dissolve 173 grammes of sodio-potassium tartrate (Rochelle salts) in 100 c.c. of a solution of caustic soda, having a specific gravity 1 Small quantities of glycerine and succinic acid are also formed, and were regarded by Pasteur as derived from the sugar on which the yeast acts {Ann. Chim. Phi/s. (3) xviii. p. 323). v. Udranszky (Zeif. jihijsiol. Chem. xiii. p. 539j, however, states that the glycerine certainly, and probably the succinic acid, is derived from the substance of the yeast itself, probablj' from the lecithin it contains. - A full description of humous substances will be found in a paper by Hoppe-Seyler, Zeit. physiol. Chem. xiii. C6. ^ Ann. Chem. Pharm. xxxix. (1841) p. 360. 96 THE CHEMICAL Cr)NSTITUENT.S OF THE r)RGANISM of 1"34:, and dilute with water to 500 c.c. These solutions should be kept in well-stoppered bottles, and before using equal volumes of A and B mixed together. The result is a dark blue solution, the Rochelle salt holding the cupric hydrate in solution. The solution should be freshly made, because tartaric acid tends to become converted into its isomeride, racemic acid ; and racemic acid itself reduces cupric salts like sugar. One should always ascertain that it is absent by lx»iling the Fehling's solution, which should remain unaltered by this treat- ment. On adding a little solution of sugar and boiling, a red precipi- tate of the cuprous oxide or hydrate occurs. 3. Bottfjer's test} — Take 5 grammes of basic nitrate of bismuth, 5 grammes of tartaric acid, and 30 c.c. of distilled water. To this add slowly, and with constant stirring, some strong caustic soda solution, until a clear solution is obtained. To a little of this add some solution of dextrose, and boil. A black precipitate of metallic bismuth separates. Or the test may be performed as follows : — The solution of dextrose is- mixed with an equal volume of sodium carbonate solution (1 part to 3 of water) ; a few fi-agments of bismuth subnitrate added, and the mixture boiled. A grey or black precipitate of metallic bLsmuth is thrown down. 4. Silver test. — Add ammonia in excess to a little strong solution of silver nitrate : add some dextrose and boil, metallic silver is deposited as a mirror at the bottom of the tube. Aldehyde and tartaric acid behave like sugar in this test. 5. Moore s test? — Heat the solution of dextrose with a solution of caustic potash. The mixture becomes yellow, and, on exposure to the air, brown from the formation of melassic and glucic acids. 6. Picric acid test. — Heat the solution of dextrose with a few drops of solution of picric acid, and heat. Add a little caustic potash, and a brown-red solution is obtained. 7. Indigo-carmine test. — A solution of dextrose is rendered faintly blue with indigo-cai'mine, and faintly alkaline with sodium carbonate. It is then heated to boiling without agitation ; it turns violet, then yellow, but if it is shaken the blue colour is restored. 8. Fermentation test. — A test-tube is half filled with solution of dextrose and a little dried German yeast added. Invert the tube over mercury, and leave it in a warm place for 24r hours. The sugar will undergo fermentation; carbonic acid gas accumulates in the tube, and the liquid gives the tests for alcohol. A control experiment should be made with yeast and water in another test-tube, as a small yield of carbonic acid is often obtained from impurities in the yeast. ' Bottger, -lourn. praJct. Chem. Isx. (1857), p. 432. Nylander, Zeit. physiol. Chem^ viii. (18841, p. 175. - Moore, Lancet, 1844, ii. ; Heller, Arch.f. mikr. Chem. vol. i. 1844, p. 292. THE ('.\K]'.<»in'l)l{ATES 97 9. llie PJiH)iylhydrazine test. — This test is applied in testing for minute quantities of dextrose, especially in urine. Add a pinch of sodium acetate and a little solution of i)henylliydrazine hydrochloride to a solution of dextrose ; a yellow precipitate of phenylglucosazone crystals occurring both singly and in bundles forms in a few minutes if the mixture is kept in the water bath at 100° C. ; and in a dilute solution of dextrose the crystals should be searched for microscopically (v. Jaksch).' 10. 21ie Saccharimpter test. — A solution of dextrose rotates the plane of polarised light to the right. In testing for dextrose, as many tests should be tried as possible, as many other substances give certain of the above tests ; for example, reduce copper salts, or rotate the plane of polarised light. The best tests will be found those numbered 2, 3, 8, 9 and 10 ; and the best of all is 8. Quantitative determination of Dextrose. — 1. By the Saccharimeter. This instrument is a polarimeter, and the instrumer^ts used and methods adopted are the same as that employed in pclarimetric pro- cesses generally {see pp. 40, 53). 2. The Fermentation process. — When mixed with yeast about 95 per cent, of the dextrose in solution is converted into carbonic acid and alcohol. Small quantities of amyl alcohol, glycerine and succinic acid are formed at the same time. The dextrose originally in solution may be estimated either from the loss of weight of the apparatus from the evolution of the gas, or from the gain in weight of an absorption tube containing caustic potash connected with the escape pipe, and which absorbs the carbonic acid. 1 part of CO2 = 2-045 parts of sugar. Sir William Roberts devised a simpler process, especially applicable to sugar in urine, in which the sugar present is estimated from the loss of specific gravity a solution undergoes dui-ing fermentation. The specific gravity of the solution is accurately taken : yeast is added and after remaining 24 hours in a warm place the specific gravity is again taken. The number of degrees of density lost indicates the number of grains of sugar per ounce ; and the percentage is obtained by multijDly- ing the degrees of density by a constant factor. This constant factor is according to Worm-Miiller 0*23; according to Manassein 0*2 19. Thus in a urine whose specific gravity before fermentation is 1040, and afterwards 1010, the degrees of density lost = 30, and accordingly 30 grains of sugar are present per ounce, or 30 xO"23=6"9 per cent. This method, however, is found practically to give very inaccurate "' Zeit. klin. Med. xi. 20, sec also note on y). 110. H 98 THE CHEMICAL CONSTITUENTS OF THE ORfKVNISM results. The reason of this is, that a constant factor is an impos- sibility, and, in fact, increases as the percentage of sugar diminishes. For the mathematical demonstration of this fact see Budde (Pfliiger's Archiv, xl. 1.37). 3. FeJilinf/s inefhod. — 10 c.c. of Fehling's mixture (see p. 95) cor- responds to 0 05 gramme of sugar. The solution to be tested should not contain more than about 0'5 per cent, of dextrose. It will be found necessary to dilute strong solu- tions, and most diabetic urines,' with 9 times the amount of water; this must be allowed for in the subsequent calculation. The solution of dextrose is placed in a burette ; and 10 c.c. of Fehling's mixture diluted with 40 c.c. of water, in a white porcelain, dish. The Fehling's mixture is kept constantly boiling, and the sugar is run into it from the burette gradually. The cuprous oxide is thrown down as a red precipitate, and the blue colour of the solution gets less and less, and finally disappears. When the blue colour has gone, the burette is read, and the quantity of solution of sugar used, is that which contains sufficient sugar to reduce 10 c.c. of Fehling's mixture, i.e. 0'05 gramme of sugar. Suj)pose 9-5 c.c. of the solution reduced 10 c.c. of Fehling's mixture / A or \ Ai ii 4. e 0-05x100 5 (=: (J •Uo gramme sugar): then the percentage or sugar = := — — =0'526 ; and if the solution, or the urine had been previously diluted 10 times, the percentage of dextrose in the original solution :=0'526 x 10 = 5-26. In order to insure accuracy it is always advisable to make a second observation, using the first only as an indication, and proceeding more cautiously. Beginners often find it difficult to determine exactly the point at which the blue colour has completely disappeared. In such a dilemma, a little of the hot fluid should he quickly tiltered through a thick filter paper, the filtrate acidulated with acetic acid, and a drop of potassic ferrocyanide added. If copper is present a brown colour or precipitate is produced ; in this case, more of the sugar solution must be added, and the operation continued until the filtered hot fluid gives . no reaction for copper. Fllickiger^ recommends that a small quantity of calcium chloride should be added before filtering, in order to prevent the mechanical suspension of finely divided cuprous oxide in the solution, and Hagemann^ has pointed out that in the case of urine, it is necessary to test the first two drops of the filtrate ; for by the time 1 If the urine is albuminous, the albumin must be first separated by acidulating with dilute acetic acid, boiling, and filtering. - Zeit.2>liir'-lol. Chem. ix. 335. ^ Pfiigei-'s Arcliiv, xliii. 501. TIIK ('AIJI'.OIIYDRATES 1)9 the third drop comes through, oxidation of the cuprous oxide has taken place, and cupric oxide is in solution. Such rapid reoxidation does not occur however in solutions of pure dextrose. Hageniann further states that by this nietliod as goofl results are obtained as by Allihn's method,' which is one for determining the amount of copper in the precipitate. In making these determinations in urine, it must be borne in luiiid tli;it other substances may be present which reduce alkaline sobitions of ccjppcr saUs, such as uric acid, creatinine, pjTrosatechin, and compounds of glycuronic acid. None of these substances, however, give the fermentation test. 4. OtMr Methods. — Knapp's method^ is a volumetric one, in whicli a standard solution of alkaline mercuric- cyanide is used. (10 grammes of mercuric cyanide, caustic soda (of sp. gr. 1-14) 100 c.c. made up to a litre with water; 40 c.c. cor- responds to 01 gi-amme of sugar). The solution is kept hot, sugar solution run in from a burette, and metallic mercury is deposited. The end of the reaction is the absence of mercury in the fluid ; this is ascertained by placing a drop of the clear supernatant fluid on a piece of fine filter-paper, and exjoosing it to the vapour of ammonium sulphide ; when the drop remains unblackened mercury is absent. Hachsse's method is very similar : the standard solution is mercuric iodide 18 grammes, potassium iodide 35 grammes, caustic potash 80 grammes, water to 1000 c.c. : 40 c.c. corresponds to 0-15 gramme of sugar. The end of the reaction is ascertained by means of drops of a solution of stannous chloride super- saturated with caustic soda, placed on a porcelain dish ; as long as the mercuric salt is present, the addition of a drop of the clear supernatant fluid gives witli one of these drops a brown colour, or grey precipitate. Vogel's method is a colorimetric one, and depends on the intensity of the colouration produced by boiling the solution of dextrose with caustic potash. This is compared with a standard solution similarly treated. Dr. George Johnson has also devised a colorimetric method, depending on the depth of the tint produced by boiling a solution of dextrose with caustic ]iotash and a saturated solution of picric acid, as compared with the tint of a standard. Pavy's and Gerrard's methods are modifications of Fehling's, and being especially applicable to urine, will be described under that head {see Chapter XLV.). ^ LEVULOSE Wlien cane sugar is treated with dilute mineral acids, it undergoes a process known as inversion, i.e. it takes up water, and is co]iverted into a mixture of equal parts of dextrose and levulose. .Similar hydration changes are produced by ferments, such as the invert ferment of the intestinal juice. Levulose has been discovered in blood, urine, and nniscle. It is unci'ystallisable, very soluble in water and in alcohol ; it gives the same tests as dextrose, except that it has a powerful hevorotatory action on polarised light. (")i.= —106°. ^ Zeit. anal. Chem. vol. xxii. p. 2i8. ^ Annal. d. Chem. vol. cliv. p. 252. H 2 100 THE CHEMICAL CONSTITUENTS OF THE ORGANISM We have seen that dextrose is regarded as an aldehyde ; by some, levulose is regarded as the corresponding ketone. Pure levulose may be obtained by neutralising with lime the mixture of glucoses obtained by the action of sulphuric acid on cane sugar. The levulose lime compound is a solid, while that of dextrose is liquid. By decomposing the lime compound with oxalic acid, pure levulose is obtained. GALACTOSE Galactose is formed by the action of dilute mineral acids, or inverting ferments, on lactose or milk sugar. It is dextrorotatory. (fi)p:= 4-83'3°. Nitric acid oxidises it to mucic acid. Galactose is directly fermentable with yeast ; it also reduces Fehling's solution. INOSITE Inosite is a glucose which is found in muscle, kidney, liver, nervous tissues, and several other organs of the body. It has also been sepa- rated in small quantities from the blood, traces exist in most diabetic urines, in the urine of certain cases of Bright's disease, and according to Choetta, ' Gallois,- and Kiilz,^ in normal urine too. It is also obtained from peas, beans, lentils, potato, asparagus, dandelion, foxglove, and many other plants. Frepm-ation. — From beans. A watery extract is evaporated to a syrup, and precipitated with alcohol ; the precipitate is dissolved in water, and the inosite allowed to crystallise out. From muscle or other tissues.^ An aqueous extract is freed from albumin by acidulation, boiling and filtering ; from phosphates by the addition of baryta water and filtering. The filtrate is concentrated, and creatine crystallises out. The mother liquor is boiled with four times its volume of alcohol, and the precipitate so formed is removed. The clear liquid is set aside for twenty-four hours, and crystals of inosite often separate ; if not, ether is added, and the mixture shaken, inosite then separates in lustrous leaflets. It is purified by recrystal- lisation. From urine. Take several litres of urine, and add neutral then basic lead acetate. Collect the precipitate produced by the latter ; decom- pose it with sulphuretted hydrogen ; filter ; evaporate the filtrate to a syrup, and add alcohol and ether. Inosite crystallises out. Pro23erties. — It forms large colourless monoclinic prisms, often 1 Ann. Chein. Fharm. xcix. p. 289. - De Vinosurie, Thesis, Paris, 1864. 3 Ccntralhl. f. Med. Wiss. 1875, p. 933. 4 Boecleker, Ann. Chem. Fharm. vol. cxvii. p. 118. THE CARBOHYDRATES 101 grouped in rosettes. The crystals contain two molecules of water of crystallisation. It has a sweet taste, is soluVjle in watei-, but not in absolute alcohol, or ether. It is precipitated by a mixture of basic lead acetate and ammonia. It is capable of the lactic acid fermentation • but not of the alcoholic. Its solutions have no action on polar- ised light ; it does not reduce metallic oxides ; it gives no change of colour Mhon boiled with caustic potash, neither is it decomposed by weak acids. Tests. — (1) Evaporate a little of its solution with a little nitric acid on a platinum dish ; treat the residue with a little ammonia and calcium chloride, and evaporate to dryness at a gentle heat. A bright red or violet colour is produced. This test only suc- ceeds with pure solutions (Scherer).^ (2) Add a little mercuric nitrate to a solution of inosite, on a porcelain dish ; a yellow precipitate is pi'oduced. On heating this gently, it will become red; on cooling the colour vanish c.;. Proteids, t}Tosine, and sugar must be absent (Gallois). Constitution. — From a study of its nitro-substitution and other products, Maquenne^ concludes that the graphic formula for inosite may be thus represented. It is in other words a hexatomic alcohol with six secondary alcohol groups arranged in a ring ; this symmetrical construction excluding any power to rotate polarised light according to the theory of Le Bel and Yan 't HofF (see p. 45). It is not an aldehyde, nor an acetone, nor a polyphenol, though the closed chain suggests an aromatic structure. Fig. 42. — Inosite crystals CHOH CHOH^ I CHOH CHOH\^CHOH CHOH CAXE SUGAR This sugar is generally distributed throughout the vegetable king- dom in the juices of plants and fruits, especially the sugar cane, beet- root, mallow, and sugar maple. It is a substance of great importance ^ According to Hilger {Ann. Chem. Phann. vol. clx. p. 333) the variety of acid formed is sarcolactic. - Ann. Chem. Pharm. vol. Ixxiii. p. 322. 5 Com_pt. rend. civ. (1887), 225, 297, 1719, 1853. 102 THE CHEMICAL CONSTITUENTS OF THE ORGANISM . as a food ; aftei' abundant ingestion of cane sugar, traces may be found in the blood and urine, but the greater part undergoes inversion. Pure cane sugar holds cupric hydrate in solution in an alkaline liquid, i.e. with Trommer's test it gives a blue solution. But no reduction occurs on boiling.^ It crystallises in monoclinic prisms. Aqueous solutions are dextrorotatory. (a)jj= + 73-8°. By boiling with water, or more readily by boiling with dilute mineral acids, or by means of inverting ferments, it undergoes inversion, i.e. it takes up water and splits into dextrose and levulose. C,2H2.0h + H,0=C6H,206 + C6H,206 [caiie sugar] [dextrose-] [leviUose] With yeast, cane sugar is first inverted by means of a special soluble ferment produced by the yeast cell, and then there is an alcoholic fermentation of the glucoses so formed. Nitric acid oxidises cane sugar to saccharic acid. Cane sugar may be estimated in the following way : — Take 40 c.c. of the solution of cane sugar ; add 1 c.c. of a 25 per cent, solution of sulphuric acid, and boil for half-an-hour. Care must be taken not to char the sugar. Bring the solution of sugar to its original volume by adding water. Place it in the burette, and run it into boiling Fehling's solution, as in the estimation of dextrose. It may be necessary to add excess of soda or potash to the Fehling's solution, so that the sulphuric acid in the sugar solution may be fully neutralised. Every 95 parts of glucose found corresponds to 100 parts of cane sugar. LACTOSE Lactose or milk sugar occurs in milk. It has also been described as occurring in the ui'ine of women in the early days of lactation or after weaning. It crystallises in rhombic prisms, which contain a molecule of water of crystallisa- tion. It is soluble in six parts of cold, and 2i parts of hot, water. It is thus much less soluble than cane sugar or dextrose. It has only a faint sweet taste. Aqueous solutions are dextrorotatory. (a)D= + 59-3°. FIG. 43.-iUik sugar crystals. j^ -^ ij^^^oi^ble in alcohol and in ether. Solutions of lactose reduce Fehling's solution, but less powerfully than dextrose. If it required seven parts of a solution of dextrose to 1 Most specimens of commercial cane sugar contain other forms of sugar as impurities, and these cause a small amount of reduction. THK CAlMloll^'Di;. VTKS 103 reduce a '/nvn (juantity of Fehling's solution, it would require ten parts of a sulution of lactose of the same strength to reduce the same quantity of Fehling's solution. By boiling with water, or more readily by boiling with dilute aci,0,i(C,2rLoO,(,)g, which may be regarded as constituted of one amylon or maltose group, in combination with six amyliri or dextrin groups. Its molecular weight would on this supposition be 2286. Precijntation of Colloid Carhohydrates hy Salts. — The use of neutral salts in the precipitation and separation of proteids is also applicable to colloid carbo- hydrates. Pohl' has examined a number of these, and finds that some, like gum arable, are not precipitable by saturating their solutions with neutial salts ; others, like gum tragacanth, are preciiaitated by saturation with ammonium sulphate ; and others again, like soluble starch and dextrin, are precipitated by saturation with sodium sulphate, magnesium sulphate, and ammonium sulphate. I have mA'self found that glycogen is precipitated by saturation with ammonium sulphate and magnesium sulphate, but not at all or only very slightly by sodium chloride. GLYCOGEN Glycogen or animal starch is found in the liver, muscle, placenta, white blood corj)uscles, cartilage cells, and in embryonic tissues generally. It has also been found in some specimens of diabetic urine (Leube).^ It is a substance of great physiological importance. The methods of preparing it and estimating its quantity, together Avith a discussion of its functions, will be given under liver and muscle, the two tissues in which it occurs in the adult in the greatest quantity. Glycogen is a white, tasteless, odourless powder, soluble in water, forming a densely opalescent solution. It is insoluble in alcohol and in ether. It is strongly dextrorotatory ; (a)i,= + 211°. With Trommer's test it gives a blue solution, but no reduction occurs on boiling. Glycogen gives wdth iodine a port- wine red colour ; this easily distinguishes it from starch. The colour disappears on heating, and reappears as the liquid cools. The colour wdiich dextrin gives with iodine is very similar, but dextrin forms a clear, not an opalescent, 1 Zeit.physiol. Chcm. xiv. 151. - Virchow's Archiv, vol. cxiii. 391. THE CARKOHYDKATES 107 solution witli water, and is not precipitated by basic lead acetate, as is glycogen. Prolonged boiling witli water or boiling with dilute mineral acids converts glycogen into dextrose. The ferments of the salivary glands, of the pancreas, and glycerine extracts of liver and other organs change glycogen into maltose, intermediate products of the nature of dextrin being formed in each case, as with starch. During the processes of metabolism that occur normally in the liver, glycogen is not only stored up in the cells, but a certain amount of the stored glycogen is trans- formed into sugar and leaves the liver by the hepatic vein. The form of sugar so formed is not maltose but dextrose. CELLULOSE This is the colourless material which composes the cell-walls and woody fibre of plants ' ; it may be obtained in the pure state from cotton or linen tibre by boiling out impurities with alkali, alcohol, and ether. It is insoluble in water, alcohol, or ether, but dissolves in an ammoniacal solution of cupric oxide. From this solvent it may be recovered in an unchanged form. By the action of strong sulphuric acid, cellulose is converted either into an insoluble substance which colours blue with iodine, or into a soluble substance of the nature of dextrin. A useful material called vegetable parchment is prepared by dipping sheets of paper into strong sulphuric acid. By Ijoiling with dilute sulphuric acid cellulose is con- verted into dextrose. The various digestive ferments have little or no action on cellulose. Cellulose is, however, not confined to vegetable tissues. It is tlie chief constituent of the test or outer investment of the Tunicates, and is sometimes called tunicin. Schafer - found that the cellulose obtainable from tlie mantles of the Pyrosomida?, Salpida-, and Phallusia mam- milaris has the same elementary composition as vegetable cellulose, and has also identical properties ; for instance, it dissolves in cupram- monia, and is converted by nitric acid into an explosive nitrate soluble in ether (gun-cotton). According to Berthelot ^ tunicin differs from cellulose in being less easily convertible into dextrose by the action of dilute sulphuric acid. 1 The difEerent varieties of cellulose will be found described in Watts'' Diet, of Chem. vol. i. 1888. 2 Annul. Chem. Pharm. clx. 312. 3 AiiJi. de Chem. et de Phijs. Sc'r. 3, tome 56, p. 153. 108 THE CHEMICAL CONSTITUENTS OF THE ORGANISM The skin of the silk-worm is stated to contain cellulose (De Lucca).' The mucilaginous investing matrix or zoocytium Avhich surrounds the colonies or social clusters formed by tlie protozoon OphrydiuTn versatile is also composed of cellulose (Halliburton).^ Virchow ^ found cellulose in degenerated human spleen, and in certain parts of the brain, and more recently Freund ^ has found it in the blood and tissues in cases of phthisis. GUMS Gxim arahic, the natural exudation from several species of acacia, consists chieflj' of the potassium and calcium compounds of arabin, or arable acid (C«H,„03),, Arabin forms a thick sticky solution with water ; it is insoluble in alcohol and in ether. With copper sulphate a thick gelatinous precipitate is formed, which is a compound of cupric oxide and the gum. This is insoluble in caustic soda, and is not reduced on boiling. With ferric chloride a similar thick precipitate is formed. When boiled with dilute sulphuric acid, arabin yields a crystalline sugar called arabinose ; this has the exceptional formula C^HinOj. It is strongly dextro- rotatory, reduces alkaline solutions of cupric hydrate (Fehling's solution), but will not undergo the alcoholic fermentation. Arabin is oxidised by nitric acid to mucic acid. Dextrin or British gum. — See p. 105. Animal fjum. — This substance was discovered by Landwehr, and is a con- stituent of mucin. It has long been known that when mucin is boiled with dilute sulphuric acid it yields a reducing but unfermentable sugar (CeH,„Os). This sugar comes from the animal gum which is present in mucin in combination with a proteid. Animal gum is sticky, gives gelatinous precipitates with copper and iron salts, and has the same empirical formula, (CgHinO^X,, as vegetable gum. Fuller particulars regarding its properties and phj-siological importance will be found under mucin, connective tissue, cartilage, &c. Animal dcxtran, (CnHmOj),,, a gum-like substance secreted l)y the Schizonenra lanvginosa, a gall-producing louse that attacks elms. (a)D = + 15C-7° (Liebermann).' OTHER CAEBOHYDRATES Melitose, from eucalyptus manna. 3Ielizitose, from larch mamia. 3/i/cose or Trehalose, from ergot. These are all sucroses ; they are dextrorotatory, and do not reduce alkaline cupric solutions, Synanthrose is also a sucrose ; it is foimd in the roots of certain plants. It has no action on polarised light. Sorhiii, from sorbic acid. Scyllite, from the intestines of the hag-fish and «kate. Encalin, arising from the fermentation of melitose. These three glucoses are nearly allied in their properties to inosite. Inulin is a lievorotatory carbohydrate found in the roots of certain plants together with synanthrose. In the pure state it forms characteristic crystalline 1 Compt. rend. Hi. p. 102 ; Ivii. p. 43. ^ Quart. J. Mic. Science, July, 1885. 3 Compt. rend, xx.xvii. 492, 860. •* Wtencr med. Jahrh. 1886, 335. * Pfliigcr's ArcJiiv, x\. 454. THE CAKJJOIIYDKATKS 109 t;pliernlos. Wlien boiled with dilute acids it yields levulose. Its formula is -'(C3,ir„,.()„). liatKiwsc, a crystalline carbohydrate which can be separated from molasses. Its foruuda is CisHjoOi^; the crystals contain five molecules of water of crystallisation. Dextrane, CuH^Oj, a gummy substance occurrinfjf in imripe beetroot (Scheibler).' Lichnin, (C,;H,oO.)„, occurs in the intercellular substance of Iceland moss and certain algse. Paramijlum{7Moiu\\y\\\ru : Biltschli), (C^HmOj),,, occurs in the form of granules resembling starch in the infusorian, Euglena vii-idis, and in all gregarinffi.- Pam/jalactin and other insoluble carbohydrates in the cell membrane of seeds which occur there with celhdose, differ from it in being insoluble in ammoniacal solutions of copper oxide (Schulze).^ GLUCOSIDES The substances constituting this class occur in many plants, and a few are found in animals also. They yield on decomposition a carbohydrate, generally a glucose, together with other substances. Amygdalin in bitter almonds may be taken as an example. In the bruised almond a ferment called emulsin or synaptase produces from it bitter almond oil, hydrocyanic acid, and glucose : — C,„H,,XO„ + 2H,0 = C,H,0 + HON + 2C,H,.,0g [amygilalin] [hyilrifle of [liydrocvauic [glucose] benzoyl] aclii] The following equations represent the decompositions of a few other important vegetable glucosides : — C,3H„0, + H.O = C^H^O, + C,H,,0„ [salicin] [saligeuin] [glucose] C2,H,,0„ + 4H,0 = 3C,H,03 + C,H, A [tanuiu] [gallic acid] [glucose] C,oH„KNS,0,„ = gjsO, + g^H,}^ + ^«H,A [potassium [hycU-ogeu [oil of [glucose] myronate] potassium mustard] sulphate] Among other important vegetable glucosides are digitalin, ruberj'thric acid (which yields alizarin), coniferin (which yields vanilin), and indican ^ (which yields indigo, see p. 78). The animal glucosides are : (1) mucin, which fields a proteid and animal gum ; (2) cerebrin {see nervous tissues) ; (3) chitin (see epithelial structures) ; (4) carminic acid {set: pigments). GLYCURONIC ACID Glycuronic acid is a substance which occurs under certain circumstances in urine, and from the fact that it rotates polarised light to the right ((a)^ = + 19), and reduces alkaline solutions of copper oxide, is apt to be mistaken for dextrose. 1 Jaliresh.f. Chcm. Technologic, 1875, 790. ' Maupas, Compt. rend. cii. 1'20. •* Ber. deutsch. chem. Gesell. xxii. 1192. * The indican of uriue is, however, not a glucoside, see p. 79. 110 THE CHEMICAL CONSTITUENTS oF THE ORGANISM Its formula is CgHioGj, and it is no doubt related to the carbohydrates. When pure it is not crj-stalline, but its anhydride, CgHsOg. forms colourless acicnlar crystals. It reduces not only Fehling's solution, but also gives Bottger's bismuth test. It does not, however, undergo the alcoholic fermentation, and can by this means be easily distinguished from sugar. It is insoluble in ether, but readily soluble in water and hot alcohol, crystallising out from the latter on cooling. It is precipitable from an aqueous solution by baryta water as an insoluble bar}i;a compound. Though related in its composition so nearly to the carbohydrates, it yields with urea, decomposition products which are aromatic, such as orthonitro- benzyl alcohol (Jaffe).' It occurs in the urine in the form of the potassium salt (O^HgO.K). It is found there after the administration of certain drugs (chloral and butylchloral,- nitrobenzol,* ortho-nitro toluol.^ camphor,^ &c.). It also occurs in the urine after chloroform narcosis, and in the parah-tic secretion that takes place on section of the renal nerves." Potassium glycuronate, like dextrose, gives -with phenylhydrazine hydrochloride a crystalline precipitate. Bromine converts glycuronic acid into saccharic acid, thus showing the presence in the former acid of an aldehyde group, and also its close relation to dextrose (Thierf elder).' Schmiedeberg and Meyer'' consider that it arises from the dextrose in the body ; Kiilz* has suggested that it may originate from inosite. {See also Urine.) NOTE Hirschl (• Zeit. physiol. Chem." xiv. 377) states in reference to the phenyl- liydrazine test for sugar, more especially in urine, that it is as trustworthy as the fermentation and polarimeter tests. The mixture should, however, remain in the water-bath at least an hour before crystals are looked for. The fine bright yeUow needles of phenylglucosazone (mielting-point 204° C), either single or in stars, are then easily distinguishable from the brownish amorphous precipitate (melting-point 150°) which glycuronic acid gives. If the mixture be left in the water-bath for a shorter time than an hour, the glycuronic acid compound formed is crystalline acid, and is liable to be mistaken for phenylglucosazone. In addition to dextrose, tkree other sugars have been described in human urine :— (1) Levulose (Zimmer, ' Deutsch. med. Woch.' ii. 329: Seegen, ' Centralbl. med. Wiss.' xxii. 753) ; this has been found only in diabetics mixed with dextrose, from which it is distinguishable only by the polarimeter ; (2) Lactose (Hofmeister, ' Zeit. physiol. Chem.' i. 101) ; this is found in the urine of suckling women ; the crystals of phenyllactosazone, formed by the action of phenyl- hydrazine, are ten times wider than those of phenylglucosazone, and melt at 200° ; (3) Maltose, found in diabetic urine by Le Xobel, gives a precipitate of phenyl- maltosazone which occurs in yellow tables melting at 82° C. 1 Zeit. pliystol. Chem. ii. 47. - Musculus and v. Mering, Pfliiger's Achiv, sx. 64. 3 V. Mering, Centralhl. Med. Wiss. 1875, No. 55. 4 Jaffe, loc. cif. * Schmiedeberg and Meyer, Zeit. physiol. Chem. iii. 422. 6 Ashdown, Brit. Med. Journ. vol. i. 1890, p. 171. "< Ber. deutsch. Chem. Gesellsch. six. p. 3148. 8 Zeit. jihi/siol. Chem. iii. p. 437. 9 Zeit. Biol, xxiii. p. 475. In this paper will be found a method of prejiaring glycuronic acid from purree (a yellow substance probably obtained from the urine of camels who have eaten certain fruits) ; see also Thierfelder, Zeit. physiol. Chem. xi. 388. Ill CHAPTER X THE P R 0 T E I D S The proteitls are the most important substances that occur in animal and vegetable organisms ; none of the phenomena characteristic of life occur without their presence. They are invariable and constant constituents of protoplasm. The term proteids was oi-jginally given to these substances by Mulder {TrpoiTelov, pre-eminence), and the name is a convenient one in which to include all the heterogeneous members of the group. It must not be supposed that in adopting Mulder's nomenclature we in any way accept Mulder's theory of the constitution of proteids, which will Ije referred to later. The expressions ' proteid ' and ' albuminous substance ' are synonymous. The word ' albumin ' is restricted now to a definite class of proteids. The word ' albuminoid " should 1 le restricted to a class of compounds (gelatin, mucin, itc), which, although having certain re- semblances to the proteids, difier from them in many important j^oints. The words all)uminate, albumose, albumid, Arc, are applied to certain derivatives of the proteids ; these terms should always be most care- fully used, as their similarity to one another is apt to give rise to confusion. The following short description of the proteids must serve in lieu of a logical definition ; for although the proteids are the most important of all organic substances, they are those about which we have the least information. ' Proteids are highly complex and (for the most part) uncrystallisable compounds of carbon, hydrogen, oxygen, nitrogen, and sulphur, occur- ring in a solid viscous condition, or in solution in nearly all the solids and liquids of the organism. The difierent members of the group present difierences in physical and to a certain extent even in chemical pro^jerties. They all possess, however, certain common chemical re- actions, and are united by a close genetic relationsliip (Gamgee).i 1 Physiol. Chein. p. 4. 112 THE CHEMICAL CONSTITrENTS OF THE ORGANISM The following table from Gorup-Besanez' exhibits the proportion of proteids contained in the liquids and solids of the body : — Per cent. Per cent. Cerebro-spinal liquid . . 009 Chyle .... . 409 Aqueous humour . . Ol-l Blood .... 19-56 Liquor amnii . 0-70 Spinal cord . 7-49 Intestinal juice . . 0-fi5 Brain .... 8-63 Liquor pericardii . 2-30 Liver .... 11-64 Lymph . 2-46 Thymus 12-29 Pancreatic juice . 333 Muscles 1618 Syno^-ia . 3-91 Tunica media of arteries 27-33 M'ilk .... . 3-94 Crs'stalline lens . 38-30 The proteid constituents of the animal body are derived from vegetables, either directly, or indirectly through the body of another animal. Synthetic processes do occur in the animal body, but to a much greater extent in A-egetables. Here the proteids are built up from simpler compounds derived ultimately from the soil and the atmosphere. In animals the proteids are hrst converted into substances called peptones, in which form they are absorbed ; the peptones are re- converted into proteids similar to those originally ingested, and these proteids are assimilated, that is, become part of the living organism. During life, however, there is not only a process of building up going on, but also a process of breaking down, the two constituting what is known as metabolism. The result of the destructive metabolism of proteids is the formation of various oxides, carbonic acid and water, and certain not fully oxidised products (urea, uric acid, etc.) which contain the nitrogen of the original proteid. COMPOSITION AND CONSTITUTION OF THE PEOTEIDS The various proteids differ a good deal in elementary composition. Hoppe-Seyler gives the following percentages : — C H X S O From 51-5 6-9 15-2 0-3 20-9 To 54-5 7-3 17-0 2-0 23-n From figures of this kind various observers have attempted to construct an empirical formula for certain typical proteids, egg-albumin beintf the one usually selected. Thus Lieberkiihn assigned to albumin theformulaC^.jHiioXjgO.j.iS ; Loew^ gives the same formula ; Harnack^ gives C204H322X52O66S2 ; Schutzenberger^ C,^^Il:i^.^e,Oy,Ss, and there 1 Lehrhuch, p. 128. - Loew aud Bokomy, Die cliemische Kraftquelle i»i lehenden Protoplastna, Munich, 1882. 5 Zeit. physiol. Chem. v. 207. * Bull. Soc. Chim. vols. xxm. and sxiv. 'I'lll'! I'lv'OTl'MDS 113 have boon otliers. TIio great divergence between tliese numbers requires no eonunent. Results wliicli are e([ually conflicting have been oljtained in attempts to ascertain the molecular weight of albumin. Lieberkiihn, in 1852, attempted to esta])lisli it by analysing the copper compound resulting from the action of a soluble copj)er salt on a solution of egg- albumin. This compound has since then been analysed by six different investiga- tors and found to contain from 1'5 to 5*2 per cent, of CuO. The compound formed is thus one which contains no definite quantity of copper, or there may be several copper albuminates in the mixture. Chittenden and Whitehouse' have both with egg- albumin and myosin found equally variable results with other metals. Therefore, although the molecular weight of albumin is undoubtedly very high, no accurate measurements have as yet been made. Still more contradictory and mutually destructive theories have been formed with regard to rational formulte for the proteids. The usual method which a chemist follows in attempting to discover the constitution of any substance is first to observe the way in which it decom2:)Oses under certain circumstances (analysis), and then if possible to build up the original material from the simpler compounds so obtained (synthesis). In the case of the proteids there have been many observations of the nature of analysis, but synthesis has not yet been successful. The various theories that have been formed all depend on the results of the decomposition of proteids, and hei^e we meet with many difficulties. Fii\st, because the products of decomposition are so numerous ; secondly, because under differing circumstances they are so various ; and, thirdly, because in all probability living proteid differs in its constitution from the non-living j)roteid, with which necessarily la])oratory experiments have to be made. Metabolism is a very dif- ferent process in its results from those of experimental chemistry. Before going into the theoi'ies themselves it will be necessary to give a list of the products of decomposition which result from different treat- ment of albumin. (1) In the body. Carbonic acid, water, urea are the chief final products. Glycocine, leucine, uric acid, ifec, are probably intermediate products. Carbohydrates (glycogen) and fats may also originate from proteids. (2) Action of heat. The oily liquid (Dippel's oil) obtained by dry distillation contains ammoniacal salts of the fatty acids, amines, and aromatic compounds. (3) Putrefaction. Ammonia, ammonium sulphide, carVjoiiic acid, 1 Studies from the Lab. Physiol. Chem. Yale Univ. ii. 95. I 114 THE CHE^nCAL CONSTITUENTS OF THE ORGANISM volatile fatty acids, lactic acid, and amidu-acids (leucine, tyrosine, tkc). Indole and skatole. (4) Action of strong minei'al acids and caustic alkalis. The chief products are leucine, tyrosine, aspartic acid, and glutamic acid. (5) Action of baryta water in sealed tubes. (See further Schiitzen- berger's theory, next page. ) (6) Action of oxidising agents. "With nitric acid a yellow substance called xanthoproteic acid is first formed. As the constitution of albumin itself is unknown, that of its compounds is much more involved in obscurity. In spite of this, various substances have been prepared as the result of the action of nitric acid on albumin, and names trinitro- albumin, hydroxytrinitro- albumin, hexnitro- and hexamido-albumin sulphonic acids,' itc, with formulte have been given to them. It need hardly be said bow exceedingly uncertain all this is, and that different analysts give different results. By oxidation with potassium perman- ganate, Maly- obtained a substance to which he gave the name oxy- protosulphonic acid. These substances on fui-ther oxidation break up into simpler compounds like those already enumerated (fatty acids, amido-acids, aromatic bodies). From results such as these, in which we see that amides, aromatic substances, and fatty derivatives are the mo.st abundant, Gautier^ concludes that the different proteids differ in the arrangement, relation, proportion, and in some cases even in the nature of their contained radicles. 'We can now pass on to consider briefly the various theories that have been held with regard to the constitution of the proteid molecule. a. Mulder's theory.— ^IvIAqv* observed that by the action of caustic potash, sulphur was removed from a proteid, and he called the sulphur- free reaiiiwe protein, and ascribed to it the formula CgijHogNjOio. He considered that the different proteids were combinations of ' protein ' with different amounts of sulphur. Liebig and others pointed out that the warming of a proteid with potash removes not only sulphur, but also ammonia ; and even though the residue gives no further colour with lead salt.s, it still retains some sulpliur. It is thus possible to speak of two forms of sulphur in proteid, that which is loosely and that which is firmlv coml lined. "^ Further investigation has clearly shown that 1 Loew, J.pr. Chem. (2| iii. 180. 2 Centralhl. med. Wiss. 1885, 740. Maly's Jahresh. xviii. 10. 5 Chimie appliquee a la physiol. i. 253. ^ Ann. Chem. Pharm. Ixi. 121. ^ Danilewsky, Zeit. physiol. Chem. vii. 440. A. Ki-iiger, PjJiiger's Archie, xliii. 244. In the latter paper will be found an interesting s-eries of suggestions as to the way in whicli these two forms of suljihur are combined. TiiK i'i;(»Ti-:ii)s 115 ' Itrntein ' is an artiticial product, very much like what we now call alkuli-albuinin. The sole remnant of this theory now extant is the word 'Proteid." h. Schiifz'')ihi'rt/i'r's tlieory. — Nasse' was the first who attempted to get an insight into the molecular constitution of the proteids by treating them in sealed tubes with baryta water at a high temperature for many hours. He found that the nitrogen was differently combined, part being easily displaceable and part held firmly. Schiitzenberger '^ has carried on researches in the same direction. He found that the products of decomposition are ammonia and carbonic acid in the same ratio as would result if urea wei'e treated in the same way ; other volatile products (pyrrole, indole, acetic acid, itc), and a fixed residue in which the substances most abundantly present were leucine and tyrosine— the latter containing the aromatic radicle. Other substances also of the nature of amido-acids were found. These amido-acids he classifies into two groups — the leucines, or amido-acids of the acetic series (C„H„^.,N02), and leuceines, or amido-acids of the acrylic series {C„H.2„_iN02). Both leucines and leuceines are produced by the splitting up of bodies of the formula C^^HoniNoO^ (»i=10 or 12), which ha\ e a sweet taste and are therefore called gluco-proteins. Albumin is regarded as a ureide, or compound of urea ; the urea is combined witli gluco-proteins and the gluco-proteins split up on hydration into amido- acids. The nitrogen is thus after hydration combined as NH.2 (amidogen) ; in the proteid itself the nitrogen is probably present as XH (imidogenj. c. Pfiilyer's theonj. — Although the distinction between living and non-living proteids was emphasised by John Fletcher"' in 1837, it was not until 1875 that an intelligible theory to explain such difference was advanced by Pfliiger.^ The non-living proteids, such as are con- tained in white of ^^^^^i,., are stable and indifferent to neutral oxygen ; but when these proteids are assimilated, that is, become part of a living cell, the molecules of proteid live by breathing oxygen ; not necessarily oxygen from without, as frogs kept in chambers free from oxygen will continue to live for many hours. The assimilation of a proteid is probably due to the formation of ether-like combinations between the molecules of living proteid and the isomeric molecules of the food ^ Pfiiiger's Aj-chiv, \\. 589. - Bull. Soc. Chim. vols, xxiii. and xxiv. ; Annales cle Chim. et Phi/s. (5) xvi. 289; and a large number of papers in the Compt. rend. In a recent paper, Compt. rend. ci. 1267, the formula for albumin given is simpler than those adopted in his earher work ; it is CogHj^NgOio- More recently still (C B.. cvi. 1407) he has succeeded in preparing leuceine syntheticallj*. 5 Budiments of Phi/siology, Edinburgh, 1837. * PJJiiger's Archiv, x. 251. 116 THE CHEMICAL C0>'STITrENT8 OF THE OECtANI>;M proteid, water being eliminated, tliis process of polymerism producing lai'ge and heavy but still simple molecules. In this process the nitrogen of the non-living proteid leaves the hydrogen with which it was com- bined in the form of amidogen (XHo), and enters into combination with carbon to form the more unstable substance cyanogen (CX). We thus find uric acid, creatine, guanine, (fee, as products of proteid meta- bolism, while none of such cyanogen-containing bodies are obtainable fi'om non-li^'ing proteids. d. Loew's theory. — The researches of Loew and Bokorny ' have taken the same direction as those of Pfliiger, that is, they are at- tempts to explain the distinction between living and dead protoplasm. Living protoplasm or proteid in the cells of various alga? has the property of reducing silver from a weak alkaline solution of silver nitrate ; dead proteid has no such effect, and animal protoplasm is so quickly killed by silver nitrate that it also does not give the reaction. The conclusion arrived at is that something of the nature of an aldehyde occurs in living protoplasm. Formic aldehyde is probably formed in plants by the union of carbon and water ; if this is united to ammonia, aspartic aldehyde is formed, thus : — 4CHOH-hXH3 = XHo.CH.COH -h2H..O CH.,.COH [fonnic aldehyde] [aspartic aldehyde] By polymerisation of aspartic aldehyde, we have — XHo.CH.COH' 3J i i CHoCOH •=Ci.,H,;X30,-F2HoO and by further polymerisation in the presence of a sulphur compound and hydrogen, we get GCi2H,7X304 + HoS-f6H2=C;,Hii.2X,gS0.2.2 -t-2HoO, which represents the composition of ordinary albumin. The weak point of the theory is that the aldehyde of aspartic acid is unknown to chemists ; no doubt it is a most unstable substance. If such an aldehyde group does exist in Hving proteid, the instability of proteids is explicable, because molecular movements would be con- stantly occurring in the aldehyde group. e. Latham's theory. — Latham - considers living proteid to be com- posed of a chain of cyanalcohols, or cyanhydrins as they are some- times termed, united to a benzene nucleus. » hoc. cit ' Biit. Med. Journ. vol. i. 1886, p. 629. 'I'lIK I'ROTEIDS 117 Cyanalcdhols are substances ol)taiue(l Ijy the union of an aldehyde •with hydrocyanic acid, thus : — C2H5.OH + 0=CH3.CH0 + H2O [etliyl alcohol] [etbalileliyde] CH,.j.CHO + HCN=CH3CH(CN)0H [c'tliiildelivile] [livilrocj-auic [cvanetlivlic alcoliol] acidj Ethyl alcohol is taken as an instance in the above equations, but many other alcohols are considered to form similar cyan-derivatives, and these are united to one another and to benzene to form a proteid. The theory is a satisfactory one, inasmuch as it includes the hypo- theses both of Pfliiger and Loew. Latham, moreover, shows exhaus- tively that the various products of the disintegration of albumin can also be obtained by the condensation and intramolecular changes that these cyanalcohols, which are exceedingly unstable bodies, undergo. Instability and proneness to undergo intramolecular changes are two properties common to living proteids and to cyanalcohols. In an elaborate and painstaking manner Latham moreover adapts his theory to explain certain morbid processes ; he shows how, by a rearrangement of atoms difi'erent from that occurring in normal metabolism, excess of sugar may be produced in diabetes, excess of uric acid in gout, and certain ptomaines ' in other complaints. We can now leave these theoretical considerations and pass on to consider matters of greater practical interest. TESTS FOE PROTEIDS Solubilities. — All proteids are insoluble in alcohol and in ether. Some are soluble in water, others insoluble. Many of the latter are soluble in weak saline solutions. Some are insoluble, others soluble, in concentrated saline solutions. It is on these varying solubilities that proteids are classified. All pi'oteids are soluble with the aid of heat in concentrated mineral and acetic acids and caustic alkalis. Such treatment, how- ever, decomposes as well as dissolves the proteid. Proteids are also soluble in gastric and pancreatic juices, but here again they undergo a change, being converted into a variety of proteids called peptones. Heat-coayidation. — Many of the proteids which are soluble in water or saline solutions are rendered insoluble when those solutions are heated. The solidifying of white of egg under such circumstances is a familiar instance of heat-coagulation. Heat-coagulation must be very 1 Lancet, vol. ii, 1888, p. 751. 118 THE CHEIMICAL CONSTITUENTS OF THE ORGANISIM carefully distinguished from ferment coagulation — a process by means of which a ferment converts a previously soluble into an insoluble proteid ; as instances of ferment coagulation the formation of fil)rin in shed blood under the influence of fibrin-ferment, or of a curd of casein in milk under the influence of rennet, may be taken. The temperature at which a proteid enters into the condition of a heat-coagulum is fairly constant, and may be employed as one of the means of ascertaining what proteid is present in a given solution. The temperature varies somewhat with the reaction of the solution,^ with the quantity and nature of the salts also present,- and, under certain circumstances, especially in an alkaline solution with the concentration of the solution.-' Unless a solution is very concentrated the contained proteid is not coagulated by heat in an alkaline solution, as it is converted into alkali-albumin ; if the quantity of alkali is, however, very small, the temperature of heat-coagulation is raised. A neutral solution becomes alkaline after the separation of a heat-coagulum, and this alkalinity (produced no doubt by an alteration in the salts related to the pro- teid) may hinder the coagulation of the remaining proteid in the solution. It is generally advisable to have the solution very faintly acid ; a weak solution of acetic acid (2 per cent.) may be employed for the purpose of acidification. Acid-albumin does not form so readily as alkali-albumin, and the presence of a small amount of acid renders easier the separation of the coagulated proteid into flocculi, which can be then removed by filtration. An excess of acid lowers the tempera- ture of coagulation, or it may convert the proteid intoacid-albuinin and so jDrevent coagulation altogether. The simplest method of ascertaining the temperature of heat- coagulation is to place enough of the solution in a test-tube to cover the bulb of a thermometer. The test-tube, the contents of which should be kept constantly stirred by the thermometer, is then placed in a flask containing water and situated over a Bunsen burner. As the temperature rises the point at which flocculi separate should be carefully noted ; a few degrees below this point the liquid becomes thick and opalescent. A form of double water- bath consisting of two beakers one within the other is recommended by Gamgee,* and Schafer'' ' Halliburton, Journ. of Fhysiol. v. 165. - Limbourg, Zeit. physiol. Cliem. xiii. 450. 3 Haycraft, Brit. Med. Journ. vol. i. 1890, p. 167. * Physiol. Chem. p. 15. ^ In my own work I have found certain inconveniences in the use of Gamgee's apparatus, and have therefore used Schiifer's. A description of it will be found in my paper in the Journ. Physiol, vol. v. p. 153. TUK I'KOTKIDS Hi) has invcntoil a very convenient form of running water-bath, thnly proteids present are albumins and globulins. 4. Densimetric metliod. — Methoils have been devised for the quan- titative estimation of proteids in solution by means of multiplying the loss of specific gravity which such solutions undergo on removal of tlie ' Hoppe-Seyler, Handbuch, p. 312. 2 Ffii'igefs Archiv, xi. 10; see also Hoiimaim, Virchoiv's Arcliiv, 1879, p. 255. THE I'liOTElD.S 127 proteid by a CDUstaut factor, under the mistaken notion that the loss in density is (hrectly proportional to the amount of proteid removed.' We have already seen that the method is a fallacious one in the case of dextrose (p. 97). The supposed constant factor is from its very nature a variable one, and a simple algebraical demoiistration of this will be found in a paper by Huppert and Zahor.^ Zahor^ finds, how- ever, that with urine it yields very good practical results. For clinical work, the specific gravity is estimated by a urinometer, marked to four places of decim;ds, before and after the separation of the albumin, by means of acidification (if necessary) and boiling. The apjiroximate percentage of albumin is the loss of specific gravity multiplied by 400. 5. Metliods in icJiicJi a nitroyen estimation is made. — Ritthausen precipitates the proteids from solution with copper sulphate, collects the precipitate, and calculates the amount of proteid in it, by the amount of nitrogen t)btainable in a combustion. Sebelien ■* has tested this method, using copper sulphate, lead acetate, phosphomolybdic acid, tannin and other precipitants. Tannin gave ow the whole the best results. The nitrogen in the precipitate produced by tannin is estimated by Kjeldahl's method {see p. 23), and multiplied by 6-37 to obtain the total proteid. The method is stated to produce less error than the more usual methods involving the washing, drying, weighing, and incineration of proteid precipitates. A very similar method is adopted by Konig and Kisch,'^' who give the multiplier as 6'25. CLASSIFICATION OF PEOTEIDS The proteids may be divided into animal proteids and vegetable proteids according to their origin. There appears to be no essential difference between these two classes, and each can be subdivided in the same manner into groups. The distinction, however, is a con- venient one on which to form the basis of a classification. A. Animal Proteids. — Class 1. Albumins. —These are proteids which are soluble in water, in dilute saline solutions, and in saturated solutions of sodium chloride and magnesium sulphate. They are, how- ever, precipitated by saturating their solutions with ammonium sul- phate. Their solutions are coagulated by heat, usually at 70^-73°C. a. Serum-albumin. Not precipitated by ether. h. Egg-albumin. Precipitated by ether. c. Cell-albumin. 1 Bomliardt, Zcit. Anal. Chem. 1870, 149; lb77, 124. - Zeit.ijlujsiol. Chciii. xii. 4G7. •> Ihid. 484. 4 Ibid. xiii. 135. 5 Zeit. Anal. Chem. xxvii. 191. 128 THE che:mical constituents of the okganism d. Muscle-albumin. e. Lactalbumin. Class 2. Globulins. — These are proteids ■which are insoluble in water, soluble in dilute saline solutions, and insoluble in concentrated solutions of sodium chloride, magnesium sulphate, ammonium sulphate, and certain other neutral salts. Their solutions are precipitated by- heat ; the temperatui-e of heat-coagulation varying considerably. a. Fibrinogen. ) • i i i i , _, 11,./ , , T X • in blood plasma. 0. berum-globulin (paraglobulm) ( c. Globin ; the proteid constituent of haemoglobin. d. Myosinogen, myi)globulin, itc, in muscle. e. Crystallin ; in the crystalline lens. f. Vitellin ; in yolk of egg, not precipitable by sodium chloride. Class 3. Albuminates or derived albumins. — These are proteids derived from either albumins or globulins by the action of weak acids or alkalis. If a little solution of egg-albumin be warmed at 40°C. for 10-15 minutes with a few drops of 0"1 per cent, sulphuric acid, or 0-1 per cent, caustic potash, it will be found to have lost its typical properties, and to have been converted into acid-albumin or syntonin, and alkali-albumin respectively. The albuminates are insoluble in pure water, and in neutral solutions containing no salt. They are soluble in acid or alkaline solutions, or in weak saline solutions. They are precipitated like globulins by satura- tion with neutral salts (sodium chloride, magnesium sulphate, ammonium sulphate). Their solutions are not coagulated by heat. a. Syntonin or acid-albumin. Precipitated by neutralising its solution even in the presence of alkaline phosphates : the neutralisation precipitate dissolves in excess of alkali. b. Alkali-albumin. Precipitated by neutralising its solutions. If alkaline phosphates are present, excess of acid must be added to cause precipitation, the alkaline phosphates being converted into acid phosphates, before the acid attacks the proteids. Alkali-alljumin contains relatively less sulphur than syntonin. Some of the sulphur is removed by the alkali used to make alkali-albumin ;. what is left is more tirmly combined and is not blackened by an alkaline lead solution. A very insoluble variety of alkali-albumin (probably a compound containing a large quantity of alkali) may be formed by- adding strong potash to undiluted white of egg. The resulting jelly is called Lieberkiilin's jelly. c. Caseinogen. The chief proteid constituent of milk. Class 4. Proteoses. — These are intermediate products in the hydra- tion of proteids ; the final products are called peptones. They are TlIK rUoTHJDS 129 formed in the body by tlie action of the gastric ahd pancreatic juices ; they may lie also formed artiticially l)y heating with water, or more readily by dilute mineral acids, or superheated steam.' Tliey corre- spond to the propeptone of Schmidt-Mulheim, and to the A-peptone of Meissner. They have been chietiy worked at by Kiihne and Chittenden, and will be more fully referred to in connection Avith digestion. They are not coagulated by heat ; they are precipitated but not coagulated (see p. 125) by alcohol ; they all give the biuret-reaction (rose-red colour with caustic potash and copper sulphate), and are precijjitated by nitric acid, the precipitate being soluble on heating and i-eappearing when the liquid cools. They may be sub-divided into albumoses, globuloses, vitelloses, caseoses, myusino.ses, etc., according as the original proteid from which they are formed, is albumin, globulin, vitellin, casein, myosin, ifec, respectively. The albumoses may be taken as an instance of the class. All the other groups may be subdivided in the same way. The albumoses are of two varieties, hemi-albitmoses, those whicli ai'e converted by further digestive activity into hemipeptone, and ((titi-a/hunioses, those which are converted similarly into anti-peptone. According to their solubilities, albumoses are divided into : — a. Proto-albumose. Soluble in cold and hot water and in saline solutions ; precipitated like globulins by saturation with sodium chloride or magnesium sulphate. b. Hetero-albumose. Insoluble in water ; soluble inO"5-15 per cent, sodium chloride solutions in the cold, but precipitated by heating to 65°. The precipitate is, however, not a heat-coagulum, as it readily dissolves in dilute acid or alkali. Hetero-albumose is precipitated by dialysing out the salt from its solutions. Like the other albumoses, it is precipitated by alcohol, but, unlike them, is partly converted into an insoluble product called dys-albumose. Hetero-albumose, like proto- albumose, is precipitated by saturation with salts. Proto- and hetero- albumoses are often called the primary albumoses, as they are the first products of the hydration of proteids. c. Deutero-albumose. Soluble in cold and hot water. It is not precipitated from its solutions by saturating with sodium chloride or magnesium sulphate, but it is by ammonium sulphate. It is not precipi- tated by copper sulphate, and only gives the nitric acid reaction so characteristic of albumoses in the presence of excess of salt. It is thus in 1 Neumeister [Zeit. Biol, s.-s.xi. 57) has recently fouud that the albumose formed by the action of superheated steam differs in a few minor reactions from those formed by acids or by gastric digestion. He has apphed the name atmid-albumose to it. K 130 THE CHE:MICAL CON'STITI'ENTS of the ORr^ANISM its reactions nearer to peptone, than the other albumoses ; it is an inter- mediate stage in the conversion of the primary alVmmoses into peptone. Class 5. Ppptones} — These are the final products oi the hydration of proteids. If hydration goes further the peptone is split into simpler substances and remains no longer a proteid. They are soluble in water, are not coagulated by heat, and are not precij^itated by nitric acid, copper sulphate, ammonium sulphate, and a number of other precipitants of proteids. They are precipitated but not coagulated by alcohol. They are also completely precipitated by tannin, potassio-mercuric iodide, phosphomolybdic acid, phosphotungstic acid, and picric acid. They give the biuret reaction (rose-red colour with a trace of copper sulphate and caustic potash or soda). Pure peptone separated from all other pi'oteidsby ammonium sulphate, freed from excess of salt by dialysis, precipitated by alcohol and dried, hisses and froths with evolution of heat on being dissolved in water. Its taste is somewhat cheesy but not unpleasant. The bitter taste of a'rtificially digested food is due to some product not yet separated, native proteids and albumoses being almost tasteless. Peptones are divided into a. Hemipeptone. The form of peptone which by the further action of pancreatic juice is split into simpler products, such as leucine and tyrosine. h. Antipeptone. The form of peptone which is not decomposed in this w^ay. It moreover yields no tyrosine on treatment with sulphuric acid, and does not give Millon's reaction. Both forms of peptone are readily diffusible through animal membranes ; albumoses are only slightly difFusil)le. The utility of the formation of ditiusible substances during digestion is obvious. The table on the next page contrasts the chief reactions of the albu- moses and peptone. Class 6. Coagulated Proteids. — («) Proteid in which coagulation has been produced by heat. Insoluble in water, weak acids, and alkalis. Soluble after prolonged boiling with concentrated mineral acids. Soluble in gastric and pancreatic juices giving rise to peptones. (h) Proteids in which coagulation has been produced by ferments, i. Fibrin. See blood, ii. Myosin. See muscle, iii. Casein. See milk. iv. Anti-albumid. A comparatively insoluble byrproduct formed in gastric digestion. 3 See Kiihne and Chittenden, Zeit. Biol. xxii. 423. TIIK I'KO'I'KIDS 131 Hot and Copiier Variety of rroteid Hot nml Cold Water Cold Saline Solutions e.g. 10% NaCl ■ Saturation with NaCl or MgSO« Saturation with AmjSO^ Nitric Acid Copper Sulphate Sulphate and Caustic Potash Proto-nlbuinose Soluble Soluble Precijii- Precipi- Precipi- Precipi- Rose-red tatefl tated tated in cold ; iire- cii)itate dis- solves with heat and reappears ou cooling tated colour (Biuret re- action) Hetero-allniniose Insoluble ; Soluble ; Precipi- Precipi- Ditto Precipi- Ditto i.e. preci- partly jirc- tated tated tated pitated by cipit'ated, dialysis but not co- from saline agulated on solutions. heating to 65° C. Deutern- Soluble Soluble Not preci- Precipi- This re- Not pre- Ditto albumose pitated tated action only occurs iu presence of excess of salt cipitated PeiitDiie . . . So'uble Soluble Not preci- Not preci- Not preci- Not pre- Ditto pitated pitated pitated cipitated B. Vegetable Proteids. — The amount of proteid matter in plants is less than in animals. Proteids occur either dissolved in the juices of plants, or in the solid form composing the protoplasm in the plant cells, or often deposited in the form of granules (aleurone grains). Vegetable j^roteids do not differ in their essential characteristics from animal proteids, but unlike most animal pi'oteids they have frequently been obtained in a crystalline form. Much error and confusion has crept into our knowledge of vegetable 2)roteids from the researches of Ritthausen. He used caustic alkalis as a means of extracting the proteids from the vegetable tissues, and consequently converted the native proteids, globulin, albumin, &c., into alkali-albumin. It is necessary to remember that the substances legumin, conglutin, etc., which he thus obtained are artificial products, and do not represent what is present in the plant tissues themselves. The vegetable proteids may be subdivided into the same six classes as the animal proteids. Class 1. Albumin^. — The term vegetable-albumin is often used synonymously with vegetable proteid ; it should be properly restricted as in animals to those forms of proteid which are soluble in water and coagulable by heat. The greater part of the proteid coagulable by heat in the juices and seeds of plants is of the nature of globulin, not albumin. Small quantities of a true albumin have been described by Martin' in the juice of the papaw fruit, and by Green^ in the 1 Jourii. Phijsioh vi. 336. ^ Proc. Boy. Soc. xl. 28. K 2 132 THE CHEMICAL CONSTITUE^'TS OF THE OEGANISM latex of several caoutchouc-yielding plants of the natural orders Ajjocynece and Sajjotaceie. Class 2. Globxdins. — These are by far the most abundant j^roteids present in plants. This view, which has received the powerful support of Hoppe-Seyler/ who speaks of the proteids in buds, young shoots and seeds, as globulins, is contrary to that of Ritthausen, who on the ground of concordance in elementary analyses, regards vegetable pro- teids as consisting of legumin and other allied substances, which have been shown to be artificial products produced by the caustic alkali used in their preparation.^ The earliest observations of value on this subject are those of Weyl.'^ He compared the composition^ and reactions of animal and vegetable proteids, he showed tliat the two classes were practically identical. He did not find any albumins, and examined the proteids extracted by salt solution from oats, maize, peas, mustard, Para nuts, (tc, which consisted chiefly of plant-vitellin. A second proteid, also a globulin called plant myosin, was found in wheat, peas, oats, white mustard, and sweet almonds. No alkali-albumin (plant-casein or legumin of Ritthausen) was present in any of the plants examined. The vitellin occurring in plants is often crystalline, and a number of observations have been made by different observers on tliis crystal- lised albumin, as it is often incorrectly called by them. The proteids occurring in aleurone grains have been the subject of masterly researches by Vines' ; he found much globulin there. The proteids of the papaw fruit, Abrus jjrecatorius,^ wheat and other flours^ have been investigated by Martin ; the change that occurs in the process of germination of seeds has been the subject of a research by Green* who has also investigated the proteids in latex. These re- searches may be briefly summarised as follows : — Vines' Investiffations on Aleurone Grains.— The aleurone grains of the peony (^PceoJiia off.) contain an albumose, and vegetable myosin ; of the castor oil plant {Ricinus comm.} an albumose, a globulin insoluble in saturated sodium chloride solution (myosin), and a globulin soluble in that solution (vitellin): of blue lupin, chiefly crystalloid vitellin. Similar crystalloids were found in many other plants. The following classification of aleurone grains is given : — 1. Those soluble in water. Albumose. 1 Physiol. Chem. p. 75. - Eitthausen defends his views in Chem. Centr. 1877, 567, 586. 3 Pfliiger's Archiv, xii. 635. Zeit. physiol. Chem. i. 72. 4 For comparative elementary analyses, see A. Brittner, A". Rej}. Pharm. xxi. 66 and 129. 5 Proc. Boy. Sac. xxviii. 218 ; xxx. 387 ; xxxi. 62. ^ jijd xlii. 331. 7 Brit. Med. Journ. vol. ii. 1886, p. 104. ^ Proc. Boy. Sac. xli. 446. THE PROTEIDS 133 2, Those soluble in 10 per cent. XaCl, a. Grains without crvstalloifls. Soluble in saturaterl NaCU h. Grains with cr}-stalloifls. SoluVile in saturated NaCl. 3. Those partially soluble in 10 per cent. NaCl. Some of these are crystalloid, some insoluble, some soluble in saturated NaCl solutions. Martin's Inrestigations on Papatv. — Tlie proteids present are : — 1 . A globulin ver}- like serum-globulin. 2. Albumin. (Already alluded to, p. 131). 3. Albumoses of two kinds ; with one of which (o-phytalbuniose) a ferment (papain), in nature very like the tryjDsin of the pancreatic juice, is associated. Martin's Ohserrations on W/ieat Flour. — The flour itself contains two pro- teids— vegetable myosin and an albumose. "WTien mixed with water, these undergo certain changes, and are converted into the insoluble proteid called gluten. Martin's Ohservations on Ahrus (Jequirity). — Warden and Waddell' have given the name 'abrin ' to the poisonous principle of jequirity; this plant is used as a drug to produce conjunctivitis when applied locally to the eye. It is not an alkaloid, but a proteid. The proteids are two in number— a globulin (resembling serum-globulin) and an albumose. Both proteids have a poisonous action, which is destroyed at a high temperature.- Greens Investigations of Latex.— In Apocyneae and Sapotaceaj the proteids are two albumoses and an albumin. In the manihot (Euphorbiaceae) a globulin, in the common lettuce (Compositae) an albumose. We can now pass on to consider the chief member.s of the globulin group occurring in plants. (a) Plant-vitellin (phj'to-vitellin). This proteid is like animal vitellin, a globulin soluble in saturated solution of sodium chloride. It is coagulated by heat at about 75°C. In the yolk of the eggs of certain fishes, this substance has a semi-crystalline form, but in the aleurone grains of many plants it is distinctly crystalline, or can be made to crystallise. This is thus the purest proteid known, but even it leaves on ignition an ash consisting chiefly of alkaline phosphates. Elementary analysis gives C, 52-43; H, 7-12; X, 18-1; S, 0-55; O, 21 -s (Weyl). The observations of Vines have shown that the crystalline proteids obtainable from aleurone grains differ in solubilities and crystalline form, and thus there is probably more than one crystallisable proteid. The following are the chief observations made on the subject of crystalline vegetable proteids: — Harti?,^ in 1855, was the first to discover a crystalline proteid in plants. ' Xon-bacillar Nature of Ahrus Poison, Calcutta, 1884. 2 Martin, Brit. Med. Journ. vol. ii. 1889, p. 184. 5 Botan. Zeitung, 1855, p. 881. 134 THE CHEMIC.IL CONSTITUENTS OF THE ORGANISM Mascbke,' by extracting Para nuts with water at 50°, filtering and evaporating at the same temperature, obtained a crystalline proteid residue. WeyP identified the proteid as vitellin, and found it in a number of other likints. Schmiedeberg' obtained a crystalline compound of this proteid with magnesia. Griibler' prepared the same compound and another, also crystalline, with lime; the molecular weight calculated from the first is 5081, that from the second 8848. Griibler's method of preparing the vitellin ciystals is to dissolve the proteid from pumpkin seeds in solution of sodium chloride at 40° ; on cooling the liquid to 7° the crystals (regular octahedra) separate. Ritthaiisen,^ adopting the same method, obtained crystals (octahedra and rhombic dodecahedra) from expressed hemp cake, castor oil seeds, and the seeds of sesanuim indicum. Drechsel" introduced another method, which consisted in extracting the seeds (pumpkin) with water, and dialysing the extract into alcohol ; the water diflfuses into the alcohol, leaving crusts of microscopic crystals in the dialj'ser. Vines found that the natural crv'stalloids imbedded in the ground substance of the aleurone grains were hexagonal rhombohedra in certain plants, and regular tetrahedra in others. (b) Plant-myosin. This like animal myosin coagulates at 56'^C. It also like the myosinogen of nauscular tissue is converted into a more insoluble substance by a ferment action ; this substance is called gluten-fibrin and forms the basis of gluten {see further, next page). (c) Vegetable-paraglobulins.'^ This class of proteids coagulating at 75° and precipitated by saturation with sodium chloride was first described by Martin ; one of these proteids occurs in papaw juice, another in latex, another in ahriLS seeds. Class 3. AUmminates. — Acid-albumin or syntonin and alkali- albumin are formed readily by the action of acids and alkalis respectively on the native globulins of plants. Plant-myosin like animal myosin is especially readily convertible into these albuminates. a. Legumin, or vegetable casein. The term legumin appears to have been used synonymously for vegetable proteid by the earlier investigators.** It has been the subject of laborious examination by Ritthausen.^ We now know that it is simply alkali-albumin formed from the native globulins by the caustic potash used in extracting it from the plant. h. Conglutin is the legumin obtainable from almonds and lupines. It is more glutinous and more soluble in acetic acid, and richer in nitrogen than ordinary legumin (Ritthausen).'° 1 Journ. jJt'akt. Chem. Ixxiv. i'd6. ^ Loc. cit. 3 Zeit.plujsiol. Chem. i. 205. * Joiirn. ■prcikt. Chem. cxxxi. 105. 5 Ihid. p. 481. <^ Ibid. (2) xix. 331. 7 Martin, Proc. Physiol. Soc. 1887, p. 8. 8 Einhof, N. aUgemein. J. d. Chem. v. A. Gchleii, vi. (1805), pp. 126, 548. Dmnas and Caliours, Liebig, and others, also examined this substance. 9 Zeit.f. Chem. (2) iv. 528, 541; vi. 120; J.pr. Chem. ciii. C5, 78, 103, 273. JO J.pr. Chem. (2) xxvi. 440. 'I'lIK rUOTKIDS 135 Class 4. J^rotcnsrs. — Previuus to Viues's observations these were spoken of as vegetable peptones. Vines recognised that the aleurone grains did not contain true i)ej)tone, but a substance which he spoke of as hemi-albumose. Two albunioses of doubtful nature are described by Green in latex. The following albumoses in plants have been more fully described (Martin): — a. a-Phyt-albuniose. Ver3' like proto-albuniose, and probably iden- tical with Vines's hemi-albumose. Found in papaw juice, wheat- flour, abrns. Associated in papaw juice with the ferment papain. 6. />Phyt-albumose. Very like hetero-albumose. Pound in papaw juice. ('. Insoluble phyt-albvniiose. A constituent of gluten. d. Vitelloses.^ Intermediate products in the hydration of vitellin analogous to albunioses, and subdivided like them into anti-, hemi-, proto-, hetero-, dys-, and deutero-vitellose. Class 5. Peptoneii.- — ^True peptone does not appear to be found native in plants. It is formed from vegetable as from animal proteids by hydration processes, such as is brought about by boiling with dilute mineral acids, or treatment with gastric or pancreatic juices. The intermediate products are proteoses. Papain like panci-eatic juice acting on animal proteids converts them in an alkaline medium into proteoses, and finally peptone ; acting on vegetable proteids, it stops short at the proteoses, no true peptone being formed. Leucine and tyrosine are, however, found in the tissues of the plant (Martin).- Probably circulating proteid in the plant consists of albumoses. Class 6. Cocujulated proteids. — a. Proteids in which coagulation has been produced by heat. Albumin and globulin of vegetable origin, like the same substances of animal origin, are converted at a high temperature into an insoluble heat-coagulum. b. Proteids in which coagulation has been produced by a ferment - action. i. Gluten (the sticky constituent of dough which may be washed free from starch by kneading in a stream of water) is probably formed by a ferment-action from the proteids pre-existent in flour. This is sup- ported by the fact that washing flour with water at a low temperature (2°C.) does not lead to the formation of gluten. The ferment has, however, not been separated.-^ ' Neumeister, Zcit. Biol, xxiii. 402. - Journ. Physiol, v. 213; vi. 336. 3 Joliannsen {Ann. Agrononi. xiv. 420; Absf. J. Chein. Soc. 1889, p. 296) has advanced certain facts which tell against the ferment theory. 136 THE CHEMICAL CONSTITUENTS OF THE ORGANISM Boiling water or alcohol extracts from gluten a sticky substance, called insoluble phyt-albumose by Martin, and con-esponding to two substances, called gliadin and mucedin by Rilthausen.' The insoluble non-sticky residue is called gluten-tibrin. We have already seen that the proteids in the flour itself are (1) vegetable-myosin, and (2) a soluble albumose. Proljably they are the precursors of gluten, according to the following sclienie (Martin) : — .(, /Gluten-fibrin precursor, myosin. I Insoluble albumose precursor, soluble albumose. ii. Anti-albumid, anti-vitellid, il'c, are substances of a compara- tively insokible nature formed during the earlier stages of gastric digestion. Formation of and cJiarKiPH in tJie pt'ofeids in plants. — The formation of proteids in plants is undoubtedly a synthetical process, the elements and simple compounds, which are combined together to form them, being ultimately derived from the soil and air. An exception to this rule occurs in the carnivorous plants, and in parasitic plants, which live vipon the materials formed by other plants. In the carnivoi'ous plants^ (Drossera, Diona?a, ifec.) a juice is secreted which has the jiower of converting into peptones the proteid matter in the flies and other small creatures caught by the plants. Many nitrogenous bases are found in i^lants, such as asparagine, leucine, tyrosine, adenine, ttc. It is possible that these substances are not always products of the breaking down of j^roteids, as they are in animals, but in certain cases, at any rate, are stages in the building up of the jjroteids. Thus asparagine is probably formed by the union of inorganic nitrogen compounds with malic acid within the plant, the malic acid being derived from the carbohydrates,^ which are formed by the union of carbon aiid water under the influence of chlorophyll. But under certain other circumstances it has been shown that asparagine ai'ises from the decomposition of proteids.'* A large number of observations on these nitrogenous bases, and the changes they undergo in germination, have been made by E. Schulze,''' but comparatively few on the changes that the proteids undergo. We have in plants certain reservoirs of 1 J. ])r. Chemic, Ixxiv. 193, 384. For earlier observations on tjhiten, see Bouchardat, Comjpt. rend. xiv. 962; Taddei, Giornale fisica di BriujiudrUi, xii. 300. See also Giinsberg, J. pr. Chem. Ixxxv. 213. - See Darwin, Carnivorous Plants. Article ' Camiv. Plants ' in Encydop. Brit. (P. Geddes). 3 Miiller, Landiv. Versiichs. Stats. 1880, 326. ■* E. Scliulze and E. Kisser, Ibid, xxxvi. 1. * Numerous papers in Zeit. j)^iysiol. Chem. See especially xii. 405. TIIF ?K(VrKll)S 137 food material ; this is especially seen in the cotyledons ; this food material to be available for the needs of the plants must be converted into a soluble form. Starch, for instance, is converted into a soluble sugar, in most cases by the activity of an organised ferment, e.g. in malt; the proteids must be similarly changed, either into an albumose, peptone, or soluble nitrogenous base (asparagine, leucine, &c.), before it can be carried by the sap to other parts of the plant. Gorup- Besanez' stated that the changes in the reserve jDroteid materials during germination ai*e probably due to the action of a ferment, and though this Avas disputed by Krauch,'^ subsequent experimenters are agreed that the ferment theory of Gorup-Besanez is probably correct. Green^ considers that the nitrogen travels from the seed to the growing points in the form of amides, not in that of peptones or other proteids. Martin on the other hand is inclined to believe that the circulating nitrogen is chiefly contained in one or more soluble proteids of the albumose class. One of the best known ferments that effect these changes is papain or papayotin, obtainable from the juice of the papaw plant (Wurtz, Martin) ; but as the subject is more investigated, it becomes more and more strikingly demonstrated that papain is no single instance of a proteid -splitting ferment occurring in plant tissues, but that such ferments are practically ubiquitous."* PROTEIDS AS POISONS Albuminous substances form a most important element of food, but it is only within the last few years that the fact has been established that there are certain proteids which, when introduced into the circu- lation, are poisonous. This fact, remarkable as it is in itself, becomes of greater significance when it is considered that the poisonous proteids are not distinguishable by any well-marked cheinical or physical pro- perties from the non-poisonous or food proteids. The most important of the vegetable proteid poisons are : — 1. Those contained in the seeds of jequirity, allusion to which has already been made (p. 133). 2. The proteid associated with or identical with papain -^ 3. Lupino-toxin (?), from lupimis hcteus.^ ' Bericlite deiitsch. clieni. Ges. 1874, p. 1478. 2 Ahst. Chem. Soc. Journ. 1878, p. 996. ^ Proc. Boy. Soc. xli. 466. * See Thiselton Dyer's Presidential Addi-ess, Section D. Brit. Assoc. 1888 (Bath meeting); also Hansen, Botan. Zeitung, 1886, p. 137. Ellenberger and Hofmeister Bied. Centr. 1888, p. 319. = Martin, Brit. Med. Journ. vol. ii. 1889, p. 184. <5 Schmidt's Jalirh. 1888, cciv. 10. 138 THE CHEMICAL CONSTITUENTS OF THE ORGANISM The most important of the animal proteid poisons are : — 1. Snake poison. Bacteria and alkaloids are here absent,' and the proteids obtained in a pure condition are as poisonous as the original veuom. Fayrer, Brunton, Weir-^Mitchell, Wolfenden, Reichardt, and others, are unanimous on this point. The poison has been examined in the cobra, viper, crotalus, copperhead, mocassin, and the same result arrived at in all cases. Wolfenden,- who has examined the venom of the cobra and viper according to the most recent methods of separating proteids, finds in the former (1) globulin, (2) albumin, and (3) syntonin ; and in the latter (1) globulin, (2) albumin, and (3) an albumose. All these are poisonous. The chief symptom produced is asphyxia. 2. The proteids in the seinim of certain fishes (conger eel, muraena, Jcc.).3 3. Proteid poisons found in certain spiders.^ i. Proteids foi-med during natural digestion in the stomach and small intestines — albumoses and peptones. 5. Wooldridge's tissue-fibrinogens which produce intravascular coagulation of the blood. 6. Fibrin-ferment. The poisonous proteids numbered 4. 5, and 6 will be more fully discussed in connection with the blood. Poisonous proteids produced by bacterial acti^-ity will be referred to under Fermentation (Chaps. XII and XIII). In the following table Martin^ compai'es the activity of some of the most important proteid poisons : — Fatal Done Venom of common adder* . . 00021 gramme per kilo of body weight Venom of AnstraUan tiger snake* . 0*0049 ,. „ Venom of cobra* .... 0000079 Abrus poison : — Globulin 001 Albumose 006 ,, Peptic albumoses* .... 0*3 „ „ ^ In some cases alkaloids are present, but they are non-poisonous ones (Gantier). - Journ. Physiol, vii. 327. References to other writers will be found here; the cobric acid of Blyth [Analyst, i. 204 1 is shown to be non-existent. ^ ilosso, Mah/s Jahresberichf, xviii. 92. * Kobert, Sitzungsh. der Dorjjaterr Naturforsch. Gesell. 1888; abstracted in Centralbl.f. d. med. Wiss. 1888, p. 544. ■' Proc. Boy. Soc. xlvi. 108. ^ Fontana, quoted in Marx. Giftlehre, ii. 74. ' Report of Special Commission on Snake Poisoning, Austral. Med. Journ. 1876, No. 21, p. 104. 8 Vincent-Richards, Landmarks of Snake-poison Literature. 9 Pollitzer, Journ. of Physiol. 1886. TIIK I'HO'I'KIDS 189 TABLES ILLUSTRATING METHODS OF TESTING FOR PROTEIDS The following tables present in a compact form the chief analytical methods of separating and identifying the most inipoi'tant proteids when in solution : — • Table I. — One proteid only pref'TS OF THE DRGANISM occurs only when the orf^anisms are growing, and stops when the organisms are removed or killed. This vitalistic theory of fermentation becomes especially important to the physiologist and pathologist when applied to disease. The 'germ theory,' as it is termed, explains the infectious or zymotic diseases by considering that the change in the system is of the nature of fermentation, and like the other fermentations we have mentioned, produced by particular forms of bacterium ; the transference of the bacteria or their spores from one person to another constituting infection. This theory has not been fully verified for every infectious disease by the discovery of a specific microbe ; many able investigators, however, consider it likely that the pathogenic germs of these maladies will be discovered, as in the cases of splenic fever, and relapsing fever, and a few others in which the specific bacterium has been already identified. There is, however, another class of chemical transformations, which differ very considerably from all to which we have hitherto alluded. They, however, resemble these fermentations in the fact that they occur independently of any apparent change in the agents that produce them. The agents that produce them are not living organisms, but chemical substances, the result of the activity of living cells. As instances of this class of chemical transformations, the following may be taken : the change of starch into sugar by the ptyalin of the saliva, the change of proteids into peptones by the pepsin of the gastric juice, the change of fibrinogen into fibrin, when blood is shed, &c. &c. These changes are also included under the term fermentation.^ Fermentations may therefore be divided into two classes : first, those brought about by the organised ferments (torulce, bacteria, &c.), and, secondly, those brought about by the unorganised ferments (pepsin, diastase, &c.). Each of these classes may be again subdivided, according to the nature of the chemical change produced. Previous to 1838, the action of yeast was regarded as a catalytic one (Berzelius) ; that is to say, the influence of its mere presence causes a separation of the constituents of sugar, just as platinum black causes peroxide of hydrogen to give up an atom of its oxygen. A modification of this theory was proposed by Liebig in 1848 ; he gave the organisms associated with the change a secondary position, hold- ing that they produced substances of a chemical nature which were the true ferments ; and he considered that the molecular vibrations of these ferments caused a rearrangement of the atoms of the .substance 1 Sheridan Lea suggests the term zymolysis for this variety of fermentation iJourn. of Physiol. 1890, p. 254). Sir W. Roberts suggested the term enzymosis {Proc. Boy. Soc. vol. xxxi. p. 145) many years ago for the same processes. FERMENTATION 153 undergoing fermentation. He compared this action to the decomposi- tion of acetic acid into acetone and carbonic acid produced by heat, or the change of cyanogen dissolved in water into oxamide, produced by the \ibrations of a trace of aldehyde. This action is also comparable to the action of the unorganised ferments, in which the living cells, for instance, of the stomach, produce a chemical substance, pepsin, the active agent in producing the fermentative change of albumin into peptone. In certain cases this view of Liebig has been justified ; soluble ferments have been separated from the organisms, and these have the same action when the organisms are absent as when they are present. Thus yeast cells, in addition to causing the alcoholic fermentation, pro- duce also an in^'erting ferment, that is, a ferment which transforms cane sugar into glucose ; this ferment can be readily separated from the organisms (Barth,' Donath,- Lea,^ ikc). The alkaline fermenta- tion of urine, in which urea is converted into ammonium carbonate, is brought about by an organism very similar to yeast, and to it the name torula iirece has been given. Here, again, a soluble ferment with the same power has been separated from the cells (Musculus,* Lea). But in the greater number of cases, attempts to separate such soluble chemical ferments have been unsuccessful^ and thus attention has been more concentrated on the biological side of tl:ie problem. In the case of the alcoholic fermentation, Helmholtz, Mitscherlich, and others, showed that if the yeast cells were prevented from passing into a fer- mentable liquid by the interposition of an organic membrane, fermen- tation did not ensue. That the organisms themselves are absolutely necessary, is also shown by experiments with the bacillus anthracis, the specific microbe of anthrax or splenic fever. A cultivation of the bacillus inoculated into an animal causes the death of that animal by splenic fever ; but if the bacilli be first carefully filtered oflf from the cultivation fluid, the filtrate is innocuous.'^ If, however, it be freely admitted that the organisms themselves are the cause of the fermentation, the question still remains, how do they act 1 Do they live on the fermentable matter, and then excrete what we call the products of fermentation 1 This view is not tenable, because of the immense volume of the substances in which they pro- duce changes ; Pasteur considers that of the sugar acted upon by yeast only one per cent, is taken up by the yeast itself. Another view, which > Ber. d. deutsch. chem. Gesell. 1878, p. 474. » Ibid. 1875, p. 795. ^ Journ. of Physiol, vi. 1S6. ^ Comj)t. rend. Ixxxii. 333. P/}iiger'sArchiv,xn.214. * In such experiments the culture fluid employed has been beef-tea or a similar infusion. More recent experiments [by Wooldridge, Hankin, and Martin have shown that if the bacilli be grown in a fluid rich in proteids, they produce a poison, a solution of which causes anthrax [see p. 1C8). 154 THE CHEMICAL CONSTITUENTS OF THE ORGANISM is probably more correct, is that the organisms produce, very much as Liebig supposed, a soluble ferment, which acts on the fermentable matter. This view, which has received the powerful support of Hoppe- Seyler, is at once confronted with the difficulties already mentioned, the chief of which is the inability of various observers to separate such soluble ferments from the organisms. It is, however, always unsafe, when results of experiments on any subject are negative, to assume that our knowledge upon that subject is complete and final. The inability of observers in the past to perform an experiment may be from lack of means or of knowledge ; and it is possible that the presence of soluble ferments in places where their existence has been hitherto denied, may be demonstrated in the future. The separation of the inverting ferment from yeast, and of the urea- ferment from the torula urexp., is a step which may be the first in a series of discoveries. Sheridan Lea in his experiments, indeed, pointed out a possible explanation of the negative results of previous investigators. Both the urea-ferment and the inverting ferment were obtained by precipitation of the cells with alcohol, and subsequent extraction of the alcoholic precipitate with water, but neither is present in the fluid sur- rounding the cells during the progress of the change which they produce. This is probably due to the fact that ferments, being non-diflTusible, are unable to pass from the protoplasm of the torula, through its surrounding investment of cellulose. It has already been surmised that ferments are of the nature of the living proteids (p. 146) ; like other proteids they are indifiusible ; this readily accounts for the fact they are not discoverable outside the cell wall ; and like all living things their properties during life are different from those after death ; this readily accounts for the fact that, with a few exceptions, they are not discoverable inside the cell wall, after the cell has been killed by alcohol. The few exceptions are probably those which are more robust, and withstand the action of alcohol better. If this hypothesis be admitted, and until it is replaced by a better it must l)e admitted, the difierence between organised and unorganised ferment action is this : an organised ferment is one which does not leave the living cell during the progress of the fermentation ; an un- organised ferment is one which is shed out from the cells, and then exerts its activity. Probably the chemical nature of the ferment is in the two cases the same, or nearly the same. If it be admitted that the ferments are proteid in nature, or some- thing closely akin to proteid, and it be also remembered how imper- fect our knowledge of the proteids is, it may seem a task from which one would shrink, to attempt to explain any further how the ferments PEKMENTATIOX 155 act. The ferment actions however consist very largely in the trans- ference of water, or of oxygen ; and we happily have in the simpler regions of chemistry, examples of action which seem to be analogous to what we call ferment action in the vaguer regions of organic chemistry. The most striking of the phenomena of fermentation are these : — (1) A small amount of the ferment produces a change in an, over- whelmingly large quantity of material. This is even more puzzling in the case of the unorganised than in that of the organised ferments. A needle prick, if the point of that needle is infected with the bacillus nntltracis, will cause the animal so inoculated to die of splenic fever. The inoculated bacilli have the power of rapid multiplication, and so rapidly poison the whole of the blood. A minute fragment of rennet will cause curdling throughout a huge volume of milk. There is here, however, no such power of self-multiplication. (2) The ferment itself takes no apparent part in the change pro- duced, but, after having produced its action, can be used again to produce the same action in another mass of material. The vibration theory of Liebig is only to a certain extent an explanation of these phenomena ;^ the changes taking place among the atoms composing the molecules of the ferment produce vibrations, which, acting on the molecules of the substance with which the ferment comes in contact, set up there similar molecular vibrations and rearrangements. This is quite comparable to what is taking place around us every day in a social capacity. An irritable quick-tempered individual enters a room filled with pleasant people. The influence of his presence soon causes the whole assembly to become changed, ax^d bad temper to rule supreme. The analogy to the case of a ferment is com- pleted by the fact that the author of the change is himself unaltered, and is capable of producing the same action on another mass of material. This homely comparison however helps us very little ; it leads us into the regions of psychology, where the problems are even more com- plicated than in physiology. It will be of greater help to find com- parisons in simpler chemical reactions which are well understood. Take the case of the ordinary way in which oxygen is made. If one heats potassium chlorate (KCIO3), the oxygen comes off, and potassium chloride (KCl) is left behind. If, however, a little manganese dioxide be mixed with the chlorate in the first instance, the oxygen ^ A ' contact theory ' more recently advanced {Watts' Dictionary , 1889, vol. ii. p. 540) is that the enzymes raise the molecular temperatures of the decomposing molecules to the point at which their molecular equilibrium is destroyed ; their decomposition is pro- duced by rearrangement of energy, not by any increase or decrease of the amount present in the system. 156 THE CHE:snC.\L CONSTITUENTS OF THE OEGANISX comes off much more easily, but the manganese dioxide is unaltered at the end of the experiment, and this is quite comparable to what occurs in the case of a ferment. Take another example ; in the manufacture of ordinary oil of vatriol, sulphurous acid, atmospheric air, and steam are brought into contact M-ith one another in a large leaden chamber. These three substances alone would suffice to form sulphuric acid (SOo + O + HgO ^rHoSO^), but the action would be a slow one. The combination is hastened by the presence of a small quantity of nitrous acid (NjOg). The sulphurous acid (H.^SOg) combines with the nitrous acid, and is then decomposed into sulphuric acid (HoSO^) and nitric oxide (X2O2). HoSOg + N2O3 = H,S04 + N2O2 [sulphurous acid] [nitrous acid] [sulphuric acid] [uitric oxide] The nitric oxide left combines instantly with oxygen, to form nitrous acid again, which in turn undergoes the same decomposition with sulphurous acid. Thus the nitric oxide serves as an oxygen carrier, and as it is continually being recovered, and itself taking no part in the composition of the final product (sulphuric acid), a small quantity will last an indefinite time, and always be ready to perform the same office. Here again it plays the part of a ferment. Take another example, this time from organic chemistry ; namely the action of sulphuric acid in the manufacture of ether from alcohol. If one distils together alcohol and sulphuric acid, ether and water will be found in the distillate, and the sulphuric acid apparently unchanged in the retort ; and the same quantity of sulphuric acid can be used over and over again, to break up an indefinite quantity of alcohol. Xow if the action of the sulphuric acid had not been understood, as it was not until comparatively recent times, the reaction would have been still looked upon as puzzling, and described as catalytic. ^Ve do, however, understand how sulphuric acid acts. The first reaction that takes place may be denoted in this way. We start with alcohol and sulphuric acid : — HHSO4 sulphuric acid, OH(CoH7) alcohol. When these come together, the vertical line represents the pro- ducts of their interaction ; they split into H 1 HSO4 OH ; (C,H,) water ethTl-hydrogen sulphate water, which comes over in the distillate, and ethyl-hydrogen sulphate. The ethyl-hydrogen sulphate reacts with more alcohol, and, the FEKMENTATJON 157 vertical line ajj;ain indicating the way in which the atoms are re- arranged, we have CaHftO H80, H sul]iliuric acid sulphuric acid, again ready to be split up as before, and ether, which distils over. In this reaction the sulphuric acid acts as probably ferments do in fermentation. Apparently they are unchanged at the end of the reaction ; probably they have acted in some such way as the nitric oxide or the sulphuric acid do in the examples just given. Probably they play the part of an oxygen carrier, or a water carrier, and then in the later stages of the reaction are deprived of their extra oxygen or water, and thus appear the same as before the reaction began. Lastly, an example may be taken from physiology itself ; the example is that of the action of htemoglobin ; it comes to the lungs in the venous blood, is converted there into oxyhsemoglobin, takes the oxygen to the tissues, and returns as it started, in the condition to act over and over again as an oxygen carrier. This action of hsemoglobin is not generally called a ferment action, but it appears to me to be clearly in the same category of phenomena. We do not call it a ferment action, because we understand it ; when we attain to a similar accurate understanding of the action of pepsin, and of bacteria, we shall probably cease to call them ferment actions, and reserve that term for what we do not understand as a convenient cloak for ignorance. The action of sulphuric acid in etherification is no longer cloaked under the similar term catalysis. There seems no reason why in the future we may not attain to as accurate knowledge concerning ferment actions as chemists have arrived at in connection with many formerly so-called catalytic iDhenomena. We have thus a series of occurrences in chemistry, starting with the simple catalytic processes of inorganic chemistry, and ending with the ferment processes of physiological chemistry, probably differing only in the complexity of the substances taking part in them. Ferment activity is a manifestation of protoplasm in a living condition ; and I regard it as possible that, by working out ferment actions in the light of the simpler catalytic actions, we shall obtain an insight into the explanation of other still more complex vital actions. A step to the better knowledge of fermenting processes has been made by Hoppe-Seyler,' who has pointed out that the oxidation in which the action often apparently consists is not a direct one, but rather of the nature of reduction. 1 The most recent exposition of Hoppe-Seyler's views in this direction will be found in the Zeit. physiol. Chem. x. 36. 158 THE CHEMICAL CONSTITUENTS OF THE OKGANISM Thus in the lactic and alcoholic fermentation, and in putrefaction, there is a liberation of hydrogen, and this nascent hydrogen combines with an atom of oxygen from ordinary oxygen (O.^) to form water (H2 + 02 = H20 + 0). The nascent oxygen (O) thus liV)erated, oxidises any oxidisable substance present, or it may unite witli hydrogen to form water, or oxygen to form ozone (O3). But if on the other hand the nascent hydrogen meets with no free oxygen, it takes the oxygen from organic substances, that is, reduces them. Thus in putrefying liquids, oxidation may be proceeding in the upper portions whei'e there is free access of atmospheric oxygen, and reduction in the lower layers where free oxygen is absent. It is probable that some of the changes occurring during the nutrition of living cells are similar to these fermentations. The nascent hydrogen liberates nascent oxygen, wliich then oxidises oxidis- able material. The following hypotlietical formula would represent what occurs ; supposing n is oxidisable material, then HH + 02 + w=H20 + 0». THE UNOKGANISED FEEMENTS These substances can be extracted from the cells in which they occur by water, dilute acids or alkalis, salt solution, or glycerine. They are precipitated from such extracts, or from the secretions in which they occur, by alcohol, or by saturation with ammonium sulphate, ' or by lead acetate. The precipitate so obtained is proteid in nature,^ or closely allied to proteid. On drying this precipitate a colourless, tasteless, amorphous powder is obtained. These ferments may be arranged, according to their action, into the following classes : — 1. Proteolytic : those wliich change proteids into jjeptones. This is probably a process of hydration, as it can be also brought about by other hydrating agencies, such as boiling with dilute mineral acids, or superheated steam. Examples : pepsin, trypsin, papain. 2. Amylolytic : those which change amyloses (starch, glycogen) into sugars. This also is a hydration. Examples : ptyalin, amylopsin, diastase. 3. Steatolytic : those wliich split fats into fatty acids and glycerine. Examples : ferments in pancreatic juice and bile. 1 Krawkoff, J. Buss. Chem. Soc. 1887, p. 387. 2 Elementary analyses have been made of various ferments by Sclunidt, Schlossberger, Hiifner, and others. Much the same results have been thereby obtained as in the case of proteids. FEKMKNTATION 159 4. Inversive : those which convert cane sugar into glucose. Examples : iuvertin of intestinal juice, and of yeast cells. 5. Emulsin ' or synaptase : a ferment which converts glucosides (amygdalin, salicin, Arc.) into glucose, and other com])ounds. Myrosin is a very similar ferment. 6. Coagulative. Examples : fibrin ferment, myosin ferment, rennet, ferment from Wuhania cooyuJans which acts like rennet (Lea). A rennet- like ferment is obtained from certain other plants,^ and certain bacteria.^ There are other fermentations, such as the conversion of glucose into mannite, or of glycerine and mannite into alcohol by the action of putrefying nitrogenous organic matter, which have been described, but which are of little importance to the physiologist. The preceding classification is found to be very useful from a physiological standpoint. In many instances the same chemical change, which in all cases appears to be of the nature of hydrolysis, may be eS'ected by the action of ordinary chemical reagents, such as dilute mineral acids, or caustic alkalis. Hoppe-Seyler ^ has accordingly classified ferments from a chemical standpoint as follows : — a. Ferments wlucli act lihe dilute mineral acids at 100° C. : — i. Change of starch or glycogen into dextrin and grape sugar.* C,,H,„0,„ + 3H,0 = C«H,„0, + 3C,H,,0, [starch] [ilextrin] [glucose] ii. Change of cane sugar into dextrose and levulose (inversion). C,„H2,0„ + H„0 = C.H.^O, + C.H.^Og [cane sugar] [dextrose] [leTulose] iii. Change of various benzol-glucosides into sugar, and simpler benzol-dei-iva- tives by the action of emulsin {see p. 109). Example : C.^H^O, + H,0 = C,H,„0, + C^H, | q^"^^ [salicin] [sugar] [saligenin] iv. Decomposition of sulphur- containing glucosides into sugar, sulphuric acid^ and oil of mustard, by the action of myrosin.*^ Example : C,„H„NS,,0,oK = CgH.jO, + HKSO^ + C^H^NS [potassiixm [sugar] [hydrogen, [oil of myrouate] potassium nuistanl] sulphate] 1 Emulsin was prepared in a very pure condition by Aug. Schmidt {Inaug. Diss. Tubingen, 1871). He found that it had the following percentage composition: C, 48"76;, H, 7-13; N, li-16; S, 1-25; O, 28-70. 2 E.g. artichokes, black pepper, &c. See Watts' Dictionary, vol. ii. (1889), p. 545. ^ Warington, Jotirn. of Chem. Soc. 1888, p. 737. 4 Physiol. Chem. (1881), p. 116. 5 The above foiTUula is Hoppe-Seyler's after Musculus. Brown and Morris give a different equation (see p. 104). 6 In the above.equation the process is apparently not one of hydrolysis, but it seems. 160 THE CIIE:^rirAL CONSTITn:NTS OF THE ORGANISM b. Ferments irhich act like caustic alhalis at a hiyher temjierature. Fermenta- iire saponification : — i. Decomposition of ethers (fats) into an alcohol (glycerine) and an acid (fatty acid). Example : C3-H,„,06 + SHjO = 3(C,sH3,02) + ^^S>i [tri-olein] [oleic acid] [glycerine] ii. Decomposition of amido-compounds with absorption of water. Examples : ( 1 ) CONjH, + H,0 = (NH JX'Oj [urea] [ammonium carbonate] (2) CgHgNOj + H,0 = an^XO., + C-HgO, [liippuric [glycocine] [benzoic acid] acid] (3) C2sH,5NS0, + H,0 = C,H,XS03 + C.^H^G^ [tanrocholic [taurine] [cbolalic acid] acid] (4) The decomposition of proteids and albuminoids (gelatin, chondrin, &c.) into lencine, tyrosine, &c., brought about by the pancreatic ferment— trypsin. It will be seen, on glancing through these enumerations of the unorganised ferments, that the greater number of those occurring in animals are found in the alimentary- canal, and are concerned in the digestion of food. They all act best at a little over the temperature of the body (40° C), their activity is hindered by a lower temperature, and thev are destroyed by a high temperature. In a dry state pepsin and trypsin may be heated to 170° without harm,' but in a moist state a temperature below 100° C- is sufficient to destroy them. All fermentative processes require the presence of a certain amount of water. Free acid is harmful, except in a few cases, e.g. gastric digestion. The caustic alkalis also when present in more than very minute proportions hinder fermentation : as also do salts of the heavy metals, and ether, and chloroform in some cases. Quite small percentages of neutral salts (such as Q-OOl per cent, of the sulphate of sodium, potassium, ammonium, or magnesium, 0-02 of various urates, O'Ol of sodium chloride or phosphate) have a very considerable inhibitory effect when pure solutions of pepsin or trj'psin are used (Nasse,' Heidenhain,^ A. Schmidt,-^ E. Stadelmann ^). Bert and Kegnard state that organised ferments are killed by probable on further investigation that the formula of myronic acid may be modified, and that the ferment change will be found to be also one of hydrolysis ("Will and Kijmer, Liehig^s Annalen, cxxv. 263). By hydrolysis, one means the fixation of the elements of water, followed by decomx)osition into simpler products. The term should be distinguished from hydration, in which there is no such subsequent decomposition. - Huppe, Chem. Centralblatt, 1881, p. 745. ' The critical temperature at wliich the soluble ferments are destroyed varies with the different enzj-mes; the range of temperature is approximately 50°-75°C. 5 Pfiiiger's Arch. xi. * Ibid. x. * Ibid. xiii. « Z^t. Biol. xxv. 208. FKH.MEXTATION 161 peroxide of hydrogen, and unorganised ferments not. Schiitzenberger and Dumas state that borax destroys the activity of the unorganised, but not that of the organised, ferments. THE ORGANISED FERMENTS Gay-Lussac showed that boiled grape-juice introduced into the Torricellian vacuum of a barometer remained free from fermentative change for an indefinite time, but that on the admission of a bubble of air, fermentation soon commenced. Schwann (1838) showed that, if the bubble were admitted to the vacuum through a red-hot tube, fermen- tation did not occur. This experiment demonstrated clearly that, what- ever it was in the air that caused fermentation, it was destroyed by heat. In the same year Schwann polluted out the vegetable natui-e of the yeast cells which had been seen by Leeuwenhoek so long ago as 1680 ; Schwann showed too that they grew in saccharine solutions, and for the first time it was asserted that fermentation depended on the action of living things. In the seventeenth century Stahl had remarked that fermentation and putrefaction were essentially the same ; this, how- ever, was not verified until Schwann demonstrated (1) that if air were excluded from boiled putrescible fluids, they did not putrefy: (2) that if air were subsequently admitted, putrefaction soon set in ; (3) that if the air had been previously passed through red-hot tubes, no putrefaction oc- curred; (4) that the putrefying fluids always contained bacterial growths; (5) that certain substances which we now call antiseptics, such as corrosive sublimate, which destroy organic life, put a stop to putrefaction, pre- sumably by destroying the bacteria. There were many other observations made, all tending to the same end, namely, that it was not air, but something in the air, that caused both fermentation and putrefaction ; among these may be mentioned that of Hofmann, who showed that cotton wool will filter off from the air the material in question; and that of Mitscherlich, who showed that yeast loses its power after thorough filtration and removal of the yeast •cells. In 1857 Pasteur showed that each particular kind of fermentation was connected with the growth and development of a special organism; one organism producing the alcoholic, another the lactic, another the acetous fermentation, and so on. By introducing the method of culti- vating organisms in certain special fluids particularly adapted to the wants of one kind, and only one kind, of organism, he was able to separate different organisms from one another, and thus to investigate their individual properties, by inoculating them into previously sterilised putrescible or fermentable fluids, or into the body of certain animals. 162 THE CHEMICAL CONSTITUENTS OF THE ORGANISM This method has in the hands of other investigators (Koch, Klein, &c.) been much elaborated, and bacteriology is now a science in itself. The method of pure cultivations has attained to a great pitch of per- fection, and by it organisms which are apparently the same to the microscope can be divided into different kinds, according to their manner of growth and their physiological effects. The value of the method has been of most value in connection with the microbes of disease. The general principles of the method may be briefly summarised here, but for full particulars the reader is referred to any of the excellent works on the subject now published. The medium in which bacteria are to be grown may be either solid or liquid. Good liquid media are sterilised milk, serum or hydrocele fluid, or a bouillon made by boiling 500 grammes of beef in a litre of water for 45 minutes ; this is- rendered alkaline by carbonate of soda, filtered, and sterilised by exposure to the temperature of 100° C. for about an hour on three successive days. ^ Some culture liquids are simpler ; thus Pasteur's fluid is 1 part ammonium tartrate, 10 parts cane sugar, and the ash of 1 part of yeast in 100 parts of water. Colin s fluid is 0-5 grm. potassium phosphate, 0'5 grm. magnesium sulphate, 0-05 calcium phosphate, and 1 grm. ammonium tartrate in 100 grms. of water. Among solid media, which are more suitable for pure cultivations, the follow- ing may be mentioned : — a. The cut surface of a boiled potato or boiled white of egg sterilised by being washed with a solution of corrosive sublimate. i. Meat juice, to a litre of which is added 10 grms. of commercial peptone, 5 grms, of sodium chloride, and 100 grms. of pure gelatin. The mixture is heated, made slightly alkaline with sodium carbonate, filtered hot into test-tubes sterilised by discontinuous heating, and the tubes plugged with sterilised cotton wool. c. Instead of gelatin in the above, agar-agar (prepared from seaweed) may be used. This gelatinises at a higher temperature than gelatine, and so is suitable^ for the cultivation of organisms which grow only at temperatures approaching- that of the body. d. Blood serum made firm by heating to 68° C. for an hour. This is sterilised' by raising it to 56° C. for two hours daily for eight days. All instruments used in e.xperimentation must be sterilised — metallic instru- ments bj' the Bunsen flame, glass instruments by placing them in an oven at 160° C. To inoculate a new tube or flask with a definite organism that has been grow- ing previousl}' in a culture tube : push the point of a freshly drawn out capillary pipette through the cotton wool plug until it reaches the culture fluid or solid ; a small drop ascends the tube of the pipette. Withdraw the pipette and similarly 1 This 'discontinuous sterilisation,' as it is called, was introduced by Tyndall {Floating Matter in the Air, 1881), who found it more efficacious than i)rolonged heating. The reason is that tlie spores resist heat much more j)owerfully than the fully grown bacteria ; the spores not killed by the first heating germinate before the second boiling when they are killed, while some which may not have germinated will have done so by the time of the third boiling. FERMENTATION 163 push it into the material at the bottom of the second tube — that which is to be inoculated. The pipette is withdrawn, and the tube placed in an incubator at the temperature at which the micro-organism grows best. The separation of organisms may be effected by one of three methods : — 1 . Kleb's method of fractional cultivation. 2. Lister's and Niigeli's dilution method. 3. Koch's method of plate cultivation. 1. Fractional cultivation. — If a trace of culture fluid containing several organisms be inoculated into a series of new tubes containing different nourishing materials, it will be found after 24 to 48 hours that probably one species in each tube — i.e. the one that grows best in that particular medium and at that particular temperature — will have increased enormously, and that the others have made little or no progress. Inoculate a new culture tube with a trace of this cultiva- tion ; the chances are that you inoculate one kind of organism only, viz. the most abundant ; but, to be certain, the process should, after 24 hours, be repeated, and if necessary repeated again. The naked eye appearances of the cultivation, coloration, or liquefaction of the medium, the formation of a pellicle, micro- scopic appearance, and many other conditions, soon indicate when a desired single species is obtained. 2. The method of dilution. — The original culture fluid containing several species is greatly diluted with sterile salt solution, or some other indif- ferent fluid : with droplets of this new tubes are inoculated. It is probable that, owing to the great dilution, the droplet used contains only one organism. This chance is increased by repeating the process several times in succession. This method may be verj^ successfully combined with that of fractional cultiure. 3. Plate cultivation.— A test-tube of sterile nutritive gelatine is liquefied by gentle heat, and inoculated with a trace of the bacterial mixture, either by a capillary pipette or by the point of a previously over-heated and cooled platinum wire ; this is well mixed and expeditiously poured into a sterilised shallow glass dish, and covered with another glass dish ; both are then placed under a bell-jar, the interior of which is kept moist by a piece of wet blotting-paper, and the whole incubated at a suitable temperature. In a few days each species of bacterium will start a separate colony, difi'ering in shape, colour, size, and general appear- ance, from the others. By reinoculation of gelatine tubes from these, pirre sub- cultures of the different species can be obtained. Modifications of these various methods are used for the inoculation of nutrient material with blood, juices, and tissues of animals, and in the examination of water and air for micro-organisms.' Another large branch of the science of bacteriology is that relating to the microscopic preparation and methods of staining of micro-organisms, which has now reached a great degree of elaboration. Micro-Organisms may be classified in many different ways. They may be classified morphologically ; they all belong to one of the lowest groups of fungi called the Schizomycetes. They are devoid of chlorophyll, and multiply usually by fission, but in some (many bacilli) by a process of spore formation. The yeast cells, which are considerably ^ For full particulars see Klein's Micro-OrQanisms and Disease. M 2 164 THE CHE:\nC.\L CONSTITUENTS OF THE ORGANISM lari^er than most other globular forms, multiply usually Vjy budding. The forms assumed by bacterial growths are : — {a) Globular; termed micrococcus. (h) Rod-like; or bacillus. (c) Filamentous; either .single filaments, or composed of bacteria remaining attached after division (Leptothrix). {d) Spiral; termed vibrio, or if the sinuosity is very great, spirillum. (e) Plates or tablets formed by the irregular branching of cells, the branches remaining attached (sarcina). As a rule a microbe retains the same form generation after generation; but occasionally, as in cladothrix, a micrococcus form may become rod- like or filamentous at another stage (pleomorphism). Dense swarms sometimes occur in which the bacteria become fixed in a matrix of their own swollen, contiguous cell walls, and pass into a resting state as a so-called zooffloea. Another classification of these growths may be made according to whether the organisms are aerobic, or anaerobic. In 1864 Pasteur observed that the butyric acid ferment can Kve and multiply in a saHne fluid containing sugar and calcium lactate in the absence of free oxygen (anaerobic); on the other hand, other growths Uke the bacterium aceti require oxygen (aerobic). In a mixture of bacteria Engelmann showed that some species gather close to a bubble of air, others come near it when it has lost some of its oxygen, and others keep away from it altogether. Most fermentative organisms are capable however of assuming two conditions : one aerobic, the other anaerobic ; it is in most cases in the latter condition that an organism carries on the work of fer- mentation, as it has to remove oxygen from the fermentable material. A third classification maybe made on the basis of theefiects caused by the gro^%"ths of the micro-organisms : — (a) Those associated with known chemical processes ; such as the yeast plant, the bacterium lactis, the bacterium aceti, the micrococcus ureae, &c. (b) Those associated with the putrefaction of organic matter ; these are various forms of micrococcus, bacterium termo, bacterium subtile (the Hay bacillus), kc. (c) Those chiefly remarkable for producing colour ; such as bac- terium rvhescens, the peach-coloured bacterium, B. syncyanuinoi blue milk, B. cp.ruginosum of green pus, the micrococcus prodigiosus of red bread, and many others. {d) Those which produce disease when grown within the living FERMENTATION 165 botly ; among these may be mentioned the bacillus anthracis of splenic fever, the spirillum of relapsing fever, bacilli in various forms of septicivmia, in fowls' cholera, the comma bacillus of Asiatic cholera, and many suspected growths in various zymotic diseases, as smallpox, diphtheria, scai'let fever, typhoid, leprosy, rabies, etc. [See also Chapter XVI, Blood in Disease.) As is seen from the preceding list, a chemical classification is at present impossible ; in a few cases, as in the alcoholic fermentation, the lactic fermentation, or the acetic fermentation, &c., the decom- position can be represented by means of a chemical equation ; in other cases the substances produced may be identified, but the chemical decompositions by which they are brought into existence are unknown; this is particularly the case when we have to deal with proteids. In other cases still, particularly in disease germs, even the products of fermentation are still unknown. The recent discovery of the im- portance of animal alkaloids (ptomaines and leucomaines) has led in certain cases to the discovery that it is these substances which are the chemical poisons so long sought after. In other cases the poisons are proteid in nature. The following classification of the action of organised ferments according to their chemical action, is a completion of Hoppe-Seyler's arrangement, part of which has already been given in connection with the unorganised ferments (p. 159). I. Ferments whioli change mihydrides Into iDjdrates or cause hydrolt/sis : — a. Ferments acting like dilute mineral acids at 100°. These appear to be all unorganised. h. Ferments acting like caustic alkalis at a higher temperature. i. Decomposition of fats into glycerine and fatty acids. This appears to occur not only with the unorganised pancreatic ferment, but also during putrefaction, presumably by the action of bacteria, ii. Decomposition of amido-compounds with absorption of water. The four examples already given (p. 160; are brought about also by putrefactive organisms ; the decomposition of urea by the m. urete, through the intermedia- tion of a soluble ferment ; the decomposition of hippuric acid, taurocholic acid (and glycocholic acid more slowly) by putrefactive bacteria ; and the decomposi- tion of proteids and albuminoids into leucine, tyrosine, &c., is brought about, not only by the unorganised ferment trypsin, but also by putrefactive bacteria, such as occur, for instance, in the intestinal canal. II. Fermentations in ivMch there is transference of oxygen from the hydrogen to the carbon atoms. a. The lactic acid fermentation. The decomposition of milk-sugar, inosite, and other carbohydrates, into lactic acid. The ferment is associated with the presence of a micro-organism — the bacterium lactis. But other fungi — e.g. the spores of jjenicillium — will bring about the same result (^see also Milk). 166 THE CHEMICAL CONSTITUENTS OF THE ORGANISM h. The alcoholic fermentation.' The transference of the oxygen from the hydrogen to the carbon atoms is thus depicted by Hoppe-Seyler : — CH.,.OH I CH.OH C0(0H)2 Hydrogen carbonate. CH.OH CH^.OH-l I +2II.,0= I I Alcohol. CH.OH " CH3 J Alcohol. CH.OH CH„ -1 COH CH^.OHj [Grape Sugar] C0(0H)2 Hydrogen carbonate. c. Many cases of putrefaction come under this head- : — i. Of simple inorganic compounds : Examples: (1) (CH02),,Ca + H30 = CaC03 + COj + 2H2 [calcium [c.ilciuiii formiite] carbonate] (2) (C,,H30,)._,Ca + H.O = CaCOj + CO., + 2CH, [calc. acetate] [marsh g;is] ii. Of organic compounds : — (1) (CgHsOa^Ca., + H.,0 = CaCO, -f 3C0., + 4H„ + (C,H,0,).,Ca [calc. lactate] [calc. butyrate] (2) wCsH,„0, + 7iE..,0 = SrtCO,, + ;'>wCH, [cellulose] [carlionic [mar.sli acid] gas] (3) Calcium malate yields carbonate and lactate of lime. (4) Glj'cerine with calcium carbonate and putrefying fibrin yields, on exclu- sion of oxygen, no alcohol, but butyric, butyro-acetic, and succinic acids ; pro- bably lactic acid is first formed, which is then decomposed into carbonic and butjTic acids ; the butyro-acetic acid arises from reduction ; the formation of succinic acid is difficult to understand. (5) Proteids are easily decomposed by putrefactive organisms. Insoluble proteids, like fibrin, are first dissolved, forming a solution of globulin. Solutions of proteid appear to undergo first a change lilce that produced by digestion with the formation of albumoses and peptone. Then amido-acids (leucine, tyrosine, &c.), fatty acids, ammonia, carbonic acid, amines, and in certain cases indole and skatole, are formed. Another classification of ferments has been made according as they produce acid bodies (acetic acid from alcohol, lactic acid from sugar, &c.), or basic products (urea from ammonia, ptomaines from proteids). Such classifications are neces- sarily incomplete, but Hoppe-Seyler's is especially useful as showing what the basis of a scientific chemical arrangement should be. In addition to these forms of fermentation the list may be completed by the mention of : — 1. The acetous fermentation; the conversion of alcohol into acetic acid ' Other alcohols (propyl, butyl, etc.) may be produced under suitable conditions. * These cases have been examined by Hoppe-Seyler by mixing the substance in question with small quantities of sewer mud ; the gases that come off are collected and analysed. FERMENTATION 107 •(C.,HgO + 0.. = C.,H^O.^ + H._.0), brought about by the mycoderma aceti aiiody after death ; also in eggs, milk, and cheese, and other forms of food. It is, however, fully proved that ordinary proteids will also, under the influence of certain bacterial growths, produce neurine, putrescine, cadaverine, as well as the simpler bases, such as methylamine, ethylamine, and ammonia. The question, how the bacteria do it ? is a more difficult matter to answer. The decomposition of lecithin produced by these organised ferments is no doubt similar to that brought about by ordinary chemical reagents ; but we cannot describe the decomposition of proteids until we know their composition. The different views now held as Uj the constitution ' Zeit.phijsiol. Chem. x. 146. ^ Inaug. Diss. Dorpat, 1869. ^ Virchow's Arcliiv, 1863, vol. xxvii. p. 240 ; vols, xxviii. xxix. and others. * The probability that cholera is caused by an alkaloid was first pointed out by Lauder Bnmton {Brit. Ass. Beports, 1873); he deduced it from the similarity of the symptoms of cholera and alkaloid (muscarine) poisoiiing. Cadaverine and putrescine are not markedly toxic. PTOMAINES AND LEl'COMAINES 173 of a proteid will each involve a sepai-ate theory as to how an alkaloid may be formed from it. It may, however, be regarded as settled that animal alkaloids, whether of the ptomaine or leucomaine series, are produced anat-robically (see p. 164). The priority of describing an alkaloidal substance in animals is claimed by Dupr^ and Bence Jones. These observers in the year 1866 described an alkaloidal substance, which they separated from the solid and liquid tissues of animals, and they named it ' Animal Quinoidine.' ' The honour is also claimed for a chemist who worked at Stettin, named Marquardt,2 who described an alkaloid obtained from a corpse, to which he gave the name ' septicin,' and which he found was similar in its action to coniine. The work of Panum, Schmiedeberg, Bergmann, and Schmidt on sepsin has been already alluded to. These Avere all more or less gropings in the dark until the master hand of Selmi ^ placed the matter on a satisfactory basis ; it was he, too, who invented the word ' ptomaine.' The details of methods of separation and of analysis have, as usual, been left to a multitude of German workers, but Brieger stands head and shoulders above all the rest. In France the subject has been taken up by Gautier, Avho has produced numerous memoirs on the subject.^ Brieger was the first to obtain from the uncrystallisable extracts and syrupy products of previous investigators pure materials in a crystalline form. He found it necessary to adopt considerable modifi- cations of the Stas-Otto process, which is the best for the extraction of the vegetable alkaloids. The pure crystalline alkaloids were not only analysed by him, but in many cases their constitution was worked out also. He found that the bases isolated from putrefactive mixtures w^ere less poisonous than those produced by pathogenic bacteria. These latter poisons include such substances as typhotoxine (from cases of typhoid fever), tetanine-^ (from cases of tetanus), and several others ; on account of their powerful poisonous properties Brieger has separated them from the other ptomaines, and calls them toxines. It has been recognised that it is very difiicult to draw the limits of the word ptomaine ; the products of metabolism of bacteria are not, in general, difierent from those of the higher organisms ; thus choline, neurine, creatinine, &c., are normal products occurring in, or separable from, healthy animal tissues. Gautier has invented the word leucomaine 1 Froc. Bo7j. Soc. xv. 73. Zeit.f. Chem. 1866, p. 348. * Schuchardt in Maschka's Sandb. d. ger. Med. ii. 60. ^ Deutsch. chem. Gesell. xi. 808. * For the latest see Bull. Soc. Chim. xi. 6. 5 For the last paper on tetanine from a case of tetanus, see Brieger, Berliti. klin. Wochenschrift, 1888, No. 17. 174 THE CHEMICAL CONSTITUENTS OF THE ORGANISM for the basic products produced in the tissues of living animals by metabolic processes, while he reserves the word ptomaine for those formed by putrefaction after death. It must, however, be remembered that many leucomaines are powerful poisons. If ptomaines and leucomaines are to include all bases produced in animals, the simpler substances, like methylamine, trimethylamine, (fee, must all come under either one or the other heading. This is especially necessary, since it has been sho^yn that probably the diamines, like putrescine' and cadaverine,^ are derived by oxidation from the monamines.-'* Such, then, is a sketch of the ptomaines, in which their importance has been indicated by a few examples. The subject is yet in its infancy, and many more facts must be collected before positive general conclusions can be drawn. Without at all wishing to minimise their importance, it is, however, necessary to insist on one point, and that is that all poisons produced by bacteria are not necessarily ptomaines, that all mysterious symptoms in obscure complaints cannot be as yet attributed to leucomaines. There is always a tendency after any great discovery is made to attribute to it wider importance than it really possesses. We have many instances of this in pathology ; the doctrine of the solidists, which totally excluded humoralism, was an outgrowth of Schwann's great generalisation we call the cell theory. Similarly there can be little doubt that under the influence of the germ theory many premature conclusions were jumped at, concerning the association of organisms with disease. Ptomaines are now displacing somewhat the microbe, which was formerly regarded as all-important, but this must not be pushed too far. The discovery of ptomaines is complemental, not antagonistic, 1 Putrescine (C4Hi2N.2) is chemically tetramethylenediamine (Ber. deutsch. cliem. Gesell. xxi. 2938). 2 Cadaverine (C5H14N2) is pentamethylenediamine (Ladenburg, Ibid. xix. 2585). Two other alkaloids named neuridine and saprine have been separated by Brieger, which are isomeric with cadaverine. 5 A full explanation will be found in an interesting paper by Baumann and V. Udranszky [Zeit. jjhysiol. Chem. xiii. 562). These observers show that the two diamines which are found in cholera, and in pure cultivations of the cholera bacillus, are also found in the urine and fseces of patients suffering from cystinuria, a condition apparently very different from cholera. Normal urine is toxic, but this is probably due to the inorganic potassium salts in it (Stadthagen, Zeit. klin. Med. xv. parts 5 and 6), not to any alkaloid. Ptomaines have never (until these cases of cystinuria were described) been satisfactorily demonstrated to exist either in normal or pathological urine, though theoretically their presence there is possible, for the ptomaines formed by putrefaction in the intestine might conceivably be partly reabsorbed and then excreted in the urine. Ponchet {Gompt. rend, xcviii. 1560) has stated that normal urines contain poisonoas alkaloids; his methods and results are, however, full of fallacies. Hunter has found diamines in the urine in cases of pernicious aneemia. PTOMAINES AND LEUCOMAINES 175 to the germ theory. We must remember that there are many powerful poisons which are not alkaloids at all. Snake poison is a striking example of this ; it is a poisonous proteid indistinguishable from other proteids by its reactions. The products of digestion produced normally in the alimentary canal (albumoses and peptones) ai-e also powerful poisons. Recently it has been surmised that the bacillus anthracis produces a poisonous albumose, which also has the power of conferring subsequent immunity from the attacks of the bacillus.' Not doubt the poisonous proteids, as well as the ptomaines, will have to be very largely reckoned with in the investigations of the poisons of diseases. METHODS OF SEPARATION OF PTOMAINES The first method of any importance for the separation of alkaloids from- organic mixtures was proposed by Stas,''' subsequently modified by Otto,^ and now known as the Stas-Otto jirocess. Other methods have been introduced by Dragendorii,^ Sonnenschein,^ Sehni," and Brieger." The last-named observer's jirocess is specially adapted for the separation of ptomaines. The Stas-Otto process.^ — The substance to be operated on, if solid, is finely divided, and repeatedly digested for many hours with fresh quantities of rectified spirit at a temperatui-e of 55° C. Liquids are also treated with twice their volume of spirit. The residue is finally digested at 35° C. with spirit faintly acidified with acetic acid ; it is then once or twice more digested with unacidified spirit. The alcoholic liquids obtained before acidification are mixed together,, and rapidly and momentarily raised to 70° C, cooled, and filtered ; those obtained with and after the use of acetic acid are mixed together and similarly treated. But the two liquids, the unacidified and the acidified, are not mixed with one- another till later. Each infusion is then concentrated to a syrupy consistency at a temperature of 35° C. To the syrup about 30 c.c. of absolute alcohol are added^ with constant stirring and grinding in a mortar. The alcohol is poured off from the pasty mass, and replaced by successive portions of 15 c.c. of alcohol, so long as a colour is imparted to it. The alcoholic extracts are mixed, filtered, and the- filtrate concentrated as before at .S5° C. We have again a syrupy residue, both from the unacidified and the acidified original extracts ; each is diluted with water, filtered, and the filtrates mixed. They should now measure 15-20 c.c. This is jmrtially neutralised with soda, but, still faintly acid, is placed into a well- .stoppered tube. This liquid contains all the alkaloids present in the original material, and is free from proteids. This aqueous liquid (A) is now covered with twice its volume of ether, and the whole is mixed by gently and repeatedly inverting the tube. The ether is allowed to separate, and is pipetted off. A fresh quantity of ether is then used, and the extraction with ether repeated until a few di'ops on evaporation leave no residue ; four or five extractions generally suffice. Each ethereal solution is washed by shaking it with 5 c.c. of water to ^ See p. 168. ^ Stas, Liehig's Annalen, Ixxxiv. 379. 3 Otto, Ibid. c. 39. * Dragendorff, Gerichtl. clicm. Emnit. v. Gift, 1876. ^ Sonuenschein, Liehig's Ann. cv. 45. ^ Selmi, Journ. of Chem. Society, 1877, p. 93. '' Brieger, Die Ptomaine, part i. 1885; part ii. 1885; part iii. 1886. 8 As modified by Dr. Stevenson, Watts' Diet. vol. i. 1888. Art. 'Alkaloids poisonous." 176 THE CHE:\nCAL CONSTITUENTS OF THE ORGANISM ■which a drop of sulphuric acid has been added : they are then mixed, and on evaporation may leave an oily residue, which should be reserved for further ex- amination. The bulk of the alkaloids, however, remain insoluble in ether. The acid liquid (A) (after washing with ether), and the acidulated water used in washing the ether extracts, are mixed, made alkahne with sodium carbonate, and exhausted, once with a mixture of chloroform and ether (1 : 3), and subsequently three or four times with ether alone. These extracts are successively washed with water, then acidulated water, and lastly water again. The alkaloids are thus first liberated by the alkali, then dissolved in the ether-chloroform, then again converted into sulphates, which, being insoluble in ether and chloroform, pass into the acid solutions, impurities being left behind in the ether. The acid liquid and the final wash water are mixed, washed with ether, once more made alkaline, and again extracted with chloroform-ether and ether. These extracts are washed with water, made barely alkaline with sodium carbonate, filtered, and the filtrates evaporated to dryness below 35° C. This may be then dried at 100°, cooled and weighed, the weight being that of the total alkaloids. If a volatile alkaloid is present, the residue wiU be oily ; whether these are present should be discovered by first evaporating a few c.c. only. If they do occur, the extracts must be acidified with hydrochloric acid, and then on evaporation the non-volatile hTdrochlorides are left. The free alkaloids obtained in the first instance are con- verted into hydrochlorides, dissolved in water, and then separated and tested for according to their various properties. They are best separated by the use of different solvents (petroleum ether, benzene, chloroform, alcohol, &c.), in which some are and some not soluble. Brieger's irvethod. — The mass of putrefying material is boiled with water, filtered, and the filtrate precipitated with subacetate of lead. This precipitate is filtered off ; a stream of hydrogen sulphide is passed through the filtrate, and the lead sulphide separated by filtration. The filtrate is evaporated to a thin syrup, and this is extracted with amyl alcohol. The extract is repeatedly treated with water, and then concentrated, then made strongly acid with sulphuric acid, and repeatedly shaken with ether, which removes oxy-acids. Freed from ether it is evaporated to a quarter of its bulk, and thus volatile fatty acids are driven off. The sulphuric acid is precipitated by baryta, and the precipitate removed by filtration : the excess of baryta is precipitated by a stream of carbonic acid, and this is also removed by filtration. The fluid is warmed for some time on the water-bath, cooled, and precipitated with mercuric chloride. The precipitate is well washed and decomposed by sulphuretted hydrogen ; the mercuric sulphide is filtered off, and the filtrate concentrated. Inorganic substances crystallise out first, which are filtered off and washed with absolute alcohol. Subsequently long needles form, of organic nature, which are soluble in water and dilute alcohol, but insoluble in absolute alcohol, ether, benzene, and chloroform. These substances consist of the ptomaines, and they are then separated by fractional precipitation with the chlorides of platinum or gold. Baumaim and v. Udranszky have separated cadaverine and putrescine by the different solubilities in ether and alcohol of their benzoyl-compounds.' In some of his researches Brieger has shortened the procedure by precipitating the putrid fluids after boiling and filtering directly with mercuric chloride, i.e. the fijst precipitation, that with lead acetate, is omitted. As mercuric chloride does not precipitate all alkaloids, both precipitate and filtrate must be examined. • Baumann and v. Udranszkj-, Zeit. plitjsiol. Chem. xiii. 562. I'Tn.MAINF.S AM) LEIC* >MA1NKS 177 GENERAL PKOPEKTIES OF THE ANIMAL ALKALOIDS The ai\iaial like the vegetable alkalouls maj' be divided into two groups : those which do, and those whicli do not contain oxj'gen. Those which do not contain oxygen are the ptomaines proper, and, like the non-oxygenated vegetable alkaloids, are liquid, volatile, and odorous. The oxj'genated alkaloids are crystal- line and fixed. They all have an alkaline reaction. Tf>ey are oxidisable and unstable, especially under the influence of an exces.s of mineral acid, wliicii colours thera red, and then converts them into a resinous mass. Tlieir chloroplatinates and chloroaurates vary much in solubility. Picric acid precipitates most of them, the colour of the precipitate usually being pale yellow. Tannin, mercuric chloride, \;c., also produce insoluble precipitates as a rule. Pliosphomolybdic acid precipitates all the alkaloids. The ptomaines are energetic reducing agents, decomposing chromic acid» iodic acid, and silver nitrate. With f erricyanide of potassium and ferric chloride they give Prussian blue. This was at one time considered to be characteristic of the animal alkaloids, but it has been found that manj- vegetable alkaloids give the same test, and a few of the animal alkaloids (especially those containing oxygen — Brieger) do not give it. There is, so far as is at present known, no class reaction by whicli the alkaloids of animal can be separated from those of vegetable origin. ENUMERATION OF THE ANIMAL ALKALOIDS The animal alkaloids which up to the present have been obtained in a pure condition may be arranged as follows : — 1. Non-oxygenous ptomaines — Hydro-collidine Saprinc Collidine Cadaverine Parvoline Putrescine Xeuridine Mydaleine 2. Oxygenous ptomaines — Neurine Mytilotosine Choline Tetanine Muscarine Typhotoxine Gadinine S. Leucomaines of tlie uric acid group — Carnine Sarcine or Hypoxanthine Adenine Xanthine Guanine Pseudo-xanthine 4. Leucomaines of the creatinine group — Creatinine Cruso-creatinine Xantho-creatinine Amphi-creatinine 178 THK C'HP:MICAL CONSTrn'ENTS OF THE OROANISM 1. The non-oxygenous Ptomaines nreluiulA and volatile. The properties of the chief members of the liTonp are :- -- a. Purroline, CHj^N. This was lirst separated from the putrid flesh of the mackerel and horse. It is an oily base, of a yellow colour, boiling a little below :200° C. Its chloroaurate and chloroplatinate have been prepared. These are crystalline, and the latter is the more insoluble of the two (Gautier and Etard). b. Hydro-colUdiiie, C'^H,3N (boiling-point 210° C), and Collidine, CgH|,N ; the first-named of these two other bases derived from the same source as the preceding, the latter from the putrefied products of ox pancreas and gela- tine (Nencki, Gautier). Nencki considers collidine to be iso-phenyl-ethylamine, /CH, C.H- — CH<^ . These three bases are all highlv toxic. \NH, c. Neuriflinc, CjIIj^N.,, is a constant product of the putrefaction of proteids. Its hydrochloride, platino-chloride, and auro-chloride have been crystallised and analysed, but the free base is so unstable that it has never been obtained pure. A solution of sodium hydrate breaks up the hydrochloride of neuridine into dimethylamine and trimethylamine (Brieger). d. Saprine, C-,H,4N.^, though isomeric with the preceding, differs from it in the solubilities of its salts, and probably also in chemical constitution. e. Cadarerine, C5HHN2, a third isomeride, generally appears late in ordinary putrefactive processes, but readily in cultivations of the cholera bacillus and Finkler-Prior vibrio. Its choi.ical constitution has been worked out by Ladenberg,' who has found that it belono-s to the group of diamines, and that it is penta-methylene-diamine. It has a spermatic odour, and boils at 115°-128° C. f. Putresoine, C^U^.,^.,, is also a diamine, being tetra-methylene diamine. It is usually found acccnipanying cadaverine, but makes its appearance rather later. The chief work on this diamine has been done by Brieger,- Bffiklisch,=' and its constitution was discovered by Baumann and v. Udranszky.* These two diamines also are found in the fteces and urine in cases of cystinuria (Brieger and Stadthagen.^ Baumann, and v. Udranszky"). They are both poisons, but not virulent ones, and the symptoms they produce are very similar to some of those of cholera (h.-emorrhages and necrosis),' but the muscular cramps and other prominent symptoms of that disease are probably produced by other poisonous alkaloids (toxines, as Brieger would call them) not yet separated. Other poisonous alkaloids belonging to this group have not yet been fully examined ; thus a base named mydaleinc was described by Brieger, and this pro- bably is also a diamine ; it is markedly toxic.^ 2. The oxygenous Ptomaines may in many cases be obtained from healthy tissues • and tliev are also formed from those tissues on the occurrence of putre- 1 Ber. (h'utsch. cheiii. Gcs. xix. 258.5. The original formula assigned to cadaverine Ly Brieger was C5H16N2. - Berlin. Miu. Woch. 1887, No. 44. " Bdeklisch, Brr. d. GeseU. xx. 1441. * Ibid. xxi. 2938. ^ Arrh.piitli. Anat. exv. part :!. 6 Zeit.phiisiol. Chem. xiii. 5(52. " For symptoms see v. Behring, Dr^f.scA. iiied. Woclienscli. xiv. No. 24. Sclienerling, Mahfs Jahresb. 1887, 491. Fehleisen and Grawitz, Virrhov's Arch. ex. 1. « See Articles by Lauder Brunton on Food and Poison, Practitioner, vol. xxxv. Aug. Sept. and Oct. 1H85. I'l'd.MAlNKS AMI LKrco.MAlNKS 170 fnction. Tliey may tlius 1)0 in iii;niy cases Iciicnmainns a,< well. It is very tlniil)tfiil if thi'\- exist in a free state in healthy tissues ; pr()l)at)ly tlicy are lorined V)y tlie action of the reagents used on analysis, e.jf. the neurine is derived from Icvithin (ivr Chap. XXIV). a. Xenritic, C.,H,.,NO. is triniethyl-\inyl aninioniuni hydroxide. Thi> is a syrujiy base anvo is termed the life-history of an organism (ontogeny), and this develop- ment is, according to the Darwinian hypothesis, in its essential features similar to the historical development (phylogeny) of the higher or- ganisms from simpler and ultimately from unicellular forms, which has occupied untold ages in the past. Physiology may be described as the science which treats of the functions of protoplasm and its modifications. Some of these functions can Ije accounted for by chemical laws ; this constitutes the depart- ment of the science known as Chemical Physiology, or Physiological Chemistry ; other functions are physical manifestations ; the greater our advance of knowledge of protoplasm becomes, the more does it become evident that all vital phenoinena may be classed under one or other of these two heads ; the unexplained residue we must classify as vital, using that word simply for want of a better, and not as implying any belief in the existence of a special or vital force. If we take a single animal cell, either a unicellular oi-ganism like an Amceba, or a white blood corpuscle, which is an instance of a cell re- taining its primitive structure in the adult form of higher animals, we find that it has the following properties : 184 THE TISSUES ANIJ OROANS (iF TliE BODY 1. Pniiy r i>f hwvement. — The shape of the cell is continually chang- ing, processes being extended and withdrawn. Movement may l)e sometimes apparently spontaneous, but usually it is excited by the in- fluence of external agencies — heat, foreign particles, itc. These agencies are termed stimuli, and the power of responding to a stimulus by con- traction is known as irritability . '2. Fov-er of assimilation, that is, of al)sorbing dead matter, and converting it by chemical changes into a part of itself, i.e. into proto- plasm or living matter. 3. Poirer of grov:th : this follows from the last. •4. Poller of secretion ; or the elaboration from protoplasm of new substances ; this is seen in the formation of vacuoles, but to a greater extent in the higher animals, where certain cells, in organs called secreting glands, are set apart specially, for the formation of higlily elaborate materials, known as enzymes or ferments. Some secretions are merely discharged from the cells as waste products ; these are known as excretions. •"). Potcer of reproduction, or the giving ofl' of living things similar to themselves ; in the simplest condition this is Ijrought about by budding — the detachment of minute particles of protoplasm which grow into adult cells ; or hy fission, due to the splitting of the cell, including its nucleus, into two, each daughter cell growing into an adult, which in its turn undergoes a similar di^dsion. In the development of a higher animal from a single cell, or ovum, there is, after fertilisation, first a division of that cell into two, each of which again divides, so that four, and then l)y a similar process eight, sixteen, and so on, cells are formed. The cells so fonned do not become detached from one another, but remain adheient, so that a little mass of simple cells, each like the original, is formed. These become arranged in the fonu of a little sphere, at first solid, and then contain- ing liquid shed out from the cells ; a little latei- it will be found that the layers of cells are three deep ; the outermost layer is termed the epiblast or ectoderm, the innermost the liypohlast or endoderm, or entoderm, while the middle one is the mesobhist or mesoderm. From these three layers all the tissues and organs of the adult are formed : the epidermis and nervous system from the epiblast : the lining membrane of the alimentaiy and respiratory ca^ities, with the cells of the diges- tive glands, from the hypoblast, and the lest of the body from the mesoblast. In the further development of the adult from the three primaiy embryonic layers, there is not merely subdivision of the cells, but the cells in certain parts become modified or altered from their primitive TIIK ('KI>L 185 vonditiou ; some become liollow aiul adhoi-ent to one another to form l)lootl vessels ; others l)ecome elongated and thi-ead-like, to form muscular fibres ; in other pai-ts the cells l)ecomc modified chemically, as in the horny layers of the epidermis, or the mucin-yielding cells of salivary gland;-:. In other parts, as in the connective tissues, the cells may become separated by an intercellular substance, in which fibres may form, or in which, as in l)one, calcareous matter may be deposited. These are merely instances of the variations that may occur. In the chapters that follow this, the several tissues and organs so formed, will be taken xeriatlm. For the present we have more especially to deal with the structure of the primitive cell. Chemical investigation of such an object is fraught with difficulties, and must in many cases be performed on a microscopic slide ; in certain other cases, however, as with pus cells, liver cells, itc, it is possible to obtain large collections of cells, and then the methods of macro-chemisti'y can Ije apj^lied. The cell theory is associated in greatest measure with the name of Theodor Schwann ; and I translate here the following sentences from the life of that scientist written by Leon Fredericq of Liege.' ' Previous to Schwann's time, it was known that in animals there were example of oi-gans formed of cells. Midler had described them in the spinal cord ; Henle had studied them in the epidermis ; Henle and Purkinje in glands ; Ehrenberg and Valentin in nerve centres, etc. But these were isolated facts, and indeed certain savanttt looked upon them as exceptions. No one had yet thought of carrying into the domain of animal histology the general notions derived from the microscopic study of vegetable structures. Schwann has himself told the accident which gave him the first idea in his great discovery. ' " One day I was dining with M. Schleiden, the illustrious botanist, and he was telling me of the important part played by the nucleus in the development of vegetable cells. I suddenly remembered having noticed a similar appearance in the cells of the spinal cord, and the same moment I gi-asped the importance of being able to show that this nucleus plays a part, similar to that observed in plants."' ' The two scientists immediately repaired to the anatomical theatre to examine the nuclei in question, and Schleiden recognised their perfect resemblance to vegetable nuclei. ' " Since that moment," Schwann continues, " all my efforts tended to try to prove the pre-existence of the nucleus in the cell. ' " Once arrived at a satisfactory conclusion concerning the cells of the spinal cord, and of cartilage, the origin of the elementary parts of other tissues by the same mode of development, that is to say, by means 1 Li('ge, 1884. 186 tup: tissues and oRfrANS OF THE BODY of cells, was no longer a matter of doubt to me, and further ob.servati ('■) 0.ri/(/>ii.- Witli«li;i\val of oxygt'ii ultimately produces death, but in media free from oxygen, protojjlasmie movement will continue foi- some houi'S, the cells giving oil' carlionic auliydride. The oxygen i)re- viously taken into the cell is in a state of loose combination ; when how- ever this storage oxygen is exhausted the cell dies. ((/) I'oltionti. — A slight excess of acid, and a rather larger (|uantity of alkali, causes a cessation of protoplasmic movement, which can be counteracted for a time liy neutralisation. A \cry weak alkali stimulates the movement. Carbonic acid gas, ether, and chloroform vapour stop it. Veratrine (Kiihne) and quinine (Binz) ' act similarly. (e*) Ardjic'ml stivudatiou. — The following may be used as stimuli to movement : weak electrical currents, or sudden alterations of tem- perature, within the temperature range of contractility ; in the case of most cells, light does not act as a stimulus ; in other cases it does ; for instance the Pelomyxa moves actively in the dark, and becomes spherical when exposed to light ; plant cells containing chlorophyll are most sus- ceptible to the influence of light. Mechanical stimuli like pressing, bruising, tearing, itc, and chemical stimuli like anuuonia vapour, various strengths of saline solutions, kc, may also be employed ; as a rule, however, in chemical .stimulation, accessory phenomena like shrinking, swelling, or coagulation interrupt and u'.ask the efl'ect of the excitation. Theoretical conclusions. Protoplasm must be regarded as an aggregate of exceedingly minute, contractile, excitable form-elements, and the raoAement as a Avhole is the result of the changes in form of these veiy small elements. The nature and cause of the changes in form of the latter remains provisionally undetermined. With regard to their form we may take it for granted that when in a condition of maximal excitation they are almost spherical, and when not excited are generally elongated or thread-like. The mechanic;il behaviour of naked protoplasm teaches us that the changes in form must take place with a force which exceeds, as a rule, the force which the elements, if they were fluid, would put forth, in order to assume a spherical form. These contractile elements may be called ' Inotagmata.' Pidbablv they are positive uniaxial doubly refracting. - The active as well as the passive phenomena of protojilasmic move- ment compel us further to make the assumption that the inotagmata of protoplasm aie not like those of muscles and cilia arranged in a relatiyely Arm manner with their axes ii.i (me deflnite direction, but are fastened together loosely and are capable of moving one against ' Arch, iitikr. A)taf. iii. p. 383, 1H()7. - Contractilitat und Doppelbrechuiig, Fflii(/cr's Arcli. xi. 1875. 100 THE TISSrES AM) OROAXS OF THE T-oDY the other in all directions ; still tlie possibility of a temporary or per- manent grouping of a greater or less number of inotagmata into definitely shaped larger masses (fibres, networks, membranes, &,c.) is not excluded. As a reason for the possibility of alteration of arrangement of the protoplasmic particles, and in connection with the prevailing views concerning the molecular structure of organised masses, we must assume the existence of a capability for the imbibition of important quantities of water between the inotagmata, and the larger masses or inotagma groups. The motility, as already shown, increases or dim- inishes with the quantity of this water. PHYSICAL AND CHEMICAL PEOPEETIES OF PROTOPLASM Engelmann ^ describes contractile protoplasm as a liomogeneous, transparent, almost always colourless mass, with a higher refractive index than water, but lower than oil. In some cases where it has the form of fibres or thin layers with a prevailing movement in one direction, it is doubly refracting, and as in muscles and cilia with a single positive axis, the optical axis coincides with the direction of the movement. Different portions of the same protoplasmic mass may have different refractive powers, and during movements the refractive power of the same portion changes to a considerable extent. Protoplasm is semifluid, does not mix, but swells up with water ; it is cohesive and extensible. Though the superficial layers of many cells are firmer than the interior, a distinct niembrane is absent as a rule in animal cells. Protoplasm, almost without exception, contains granules which play a passive role in movement. The granules are albuminous, fatty, and in some cases inorganic (e.g. calcium carbonate in certain Myxoplas- modia) in nature. Often the exterior portions of the cell (exoplasm) are free fi'om granules, while they are present in lai'ge quantities in the internal regions (endoplasm). The irregular shaking, dancing motion of these particles, called the 'Brownian movement,'- must not be mistaken for Altai movements. In addition to granules, protojilasm in vegetal)le cells always, in animal cells often, exhiliits vacuoles or spaces filled with a watery liquid. These are globular in resting protoplasm, but may l)ecome drawn out ■during movement. The same holds good for gas bubbles,- which oal])histtii : is if Itoinogenpous?- AcoovA'nv^ to the observations of Heitzmann, Froman, Klein, Carnoy, and others, the cell-protoplasm (or cytoplasiii as it is often called, to distinguish it from the nucleo-plasm or substance of the nucleus) is composed of a line network, reticulum, or spongework of fibrils, with a more fluid material in its interstices. These observers consider that the graimles observed in protoplasm are chiefly the optical sections or crossing points of the fibrils ; at the same time true granules or microsoxies may occur in the interstitial fluid. Other histologists (8chafer, Rabl, etc.), while not denying that such a reticulum may sometimes occur, yet consider that it is, as in the Amueba, often absent. The protoplasm of cells is often highly -vacuolated ; it often contains rods (as in cartilage cells), or has a striated appearance (as in cells of the ducts of the salivary glands). In other cases again the appearance of a reticulum appears only after treatment with certain reagents, and may be a kind of coagulation produced by tliose reagents. Carnoy believes that the reticulum consists largely of a substance called jilnsfin, which we shall describe more fully in comiection with the nucleus, where it is also found. The following terms for these two different portions of protoplasm .jire used by different observers : — The reticulum is called : Protoplasma by Kupffer ' (including also granules). 1 Sitziinysh. d. iiiiitli.-phijs. Klafrnc d. h. Bdijr. AkaiJ. MiiiicJi/'ii, l.ss'i, vol. \i. Baijr. Fiscltrrei-Zeitnug, IKHO. 11)2 THE TISSFES AND OROANS OF THK I'.oDV Cytoliyaloplusina by Strasburgei'. Substantia opaca by Leydig. Mitom by Flennning. The iiiter-i-etieular, inore fluid substance is called : Paraplasma by Kupffer. Cytochylema by Strasburger. Substantia hyalina by Leydig. Paraniitom 1)}- Flemming. Strasburger further distinguishes in the cytochylema : Plasniochyma, the portions rich in proteids. Cytochyiua, the more watery sa]i in the vacuoles. Schwartz,' who has examined protoplasm and nuclei micro- chemically, describes the following constituents in cell protoplasm or cytoplasm. 1. Plastin, a sticky, thready mass which resists peptic and tryptie digestion ; and is insoluble in concentrated potassium hydrate and sodium chloride solutions (1 in 10). He further distinguishes between cytoplastin (the plaslin of cytoplasm), and chluropListin (a similar sub- stance found in chlorophyll grains). 2. Microsomes, the granules of the protoplasm : insoluble in water. These may be absent. 3. The materials 'dissolved in the vacuoles, Avhich in plant cells are always present. This fluid is stated to be sometimes acid, sometimes alkaline. The contents of vacuoles will be discussed more fully later on. Few histologists deny that protoplasm often shows reticulum and enchylema, to adopt Carnoy's terms. This view of the structure of cells is specially interesting to the physiologist, as in the cells which become muscular fibres, the reticulum takes on a definite arrangement, different fi'om the irregular disp(jsition it has in ])rimitive cells. This orderliness of arrangement is accompanied l)y the ordered and specialised movement known as muscular contraction. It is, however, necessary to guard against the assumption that the reticulum is firm, for it is only slightly less fluid than the enchylema. 'rhe FrotHds of Protoplasm. — The largest chemical constituent and the only constant one in protoplasm is jDroteid matter. Various theories to account for the differences between living and non-living proteids have been already dealt with (p. 115). 1 Uic iiiorjiltol. u. cJiciii. Ziisainmotsetziouj dcr Frviop. Bi-e>,liUi, lss7. TIIK CELL 193 In the case of the lymph cells occurring in lymphatic glands, and of the liver cells, large masses of cells, very little altered in structure from primitive cells, can be obtained ; here the methods of macro- cliemistry are applicable. These and other cases will be more fully considered in connection with those tissues, but for the present it may be here stated that as a general rule the proteids of cytoplasm are : ( 1 ) Xiicleo-albumins ; compounds of a phospliorised substance with a proteid. This substance is probably identical with the substance called plastin Ijy those wlio have employed the methods of micro-chemistry. (2) Globulins : one in small amount with a heat-coagulation tem- perature of about 50°C. ; another (cell globulin) in larger amount, which resembles serum -globulin in its heat-coagulation tempei'ature (75°C.). (3) An albumin in small quantities resembling serum-albumin in its characters ; this, however, is often absent. (4) Albumoses and peptones in small quantity, and generally produced as a result of post-iiu>rtem changes, or of retrogressive changes occurring within the body, as in the degenerative changes that occur in pus cells. The contents of Vacuoles. — A vacuole is a globular cavity con- taining a watery fluid. A'^acuoles appear to be always present in vegetable cells ; in animal cells they also frequently occur. In unicellular organisms like the amoeba, solid particles when ingested are surrounded with liquid ; this liquid may be taken in with the solid, but generally it is poured out by the cell around the solid, and appears to play the role of a digestant. Other vacuoles, such as the contractile vacuole of the amoeba, are excretory. Engelmann found that blue litmus gTains wlien ingested by amoebae and other protozoa became red after a time. A. G. Bourne ' suggests that this may be due to an acid secreted in an attempt to digest the particles. If vorticelke take in aniline blue, the protoplasm becomes filled with blue vacuoles, the contents of which are gradually absorbed, the pigment reappearing of a different tint in the contractile (excretory) vacuole. Schafer and Eckstein ^ found that blue litmus granules taken in by white blood corpuscles remain unchanged in colour. Miss Greenwood^ has made a very exhaustive study of the processes of digestion in Rhizopods, especially in amoeba and actinosphserium, with the following results : — 1 Quart. Journ. Microsc. Science, xsiv. 377, foot-note. 2 Qiiain's Anat. vol. ii. p. 5. ^ Journ. of Physiol, vii. 253; viii. 2G3. 194 THE TISSl'ES AND OIJO AXS oF THP: T.(^J>Y (1) The ingestion of solid matter is promiscuous in amceba ; that is, nutritious and innutritious particles are taken in with equal readiness. Actinospharium, on the other hand, rarely ingests innutritious par- ticles. (2) The nuti'itious particles are in both animals digested by fluid poured out around them. This fluid has no action on the cuticle of organisms, on cellulose, on siliceous eel] -walls, on fat, or on starch. It is colourless ; it digests proteid matter, especially uncoagulated pi-oteid. It changes chlorophyll to a dark-brown colour (especially in amoeba). It has no action on litmus or carmine particles accidentally inclosed with nutritious matter, and is therefore neutral in reaction. Innutri- tious matter does not become surrounded by this fluid. (3) The secretion is more active in actinosphferium than in amoeba. (4) Ejection is performed at the hinder end of the amceba, either by means of a vacuole, or often (for instance, when algse are taken in) without one. In actinospheerium an excretory vacuole is always pi^esent. (5) The time between ingestion and ejection is difficult to deter- mine, and varies with the size and digestibility of the ingesta from 3-4 days in amceVja, from 1^ to 8 hours in actinosphferium. THE NUCLEUS A large amount of original work has during the last twenty years been carried out, bearing on the structure and changes that occur in the nucleus of cells. The existence of the nucleus was discovered by Robert Brown in vegetable cells in the year 1821, and in animal cells by Th. Schwann nearly twenty years later. It was at first described as a more solid structure in the interior of the protoplasm or vitellus of the cell, con- taining in its interior, one or several still more solid particles called nucleoli. We now know that the nucleus consists of a spongework of fibrils, permeating which is a more liquid substance, the nuclear matrix. The particles called nucleoli may be thickened portions of these fibrils, or they may float free in the nuclear matrix. This is universally true for all nuclei, but these structures vary in size and shape in difierent cells of the body. Spontaneous changes in form may occur in nuclei liberated by the inipture of cells (Strieker, Flemming, Klein), which have been compared to the amceboid movements of protoplasm. Histologists distinguish between the ' resting ' or non-dividing nucleus and the ' di^-iding ' nucleus. "When cells divide, the nucleus first undergoes certain well-marked and de^nite changes ; the fibrillar TTIE ("ELL 195 inateiial is arranged into definite patterns (skeins, stai'S, rosettes, iVrc); these separate into two groups which form the foundation of the daughter nuclei. The name given to this series of changes is k(irijokint'si.raiie. 2. Xnclear network. 3. Xet-kaots. 4. Xucleohis. 5. Xuclear matrix. (After Waldeyer.) Tliese different parts must be taken one by one : — llie tuidear network. — This consists of fibres thick in parts, thin in other parts, arranged according to most observers irregularly. Balbiani describes this network in the chironomus larva as being composed of a o 2 li)f; THE TISSUES AND ORGAXS OF THE EDDY .single, much twisted thread. Flemnung has, however, shown that this is hy no means a widespread occurrence ; and some regard it still doubtful whether the numerous threads which are present, do or do not anastomose. Rabl ' considei'S that even in the resting nucleus, the fibres have a regular arrangement ; he distinguishes between primary and secondary fil>res ; the primary are the thicker ones, which run from one aspect of the nucleus, called the polar fii^Jd, where they loop round and part from one another ; they spread over, and throughout the nucleus in a Fig. 4G. — Selicme of resting nucleus (after Kabl). P, Polar area. A, Antiiiolar area. The" left- hand part of the figure shows primarj" fibres (1, 1 ) only ; tlie right-hand part of the figure shows- the primary fibres (1', V) connected into an anastomosing network by secondary fibrcr^. 2. 2, JCet- knots ; 3, Nucleolus. radiating way, and at the opposite end of the nucleus, or cmtipolar field, they are free and .show no special arrangement. They are depicted in the left-hand half of fig. 46. The secondar)- fibres are finer, and by connecting the primary fibres form the network, as we have already described it, and thus render it very difficult to identify the primary fibres in the resting nucleus. Dilute acids (acetic, formic, lirc.) render the whole nucleus more apparent ; water causes it to swell. Kearly all staining reagents (acid carmine, logwood, safranin, itc.) colour the network and nucleoli very intensely, while the interfibrillar substance remains uncoloured, or is only coloured faintly. From this tlifierence in behaviour, to .stains, Flen\ming distinguishes in the nucleus between chromatic and achro- matic substances. 1 MorpU. Jahrb. x.'(1885), p. 214. THE CKLL 1<)7 Tho chroiiiatic substance, or cliioinatin, includes the network and the nucleoli; and is, as E. Zacliarias' has shown, identical with nuclein. Th(^ achi'oniatic substance is the intertibrillar material ; durin<( karyokinesis, part of this becomes arran<4('d into a spindle-shapcul collection of fibres. Ptitzner- uses these terms more fully as follows : — Chromatin = substance of the nuclear network. Prochromatin (later, pseudo-chromatin) = nucleoli. Achromatin = nuclear matrix. Parachromatin = spindle ligure. Schwartz'' uses the following terms : — 'Chromatin = the nuclear network. Linin = the spindle ligure. Paralinin = Flemming's achromatin (probably a globulin). Pyi-enin = nucleoli. Amphipyicnin := nuclear membi-ane. These names are given, not from the chemical properties, but chieliy from the microscopic appearances of the structures in ((uestion. A very important step in our knowledge of the chruniatic fibres w^as the discovery of Balbiani and Pfltzner, that they are made up of a number of granules or discs (their foi'm is still uncertain) regularly arranged in single or multiple rows. Carnoy ' believes that tlu; tila- ments though chiefly composed of nuclein have an outer shell of plastin, an observation confirmed by van Bambeke.'' T/ie nucleoli present many difficulties ; the chief doubtful point c'on- cerning them is their relationship to the network. Flemming and Pfltzner regard them as different from the network, and not connected to it ; others (Klein^) consider them as merely thickened portions of the network, and composed of the same material. No doubt such nucleoli (net-knots in tigs. 45 and 46) do occur. But in addition there appear to be true nucleoli in Flemming's sense — rounded bodies free in the meshes of the network, floating in tlie nuclear matrix, and behaving differently to reagents. Zacharias states that they consist of a shell of plastin, and their, intex'ior of proteid matter ; they are apparently not composed of nuclein 1 Botan. Zcittunj, 1H81, 188'2, 188.5, 1887. ^ Morph. JaJtrb. vii. 1881, p. '280 ^ Die morphoJ. n. cheiii. ZusammcHsctz. d. Protop. Breslau, 1887. 4 La Cellule, vols. i. and ii. ■' Arch, tie Biol. viii. 1887, p. ;M!). ■^ Quart. J. Mic. Science, xviii. July 1878; xix. p. 1'25. 198 THE TISSUES AND OKOANS OF THE BODY Ogata,' Lukjanow,- and Stolnikow •' distinguish nucleoli into karyo- somes, plasmosomes/ and hyalosonies, according to their behaviour to eosine, nigrosine, safranine, and other stains. During cell division the nuclei dissolve in the nuclear matrix. The nuclear 'matrix is not a simple watery Huid, but is rich in proteids. Various I'eagents cause the appearance in it of a line preci- pitate, which one must guard against looking upon as a structure. Carnoy has apparently fallen into this error, when he describes a fine network of plastin in the nuclear matrix. The nuc/ear membrane is regarded by some as the optical appear- ance presented by the termination of the nuclear network } but most observers agree that a true membrane is present. This is achromatic (Flemming, Strasburger, Pfitzner) ; it is formed from the cell proto- plasm (cytoplasm) which lies next the nucleus, and is sometimes termed the inner cell membrane. The Dividing Nucleus It has been known since 1824 (Prevost and Dumas) that cells- multiply by the subdivision of existing cells, v. Mohl, Remak, and Virchow showed that spontaneous generation of cells does not occur,, the latter summing up the situation in the now classical phrase, ' omnis cellula a cellula.'"' Among the earliest who watched the com- plete process of cell division in amreba? and white blood corpuscles under the microscope were Strieker, Klein, Hchulze, Ranvier, and Waldeyer. Reniak's scheme of division was very simple : he stated that first the nucleolus, then the nucleus, and lastly the cell split into two parts, and so formed two daughter cells. Direct nuclear subdivision (a la Remak) is called by Flemming amitotic, as distinguished from the mitotic or karyomitotic subdivision of the same author. In the latter there is a highly characteristic arrange- ment of fibres in the form of figures, which replace tire nucleus ; this separates into two parts, and from each of these parts a daughter nucleus is formed, and the division of the cell protoplasm follows. The more carefully the cases of so-called direct cell division are examined the fewer do they appear to be. There are certainly variations in different cases, and, as Carnoy says, no stage seems to >)e absolutely essential : some nuclei, for instance, are very poor in chromatin ; and the differences that do exist are only of degree, not of kind. The chief characteristics of a dividing nucleus (the chromatic 1 Arch.f. Physiol, n. Anat. Physiol. Ahfli. 1883. ^ Ibid. 1887, p. GO. "■ Ibid. 1887, p. 1. * The plasmosomes are stated to wander out from the nuclei in certain cases to form para-nuclei (Lukjanow). ^ Arch. /.path ol. Anat. viii. 23 (1855). 'niK (,'i:i,L 199 nucleai' figuie, the acliroiuiitic .spiudlc, and the cytasters) were first described by A. Schneider,' and sulisequently rediscovered by Butschli^ and Fol.-' The name htri/okutcsis was applied to the series of changes by Schleiclier.^ Since then our knowledge of the process has been ct^. c. Tig. A7. —KarmkiuPMx. The skein or spirem stage, a. View of the nucleus from the poUir area : 6, from tlie antipolar area (dense skein); c, later stag:e (loose skeiu) ; the chromatic loops are thicker, shorter, and less twisted ; tlio spindle makes its appearance ; rf, end of the skein stage. The chromatic loops are splitting longitudinally into sister threads ;^ the spindle has taken up the position in wldch it will remain till cell ilivision is over. (After Waldeyer. ) furthered by numerous investigators, but especially by Strasburger,""' Flemming,'' E. v. Beneden,'' and more recently by Rabl.^ A full account of karyokine.sis is obviously out of place in this work, and therefore the following brief account (after Waldeyer)-^ must suffice. 1 'Unters. ii. Plathelmintheii,' Jalirbiich der Oherhessischen Ges. fiir Natttr. unci Heilkunde, 1873. - Zeit.f. iviss. ZooJ. 187.", xxv. 201. ^ Atrh. dc Zool. iv. (1875). 4 Centralhl. med. Wiss. 1878, p. 418. Carnoy's term is Cytodieresis. 5 Strasburger, Zellbildnng u. ZeUtheihing, 3rd edit. 1880. Kern und Zellthrilnng, 1888, Jena (see Nature, Nov. 1, 1888). Arch, mi'kr. Aunt. xxi. 476; xxiii. 240. •^ Flenuiiing, ZcU-Subsfanz, Kern ii. Zelltheihmg, Leipzig, 1882. Arch.nrikr. Anat. XX. 1 ; xxiii. 141 ; xxiv. 50 and 338; xxix. 389. Biol. Centralhl. iii. (541. Arch. f. Anat.u. Phijsiol. Anat. Ahth. 1885, 223. Zool. Anzeiger, 1886, no. 216. 7 E. van Beneden, Bull. arad. rni/. de BeJgi.que, 1870, 1874, 1875, 1876, 1884, 1887. ® Morjjh. Jahrl). x. 214. 9 An excellent summary of the present state of our knowledge on this important question will be found in Waldeyer's paper ' Ueber Karyokinesis,' Arch, viikr. Anat. xxxii. This paper has been translated by Dr. Benham {Quart. J. Micros. Science, xxx 159, 215). 200 THE TISSUES AND OEGANS OF THE Y,ODY The process may be divided into the following stages : — 1. The resting nucleus. 2. The skein or spireni stage ; the nucleoli dissolve, and the nuclear matrix then becomes moi-e stainable ; the secondary fibres (Rabl) dis- appear, and the primary loops running from polar to antipolar regions remain (figs. 47 « and h). 3. Each loop splits longitudinally into two sister threads, and the achromatic spindle' appears (fig. 47 c and d). The direction of the axis of the spindle when it first appears is often different from that it subsequently takes up. Strasburger considers it is formed of cyto- KlP-m Fig. 48. — Karmkinens. Monaster or equatorial stage. The nuclear membrane lias disappeared ; the protoplasm of the cell is lUvideil into a clear inner (5) and grannlar outer zone (6). The spindle (3) terminates at each pole in the polar or central corpu;=cle (2) roiuid which the granules of the protopliism are radially arranged to form a c\^aster (1). The chromatic fibres (4) now each longitudinally split into two sister threads are grouped around the equator of the spiudle. a is a view from the side ; 6, from one pole of the nucleus. (After Waldeyer.) plasm which has inti'uded into the nucleus (in some cases through pores in the nuclear membrane). Carnoy thinks the dissolved nucleoli contribute to its formation ; Flemming, that it is formed from the achromatic substance ; Platner, that it may have a double origin, i.e. from both achromatic substance and cytoplasm. 4. The equatorial stage ; monaster. The nucleus has now two poles — those of the spindle. The spindle terminates in two polar corpuscles.- The nuclear membrane is lost, and thus cytoplasm and ' The spiudle or karyaster is better marked in vegetable than in animal cells, it is occasionally cylindrical in shape, it is but little affected by stains, it is rendered more apparent by dilute acids, but readily dissolves in artificial gastric juice. - These are conijjosed of nuclein and are pai'tly derived from cytoplasm (Canioy). They are absent in x^lant cells. THE CELL 201 nuclear matrix become continuous ; tlie cytoplasm separates into a -clear and a granular zone, and the granules arrange themselves radially from the polar corpuscles (cytasters).' The chromatic fibres sink to the equator of the spindle, and arrange themselves so as to j)roject horizontally from it (scf. fig. 48 a and b). 5. Metakinesis. The sister threads separate, one going towards one pole, the other to the other pole of the spindle (fig. 49) ; one set of sister threads form one daughter nucleus, the other the other. CO. h. o. Tiu. iQ.— Juiriitdiiip.iix. Separation of sister threads ; (Metakinesis) one set moving toward one pole of tlie spindle, the otlier towards tlie other, a, 6, and c, sliow successive steps in the process. In (• (1) the uniting filaments (v. Bencden) are seen ; and tlie appearance from each pole is like that in fig. 48 h, except that the chromatic fibres are single not double. This stage (e) is called the Dyaster or daughter star stage. 6. Dyaster, or daughter star stage ; this stage occurs when the two sets of sister threads are sej)arated, as in fig. 49 c. The fibrils which still unite them are regarded by v. Beneden as difterent from the spindle, ■which gradually disappears ; Strasburger believes these are the spindle fibres along which the chromatic filaments shift. Each daughter nucleus then goes backwards through the same series of changes ; the dyaster is followed by the 7. Dispirem or daughter skein stage (upper part of fig. 50). The new nuclear membrane begins to form in this stage at the antipolar region, and the polar corpuscle disappears. The cell itself then divides ; the cell membrane being formed in plants by thickenings or knots in the equatorial region of each spindle fibre ; these thickenings coalesce. They are called dermatosomes, and are absent in animal cells. 8. The resting daughter nuclei ; when the cytoplasm has divided, the remains of the spindle disappears, the chromatic fibres become more "twisted, lose their equal calibre, and l)ecome connected by secondary fibres, as is shown in the lower nucleus (figure 50). 1 Also called aureola and lielioina. 202 Tin-: TISSUES and organs of the body In the egg cells of certain animals when dividing (e.g. Ascaris megalocephalus, v. Beneden), the chromatin filaments are but little marked ; the whole or nearly the whole of the granules of the cell protoplasm are arranged in a radial way round the ex- tremities of the spindle. At each end of the .spindle is a polar corpuscle, and a spherical mass of protoplasm which acts as an attrnclion spliere surrounds it. Each attraction sphere consists of protoplasm arranged in two zones. The cytasters, as generally seen in kaiyo- kinesis, are probably due to a less highly developed condition of the same state of things. V. Beneden calls the polar cor- puscle the central corpuscle, and he be- lieves that it is in the central corpuscle and its surrounding attraction sphere that we must seek the cause of sub- division, not in the nucleus. We must next proceed to examine the properties of the various chemical .substances found in the nucleus, many of which have been alluded to in the foregoing account of its structure. Tig. 50. — Karyokinf!is,^na.\ stages. Tlie place of (livisioii of the cell proto- plasm is seen. The upper nucleus still shows the remains of the spindle. The chromatic loops are now twistefl (ilaugliter skein). The lower nucleus is further ailvancee-Seijler'$ Med. Cliem. Untersuchungen, Heft iv. C1S71), 460. 3 Ibid. p. 441. 4 Verhandl. der nat. Ges. Basel, vi. (1874j, Heft i. c 49-58 H 7-10 N 15-02 P 2-28 TlIK ('KM- 20S" of different animals and from tlu; yolk of hens' eggs, Hoppe-Seyler, ' Kossel,- and Loew^ from yeast, Plosz' from the liver, von Jaksclr* and Geoghegan '' from the brain, Lubavin " from cow's milk, and Worm- Miiller** from yolk of egg. Tn fact, whei'ever nuclei are present, a substance is found which is rich in phosphorus, soluble in weak alkalis, insoluljle in weak acids and in artificial gastric juice, and with the sticky character of mucin to a certain extent. A similar body is also found in the substances, like milk and yolk of egg, which form the food of the young animal. This substance is termed nuclein. Nuclein is a compound of carbon, hydrogen, niti-ogen, sulphur, phosphorus, and oxygen. Elementary analyses of nuclein from dif- ferent sources yield very discordant results. The following examples- may be quoted : — From Pus (Hoppc-SeyU'r) r,-oiii Siieruiatozoa nf From Human Eraiu Sa mou (Miesc'liw) (v. .Jaksch). 36-11 50-6 5-15 7-6 1:3-00 13-18 9-59 1-89 The nuclein from spermatozoa differs from other nucleins in con- taining no sulphur. Miescher's formula for it is C.,9H49N9P3029. From these results we must conclude, either that nuclein is not a chemical unit, but a mixture of organic phosphorus compounds with ^vo- teids or proteid-like substances (Worm-Miiller), or more probably that several varieties of nuclein exist (Hoppe-Seyler).'-^ Miescher himself found that some nucleins were more insoluble in alkalis than others. Kossel '" confirms Hoppe-Seyler's view of the case, for he finds that on heating yolk-nuclein and milk-nuclein with weak acids, no bases rich in nitrogen like guanine and hypoxanthine are formed, whereas such bases are obtainable from cell-nuclei. Yolk-nuclein and egg- nuclein contain iron, cell-nuclei do not. A compound of nuclein with iron, called hepatin, is also found in the liver (Zaleski)." An iiitermediate product between nuclein and hypoxanthine is called adenine (C-^'R-.l^^ + 'SH^O) by Kossel, '^ its discoverer. It crystallises in the rhombic system, forms compounds with bases, acids, and salts. On heatii\g it with sulphuric acid, NH is replaced by O, and 1 Mcut exhibit points ftf difference in structure, of which the most striking is the presence of a cell wall in most vegetaVjle cells ^ and its absence in most animal cells ; the amount of vacuolation is also generally greater in vegetable cells. Both animal and vegetaVjle pro- toplasm breathes oxygen and gives off products of oxidation, like carbonic acid. This process is, however, in the light counteracted in most vegetable cells by the activity of the green pigment cUorophyll. Plants containing chlorophyll require simple chemical suVjstances as food. Oxygen, traces of ammonia, and, under the influence of sunlight, carbonic acid are absorbed from the atmosphere. "Water, ammonia and its salts, nitrates and other salts are taken up by the roots from the soil. Carbon is obtained by the decomposition of carbonic acid ; this is brought about in the light by the agency of the chlorophyll. Fungi and other plants devoid of chlorophyll obtain it by decomposing com- pounds in which carbon is combined with hydrogen. Hydrogen is obtained from water. Oxygen is obtained from the air. Nitrogen from ammonia and its salts Ijy lower plants, from nitrates by the higher plants. Sulphur is obtained from sulphates. Phosphorus is obtained from phosphates. Potassium, magnesium, sodium, calcium, iron, etc., from various salts in the soil. From these simple materials the plant builds up complex nitro- genous and non-nitrogenous bodies. Synthesis is in fact character- istic of all plants containing chlorophyll, but analytic processes also occur. The formation of non-nitx'ogenous substances, such as starch, is directly connected with the action of chlorophyll under the stimulus of lifht. Kraus found that starch grains appeared in the chlorophyll corpuscles of Spirogyra within five minutes after exposure to bright sunlight, within two hours in diffuse daylight ; in Funaria they were slower in appearing.^ It is probable that chlorophyll acts by causing the unirm of car- 1 Composed chiefly of cellulose. Paragalactin and other insoluble carbohydrates are also present {see p. 109). 2 Vines, Physiology of Plants, p. 147. TIIK CKLL 200 bonic aciil and water to make furinic aldehyde, oxygen beiii^ cliiniiiated, thus :— C02 + H,0=CH,0 + ()., ]iy |iulyuu'risati(>n and dehydration the ahh-hydc l)ecomes starch, thus : — 6CH20 = C«H,,0,i (grape sugar) C6H,206-HoO=C6H,o05 (starch) Cellulowe, fats, non-nitrogenous acids, and nitrogenous substances like asparagin, leucine, proteids, itc, are built up proljably by similar synthetical processes, but these have not yet Vieen worked out. The energy that enables the plant cell to do this work is undoubtedly the radiant energy f>f light, and more especially of those rays absorbed by the chlorophyll. When examined spectroscopically a solution of chlorophyll or a green leaf presents cei'tain absorption bands (fig. 51). It is the light of just those parts of the spectrum that is most active in the decomposition of carbonic acid ; the position of the maximum energy of light coincides with the maximum of absorption (Langley). Engelmann has shown that bacteria placed with a filament of Clado- jihora on the solar spectrum under the microscope, collect around the filament in the regions of the chlorophyll bands, that is, in the regions where most oxygen is being e\'olved. ' A green plant kept in dark- ness soon dies, owing to its not being able to obtain carbonaceous food. A somewhat elevated temperature is essential to the life of all plants. Light has apparently no influence on the true respiration (taking in oxygen and giving off^ carbonic acid) performed by the plant protoplasm. In these synthetical processes the kinetic energy of the sun's rays is stored up and becomes potential. Energy is, of course, expended during plant-life, but in less amount than it is stored. This stored energy is again liberated as heat in the 1 More recently Engelmann {PfliUjer''s Archiv, xlii. 186) has demonstrated tlie same fact in another waj'. A spray of Spirogrjra was mounted in a drop of diluted ox blood rendered venous by a stream of hydrogen. In one minute in direct sunlight, in fifteen minutes in diffuse daylight, the blood in the neighbourhood of the spray became arterial, and returned to its venous tint in the dark. If a spectrum was projected under the preparation, the change to the arterial tint occurred in the neighbourhood of those parts of the spray lying over the parts of the spectrum where the absoi-ption bands of clilorophyll are situated, especially in the neighbourhood of the C line. There are other pig- ments which play a similar role to chlorophyll, i.e. decompose carbonic acid; one of these is bacterio-purpurin, a red pigment produced by the activity of certain bacteria (Ray Lankester) of the sulphur-bacteria class (Winogradsky, Botan. Zeit. 1887, no. 31-37). These pigments are all termed chromophylls ; they act with regard to the spectrum mutatis mutandis similarly to chlorophyll. P 210 THE TISSUES AND ORGANS OF THE 150DY combustion of fuel, or as heat and motion in the bodies of living animals. The animal cell, on the other hand, receives its energy in only a small measure from the sun's heat, but the greater amount is received in the form of vegetable food, i.e. of the substances formed by synthetical pi'ocesses during plant life. In the case of carnivorous animals, the supply of vegetable iood is an indirect one through the body of another animal. The complex food stuflfs either directly or indirectly enter into the composition of the cell protoplasm, and are there burnt off as simpler products (carhtonic acid, water, urea, »fec.). During this destructive metabolism, kinetic energy (heat, motion, electricity, itc.) is liberated. The animal cell stoi-es a certain amount of potential energy, but this is less than that which is expended. In other words, the conditions of the green plant cell in sunlight are reversed. In green plant cells in the dark, and in plant cells without chlorophyll, the A"ital processes resemble those of animal cells. In those exceptional animals, the cells of which contain chlorophyll (e.g. hydra viridis), their behaviour in sunlight is like that of plant cells — they decompose carbonic acid, liberate oxygen, and store the carbon. Speaking generally, the plant is chiefly concerned in synthesis, and .so funiLshes potential energy to the animal. The animal liberates this as kinetic energy, which, however, is not retransfomied into potential energy for the plant. The plant receives new supplies of energy from the sun's rays.^ In a recent paper Pfliiger ^ has pointed out that this contrast must not be pushed too far. The cell protoplasm itself acts in the same way in both animal and plant cells, breathing oxygen and giving out car- bonic acid, water, and amido-compounds. Synthetic processes are undoubtedly more highly developed in plants containing chlorophyll, but they are present also in animal cells. The formation of hippuric acid from benzoic acid and glycocine, or of ethereal sulphates from phenol and sulphuric acid, are typical instances of syntheses occurring in animal cells. He also shows that syntheses must occur in the retrogressi^•e metamorphoses that lead to the formation of uric acid and its congeners from proteids, in the formation of fat from proteid, or from carbohydrates, and in the formation of carbohydrates (glycogen) from proteids. These chemical operations performed by the living cell cannot be imitated in the laboratory or explained by any known chemical laws ; there is no doubt, at first, an extensive breaking down of the complex molecules, and then the cells build up entirely new 1 The foregoing account of the cell processes is very largely taken from McKendrick's Physiology, pp. 23-27. * Pflilger's Archiv, xlii. 144. 'I'lIK CKI.I. '211 materuils aj,'ain, of a c-oniplfx iialiirc timn the simple cai-ljon coiii]h)U1h1.s so liberated. Tlie close reseinhlaiicT between animal and ve^(;table cells is furthei- shown by the fact that many lower plants (bacteria, moulds, «fec.) not only flourish in solutions of albumin and sugar, but actually shed out ferments to convert proteid into peptone, and starch into sugar, to aid absorption. They breathe oxygen, produce carbonic acid, amido- derivatives, and, without the aid of sunlight, fat, carbohydrate, and proteid. Nageli ^ has shown that these fungi will assimilate carbon from compounds in which it is combined with hydntgen (amines, ifcc), but not from those in which it is combined with nitrogen (cyanogen, &c.). The (juestion whether- light lias any influence in accelerating the chemical pi-oeesses in animals, was answered in the affirmative by Moleschott 2 and v. Platen."* Speck "* and Loeb '^ have, how^ever, shown that light of itself does not cause the increased pi'oduction of car- bonic acid, but acts reflexly through the nervous system, especially through the retina, whereby increased muscular movements occur, and so an increase in the chemical processes takes place. Loeb took lepidopterous larvaj in the chrysalis stage when movements are absent, and found that oxidation processes were practically equal in those exposed to light and those kept in the dark. APPENDIX,— CHLOEOPHYLL ''■ The term chlorophyll was inventefl by Pelletier and Caventon ; ^ it is the substance or mixture of substances to which the green colour of leaves and other vegetable organs is due. It is an exceedingly unstal)le body, and most attempts to isolate it have failed, because in the processes adopted for the purpose decom- position has been brought about. Berzelius, Mulder, and Fremy employed strong mineral acids to extract it fiom leaves, under the mistaken im- pression that it was a stable body, but solutions of chlorophyll are destroyed by the action of air and sunlight, much more than by strong acids. 1 Sitzungsh. Bair. Akail. W/.s.s. I.s7>.». - Wien. med. Wochensch. 1H85. 3 Pfliic/er's Archiv, xi. 272. * Arch. f. exj). Path. n. Phar)tiak. xii. ^ Pfiicjer's Archiv, xlii. 393. 6 Tlie following account of the chemistry of chlorophyll is almost entirely an abstract of a paper by Dr. Schunck on that subject in the Annals of Botany, vol. iii. pp. 65-120. ' Annalrs dc chiinic ct de ph unique, ix. 194. V 2 212 THE TISSl'ES AND ORGANS ()K 'I'HK P.ODV Gautier' obtained a st)lutioii of chloiDphyll Ijy the use of neutral solvents, like alcohol and ether, and stated he obtained green crystals which he considered to be composed of the pure pigment, but Hansen^ did not succeed in ol)taining them. Hansen employed caustic soda as a solvent ; this saponities the fat which accompanies the chlorophylL A yellow pigment is removed from the mixture l)y light petroleum, and then the chlorophyll is dissolved out by a mixture of alcohol and ether. On evaporating the solvent, dark green sphaero-crystals of ' chlorophyll-green ' are left. These crystals can, however, hardly be composed of pure chlorophyll, as they are easily soluble in water, a medium in which chloroj^hyll is insoluble ; and in a later communication Hansen himself has admitted that his crystals contain .sodium. In view of the difficulty found in isolating chlorophyll, our know- ledge of its chemical and physical properties is necessarily limited. It is insoluble in water, and soluble in substances which, like alcohol, ether, carbon disulphide and chloroform, dissolve fats. These .solutions show a green colour with a red fluorescence. Spectroscopically a solution shows four distinct bands and two indistinct bands. The two latter, distinguished as bands V and VI, are situated as is seen in figure 51 at the blvie end of the sjaectrum, and are only visible by sunlight in dilute solutions. Some observei'S consider that these are not true chlorophyll bands, but belong to a yellow colouring matter which accompanies chlorophyll, and which is called xanthophyll. Kraus^ and Sachsse'' have partially succeeded in separating the two pigments. Elementary analyses of chlorophyll have yielded most discordant results ; two of the latest determinations that have been made will serve to illustrate this statement. Carbon 73-97 per cent. 67-26 per cent. Hydrogen , 9-80 „ 10-63 „ Nitrogen 4-15 „ 5-r^ Ash . 1-75 Oxygen is also present ; when burnt, chlorophyll leaves an ash which contains phosphates of calcium and magnesium and a little ferric oxide. The ash has an acid reaction due to acid phosphate. The phosphates may be derived from phosphorus in the chlorophyll, or in an impurity ;, there is equal doubt with regard to the iron, whether or not it is con- 1 Comjjt. rend. Ixxxix. 861. 2 Arbeiten d. Bot. Inst. Wiirzhurg, iii. 12o and 430. 5 Ziir Kentniss d. ChlorophtjUfarbstoffi', Stuttgart, 1.S72. ■» Die CJiemie u. Plujuiol. d. Farsht. Leipzig, 1877. THE C'Ki.L 21;) taiiuMl in the i-liloropliyll moh^cule. Most observers agree in regarding chloropliylK-is a substance, the nioh^cules of which ai-e in a state of un- stable e(iiiililiriuHi. Dfcoiii/xixifion products oj cltloroiilij/ll . Hoppe-Seylei-' extracted fresh grass with boiling absolute alcohol ; tlui extract on being allowed to stand, deposited crystals which wei'(! ])uritied by i-ecrystallisation ; the substance so obtained, he termed clilorojiliijlluii. Tt melts at llO'C to a black licjuid, which on further heating burns with a luminous flame. It is easily soluble in ether, light petroleum, benzol, an that there is a 'diurnal variation " in the specific gravity of the blood, consisting of a fall during the ilay and a rise during the night. The specific gravity of the blood is higher in the male than in the female ; and that during pregnancy, after exeR>ise, or after the ingestion of food, there is a fall. In a passively congested part the specific gravity of the blood is high. Tiie specific gravity of delibrinated blood varies considerably, the average for human blood being ]0r>5 (Becquerel and Kodier)' ; for dog's blood, 1060 (I'tiiiger)-; for rabbit's blood, 1042 to 1052 (Gschleidlen).-' The specific gravity falls in aniemia and wasting diseases generally. It also falls after hiBmori-hage. The specific gravit}' of defibrinated lilood may be ascertained by the use of the hydrometer, or more correctly by actual weighing (sec p. 15). React ion. — The reaction of vertebrate blood is alway.s alkaline. This is due to the alkaline salts which are present. The demonstration of the alkalinity of the l)l()od is very simple. A drop of blood is placed f)n the smooth, faintly reddened surface of a piece of dry, glazed litmus j^^per,'' and after a few seconds is wiped off with a piece of clean linen rag moistened with water. The place where the blood has been standing is marked out as a well-defined blue jDatch (Schiifer '). The manufacture of glazed litmus paper of the kind just alluded to renders unnecessary the somewhat elaborate methods adopted by older observers to demonstrate the alkalinity of the blood. Thus Kiihne*' placed the blood in a small dialyser suspended in a watch-glass full of water ; some of the salts pass into the water, the alkalinity of which can be then shown. Liebreich' recommended porous slabs of plaster of Paris coloured by neutral litmus instead of litmus paper, and Zrrntz" used litmus paper previously moistened with a strong solution of sodium sulphate or sodium chloride. The alkalinity of plasma or of serum, where there is no ditRculty arising from the presence of a mass of deeply coloured corpuscles, can be always demonstrated by the use of ordinary litmus paper, or of litmus solution. Taste and odour. — The salts present in the blood give it a saline taste. Blood has also a slight but peculiar odour dependent on the presence of minute quantities of volatile fatty acids. This odour, known as the haJitus sanyuinis, differs in different animals. It may be further 1 Becquerel aud Rodier, Becherches sur les alterations dii sang, Paris, 1S44. Traitc dc chimie jMtliologiqtie, Paris, 1854, p. 41. - Pfliiger, Pfli'iger's Archiv, i. 75. ^ Gschleidlen, quoted in Gamgee's Physiological Cheniistr;/, p. 20. * Such as are prepared by Messrs. Towiison & Mercer, Bishopsgate Street. ^ E. A. Schiifer, Journal of Pliysiology, vol. iii. '' Kiihne, Virchow's Archiv, vol. xx.xiii. (18(55), p. 95. ^ Liebreich, Berichtc d. deutschen diem. Ges. zit, Berlin, 1868, p. 48. » Zuntz, Centralbl.f. d. med. Wissensch. 1867, no. 34. -220 THE TIS.'SUE.S A^"D ORGANS OF THE BODY develitped by adding to the blood a mixture of equal parts of sulphuric aci !i 2 to 1| minute „ sheep „ „ „ T to li ,, „ rabbit „ „ „ ^ to 1^ dog ,, „ M 1 o'ig salt solution exerted on the formation of fibrin, is removed. 3, Contact with living vascular walls. After death the bk>od remains fluid in the smaller vessels for many hours. Briicke (l{>o7) kept the blood fluid in the interior of a tortoise's heart (removed from the body) for eight days. A more convenient vessel is the jugular vein of a large animal like the horse. If the vein be ligatured in two places, so as to^ include a quantity of blood within it, and then removed from the animal, the blood will remain fluid for hours or even days, provided that the vessel be hung in a cool place, and that the inner lining- of the vein preserves its integrity for that time. If the vein be opened and the contents be allowed to come into contact with foreign matter,^ coagulation will ensue in a few minutes. This experiment, often spoken of as the 'living test-tube experiment,' has been employed in the researches of Hunter,' Hewson,- Lister^ and Fredericq.^ If the double ligature be applied antiseptically, and the vessel be allowed to remain in the body, the wound heals, and the included blood-column vrill be found uncoagulated months afterwards (Baumgarten,^ Senftleben ^). Blood, however, which escapes from the vessels into the tissues soon, coagulates. 4. Injection of peptone into the circulation. If a certain quantity of commercial peptone, such as Wittes or Griibler's, be injected into the circulation, and the animal then killed by bleeding, the blood will be found to have lost the property of coagulating (Schmidt-Mulheim,'^ Fano ^). The blood becomes nonnal again about three hours after the injection. The dose for a dog is 0*3 gram of peptone for every kilogram of body weight. In rabbits peptone has no such effect in liindering coagulation. Pollitzer ^ has shown that this property of so-called pep- tone is really due to the albumoses of which commercial pi-eparations of ' peptone ' chiefly consist, and especially to one of these called hetero- albumose. Pure peptone has little or no such effect. He has also ' Hunter, Worls, vol. iii. p. 29. - Hewson, Works, p. 22. 5 Lister, Proc. Boy. Sac. vol. xii. (i860), p. 5»0. * Fredericq, Becherches siir la constiiution du plasma sanguin, Gand, 1878. 5 Co7iH7ie/;HS Pfl^/joZo^y, New Syd.Soc. 1889, p. 177. « Yirchou's ArcliAsrtxn. ^21 ~ Schmidt-Mulheim, Du Bois BeijmomVs Arch.f. Anat. tind Physiol. 1879. 8 Fano, Ihid. 1881, p. 277. ' PoUitzer, -Joiirn. of Physiol, vii. 282 THE P.LOOI) 225 shown that these albumoses delay tlie coagulation of the blood aftei- it is shed. They also cause a similar delay in the clotting of dilute salted plasma.' Various diastatic ferments injected into the blood stream act in the same way (Salvioli^). In all these cases the presence of peptone, either free or when injected into the blood stream, possibly in a loosely combined condition,^ acts in all probability by inhibiting the activity of the fibrin-ferment. This inhibitory influence can be removed by passing a stream of carbonic acid through the blood. 5. Contact with oil. When blood is surrounded by fluid, of a surface- tension difterent from its own, and which does not mix with it, its coagulation is much delayed. Thus Freund found that if he smeared a glass vessel with vaseline, and carefully received blood into it through a greased cannula in direct communication with the artery of an animal, he could by covering the blood so obtained with a layer of liquid paraftin, keep it from coagulating for several hours. Haycraft^ obtained a similar result by allowing blood to drop through a layer of liquid paraflin on to greased mica plates. Haycraft and Carlier'^ received blood directly from the finger tip into a tall cylindrical vessel filled with castor oil, which is very viscid. The drops sink slowly through the oil, and by occasionally inverting the vessel, the blood may be kept from coming in contact with its ends for a considerable time. The microscopic characters of uncoagulated blood may be examined on a greased slide, if great care be taken to prevent the blood from coming in contact with anything solid or ungreased after it is shed. 6. Addition of small quantities of caustic alkalis or ammonia. In these cases the reagent used is, however, so strong as to alter very considerably the natural condition of the constituents of the blood. 7. Addition of acetic acid or excess of carbonic acid. There is here again the objection to the use of a strong acid like acetic acid, which readily converts globulins like fibrinogen into acid-albumin. Passing a stream of carbonic acid through the blood after dilution precipitates the fibrinogen, and so of course prevents the formation of fibrin. The greater quantity of carbonic acid in venous as compared with arterial ' Halliburton, Proc. Botj. Soc. vol. xliv. (1888), p. 261. - Salvioli, Centralbl. f. d. med. Wiss. 1885, p. 913. 5 It is difficult to find ' peptone ' after injection into the blood, perhaps because it is, as Hofmeister considers, held in combination by the white corpuscles. Neumeister has, however, shown that after a short time these substances (albumoses and peptones) are excreted by the kidneys, so they must be present as such in the blood, or only very loosely combined for a time at least. Further remarks on the presence and fate of peptones in the blood will be found in the Chapter on Absorption. * Haycraft, Proc. Roy. Soc. Edin. July, 1887. 5 Haycraft and Carlier, Brit. Med. Journ. vol. ii. (lS88j, p. 2-2'.l. Q 226 THE TISSUES AND ORGANS OF THE r,ODY blood is said to be the. cause of tlie slower coagulation oljserved in the former. 8. Heating blood to 56°C. immediately it is witlidrawn from the body also prevents any formation of fibi'in, as the tempei'ature mentioned is sufficiently high to cause a heat-coagulum of the proteid fibrinogen, the fibrin precursor. 9. The addition of an equal volume of a 0*5 per cent, solution of cane sugar delays the coagulation of blood for about an hour (J. Miiller'). The addition of other viscous substances, such as egg-albumin or glycerine, has the same effect. 10. The addition of much water to the blood delays its co- agulation. 11. A watery extract of the medicinal and ccmimon leech prevents the coagulation of the blood (Haycraft^). This is interesting in view of the difficulty often experienced in controlling hpemorrhage from leech bites ; the secretion from the leech evidently prevents the formation of a clot, which usually performs the part of a plug in small wounds of this kind. 12. Blood which naturally clots slowly or not at all. (a) The blood of embryo fowls does not coagulate before the 12th or 14th day of incubation.^ (b) In certain moi'bid conditions the blood also clots slowly (see Chapter XVI). So far then we have described the naked eye and microscopic phenomena of coagulation ; we have enumerated the various conditions which hasten or delay clotting ; we have arrived at the conclusion that the essential fact in the process is the separation of the substance fibrin from the plasma, and it has been incidentally mentioned that the formation of fibrin is due to the activity of an organised ferment^ called the fibrin-ferment. It will be now convenient to take up the substances fibrin, plasma, and serum, and discuss their properties more fully. After we have considei-ed at greater length the properties of the constituents of the plasma, it will be easier to understand the theory of the cause of the coagulation which is now generally held, and the grounds upon which it 1 J. Miiller, Poggendorff's Annalen, xxv. 540. 2 Haycraft, Proc. Physiol. Soc. 1884, p. 13. 2 Tiegel has stated that the blood of certain snakes does not clot for many hours after being shed ; but other observers have not found any such delay in the coagulation (see Journ. of Physiol . vii. 322). ■* By an unorganised ferment is meant a chemical agent which produces certain changes in the materials with which it comes into contact, without itself undergoing any change. The term organised ferment is applied to those which, like yeast and bacteria, consist of living organisms. THE I'.LOOD 227 rests. The theory is that of Hainniarsten, and may l)c l)riefly stated as follows : — ]V/ii'ti till' blood is wifJiin the ressf/s, one oj' the constituents oj" j)I am na, a proteid of the f/fobulin class called jibrhiogen, exists in a soluble form. When the blood is shed, fbrinogen is converted into the comparatively insoluble substance fb^'in. This change is brought about by the activity of a special unorganised ferment call cd thefihrin-ferment. This ferment does not exist in healthy blood contained in healthy blood vessels, but is one of the products of the disintegration of the white corpuscles and probably also of the Ijlood tablets, that occurs when the blood leaves the vessels, or comes into contact with foreign matter. The Plasma or Liquor Sanguinis The liquid in which the corpuscles float can be obtained by employ- ing one or other of the methods already described for preventing the blood from coagulating. The corpuscles have a higher specific gravity than the plasma ; they therefore sink, and the supernatant plasma can be removed by a pipette or siphon. It may then be more thoroughly cleared from corpuscles by the use of a centrifugal machine {see p. 17).^ The following are the forms of plasma that may be obtained : — a. Fure plasma. — This may be obtained from horse's veins by what has already been described as the living test-tube experiment. The plasma removed from the top of the vein clots slowly, at the tempera- ture of the air (usually in 15-30 minutes) ; that deeper down, nearer to the corpuscular sediment, itself contains more corpuscles, and coagu- lates more rapidly. In all cases the plasma coagulates more quickly at a temperature of 40° C. The process of clotting consists of the same stages as the clotting of blood already described ; the clot itself con- sists of pure fibrin, or fibrin with only a slight admixture of corpuscles. b. Cooled plasma.- — This is another form of pure plasma. Blood is allowed to flow into the middle compartment of a vessel containing three concentric chambers, the inner and outer of which are filled with ice (Burden Sanderson). - The corpuscles settle ; the plasma can be removed, and it is found to possess the same characters as those already mentioned. c. Transudation fluids. — Pericardial and hydrocele fluids, and similar transudations into serous cavities, resemble pure plasma very closely in composition. As a rule, however, they do not coagulate spontaneously ; but after the addition of fibrin-ferment (or liquids like sei^um which contain fibrin-ferment) they always yield fibrin. In cases, 1 In the case of pure plasma, the tubes during centrifugalisiiig must be kept cool by enclosing them in larger tubes filled with powdered ice. - Handhooh fur the Plujsiological Lab. p. 1(58. Q2 228 THE TISSUES AND OKGANS OF THE F.ODY however, in which the serous sac is inflamed, as in pericarditis, the fluid is found to contain a large number of white corpuscles ; it then clots after removal from the body without the addition of any ferment. d. Salted ])!asma. — This may be obtained by mixing the blood immediately when shed with the necessary amount of strong saline solution (p. 224). This is found better than Hewson's original method, in which he stirred the blood with the solid salt (sodium sulphate). The corpuscles settle somewhat slowly in the case of the blood of oxen and sheep, but more rapidly in that of the horse. After twenty-four hours' standing, a good supply of salted plasma can be generally obtained readily. This settling can, however, always be hastened by the centri- fuge. In sodium sulphate plasma, a few strands of fibrin may sometimes form, but as a rule it remains many days unaltered. On diluting it with four or six times its volume of water, coagulation ensues slowly ; fibrin often not appearing for many hours, but always more quickly at a tem- perature of 40° C. and always within a few minutes after adding fibrin- ferment. Sodium chloride plasma acts similarly. Magnesium sulphate plasma is preferred by some workers, because, when diluted simply with water, it clots more slowly than other forms of salted plasma, or sometimes not at all. It, however, never fails to clot rapidly with fibrin-ferment ; on this account it is generally employed as a test for this ferment. Salted plasma when diluted with water, without the addition of fibrin-ferment, clots because the dilution renders the proportion of salt present, insufficient to inhibit the activity of the ferment ; the ferment is present in small quantities, either from the presence of some few corpuscles not removed by the process of centrifugalising, or because some of these corpuscles have already undergone disintegration. Diluted magnesium sulphate plasma is least prone to undergo this slow spontaneous clotting, because the proportion of salt used has precipitated some of the precursor of fibrin, fibrinogen, and perhaps other globvilins as well ; this precipitate is a flocculent one, and settles with the corpuscles to the bottom of the vessel. The proportion of this salt usually employed is that recommended by Hammarsten, ' four parts of blood to one of saturated solution of magnesium sulphate. Magnesium sulphate plasma therefore possesses the disadvantage of not containing all the proteid matter of the original plasma ; a minor disadvantage in its use is that it is often stained with hfemoglobin.- 1 Hammai-Hten, P/7v(^er's ,4?-c7(/!', xiv. 220. 2 Hamburger {Zeit. Biol. xxvi. 414) has sliown that when blood is mixed with salt solutions of different concentrations, there is for each salt a certain concentration when no hremoglobin is dissolved out from the corpuscles, while a saline solution of less concentration becomes tinged with the pigment. The mean of these two limits gives THE r.Loni) 229 The process of clotting in all these forms of salted plasma leads to the formation of fibrin, and ultimately to the expression of a mixture of serum and saline fluid, which may be called salted serum. f. Si/rtipy plasma. — This may be obtained by filtering blood in which the coagulation has been delayed, by mixing it when shed with its own volume of a 0"5 per cent, solution of cane sugar. It coagulates in from 30 to 60 minutes. /'. Peptone jthisma. — This may be obtained by centrifugalising peptone blood, the preparation of which has been already described. The inhibitory influence of the peptone is removed by passing through the plasma a stream of carbonic acid gas ^ (Fano), or by the addition of lecithin (Wooldridge). On cooling this fonn of plasma to 0° C. a proteid precipitate consisting of rounded granules occurs (Wool- dridge). Such a precipitate produced by cooling is not obtained in pure plasma, nor in salted plasma. g. BiJe-saJt plasma. — This form of plasma obtained by mixing blood and a certain proportion of bile salts has been used by Nauck and Samson-Himmelstjema.- These observers find that with this form of plasma the accelerating influence of small quantities of lecithin and of many other organic substances (glycocine, uric acid, &c.) on coagulation -can be demonstrated. Conclusions based, however, on forms of plasma where there has been an admixture with complex organic substances, like bile salts or commercial peptone, should be received with caution, unless supported by corroborative experiments with pure plasma. h. Lpech plasiita.- This fonn of plasma is obtained from blood in which coagulation has been prevented by mixing it with extract of leeches. General Characters and Composition of the Plasma The plasma is alkaline in reaction, and yellowish in colour ; in the •case of man, its specific gra.xitj varies between 1026 and 1029.^ Like the blood it clots, and the process of clotting leads to the formation of numbers identical with the isotonic coefficients of de Vries (Pringsheim's Jahrb. iciss. Botanik, xiv. Heft iv.) 1 In connection with this action of carbonic acid gas, it should be noted that Lahousse {Archiv f. Physiol. Anaf.; Physiol. Ahth. 1889, \i. 77) has found that 'peptone blood' contains only about half the normal quantity of this gas ; the oxj-gen is rather higher in amount in peptone blood than in normal blood. Bohr {Centralhl. f. Physiol. 1888, no. 11) finds that in a dog mto which either peptone or|Ieech infusion has been injected, the out- put of^carbonic acid in the expired air is much diminished. - Inaugural Dissertations, Dorpat, 1882 and 1886. -* Gamgee, Physiological Chemistry, p. 34. Gautier, Chiiiiie appliqiiee a la physiologie, 1874, vol. i. p. 489. C. Schmidt { see Gamgee, p. 127) gives the specific jgravity of healthy plasma rather higher, viz. 1031. 230 THE TISSUES AND OEGANS OF THE BODY fibrin ; this process can be hindered or hastened by the same agencies as have ah'eady been mentioned in the case of blood. The plasma consists largely of water ; the solids, dissolved in it, fall into three classes : jDroteids, extractives, and inorganic salts. Gamgee ' gives the following table, deduced from the observations of C, Schmidt and Lehmann, which shows the relations of these sub- stances to one another in human liquor sanguinis, 1 AAA 1. 2 ^ ^ 1 -it' •'il3'02 parts of corpuscles 1,000 parts or blood yield ^ '■ i486 "98 parts of plasma 1,000 parts of plasma contain : Water 902-90 Solids 97-10 Proteids — -1. yield of fibrin .... 4-05 2. other proteids .... 78-84 Extractives, including fat .... 5-66 Inorganic salts ...... 8'5^ In round numbers it may be stated that the plasma contains 10 per cent, of solids, of which about 8 per cent, are jjroteid in nature. Besides these solids, the plasma has dissolved in it certain gases (oxygen, carbonic acid, and nitrogen), the consideration of which will be taken with respiration. General Characters of the Serum Serum may be obtained by either of the following methods : — 1. By allowing plasma to coagulate; the clot is filtered off"; the filtrate is serum. 2. By allowing blood to coagulate. This is the method more usually employed. The fluid squeezed out by the contraction of the clot is serum, which may be removed by a pipette or siphon ; and then completely freed from corpuscles by the use of the centrifugal machine. The serum has the same colour and, approximately, the same specific gravity as the plasma. It is rather more alkaline than the plasma from which it has separated. It however does not clot spontan- eously. In other words, the essential difference between it and plasma is, that in serum the fibrin-yielding constituent of the plasma (fibrinogen) has been removed in the form of fibrin. Serum also contains the disintegration products of white corpuscles and blood tablets, of which the two most important are globulin and fibrin ferment. 1 Gamgee, IbiiJ. p. 12S. 'I'll!'. I'.Looi) 231 Serum contains the same tlii^ee classes of constituents as the plasma, viz. proteids, extractives, and salts. The extractives and salts are the same in the two liquids. The proteids are different, as is shown in th(» following tal)le : — PROTEIDS OF F/dsma I Serum Fibrinogen ^subsequently changed to fibrin) . Serum-globulin Serum-globulin j Serum-albumin Serum-allnimin 1 Fibrin-ferment We have now to take up these different proteids one by one. The fibrin-ferment has been included among the proteids for i-easons which will be fully dealt with later on. Fibrin The microscopic characters of fibrin have been already described. A supply of fibrin in sufficient quantity for chemical investigation may be obtained (1) By allowing plasma to coagulate. (2) By allowing lymph to coagulate. The amount of fibrin formed from lymph is 04 to 0-8 per 1,000. (3) By inducing coagulation in fluids which contain fibrinogen ; such for instance as hydrocele fluid, pericardial fluid, and exudations into other serous sacs. This may be done by adding fibrin-ferment to such fluids. (4) By the addition of fibrin-ferment to solutions of pure fibrinogen. (5) By whipping freshly-drawn blood with a bunch of twigs ; the fibrin adheres to the twigs, and entangles but few corpuscles ; it may then be washed by a stream of running water. This is the usual method by which fibrin is obtained. Tlie amount of fibrin formed from Imman blood is ■I-2 to 4 per 1,000. Fibrin is a white stringy solid when fresh ; when dried it becomes greyish in appearance. It is extensible and elastic ; and it is owing to the retractility of its fibres that a blood clot contracts. It is exceedingly difficult to prepare fibrin free from white blood corpuscles ■,^ and certain con.stituents of those corpuscles are nearly always adherent to it. Even after prolonged washing with water, there is always a considerable quantity of fibrin-ferment adherent to it. 1 A stringy substance maybe obtained from white corpuscles which is not true fibrin. It is a nucleo-albumin. 232 thp: tissues .-^'d oegans of thp: body It may be purified by repeated extraction ^^-ith alcohol and ether. It has then the following elementary composition :' C, 52-68; H, 6'83; N, 16-91 ; S, 11 ; O, 22*48 per cent. Fibrin is a proteid, though it is not nearly so soluble in neutral liquids as most proteids are. Like many other organic substances fibrin decomposes solutions of hydric peroxide. It is soluble with difficulty in a 6 per cent, solution of potassiiun nitrate; in 5-1.5 per cent, solutions of sodium chloride; in 5-10 per cent, solutions of magnesium sulphate : and in similar solutions of other neutral salts, such as sodium sulphate and ammonium sulphate. This occurs most readily at a temperature of 40° C Denis described three varieties of fibrin: (I) fibrine concrete modifiee; (2) fibrine concrete globnline; and (3) fibrine concrete pure. Hammarsten has repeated these obser%-ations of Denis, and, in the main, confirms his conclusions : ^ the first variety is that obtained from arterial blood, and is ordinary fibrin ; the second variety is the slimy substance which forms when a 10 per cent, solution of sodium chloride is added to certain varieties of fibrin : it is, in fact, not true fibrin at all, but a nucleo-albumin which swells up with sodium chloride, and which is contained in white blood corpuscles, and which we shall have to describe fully with the white corpuscles ; this form of fibrin (so-called) is obtained when- ever white corpuscles or pus corpuscles are present in excess. The third variety of fibrin is that which forms in venous blood, and this dissolves more readily in saline solutions than that obtained from arterial blood. The substance which goes into solution when fibrin is dissolved in saline solutions is undoubtedly a proteid of the globulin class. It is coagulated by heat, it is precipitated from its solutions by saturating them with magnesium sulphate, and also by dialysing away the salt from such solutions. This globulin, however, cannot be reconverted into fibrin by the addition of fibrin-ferment. The temperature of heat coagulation is 60°-65° in a sodium chloride solution ; 73°-75° in a magnesium sulphate solution. If putrefaction is not prevented by the addition of a few crystals of thymol or some other antiseptic, there is a more rapid and thorough solution of the fibrin. Indeed, some observers have supposed that fibrin never dissolves in saline solutions, except by the process of putrefaction. Salkowski ^ has shown that a ferment, the result of bacterial life, can be actually separated from the microbes ; this acts upon fibrin like trypsin, producing first globulins, then albumoses, and finally peptones. Weak hydrochloric acid (0'2 per cent.) causes fibrin to swell up into ' Hammarsten, Pfluger's Arcliiv, xxii. 484. - Ibid. XXX. 437. ^ Salkowski, Zeitschr. Biol. xxv. 92. THK r.T.odi) 233 a transparent jelly. Stronger acids dissolve it in time with the forma- tion of acid-albumin or syntonin, and albumoses. Digestive ferments act readily on fibrin, so that fibrin is a convenient substance to use when demonstrating the activity of the digestive juices. Pepsin in an acid solution, and trypsin (from the pancreas) in an alkaline solution, cause first a splitting of the fibrin into two globulins, one coagulating at 06°, the other at 75° C. ; then the formation of albumoses and peptones. The subject of the solubilities of fibrin has been worked at by numerous experimentalists. The following is a resume of the different views that have been held : — The researches of Denis and of Hammarsten have been already referred to. Gautier' (1874) speaks of the proteid which goes into solution as an albumin. Hoppe-Seyler- has shown that it is a globulin, but considers that putrefaction plays an important part in the process of solution. Otto' also states that the sub- stance in solution is of the nature of paraglobiilin (now more commonly known as serum-globulin). Green* has (carefully preventing putrefaction) demonstrated that the globidiu in solution is not exactly like either of the two globulins of the plasma (fibrinogen and serum-globulin), but that it is in reality a mixture of two new globulins— one soluble, the other insoluble in 1 per cent, sodium chloride solution.^ I s have shown that the discrepancies in the heat-coagulation temperature, as noted by various observers, are due to the difEerent salts used for dissolving the fibrin. The solubilities of fibrin have been discussed from another point of view, viz. as a source of fibrin-ferment, by Gamgee' and by Lea and Green.* The solubilities of fibrin in digestive fluids have been a subject of investigation in all recent researches on digestion (Kiihne and Chittenden, &:c. &c., !" A. Herrmann, Ibid. p. 508. 234 THE TISSUES AND ORGANS OF THE EODY and alcohol ; it is then collected, dried, and weighed on a filter of known weight. Lastly it is incinerated and the weight of the ash deducted. In liquids like pericardial fluid, which do not coagulate spon- taneously, a small quantity of serum or of an active solution of fibrin- ferment must be added, and then the fibrin collected, washed, and w^eighed after coagulation has occurred. In blood which has already coagulated, the washing of the fibrin free from corpuscles is a long and troublesome process. In making comparative experiments of the amount of fibrin in two liquids, say in two specimens of pericardial fluid, instead of weighing the fibrin it may be stained with carmine, and then subjected to the action of equal amounts of artificial gastric juice at 40° C. The fibrin dissolves and tlie carmine passes into solution. The liquid most deeply coloured is that which had the most fibrin in it. The relative amount of fibrin in the two liquids may then be ascertained by finding out how much it is necessary to dilute the more deeply coloured one until it has the same tint as the other. The fibrin factors. — A. Schmidt considered that fibrin was formed as a union of fibrinogen and fibrino-plastin (now called serum globulin), and that this union was accomplished by the activity of the fibrin ferment. The two substances fibrinogen and ' fibrino-plastin ' were therefore termed the fibrin factors. Hammarsten has, however, shown that Schmidt's fibrino-plastin takes no part in the formation of fibrin, but that fibrinogen is the only fibrin factor, or fibrin precursor in the plasma. The term fibrin factor might veiy conveniently be dropped alto- gether, as the word fibrinogen has precisely the same meaning. Fibrinogen This may be prepared from plasma in the following ways : 1. The plasma is diluted with 15 times its volume of cold water, and a stream of carbonic acid gas passed through it ; a precipitate of .serum-globulin is obtained, and filtered off" : the plasma is then further diluted and again a stream of carbonic acid passed through it ; a further precipitate occurs which consists of fibrinogen. This is Schmidt's method ; it is, however, one which causes only aii incomplete and imperfect separation of fibrinogen from the plasma. 2, Hammarsten's method depeiids on a property in which filjrinogen differs from serum-globulin, in being completely precipitated from its solutions, by half saturation with sodium chloride, i.e. In' mixing the solution with an equal quantity of saturated solution of .sodium chloride. THE P.LOOT) 23;") The precipitate so ol)tiiinecl is washed witli a lialf-saturated solution of the salt, then dissolved in a 6-8 per cent, solution of the same salt, and again precipitated by half saturation. These operations should be performed rapidly, as prolonged contact with a half-saturated solution of sodium chloride renders the precipitate of fibrinogen very insoluble. The precipitate finally obtained is apparently soluble in water ; but it is enabled t<» dissolve in water by means of the salt adhering to it. By this method til )i-inogen may be prepared, not only from plasma and lymph, but also from exudation fluids such as pericardial and hydrocele fluids. Fibrinogen so obtained is found to have the properties characteristic of globvdins, viz. insohxbility in pure water, solubility in water con- taining oxygen, and in weak solutions of neutral salts. It is precipitated from such solutions by dialysing away the salt, or. by increasing the concentration of the salt beyond a cei'tain point, in the case of sodium chloride up to half saturation, i.e. about 18 per cent. The characteristic properties of fibrinogen are : 1. In the presence of minute quantities of certain salts of which sodium chloride and calcium sulphate seem to be the most important, the addition of fibrin-ferment causes the formation of fibrin. Without such addition, a solution of fibrinogen prepared by Hammarsten's method will remain uncoagulated indefinitely. 2. It enters into the condition of a charactei-istically sticky heat- coagulum at the very low temperature of 56° C This is true, not only with regard to solutions of pure fibrinogen ; l)ut that a heat-coagulum is formed at the same temperature in pure plasma obtained by the living test-tube experiment is a very striking proof that fibrinogen is present as such in the lilood. ' This was first shown by Hewson,^ and the fact was subsequently rediscovered by Fi-edericq.-'* Hammarsten ^ showed that in the formation of fibrin from pure fibrinogen, as well as during the process of heat-coagulation, not only is there a formation of a solid clot, but simultaneously a small quantity of a proteid (a globulin coagulating at a temperature of 65° C.) enters into solution, which is probably a decomposition product of the fibrinogen molecule. 1 This fact cannot, however, be regarded as absohite proof that fibrinogen is present as such in the circulating blood. Injection of fibrin-ferment into the circulation does not necessarily cause intravascular clotting ; it is therefore possible that certain counter- acting agencies j)revent fibrin being formed in healthy circulating blood, or that the fibrin when formed is immediately redissolved. Wooldridge supposes that in the circulatmg blood a precursor of Hammarsten's fibrinogen is present, and that this is readily changed into fibrinogen when the blood is shed. ^ Hewson, Wo7-ks edited by Grulliver, p. '2(i. •"' Fredericq, Becherches sur la constitution (hi plasma sangiiin, Gand, 1878. ■• Hammarsten, Pfliigefs Archiv, xxii. 480. 236 THE TISSUES AND ORGANS OF THE BODY 3. The fact that half - saturation with sodium chloride will com- pletely precipitate fibrinogen from its solutions is important, as it enables us to separate it from serum-gloljulin, to which it is so similar in many particulars. 4. The fact that it is necessary to dilute plasma to a greater extent than in the case of serum-globulin, in order to precipitate it by a stream of carbonic acid, is also characteristic. 5. Its specific rotatory power for yellow (i.e. sodium) light is 43° (Herrmann '). The opalescent character of solutions of fibrinogen, however, renders polarimetric observations difficult. 6. Its percentage elementary composition is as follows : C, 52"93 ; H, 6-9 ; N", 16-16 ; S, 1-25 ; O, 22-26 (Hammarsten 2) ; i.e. there is a slightly higher percentage of car})on, hydrogen, and oxygen than in the fibrins which is formed from it. Estimation. — The quantity of fibrinogen in a solution may be approximately estimated by weighing the washed and subsequently dried fibrin obtained by the addition of fibrin-ferment, or by similarly drying and weighing the precipitate caused by heating the slightly acidified solution to 56° C. Serum -globulin This substance was formerly called fibrino-plastic substance by Schmidt,^ and paraglobulin by Kulme.^ The name serum-globulin was given to it by Weyl.^ The serum-casein of Panum ^ has also been shown to be the same substance. It may be prepared from blood plasma or exudation fluids (e.g. pericardial or hydrocele fluid) after they have been heated to 56° C and the heat-coagulum of flbrinogen, which is formed at that tempera- ture, removed by filtration. It is most fi-equentl}- prepared from blood serum. The following are the various methods that have been adopted for the preparation of serum-globulin : — 1. The serum is diluted with fifteen times its Ijulk of water, and a stream of carbonic acid passed through it. The precipitate is collected and washed with ^water that contains no oxygen dissolved in it (Schmidt). 2. The serum is similarly diluted, and a few drops of 2 per cent. 1 Herrmann, Zeitsch. jjhysioL Chem. xi. 508. - Hammarsten, Pfiiger's Archiv, xxii. 480. 5 A. Schmidt, Arch. f. Anat. u. Phijsiol. 1861, p. 545, and 1862, p. 428. * W. Kiihne, Lehrbuch d. plujsiol. Chem. Leipzig, p. 174. This name is also used by Hammarsten, Pfliiger's Arch. xvii. 413 ; xviii. 35. 5 Weyl, Zeitsch. f. physiol. Chem. i. 77. ^ Panum, Archiv f. jiaihoJ. Anat. iv. Till': liLooi) 237 acetic acid juldcd. Tlierc is a .small precipitate Kolut)le in excess of the acid; a small precipitate is also produced l)y acetic acid after the remo\al of the precipitate pi'oduced by carbonic acid ; hence it was supposed to be somethint? different from serum-globulin and was called serum-casein (Panum). Hammarsten has, however, shown that both these methods produce but a very small precipitation of the globulin of serum, and that Panum's precipitate consists of the same substance as Schmidt's, There is no special serum-casein, or alkali-albumin in normal blood. 3. Mere dilution with ten to twenty times its volume of water will cause a small precipitation of the globulin from serum. In other words, globulins require a certain proportion of salts for their solution ; this may be lessened by dilution, and hence the precipitation observed. 4. The same result is brought about by dialysing away the salts (see p. 120). The precipitation, however, is never complete. 5. Saturation with neutral salts. There are many salts that pro- duce precipitation of the globulin.' Hammarsten was the first to point out that magnesium sulphate is the best to use for the purpose ; it effects an absolutely complete precipitation of the globulin, and is for this reason preferable to sodium chloride, ^ which was employed origin- ally by Denis, and subsequently by Schmidt. It precipitates none of the serum-albumin. Saturation with ammonium sulphate or potassium acetate precipitates all the proteids of the blood. It is stated by Kauder^ that half-saturation with ammonium sulphate precipitates only the serum-globulin. In all saturation experiments, the liquid should be approximately neutral to start with. Liquids like serum are, however, sufficiently near to the neutral point for the purpose, but in certain abnormal urines where globulin may be present, it is necessary to neutralise their acid reaction before saturation. In order to ensure complete saturation at the temperature of the air, it is necessary to thoi-oughly shake the mixture of salt and serum for two or three hours ; this may be done most readily by a motor- of some kind. The precipitate is collected on a filter and washed with a saturated solution of the salt used. When greater purity is required, the precipitate may be redissolved by adding distilled water ; the water is able to dissolve the globulin by means of the salt adhering to it, and then it can be reprecipitated by saturation.. 1 For a fuller account of the action of various salts see Halliburton, Journ. of Physiol. V. 176, et seq.; Lewitli, J.fiir eocperlm. Path. u. Pharmakol. xxiv. 1 ; Hofmeister, Ibid. p. 247. - A full account of the solubilities of serum-globulin in solutions of sodium chloride of different strength will be found in Pfliiger's Archiv, xviiii. 39 (Hammarsten). ' Kauder, Arch. f. exp. Path. u. Pharmakol. xx. 411. 238 THE TISSUES .\>'I) ORdANS OF THE EODY In solutions containing at least 1 per cent, of globulin it may be detected by the ring of precipitate that occurs at the junction of the two liquids, when the solution is poured on to the surface of a satui'ated solution of magnesium sulphate. Many of the distinctive characters of this globulin have necessarily been described in the foregoing paragraphs i-elating to its preparation. To these may be added the following : — It coagulates on heating to 75° C, becoming opalescent a few de- grees below that point. Its specific rotatory power for sodium light is 59-75° (Haas). The estimation of sev-inu-globulin quantitatively may be cai'ried out as follows, in such liquids as serum. The serum is saturated with magnesium sulphate and filtered, the precipitate being collected on a filter of known ash which has been previously dried and weighed. The precipitate is washed with a saturated solution of magnesium sulphate, and then the filter with its adherent precipitate is dried at 120° C. This temperature in a few hovirs renders the globulin insolu]:)le ; the salt is then washed away with water. It is subsequently washed with alcohol and ether, dried, weighed, incinerated, and the amount of ash deducted. Estimations of serum-globulin made previous to Hammarsten's I'esearches by means of the carbonic acid or dialysis methods are much too low and are practically valueless. Sources of serum-glohuHn. — The total globulin in the serum is greater than that in the plasma, but the greater part is undoubtedly pre-existent in the plasma. Schmidt considered at one time that it was derived almost entii-ely from the disintegration of the white corpviscles, that occurs when the blood is shed. It is now generally admitted that a small, but a very small, quantity of the globulin is formed by such disintegration. This may be termed cell-globulin, and is closely allied to, or probably identical with, the fibrin-ferment. Lastly, some of the globulin is dei'ived from the decomposition of the fibrinogen molecule when coagulation occurs {see p. 235). This second globulin of Hammarsten differs somewhat in its solubilities from the serum-globulin pre-existent in the plasma, and may be separated from it by fi-actional saturation with sodium chloride. The globulin of serum thus consists of a mixture of three globulins, all -closely allied in their properties to one another ; these are :■ — 1. The globulin pre-existent in the blood plasma ; this may be termed plasma-glohuJin. 2. The globulin arising from the disintegration of the corpuscles — cell-globulin. THE HLOOl) 239 'X Tlie globulin arising from the splitting of tlio fibrinogen molecule ■ — lf((iniii(irf 1 Gamgee, Jaiirn. of Phijsiol. ii. 145. - Tliis term is Buchanan's. It means fibrin obtained from blood which has been diluted with 8-10 times its bulk of water immediately it is shed. The same facts, how- ever, hold for fibrin prejiared approximately free from corpuscles in the usual way. ^ Halliburton, Proc. Roy. Sac. xliv. 255. •* Lea and Green, Journ. of Physiol, iv. 380. R 242 THE TISSUES AXD ORGANS OF THE BODY become a constituent part of the fibrin formed, though much of it remains adherent to the fibrin, as well as being dissolved in the serum. There are other globulins which have the same effect upon the formation of fibrin that cell-globulin has. For instance, the niyosinogen of muscular tissue is one of these {see Muscle). The addition of living cells, such as yeast cells, or pieces of many fresh tissues, to such liquids as hydrocele fluid or dilute salted plasma causes a rapid formation of a clot. In these cases, if neither cell-globulin nor myosinogen is present, in all probability there are similar unstable globulins in the cell-protoplasm which act in the same way. Historical account of the theories of Coagulation Xearly up tu the end of the eighteenth century the clot was supposed to consist of merely adherent corpuscles. This x'lew was held in Britain by Keill, Jurin, Thomas Morgan, John Cook, Arbuthnot, Cowper, Langrish, Berdoe, and others ; and on the Continent h\ Leeuwenhoek, Boerhaave, Van Swieten, Haller, and Marherr. Petit, Quesnay, Senac, Borelli, and Davies were the earliest to have an idea of a coagulable substance in addition to the cells, and this was fully recognised and proved by Hewson (1772), and taught by Fordyce and the Hunters.^ The reason that the blood coagulates outside the vessels and not during life was accounted for in diflerent ways ; some considered that co- iigulation was due to the action of the air on the blood (Borelli, Lower) ; others that the blood was maintained in its liquid condition during life by its continual movement ; others, again, that coagulation was due to the cooling of the blood on its removal from the vessels.'^ We how- ■ever now know, on the contrary, that blood will clot even if collected over mercury without coming in contact with the air at all, that agitation hastens and cooling hinders coagulation. Hunter considered that coagulation was an act of life, and connected with the ^•itality of the blood^ — a vague statement which implies very little ; but, as OuUiver ^ pointed out, if it is a vital act, it is equivalent to saying that we are able to pickle the life of the blood for hours or e\ei\ days, although decomposition may have begun in other parts of the body. Hewson not only showed that a coagulable substance we now call hbrin separates from the plasma, which he obtained by skimming it off from the surface of blood which coagulated slowly, but he also discovered the fact that cold, contact with living vessels, and admixture with salts are agencies which hinder or prevent coagulation. In con- nection with the influence of the Uving vessels on coagulation, the 1 For the references to the flrritings of these authors see Hetison's Woiks, edited by OuUivei-, Sydenham Soc. p. xxix. et seq. - For a rtsume of these earlier views, I am indebted to Gamgee, Phijsiol. Choii. p. 42. '' Hctcson's Woi-ks, uote V2. p. 21. THK liLooi) 243 further researches of Lister, Frederieij, unci Briickc have been aheady referred to (.v'>" p. 224). Andrew Buchanan ' was the next who made noteworthy investiga- tions into this subject. He experimented witli fluid obtained from the pericardial sac and from the tunica vaginalis in the dropsical condition of that serous membrane called hydrocele. These liquids do not •coagulate spontaneously, but Buchanan found that the addition of small shreds of 'washed blood clot ' caused the formation of fibrin in them. This power was exhibited to a greater extent still by the ' butty coat ' of a clot ; he therefore concluded that the power resided in the white blood corpuscles which are so abundant in the bufiy coat, and their action he compared to the action of rennet in curdling milk. Then came Denis,- who saturated blood plasma with sodium ■chloride, and thus obtained a proteid precipitate. This precipitate was washed with a saturated solution of sodium chloride and redissolved by the addition of water, the adherent salt rendering it soluble. This solution remained liquid for a short time, but on being allowed to stand a clot of tibrin was produced. Denis had thus obtained the precursor of fibrin from the plasma, and to it he ga\e the name phxsinine ; the pr< (teids, which were not precipitated by the salt, he called serine, ov, -as we now call them, serum-albumin. We now know that Denis' plasraine was a mixture of fibrinogen, serura-globulin, and fibrin-ferment. Alex. Schmidt ^ recognised these three substances ; he, howevei-, supposed that all three were necessary for the formation of a clot. One of the most important experiments on which he based this view was, that if serum which contains serum-globulin (or fibrino-plastic substance, as he termed it) and ferment be added to hydrocele or pericardial fluid, which he supposed contained fibrinogen but no serum- globulin, the result is the formation of tibrin. He also found that the more serum-globulin he added to a coagulable liquid the larger was the yield of tibrin from it.^ 1 A. Buchanan, London Med. Gazette, xviii. (1835), p. 50; also vol. i. new series (1845), p. 017. The latter paper was reprinted by Dr. Ganigee in the Journ. of Physiol. vol. ii. (lK7i»). The influence of leucocytes in bringing about coagulation was very strongly insisted on by Mantegazza {Centr. Med. Wiss. 1871, p. 709). - Denis, Memoire sur le sang, 1859, p. 32. ^ A. Schmidt, Arch. f. Anat. u. Physiol. 1861, p. 545 ; ISIW, pp. 428 and 533. Pjliigcr's Archiv, vi. 445. ^ In connection with the question whether or not the ferment is a globulin, it is interesting to note that the proteid present in Sclunidt's ferment solutions, and which some have considered as an impurity, was one which was jirecipitable by a stream of carbonic acid; he also found that serum minim its globulin has very little ferment Activity ; that it still possesses any is due to the fact that Schmidt's carbonic acid method ■does not completely precipitate tlie globulin. R 2 244 THE TISSUES AND OEGANS OF THE BODY O. Hamniarsten ' ascertained the cliaracters of fibrinogen, serum- globulin, and filjrin-fennent with greater exactness, and showed that serum -globulin, or paraglobulin, as he terms it, is not necessary for the formation of fil)rin, l)ut that fibrinogen is the only precursor in the plasma of the fibrin in the clot. He pointed out that pericardial and hydrocele fluids contain abundance of serum-globulin, as well as fibrin- ogen, and therefore the addition of serum causes these fluids to coagu- late, not in virtue of tlie serum-globulin, liut of the ferment it contains. He pointed out that serum-globulin is very diflicult to separate from ferment, a fact which is easy to understand, as we now know that the ferment is probably itself a globulin; the addition of apparently pure serum-globulin, prepared from serum, to hydrocele fluid causes the formation of fibrin because of its admixture with ferment. A pure serum -globulin prepared from pericardial or hydrocele fluid has on the other hand no fibrinoplastic activity. The most striking proof, however, of Hammarsten's theory is this : that a solution of the ferment added to a solution of pure fibrinogen causes the formation of fibrin. It should be mentioned that Hamniarsten does not regard the ferment as a globulin, because he is able to prepare it from horse's serum which has apparently- been deprived of all globulin by saturation with magnesium sulphate. Howells - and also Hayem ' have tried this method with the blood of other animals, but unsuccessfully, and in the course of my own work I have done the same, and again with a negative result. In the case of horse's serum, however, I ha^-e found that Hammarsten's statement is correct ; and the explanation seems to be that for some reason or other it is exceedingly difficult to ^jrecipitate all the globulin from horse's blood by the use of this salt ; but after repeated saturations one can remove all the globulin, and \s'ith it all ferment activity also. The researches of Gam gee, of Lea and Green, and of myself, into the nature of the fibrin ferment have been already alluded to {see p. 241). To Green also we owe the discovery that the presence of calcium sulphate is necessary for the proper action of the ferment to take place. This again reminds us of Buchanan's old comparison of the clotting of blood to the curdling of milk, where the j^hosphate of calcium is a sine qua nan. Ah hough Hamniarsten did not consider serum-globulin necessary for the formation of fibrin, he admitted that its presence was advan- tageous ; it can, however, be replaced by other proteids like casein, or even by salts like calcium chloride. He considers that serum-globulin possibly acts like calcium chloride in combining with the alkaline i O. Hamniarsten, Py/((5'er's Archiv, xiv. 211; xvii. 413; xviii. 08; xix. 503; xxii. 489. 2 Howells, Studies from the Fhijsiol. Lab. Johns HojiJaiis U)tii\ Baltimore, vol. ii. 3 Du sang, Paris, 1889. THE BLOOD 245 'Carbonates present in the blood, the presence of wliich would otherwise impede the activity of the ferment. The supposed action of calcium vchloride in this respect may be represented by the formula NajCOg + CaCl,=2NaCl + CaCOs. Of recent years an entirely new theory was advanced by the late Dr. Wooldridge, which may be stated as follows:' — The coagulation of tlie blood is a phenomenon essentially similar to crystallisation ; in the plasma there are three constituents concerned in coagulation, A, B, and C fibrinogen. A and B fibrinogen are com- pounds of lecithin and proteid, and fibrin results from the transference of the lecithin from A-fibrinogen to B-fibrinogen. C-fibrinogen is what hasMtherto been called fibrinogen ; A-tibrinogen is a substance which may be precipitated by cooling 'peptone plasma,' and on the removal of this substance coagulation occurs with great difficulty. The precipitate produced by cold consists of rounded bodies resembling the blood tablets in appearance. He further found that other compounds of lecithin and proteid, to which he has extended the name of fibrinogen, exist in the testis, thymus, and other organs, in the fluid of lymph glands, in the stromata of red corpuscles, and in the serum of certain animals : these substances may be extracted from the organs by water, and precipitated from the aqueous extract by acetic acid, and on redissoMng this in a saline solution, and injecting it into the circulation of a living animal, intravascular clotting occurs, which results in the death of the animal. The form of fibrinogen that acts thus, he looks upon as the precursor of A-fibrinogen. From these points of view the fibrin-ferment and the white corpuscles are looked upon as of secondary import in causing coagulation, though it is admitted that fibrin-ferment converts C-fibrinogen into 'fibrin. I have elsewhere * given at some length my reasons for not accepting this theory-, and this is not the place for debating a controversial subject. I will merely say that I still consider Wooldridge erred, first, in drawing conclusions from observations on p»eptone plasma without corroborating them by experiments on pure plasma; and, secondly, in attributing to the corpuscular elements a secondary role in the causation of clotting.' The latest theory of coagulation is that of Freund ; ^ he considers that, when blood is shed, earthy phosphates derived chiefly from the corpuscles unite with fibrinogen and thus form fibrin. Serum-Albumin Serum-albumin, or serine, is the proteid which remains in solution after the separation of serum-globulin from the serum. Now that Hammarsten's method has been adopted for the separation of serum- 1 "Wooldridge, Ludwig's Festschrift, 1886, p. 221. - Journ. of Physiol, ix. 270. Wooldridge defended liis views in the same Journal, X. 339. ^ Kriiger iZeit. Biol. xxiv. 189) and Hayem {Du sang, Paris, 1889 1, who have also recently examined Wooldridge's views, are unanimous in regarding tlie corpuscles as most important factors in fibrin-formation. * Med. Jahrb. 1888, p. 259. 240 THE TISSUES .V^'D ORaANS OF THE BODY globulin, wf know that it is more abundant than had been preWously supposetl, and may in certain serums be present in even greater abun- dance than serum-albumin. Serum-albumin may be prepared from serum which has been freed by Hammarsten's process from serum-gloVjulin in the following way : The precipitated globulin is filtered oflF ; the filtrate, which is already saturated with magnesium sulphate, is then saturated with sodium sulphate ; ^ the result is a precipitation of serum-albumin in the form of fine flocculi ; this is washed with water saturated with the two salts, and is readily soluVjle on the adflition of water. If sodium sulphate is added to serum which has not Ijeen treated with magnesium sulphate, there is little or no precipitate. Mag- nesium sulphate alone precipitates the globulin : double saturation with the two salts precipitates both the proteids of the serum. The explana- tion of this is that when sodium sulphate is addetl to magnesium sulphate,, the double sulphate of magnesium and sodium is formed, and this it is which precipitates the serum-albumin. The formtda for magnesium sulphate is MgSOj.THoO : that of sodio-magnesium sulphate is McfSO^-NaoSO^-GHoO ; that is, a molecule of sodium sulphate takes the place of one of the molecules of water of crystallisation of the magnesium sulphate. Sodio-magnesium sulphate is obtained commercially as a by-product in the manufacture of Epsom salts. Saturation of solutions of proteids, such as serum, with this salt causes a complete precipitation of all the proteids contained therein. Like ammonium sulphate it does not precipitate peptones. - This method affords us a means by which we may oVjtain serum- albumin free from other proteids ; it may be further purified by repre- cipitating it from the solution with the double sulphate, and if necessary it may be again dissolved and reprecipitate<^l. The adherent salt may be ultimately removed by dialysis. It is, however, not possible to completely remove the salts from this or anv other proteid. A small quantity of ash (0'3 to 0*5 per cent.) will always be obtained from the purest preparations, Aronstein and Schmidt stateeen able to confirm these experi- ments, and the non- coagulation noted by Aronstein and Schmidt was ' Sodium nitratft, ammonia alum, and potassium iodide act similarly. ' The action of sodium sulphate in precipitating serum-albumin has been worked out bv Denis iMemoire sur le aang, p. 3»), Schafer iJoum. of Physiol, iii. 181), and HaUiburton, Ibid. \. Ill nt Sfq. THK ]?L(>()I> 247 in all prol)ability clue to the adherence of alkaline salts in small quan- tities to the proteid.' Serum-albumin gives the usual proteid tests ; it differs from globulins in its solubility in water, and the fact that it is less readily precipitated by saturation with neutral salts. It ditters from egg-albumin in its. specific rotatory power : ^ Egg-albumin : Specific rotation for yellow light (a)j,= — 33'5°. Serum-albumin : Specific rotation for yellow light (a)i,= — 56°. Ether does not precipitate serum-all)umin : it does precipitate egg- albumin. Further investigation has shown that serum-albumin is not a single substance, Init probably a mixture of several allmmins. By fractional heat-coagulation it is possible to separate it into three proteids, coagulating a at 73°, /? at 77°, and y at 84°C.-'' (see p. 118). The results of elementary analysis also lead to the same conclusion.^ The following tables illustrate certain points in the comparative chemistry of serum and plasma. Table I Proteitls ill the Blood-Serum" Animal Man" . Total Proteids per cent. Senim-Globiilin f Serum-Albumin ' 1 7-62 3-10 4-52 1 Horse" . 7-25 4-5fi 2-67 1 Ox« . 7-50 417 3-3:i ^ Rabbit'-. 7-52 1-78 4-43 ' Pigeon". .5-01 1-32 3-6i» Hen' . . . • ■±■14 2-90 1-24 { Tortoi.'^e' 4-76 2-82 1-94 1 Lizard". 5-16 3-33 1-83 Terrapin'' o-2o 4-66 0-69 Snake'". 5-32 4-9.5 0-37 Frog'* . 2-54 2-18 0-36 1 Newt" . 3-74 3-31 0-43 ' Eer . G-73 .5-28 1-45 Dogfish" lfi2 117 0-45 1 Aronstein, Pfliiger's Archiv, viii. 173. A. Sclunidt, Liidwig's FestgaLe, 1874, p. 94 ; Pfliigefs Arcliiv, ii. 1. Heyiisius, Ibid. Lx. 514. Winogradoff, Ibid. ii. p. 605. Huiziiiga, Ibid. ii. 39-2. Haas, Ibid. xii. 378-410. - Hoppe- Sevier, Zeitsch.f. Chem. u. Pharni. 1864, p. 737. ^ HaUibiirton, Journ. of Physiol, v. 15'2. * Kauder, Arch. f. exp. Path. u. Pharmak. xx. 411. •> The total proteids are estimated by weighing the precipitate produced by adding alcohol to the serum ; the globuhn is estimated in another portion by Hammarsteu's method (p. 238) ; the difference between the two gives the amount of albumin. fi Hanunarsten, ' Ueber das Paraglobuhn,' Pfiiigt^r's Archiv, 1878. ' HalHburton, Journ. of Physiol, vii. 321. ^ May, Ibid. p. 319. ^ Howells, Studies from the Biol. Lab. Johns Hopkins Univ. Baltimore, iii. 49. '" Wolfenden, Journ. of Phgsiologtj, vii. 323. 248 THE TISSUES .\XD OKGANS OF THE BODY Table II > Heat-Coagulation Temperature of 1 Blood of 1 Fibrinogen ^erum- , .„ - Globulin teerum-Albumin 1 Man . . . . 56° C. « /3 75° C. 73° C. 77° C. 7 85° 0. Monkey Dog . 56° 56° 75° 72° 1 77° 75° 73= 78° 83° 84° Cat . 56° 75° 73^ 77= 84° Eabbit 56° 75° 73- 77= 84° Pig . Horse 56° 56° 75° 72' 77° 75° — 77° 84° 84° Ox . 56° 75° — 77° 84° Sheep Hen . 56° 56° 75° — 77° 75° 72-3= 78° 84° 86° Dove. 56° 75° 73= 77° 85° Newt 56° 75° 73° — ' — Toad. 56° 75° 75° — — ! Frog. 56° 75° 73° — — ' Lizard 56= 75° 74° — — Perch •,6° 75° 73= — — Roach 56° 75° I 73= — "' The above tables give merely illustrative examples from the diffe- rent groups of the vertebrate kingdom. From them the following •conclusions can be drawn : — 1. The temperature of heat-coagulation of the two globulins of the plasma (fibrinogen and serum-globulin) is exceedingly uniform through- out the vertebrate kingdom. The small amount of fibrinogen, as judged by the smallness of the clot in cold-blooded animals, has been already alluded to (p. 222). 2. In warm-blooded animals, mammals and birds, the serum-albumin can be differentiated into three proteids by a process of fractional heat- coagulation. In certain ungulates, however (horse, ox, sheep), only two varieties (/3 and y) of serum-albumin are present. 3. In cold-blooded animals, the proteids differ from those of warm- Wooded animals in the following points : — (a) The percentage of total proteids is smaller. [h) The serum-albumin is especially diminished, not only absolutely but relatively to the serum-globulin present. (c) The serum albumin is a single proteid, corresponding tt) that ■called serum-albumin a in the hicrher vertebrates. 1 Compiled from Papers by myself in the Joiirn. of Phi/sioL v. 159, and vii. 320; 'Quart. Journ. of Mic. Science, xxviii. 193. TilE JJLOOJJ 249 It has been stated that during starvation the serum-albumin diminishes more quickly than the serum-globulin ; in the case of snakes, Tiegel has said that the albumin altogether disappears. Burckliardt stated that much the same occurs in dogs. These observers did not, however, employ tlie only exact means of fstimating the proportion of globulin and albumin, wiiich is Hammarsten's magnesium sul])hate method. Howells and Salvioli, who employed this method, .showed tliat tlie observations of Tiegel and Burckliardt were incorrect.' In concluding the .subject of the proteids of the pla.sma and serum, it may be added that no proteo.se.s (albumo.ses) or peptones are to be found in normal l)l()()d, even in the portal circulation when absorption is taking place. Thi.s subject will be discussed at greater length in the -chapter on Absorption. The following scheme represents a method of separating the proteids of tlie plasma or of similar fluids : Plasma. Add an equal volume of saturated solution of sodium chloride. A precipitate is ])i-oduced. Filter. Precipitate : consists of FIBRINOGEX Filtrate contains the remain- ing proteids. Saturate with magnesium sulphate ; a pre- cipitate is produced. Filter. Precipitate : consists of SERUM-GLOBULIN Filtrate contains serum - ALBUMIN. Heat to TS'^C. ; a precipitate is produced. Filter. Precipitate is a heat- coagulum of serum-albumin a Filtrate contains serum-albu- min /3 and y. Heat to 77° C. ; a precipitate is produced. Filter. Precipitate is a heat-coagu- Filtrate contains serum-albu- lum of serum-albumin /3 min y, which is precipitated at 84° C. ' For reference to this subject see Journal of PhysioJogr/, vii. 322. 250 THE TISSUES AND ORGANS OF THE BODY The following is another method : — Allow either spontaneous coagulation to take place, or in the case of salted plasma, dilute and add fibrin-ferment. A clot forms, and a liquid residue called seiaim can be filtered off. Clot=tibrin Liquid residue = serum. Saturate- with magnesium sulphate ; a pre- cipitate forms. Filter. Precipitate =SERU>: GLOBULIN Filti'ate contains serum- albumin. The fcjllowing method illustrates how by the use of various salts the proteids may be separated. Plasma. — Saturate with ammonium sulphate ; a precipi- tate forms. Filter. Precipitate consists of proteids. Wash with saturated solution of am- monium sulphate. Redissolve the pre- cipitate by the addition of water. All the proteids dissolve. Saturate this solution with magnesium sulphate ; a precipitate forms. Filter. Precipitate = globulins (plasmine — Denis) (:= fibrinogen + serum-globulin). Wash this precipitate with saturated solution of magnesium sulphate ; then add water, the globulins redissolve owing to the presence of adherent salt. Heat this solution to '56°C. ; the fibrinogen is precipitated as a heat- coagulum at 56°, the serum-globulin remaining in solution. Or add an equal volume of saturated solution of NaCl ; a pp. forms. Filter. f Precipitate = fibrinogen (Filtrate contains serum globulin. Filtrate contains the other constituents of the plasma. Filtrate contains albumins which may be precipitated and separated by frac- tional - heat-coagulation. Or saturate the solution with sodium sulphate ; a precipitate forms. This precipitate = albumins, which may be redissolved in water, and then separ- ated by heat-coagulation. TllK i;i,n()l> 251 j;XTllACTIVES OF THE PLASMA AND SERUM Wo now come to the secinul ^rouji of organic substances in the li((Uoi- sanguinis. They are caHed extractives, because they can ))e extracted from the various liquids of the body by reagents like alcohol and ether ; they are present only in small quantities. The extractives of the seium are the same as those of the plasma. Ether extractives. — These consist of neutral y^.te, and cho/esterin, and in quantity vary from 0-2 to 0"6 ^ier cent. The fats are more abundant after a fatty ineal ; and minute fat globules can then be recognised in the serum by the microscope. Sometimes this is so marked that the serum looks milky like the chyle. The fats present are the same as those in adipose tissue (palmitin, stearin, olein). Rohrig' stated that soluble soaps cannot exist in the blood, as was at one time supposed ; but Hoppe-Seyler'^ has pointed out errors in Rohrig's analyses, and found ivom 0"Or)-0'l per cent, of soaps in serum. About one-tenth of the ethereal extract consists of cholesterin,"^ the chemistry of which will be taken with the nervous tissues, and bile. LprifJtin is also present in small quantities. The chemistry t)f this suljstance will be treated of in connection with nervous tissues. Nitroypnous compounds. — Urea and uric acid are found in small quantities in the blood ; the quantity of urea varying between 0*02 and 0"04 per cent, in human blood. ^ Creatine, xanthine, hypoxanthine, and hippuric acid are found in still smaller quantities. In certain diseases they are increased in quantity, and other substances belonging to the same category may appear, e.g. leucine and tyrosine in acute yellow atrophy of the liver. In certain forms of Bright's disease the amount of urea is much increased ; and in gout and allied affections there is an increase of the uric acid. It will be convenient here to mention the way in which these two substances may be estimated. Estimafion of urea, in hJood or !eniiii-iv.:e:!:. li^ere :- a ;ar&e a:—rx--:v;. o: t.'.e ".oiet eud of the spcctmnL and in tliis trivo ilarker sha>'o(oj)Iasin. — Here again microscopic methods teach us that protoplasm is not always the uniform jelly we were once led to suppose, but consists in many cases of a fine network or reiicu/iim, enclosing in its meshes a more fiuid material or enchylema (Carnoy). In the white l)lood corpuscles the granules seem to be entangled with the reticulum. On the application of dilute acetic acid, the granules and reticulum shrink around the nucleus. On the application of water, or more quickly with dilute potash, the protoplasm swells, and ultimately the corpuscle bursts and disintegrates. The partial disappearance of the granules when the l>lood is shed was observed by Haycraft to accompany the shedding out of the fibrin-ferment, or rather the formation of fibrin in the surrouaiding plasma. By the use of osmic acid, fat granules, which are stained black by 1 Miescher, Hoppe-Seijler's Med. Chem. Untersuch. Heft iv. 441. - Botan. Zeitung, 18H7. ^ Kossel, Zeitsch. f. /jhi/siol. Chem. x. '24S. 260 THE TISSUES AND ORGANS OE THE BODY this reagent, can be demonstrated to exist in the cell-protoplasm, Imt in especial al>undance in the white corpuscles and lymph cells of the intestinal vessels during absorption. The same is true with regard to glycogen, which can be detected microchemically by a solution of iodine in potassium iodide ; this stains glycogen a deep mahogany colour. Lecithin, cholesterin, and inorganic matter exist in small quantities in the white corpuscles, l)ut the bulk of the protoplasm is undoubtedly proteid in nature ; and though our present methods do not enable us to say which proteids are contained in the reticulum, and which in the enchylema, yet Ijy using extracts of lymph cells from lymphatic glands we can at least identify the proteids which are present.^ They are as follows : — 1. A mucin-like proteid similar to that described by Miescher^ in pus, and called hyaline substance by Rovida. This swells up into a jelly-like substance when mixed with 5 to 10 per cent, solutions of sodium chloride or magnesium sulphate ; on pouring such a mixture into water, this proteid extends in cohesive strings through the water, which soon contract and float on trie top. This substance is, howe^'er, not mucin, as it yields no reducing sugar on boiling it with sulphuric acid. It is also not nuclein, as the nuclei are not affected by the reagents used ; it resembles globulins in its solubilities ; it yields an ash rich in phosphorus ; and on digestion with artificial gastric juice an insoluble residue of the nature of. nuclein separates out. In all these points this proteid resembles the class of proteids named ' nucleo- albumins ' by Hammarsten.^ This is the most alnmdant of the proteids present in the protoplasm. It is probably identical with Reinke's plastin (*-ee p. 205). 2. Two globulins. These are obtained by dissolving the proteids of the lymph cells in a liquid prepared l)y mixing a saturated solution of sodium sulphate with nine times its volume of distilled water. This solution does not cause the swelling up of the nucleo-albumin like sodium chloride or magnesium sulphate solutions do. Then on saturat- ing this extract with magnesium sulphate a precipitate is obtained. This precipitate consists of the globulins, which may be washed, re- dissolved, and then separated by fractional heat-coagulation. They may be called cell-globulin a, which coagulates at about 50° C ; and cell-globulin, which coagulates at 73° C, On filtering off the heat- coagulum of cell-globulin a, which is generally only present in small quantities, the cell-globulin proper is alone left in solution, and this 1 Beports of the British Association, 1887, p. 145, and 1888, p. 'dm. Report of a Committee appointed to investigate the Physiology of the Lymphatic System. * Miescher, Hop2}e-Seyler's Med. Chem. TJntersiich. p. 441. ■> Hammarsten, Zeitsch. f. phijsiol. Chem. xii. ICiJ. •nil-: iiLooi) 261 has the properties of fibiin-fernient. Reasons liave already been given for considering the fibrin-ferment and cell-globulin as identical (.s-^'^'p. 241). 3. An albumin. After filtering oti' the globulins, the allmmin remains in solution. It coagulates at 73° C. and resembles serum- albumin a in its properties. It is present in very small quantities, and may be provisionally termed cell -alhmnin. In concluding this account of the proteids of lymph cells, it may be added that no substance like myosin or fibrin can be obtained from the cells ; there is, however, a formation of sarkolactic acid ' after death as in muscle : and if the glands be left, especially at the temperature of the body, for some hours after death, a process of self-digestion takes place, the pepsin present in the glands, as it is in most tissues (Briicke), becoming active when the reaction of the tissue becomes acid ; under these circumstances there is, in addition to the proteids already enu- merated, a small and varying amount of proteoses and peptones. The nucleo-albumin was mistaken by some of the earlier observers for myosin, from w^hich it differs markedly. Some also have mistaken it for fibrin 2 ; the way in which it extends in strings when poured into water accounts for this ; these strings subsequently contract, and here indeed is a point of resemblance between it and fibrin. But here all resemblance stops. THE BLOOD TABLETS In addition to the white and red corpuscles, a number of colourless discs averaging "002 — "003 millimetre diameter are also seen. They exist as such in the circulating blood. By some they have been supposed to be stages in the development of red corpuscles ; some** consider them to be masses of undifferentiated protoplasm, but their origin and destiny has never been explained. The action of inert solids upon them after the blood is shed is much the same as on white blood corpuscles. It causes them to become sticky, to run together, lose contour, change shape, and in many cases undergo complete dis- integration. Strands of fibrin start from collections of blood-plates, so probably one product at least of their disintegration is fibrin-ferment. In spite of the large amount of research from the histological stand- ^ It was Hirscliler who showed that tlie variety of lactic acid formed was sarkolactic acid (Zci7.sc/i. /. phy&iol. Chem.xi. 411. Berlinerblau {Chem. Centralhl. 1888, p. 757) states that lactic acid is a normal constituent of blood. But Salomon ( Virchoic's Archiv, cxiii. 356) has showni that fresh blood contains no lactic acid, but on standing a small amoiuit fomis, no doubt from changes of a fermentative nature in the wliite corpuscles. - Denis called it fibrins concrote globuline. Wooldridge also spoke of it as fibrin {Da Bois ReijmoncVsArch.f. Physiologie, 1881, pp. o87-411l. Hammarsten was the first to show that it is not true fibrin (see p. 232). ^ Haycraft, Jouni. of Anat. and Physiol, xxii. 302. '2(52 THE TISSI'ES AXI) ORCtANS OF THE I50T)Y point, on these blood tablets (Blutplattclien of Bizzozero'), we know virtually nothing of them chemically. The term hai-matoblasts has been a2:)pliecl by some to the blood tablets ; this is liable to cause confusion, as the same M'oi'd is used for the nucleated red corpuscles which occur in certain stages i)f the forma- tion of the non-nucleated red discs of vertebrates. The blood tablets are found only in mammals' Ijlood ; in fishes, birds, and amphibians they are absent, and according to Eberth and Schim- melbusch,^ and also Hayem,^ their place is taken in these groups by certain spindle-shaped nucleated cells. Lowit^ on the other hand regards the spindle cell as a variety of white blood corpuscle, and the blood tablets of mammals as something peculiar to that group ; accord- ing to him they consist chiefly of a globulin, and he considers they play an imj^ortant part in the formation of librin. THE RED BLOOD COEPUSCLES The red or coloured corpuscles give the red appearance to the blood. They are much more numerous than the white corpuscles, there being about 5,000,000 per cubic millimetre in the human male, about 4,500,000 in the female. The enumeration of the blood corpuscles is readily eftected by the hajmacytometer of Gowei's.* This instrument consists of a glass slide (fig. 5+ C), the centre of which is ruled into i\j millimetre squares and surrounded V)y a glass I'im i millimetre thick. It is proA-ided with measuring pipettes (A and B), a vessel (D) for mixing the blood with a saline solution (sulphate of soda of specific gravity 1015),^ a glass stirrer (E), and a guarded needle (F). The mode of proceeding is extremely simple. 995 cubic milli- metres of the saline solution are measured out by means of A, and then placed in the mixing jar ; 5 cubic millimetres of blood are then drawn from a puncture in the finger by means of the pipette B, and blown into the solution. The two fluids are well mixed by the stirrer, and a small drop of this diluted mixture placed in the centre of the slide C ; a cover glass is gently laid on (so as to touch the drop, which thus forms a layer i mm. thick between the shde and cover glass), and pressed down by two brass springs. In a few minutes the corpuscles have sunk to the bottom of the layer of fluid, and rest on 1 Bizzozeio, Virchow's Archil', xc. 2C1. - Virchoiv's Archil, cviii. 3tU!. 5 Hayem, Dii saiuj, Paris, 1881). ■* Archiv f. exp. Path. ii. Pharmakol. xxiv. 188. 5 Gowei-H, Lancet, Dec. 1, 1877. Malassez (Compt. rend. Is7-i) and Haj-eiu (Dii sa)ig) have invented veiy similar instruments. •• There are manv siniihir sahne sohxtions wliieh nv.iy \<^• fniployed. TiiK i;i,(H)i) 2()8 the .s(juares. The uumher on ten squares is then counted, and tliis iiiultii)lied by 10,000 gives the number in a cubic niillinietre of blood. The average number of red coi'2)uscIes in each scjuare ought, tlierefore, in normal human blood to be IH-fiO. Fkj. 54. — Hoem;ic\ tometc-r of Dr. Ciowtrt. (iliu.k' by Hiuvksley it Co., uxl'ord ,Street.) Specific gravity. — C. Schmidt gives the specific gTavity of red blood corpuscles as 1'089, Welcker as 1-105. Shape and s^'se.— They vary in size and structure in different groups of the vertebrate sub-kingdom. In Mammalia, with the ex- ception of the Camelidte, they are biconcave, circular discs ; they have no nucleus except during embryonic life, and they have a tendency to run into rouleaux when the blood is at rest, but if it is disturbed they readily become separated. In the Camel tribe they have an elliptical outline. Their average diameter in mammals is "OOT-'OOS millimetre ' (sirVo iiTLch), and al)out one-fourth of that in thickness ; there ai-e very slight variations in diSerent classes of mammals. In birds, reptiles, amphibians, and fishes, the red corpuscles are biconvex, oval discs, with a nucleus ; they are largest in the amphibia. Action of Microscojjic reagents. — Water causes the corpuscles to swell up, and at the same time dissolves out the hasmoglobin, leaving a globular colourless stroma. Salt K(iJiit(oii causes the corpusek-s to shrink. They htconie wrinl.led or ' This is often written 7-8ju. 1^ (niicro-inillimetie) = oue-thousanclth of a millimetre. 2()4 THE TISSUES A>'I) oROAXS ()]•' THE JlobV crenated on the surface. The action of water and of salt solution suggests the existence of a membrane on the surface of the corpuscle, through which osmosis takes place. The quescion, has the red corpuscle a membrane? was once the subject of voluminous discussion. An admirable summary of the positions held by the older writers is given in Gamgee's ' Physiological Chemistry' (p. 72). The matter has been finally compromised by considering the stroma to be rather denser at the surface than in the interior. The outer denser part plays the role of a membrane during osmotic phenomena. IHlute alltaVts (02 per cent, potash) slowly dissolve the corpuscles. Dilute acids (1 per cent, acetic acid) act like water; and in nucleated red coriDusclcs render the nucleus distinct. cTj Fig. 55.— a-e, successive effects of water on a red blooil corpuscle; /, a red corjiuscle crenated by salt solution ; 'j, action of tannin on a red corpuscle. Tannic acid causes a discharge of hsemoglobin from the stroma, but this is immediately altered and precipitated. It remains for a short time adherent to the stroma in the form of a round or in-egular globule of a browni.sh tinge, con- sisting probably of hajmatin. Boracic acid acts similarly, but in nucleated red corpuscles the colouring matter is jjartially or wholly collected aiTjund the nucleus, which may then be extruded from the corpuscle. Nucleus. — The nucleus of tliose red corpuscles in which one exists, has the usual reticular structure, and consists according to Lauder Brunton ' and Plcsz ^ of nuclein. Defibrinated Idood from the bird was treated with ten or twelve times its volume of 3 per cent, sodium chloride solution, and the corpuscles separated by decantation. On shaking the corpuscles with a mixture of water and ethei-, the nuclei alone remain undissolved and float at the junction of the two liquids. Xuclein rnay, however, also be prepared from red corpuscles by Mieschers method, which consists in subjecting the corpuscles to artificial gastric digestion. The nuclei alone remain undigested. Origin of blood corjiiacles in mammals. — Ihe following is a brief resume of the chief ascertained facts concerning the origin of the red discs^: — In the embryo the first formed coloured blood corpuscles are amoeboid nucleated cells. These are developed within certain mesoblastic cells which are united to form a network. The nuclei of the cells multiph', and around some of them, protoplasm coloured by lisemoglobin is aggi-egated. Finally the network is hollowed out and filled with fluid ; thus capillaries are produced ; the coloured nucleated portions of protopla.sm are set free within these as the embryonic 1 L. Brunton, Journ. of Anat. and Physiol. 2ncl series, vol. iii. p. 91. - Plosz, Hopjie-Sei/ler's Med. Chem. Z'ntrrs. Heft iv. p. -tOO. ■'' Qiiain's Anat. vol. ii. TlIK I'.hooi) 205 blood I'orpu.sc'lfs. In later emhryonic life these are rc])laecd hy the usual non- nucleated discs, wliieh are moulded witliin connective tissue cells as I lefore, except that the cell nuclei do not participate in the ])rocess. Nucleated coloured corpuscles are not seen in the blood after birth ; l)ut they continue to be formed in the red marrow, and in some animals in the spleen also. Probably the nucleus disappears from them, and the coloured protoplasm is moulded into a discoid shape. Malassez, however, considers that the red discs are formed by a process of budding from these cells, which he terms globuligenic cells.' The evidence that the red corpuscles are derived from the white, or from the nuclei of the white corpuscles, or from the blood tablets, is insufficient. Composition. — According to C. Hchmidt, lOOO ])aits of moist red corpuscles contain : — Water <;88 parts. ,, ,. 1 / Organic .... 3();5-88 „ >olids < '^ I Mineral .... 812 „ According to Hoppe-Seyler and Jiidell,- 1 00 parts of dried corpuscles contain : — Human Blood Dotr's BIoo.1 noose's Blood I. II. Froteids 12 -2-1 5-10 12-5.-, 36--11 Hjemoslobin .... 86-79 !)■! :]o 86-50 62-6.5 Lecithin 0-72 0-35 0-.3;) 0-4:6 Cholesterin 0-2.1 0-25 0-36 0-48 The mineral constituents of the red corpuscles have been C. Schmidt, and the following tables contrast those of the red those of the plasma in man. .10(^0 parts of moist corpuscles yield : — Mineral matter (exclusive of iron, which is contained in the hiemoglobin) Chlorine .... Sulphuric anhydride Phosphorus pentoxide . Potassium .... Sodium .... Calcium phosphate Magnesium phosphate . 1000 parts of plasma yield : — ilineral matter Chlorine .... Sulphuric anhyilride Phosphorus pentoxide . Potassium .... Sodium .... Calcium phosphate Magnesium phosphate . investigated b}' corpuscles with S-120 1-686 0066 1-134 3-328 I 0.")2 0-114 0073 8-550 3-640 0115 0-191 0-323 3-341 0-311 0-222 ' McKendrick's Fhijsiolocji/, vol. ii. p. 170. - Hoppe-Seyler and Jiidell, Med. Cliem. Uutfrsuch. Heft iii. p. 086. P. Manasse {Zeit. physiol. Chem. xiv. 452), gives the iserceiitage of lecithin in the red corpuscles as 1'867, of cholesterin as O'lol. •20!) THE TISSIES AND OKGANS OF THE BODY The remarkable difference in the distribution of potassium and sodium seen in the above does not, however, hold for most animals, as the following table shows (Gamgee)' : — Blootl Cells I Liquor Sanguinis .Mail . Dog . Cat Sheep . Goat . K Xa CI K Na CI . . 4U-8t» 9-71 2100 5-1'J 37-74 40-68 . . (307 36- 17 24-88 3-25 39-68 37-31 . . 7'85 35 02 27-59 5-17 37-64 41-70 . . ]4o7 3S07 27-21 65G 38-56 40-89 . . : 37il ]4S»8 31-73 355 37-89 40-41 -. Oxygen is contained in combination with the haemoglobin to form oxyhaemo- globin. The corpuscles also contain a certain amount of carbonic acid {see Eespiration). The chief constituent of the corpuscles is thus haemoglobin. According to Hoppe-Seyler ^ lecithin exists as such in the red corjjuscles ; earlier observers (Liebreich, Hermann ^) considered that it is present in the form of a substance called protagon ; of which lecithin is a decomposition product. Protagon is according to Hoppe-Seyler a mere mixture of lecithin and cerebrin. Gamgee,^ however, has more, recently shown that protagon is a perfectly definite proximate principle, and j^robably exists in nervous tissue as such, though with regard to the red corpuscles Hoppe-Seyler's view is now generally held. Lecithin and cholesterin are extracted from the corpuscles by ether. They will be more fully described under nervous tissue. T/ie protfids of the stroma. — The best method for preparing the stromata of the corpuscles is that of "Wooldridge •"' ; detibrinated blood is centrifugalised repeatedly with a 1 per cent, solution of sodium chloride until the corpuscles are obtained free from adherent serum ; they are then dissolved in 5 or 6 times their volume of water, and shaken with a little ether to assist the solution ; the white corpuscles are then allowed to settle, or may be separated by centrifugalising. Prom this solution the stromata are precipitated by the addition of a few drops of a 1 per cent, solution of acid sodium sulphate. The pre- cipitate is collected, washed, and may be readily dissolved in a o per cent, solution of magnesium sulphate. Kiibne,*" who used a rather different method of separating the stromata, found that their chief proteid constituent was a fibrino-plastic globulin. Tltis result I have 1 Gamgee, Physiol. Chem. p. 122. - Hoppe-Seyler, Med. Chem. Untersiicliungen, Heft i. p. 140. JiideU, Ihid. iii. SSO. ^ Hei-uiann, Ardtiv f. Aiiat. ti. Phi/siol. 18(56, p. 33. * Gamgee and Blaukeiihoni, Jo(o-«. of Pht/siol. Ib79. Gamgee, Phi/sioL Chem. p. tiS, * Dm Bois Beymond's Archiv f. Physiol. ISSl, p. 3S7. " Lehrbiich, p. 193. Till-: liLnoK 207 (working witli Dr. Friend ') been able to fully confirm. Tlie chief proteid present is cell-globulin, and like that obtained from white corpuscles it is apparently identical with filjrin -ferment ; though whether the cell-globulin of the red corpuscles normally takes any active part in producing coagulation appears to me to Ije very doubtful ; it may perhaps do so under certain circumstances, accounting for what Landois terms ' stroma-tibrin.' The stromata contain also a doubtful trace of cell-albumin ; but the nucleo-albumin described in white corpuscles (p. 260) is entirely al)sent from the red. Haeniog-lobin Hasmoglobin is the red pigment of the coloured corpuscles. It is a substance which gives the reactions of a proteid, but differs from other proteids in containing the element iron, and in being crystallisable. It exists in the blood in two conditions : in arterial blood it is combined loosely with oxygen, and is called oxyha?moglobin ; the other condition is the deoxygenated or reduced hiemoglobin (often called simply haemoglobin) which occurs in venous blood, that is, the blood which is returning to the heart after it has supplied the tissues with oxygen. Hjemoglobin is thus the oxygen carrier of the body, and it may be called a respiratory pigment. Distrlbiitkm. — Haemoglobin is by far the most widely distributed of the respiratory pigments. It occurs in special corpuscles in all vertebrates except Amphioxus and Leptoc€j)halvs (Lankester) - ; in the following crustaceans — Daphnia, Cheirocephalus (Lankester),^ Aijus, CppHs (Eegnard and Blancard),^ Lernantliropus, Cluvellu, and a marine parasitic crustacean (undescribed) (Van Beneden)^; in the following insects — Cheiroiwmus (Lankester),^ Mu£ca domestica (MacMunn)"* ; in the following molluscs — Plan<»-lAs, Area, and Soleii (Lankester)^ ; in the following chtetopod worms — Lumhrlciis, Luvibriculus, Limnodrilus, Eiuiice, CiiTliatulm, Kais, Xtreis, Terebella, Glijcera, Cha-togaster, Capitella, Tubife^v, Arenicola, EnchytracMis, and Aphrodite (Lankester)^ ; in the foUow^g gephyreaii worms — Phoronis, Thallascma, and Hamingia (Lankester)^; in the nemertine worm Polia (Lankester), and others of the same class (Hubrecht)^ ; in the leeches Nephelis and Hirudo (Lankester) ; in an ophiurid echinoderm (Fottinger) ; and in a holotburian (Howell)." In the above cases, however, from the iuvertebratt- kingdom, the haemoglobin does not occur in special corpuscles, but is simply in solution in the blood plasma, which has thus a respiratory, in addition to its * Journ. of Pltijsiol. x. 532. - Lankester, Froc. Boy. Sac. xxi. 187'2, p. 71. ^ Lankester, Journ. of Anat. and Physiol, ii. 114 ; PflUger's Archie, iv. 31.5. "* Regnard and Blancard, Zool. Anzeig. 18H3, j). 253. 5 Quoted by Lankester, Zool. Anzeig. 1883, p. 416, and by Gamgee, Physiol. Chein. p. 130. '' MacMunn, 'Animal Chromatology,' Proc. Birmingham Philosophical Society, vol. iii. p. 385. ' Studies from the Johns Hoj'kins Univ. Baltimore {Biol. Lab.), vol. iii. p. 284. 268 THE TISSUES AND ORGANS OF THE EODY nutritive functions. There are, however, eight invertebrate animals in which this is not the ca.se, but coloured corpuscles exist as in vertebrates ; these are the two molluscs, Solen and Area, and the five worms, Glycera, Caxjitella, Phoronis, Thallasema, and Hamingia, and the holothurian Thyonella. Haemoglobin occurs not only in the blood, but it is present in certain muscles, especially the red muscles of rodents ; and it also occurs in the muscles of certain invert eVjrates, even in some of those in which it is not present in the blood (Lankester). {See Muscle.) Hfemoglobin occurs also in the nerve cells of Aphrodite. (.SVe Nerve.) Haemoglobin may occur in the urine and other fluirls whf-re it is normally absent. (^See Urine — Haemoglobinuria.) Preparation of oxylicemoglohin f-rystals from hloo'L — The following will be found the best methods for the preparation of oxyhjemoglobin crystals. ^ 1. Defibrinated blood is mixed with its own volume of distilled water, and the diluted fluid is heated with one-fourth of its volume of alcohol. The mixture is kept for 21 hours at a temperature of O"* C. or below. The crystals which separate are dissolved in a little water, a fourth of its volume of alcohol added, and the mixture again frozen. To obtain a pure product the process of recrystallisation may l)e several times repeated. 2. Defibrinated blood is shaken with one-sixteenth of its volume of ether ; the corpuscles dissolve, and the blood assumes a hiky appearance. After a period varying from two minutes to three days, a thick magma of crystals has formed ; these are washed by decantation and centri- f ugalisation with 25 per cent, alcohol. They may then be redissolved and recrystallised as in 1 . This method is V)y far the most satisfactory one. 3. Zinoffsky ^ uses ammonia instead of ether to dissolve the stromata in the foregoing method : this has .subsequently to be neutralised with hydrochloric acid. 4. Gschleidlen ^ obtains large crystals from dog's blood by sealing the defibrinated liquid, after it has stood in the air for 24 hours, in capillary tubes. These are kept at 37° C. for some days, and then their contents are poured out into a watch-glass. Crystals then form on evaporation. 5. In the blood of some animals (rat, guinea-pig, squirrel), micro- scopic preparations of the crystals may be oVjtained by simply mixing a drop of the defibrinated blood with a drop of water on a slide : a cover-glass is then put on, and in a few minutes the corpuscles are 1 The first two methods are selected from a number of methods described by Garagee in his Physiological Chemistry, pp. 85-89. - Zinoflskj-, Zeif.jjhysiol. Chem. x. 16. 5 Gschleidlen, Physiolog. Methodik, p. 361. TiiK r.i.txti) 269 rendered colourless, and then the oxyh;enioj;;lul>in crystallises uut from the solution so formed.' G. More }>ermaneut prejiaiations may he made by Stein's - method, which consists simply in mounting a drop of blood in a drop of Canada balsam on a slide and covering it. In a few minutes crystals form. Crystals of reduced luemoglobin have been pi-epared by similar methods to those already described ; but oxygen must be carefully excluded during the experiments (Hiifner,^ Nencki and Sieber^). Blood crystals are obtained with greater difficulty from the blood of some animals than from that of others. Preyer thus classihes these varieties of blood accoi'ding to facility of crystallisation : — 1. Very difficult : calf, pig, pigeon, frog. 2. Difficult : man, ape, rabbit, sheep. 3. Easy : cat, dog, mouse, horse. 1. Very easy : rat, guinea-]iig. From my own experiments I should be inclined to put the mouse in the second class, and add tlie squirrel to the fourth class in the a])ove list. The crystals ditt'er also in solubility in water and other reagents, which is ill the inverse ratio to their facility of crystallisation. The oxyha?moglobin crystals from different animals differ sliglitlv in their percentage composition. The oxyhjemoglobin differs also in the amount of water of crystallisa- tion with which it combines. Oxyhiemoglobin is by the action of acids and alkalis decomposed, and a brown pigment called ha?matin formed. The readiness with which this decomposition is brought about, also differs in different animals ; e.g. in the blood of the dog and man it occurs easily, in that of herbivorous animals with difficulty (Korber,^ Kriiger*^). Lastly the blood crystals differ in form. As a rule they are rhombic 1 On Wivtchiug this process some of the corpuscles appear to set into miuute hexagons or other shaped crj-stals; this apparently is what Preyer called intraglobiilar ciystallisa- tion. This is, however, not true crystallisation, but simply a pai-tial creuation of the corpuscle; if any of the blood has been allowed to dry it may be well seen. Under the subsequent action of water, the corpuscle swells, and its resemblance to a ciystal disappears. * Stem, Centralhl. f. d. med. Wiss. 1888, Xo. id, and Virchow's Archie, xcvii. 483. 5 Hiifner, Zeit. phijsiol. Cheiii. iv. 88-2. * Xencki and Sieber, Berichte der deutsch. cheiii. Gesell. xix. 128 Ibid. p. 410. Copeman [Brit. Med. Journ. ii. 1889, p. 190) states that the crystals which he has obtained from human blood by adding putrid serum to it are always composed of reduced hi«moglobin. 5 Korber, Inaug. Dissert. Dorixit, ISCG. 6 Kriiger, Zeit. Biol. xxiv. 318. 270 THE TISSUES AXI) OPtGANS OF THE EOT)Y prisms ; in the squirrel and hamster ' he\a;,'ons ^ ; and in tlie guinea-pig and certain birds rhombic tetrahedia. Oxyhsemoglobin crystals thus differ in the following jioints : 1 . Readiness of crystallisation. '1. Solubility. .■'). In percentage composition (.slightly). 4. Amount of water of cry.stalli- sation. .'). Readiness to undergo decom- position by acids or alkalis. G. Crystalline form. In .spite of this, however, oxy- luemoglobin is vmiversally the same in the following points : 1. Spectroscopic properties. '1. The compounds it forms. 3. The products of decomposition, Fig. .56.— OxyliiBinoglobin crystals magnified; SUcll as ha;matin, hajmin, tfec. 1, from liiiman blood ; 2, from the guinea- pig ; 3, squirrel ; 4, hamster. r^j^^ resemblances are thus deeper than the diflFerences. Let us see if we can arrive at any conclusion concerning the differences which will explain the difficulty of there being apparently different oxyhtemoglobins. We shall approach the question Ijest by dealing at greater length with the Crystdllpgrajjliy of OxylicfinogloMn Oxyliajmoglobin crystals were first described bv Eeichert^ as occurrin<^ in tlie uterus of a pregnant guinea-pig; by Leydig^ as occurring in the alimentary canal of the leech ; and by Kolliker,* obtained from the blood of the dog, python, and other animals. Kolliker considered the crystals to be composed of a more or less modified hsematin. Funke" was, however, the first to make complete observations ' In the hamster rhombohedra are also found. - Bojanowski iZeitsch. /. iviHH. Zool. xii. 18(53, 'd'd'd) says that the blood crystals of the mouse are also hexagonal. This I have not been able to confirm ; but liave found with Kunde that they are rhombic. Still it is possiljle that they may be sometimes hexagonal. Rat's haemoglobin is also sometimes hexagonal when prepared by Stein's method. This was first pointed out to me by Dr. Sheridan Lea {see more fully Quart. •Jovrn. of Mic. Science, xxviii. lOOj. Hiifner and Biicheler obtained in one case hexagonal oxyhemoglobin crj'stals from horse's blood (Zeit. phijsiol. Chem. viii. 35b). ^ Reichert, Midler's Archiv, 1849, p. 197. * Leydig, Zeitsch.f. wiss. Zool. Bd. i. 1849, p. 116. ■' Kolliker, Zeitsch.f. wiss. Zool. Bd. i. 1849, p. 200. >' Funke, Zeitsch. f. rat. Med. N. F. Bd. i, 18.-,1, p. 184 ; Bd. ii. 18.52, p. 204 and p. 288. De sanguine venee li/nialis, Diss. Lijjsice, 18.51. riiK r.i.DOD 271 upon them, and to recognise tlicir true iiatiiri-. Kuiidt',' working at the same time, made extensive observations from a comparative point of view, and was the discoverer of the exceptional form of the crystals in the guinea-pig and squirrel. Since then, many investigators have worked at the subject, notably Lehmann," Kollett,* von Lang,' and Preycr,^ who has written an exlianstive tieatise on the subject. The tetrahedral blood crystals of tiie guinea-jiig were at one time sujiposed to liclong to the regular system, but it was von Lang who showed that they are in r(>ality rhombic. A similar rpicstion miglit arise witli regard to tlie liexagonal crystals of the squirrel and the hamster. May they not be rhombic crystals which liave what O. Fi<;. 57. Supi)ose A B r d («) to be tlie basal pUiiie of a rhombic pl.ite, ami tlie angle a b c to be approximately 120°. the lines joining a c, b d bdn^ the axe.s. Then if the angles i) a b, n c b be replaceil, as shown by the ilotteil lines, u liexagon will be i)roiluce(l differing Vmt little from a regular hexagon. mineralogists call a hexagonal habit"* {see tig. 57 «) ? or might they not be rhombic twins consisting of three parallelograms or six triangles (as shown in fig. 57 h and c) ? In order to settle this question it is necessaiy to examine the ojnical jiroperties of the crystals. Crystals may be divided, according to tlieir optical properties, into tln-ee classes : — 1. Tsotrojnc. — Those in which there is no distinction of ditferent directions as regards optical properties. This includes crystals belonging to the regular sy.stem. They haN e but one refractive index, i.e. refract light, like amorplious bodies, singly. 2. Umaral. — Those in which tlie optical properties are the same for all directions equally inclined to one ])articular direction, called the optic axis, but vary according to this inclination. This class includes crystals belonging to the dimetric system (crystals with three rectangular axes, two of them being equal) and the hexagonal system. The optic axis corresponds with the principal crystallographic axis; that is, in tiie case of a liexagon the axis perpendicular to ' Kunde, Zeifsch.f. rat. Med. N. F. Bd. ii. 1H5'2, p. '27(i. - Lchmanu, Ber. d. k. sacks. Ges. d. Wissen. IH.52, p. 22. ■■' Rollett, Sitzuu()shcr. d. Wicn. Akad. Bd. xlvi. 1802, p. 65. ■* Lang, Ibid. ^ Preyer, Die BlatkrijstaUe, Jena, 1871. " Copper glance is an instance of this occurring in tlie mineral kingdoni. In one form of mica also, crystals of the nionoclinic system simulating hexagons are found. 272 THE TISSUES AND ORGANS OF THE BODY the flat surface. In the direction of this axis a ra_v of light is refracted singly, and in other directions doubly. 3. B'laxal. — This includes the remaining three S3'stems of crystals, the trimetric or rhombic (three rectangular axes all unequal), the monoclinic, and the triclinic. In these there are always two directions along which a ray is singly refracted. The best test as to whether a substance is doubly refractive or not is this : If between crossed nicols, which consequently appear dark, a substance be inter- posed that makes the darkness give place to illumination, however feeble, that substance is doubly refractive. This action is termed the depolarisation of the ray (jtee p. 38). On submitting the squirrel's blood crystals to this test, they are found to remain dark in the dark field of the polarising microscope when they are examined with the apparent basal plane perpendicular to the axis of the instrument and rotated : nor when a quartz plate is inserted, do they produce any modification of the tint as the stage is turned. Hence the presumption is, that they belong to the hexagonal system, as rhombic crystals of hexagonal habit or rhombic twins would produce some double refraction when examined in this way. It is genei-ally stated that blood crystals are doubly refracting and pleo- chromatic (i.e. exhibit tini^s as the upper nicol is rotated). "We see it is necessary to make an exception to this rule in the case of hexagonal plates when lying flat. It is found that the hexagonal crystals from squirrel's blood are too small and thin to allow of one applying the additional crucial test of the interference figures seen in convergent polarised light. These consist of a cross and circles, which are symmetrical in uniaxal crystals, asymmetrical in biaxal crystals. We have, however, in the case of the hamster the occurrence of rhombohedral crystals : this confirms the view that the crystals are true hexagons, as the rhombohedron belongs to the hexagonal svstem. It is found, however, that after recrystallising' squirrel's oxyhaemoglobin several times, their hexagonal constitution is broken down, and the crystals obtained are either rhombic prisms or a mixture of these with rhombic tetrahedra.- This leads us to believe that whatever the difference between the various forms of oxyhaemoglobin may be, it cannot be a very deep or essential one. Have we then to deal with a case of polymorphism ? The terms dimor^jhism and polymorj)hism cannot be applied to any substance which crystallises in two or more forms, unless the compo.sition of that substance be exactly the same in all cases. Instances of dimorphism in the mineral world are carbon and sulphur among the elements, and sal ammoniac, potassium iodide, &c., among compounds. The conditions on which dimorphism depends are two : first, temperature ; secondly, the solvent from which the substance cr^'stallises. If, as in the case of many mineral salts, the compounds are united with diiferent proportions of water of crystallisation, we have to deal with different hydrates, and the case is not one of true dimorphism ; an instance of this is sulphate of soda. 1 Another peculiar result of recrystallising haemoglobin has been pointed out by Kupffer and more recently byj Kriiger (Zeit. Biol. xxiv. 47), that is, that the absorption coefficient of oxyhsemoglobin increases after recrystalUsation. In determining the abso- lute amountjof oxylisemoglobin by the spectrophotometer {see p. 50) it is best only to recrystallise once, as each recrystalUsation increases the error of observation. - HaUiburton, Quart. Joiirn. Mic. Science, xxviii. 181. Some remarkable foi-ms of oxyhaemoglobin crystals are also sometimes obtained by dissolving a mixtm'e of the haemoglobin of various animals and then ci-ystaUising. TlIK llLool) 273 The case seems to me to narrow itself down to this in the case of hirmoglobin ; either we have iiere a case of polymorphism, or the cr3-sialline forms are due to the combination with varying proportions of water of crystallisation. In the absence of a rational formula for h:emoglobin, it would be unsafe to affirm tlie former of these two alternatives. Moreover, the conditions that are known to produce dimorphism in minei'als, namely, differences of temperature and of solvent, have in the case of haemoglobin no influence. If we then fall back on the latter alternative, the question wliich arises is whether there are any facts to support it. The explanation that the varying form of oxyhemoglobin is due to varying quantities of water oE crystallisation may be otherwise expressed by saying that we have to deal with different hydrates of oxyhcemoglolin. This would account for the varying solubilities of these sub- stances in water and other reagents, and at the same time is not such an essential difference as to prevent the chief properties of oxyhsemoglobin from being universally the same. Turning to Hoppe-Seyler's researches on this subject of water of crystallisa- tion, it is seen that its amount varies considerablj'. The following is his table :— ' Percentage of Water of Crystallisation Dog's hjemoglobin . . . . . 3 to 4 Guinea-pig's „ 7 Squirrel's „ 9--t Goose's „ ..... 9'4 In an earlier paper,- the same author gives rather different percentages, viz. for guinea-pig's hsemoglobin 6, for goose's haemoglobin 7, and for squirrel's haemo- globin 9. C. Bohr' has more recently made observations on the water of crystal- lisation of dog's liffimoglobin, and as the result of thirteen experiments he finds that its amount varies from 63 to 1'2 per cent. It is thus seen that great varia- tions occur in the numbers obtained by these experiments.^ The reason for this variation seems to be the great difficulty of obtaining haemoglobin in a pure state, and also possibly because the method adopted, which is the same as that carried out in similar investigations on inorganic salts, is not applicable to such a complex and much less stable organic compound as htemoglobin ; in other words, the temperature necessary to drive off the water of crystallisation is also sufficient to cause certain decomposition changes in the pigment. My experiments have shown that squirrel's oxyhajmoglobin will under certain circumstances crj-stallise in forms other than the usual hexagonal form. A crucial experiment in order to see whether this is due to union with different amounts of water of crystallisation would have been first to ascertain the amount of this water in the hexagonal crystals, and then in the rhombic crystals obtained b)' recrystallisation. I have performed three such experiments, but the results obtained are conflicting, and exhibit variations as great as in Bohr's experi- ments, so that it is impossible to draw any conclusions from them, except the * PJiysiologische Chemie, p. 377. 2 Med. Chem. TJntersuchungen, Heft iii. 1868, p. 370. ^ Experimentale TJntersuchungen iiher die Sauerstoffaufnalime des Blidfarhstoffes Kopenhagen (Olsen and Co.), 1885. ■* The same difficulty in obtaining concordant results in the estimation of water of crystallisation was found by J. G. Otto, Pfluger's Arch. xxxi. 2i0. 274 THE TISSUES AND OIlCrANS OF THE BODY negative one that we cannot liy our present methods of research make any definite statement with regard to the water of crystallisation of oxyhtemoglobin. Even if it be found ultimately that the difference in crystalline form is dependent on varying amounts of water of crystallisation, the difficulty is only explained up to a certain point. What is left unexplained is the nature of the agency that causes the oxyhscmoglobin of some animals to unite with a certain amount of water of crystallisation, and that of otlier animals with a different amount. That some such substance or agency does exist would seem to be the inevitable result of the recrystallisation experiments which have been related. It may, however, be stated that this part is not played by any constituent of the serum. Tlie corpuscles of one animal may be obtained free from serum by centri- fugalising and then mixing with the serum of some other animal whose blood crystals have another form. But it is found on subsequent crystallisation that the characteristic form of the blood crystals is not altered thereby. One can only suggest that it is some constituent of the stroma which exerts the influence in question. Compounds of Hcemoglohin Haemoglobin forms at least four compounds with gases, viz. : With oxygen : 1. Oxy haemoglobin. 2. Methsemoglobin. With carbonic oxide : 3. Carbonic oxide haemoglobin. With nitric o.vide : 4. Nitric oxide ha-moglobin. These compounds are isomorphous, they have similar crystalline forms ; they each consist proljably of a molecule of hpemoglobin com- bined with one of the gas in question. They part with the combined gas somewhat readily ; but they are arranged in order of stability in the above list, the least stable first. 1. Oxyhcemoglohin. — This is the compound which exists in arterial blood. Many of its properties have been already mentioned. The oxygen linked to hsemoglobin, which is removed by the tissues through which the blood circulates, may be called the respiratory oxygen of hcemoglobin. The circumstances under which haemoglobin combines with and parts from its respiratory oxygen in the body will be fully described under ' Respiration.' But the same processes may be imitated outside the body, using either blood or pure solutions of haemoglobin. The respiratory oxygen can be removed, for example, in the Torricellian vacuum of an air pump. Preyer ' estimated that 1 gram of hemoglobin will combine with 1-27 c.c.^ of oxygen. Hiifner ^ gives almost the same number, viz. 1-28 c.c. of oxygen. A. Schmidt at one time considered that the respiratory oxygen of 1 Die Blutlcrystalle, p. 134. 2 Measured at 0°C. and 1 metre pressure; equivalent to 107 c.c. measured at 0° C, and 7()f' millimetres pressure. 3 Hiifner, Zeit. 2}h;jswl. Chem. i. 317. Till': iii,(»(»i) 275 hfienioglobin was ozonised, and therefore more active than atmosplieric oxygen. PHiiger ' has shown that this is not the case. Wlien dilutemoglol)in to consider. The various forms of spectroscope have been already described (p. 47). It will be sufficient here to repeat that the spectroscope is an instrument which enables us to tell the colour of a solution or transparent substance more accurately than we can with the unaided vision. White light passed through the coloured substance and then through a prism no longer gives a continuous spectrum, but certain parts of it are absorbed, hence the appearance of dark shadows or absorption bands in various parts of the spectrum. These bands remain constant in position for the same substance, and thus furnish us with a delicate test for that substance. We speak of the position of the absorption bands, either by their neighbourhood to certain of the black lines (Fraunhofer's lines) of the solar spectrum ; or more accurately still by measuring their position in wave-lengths. The sign X denotes wave-length ; in absorption spectra, the edges of the bands are sometimes so ill defined, and vary in position with the concentration of the liquid, that more often the position of the centre of the band rather than that of its edges is given. \ 500 means a wave-length equal to 500 millionths of a millimetre. The two next figures illustrate a method of representing ab- sorption spectra diagrammatically. The solution was examined in a layer one centimetre thick. The base line has on it at the proper distances the chief Fraunhofer lines, and along the right hand edges are percentages of the amount of oxyha?moglobin present in I, of reduced haemoglobin in II. The width of the shadings at each level represents the position and amount of absorption corresponding to the percentages. The characteristic spectrum of oxyha^moglobin (first observed by Hoppe-Seyler) is seen as it actually appears through the spectroscope in the next figure (fig. 59, spectrum 2). There are two distinct absorption bands between the D and E lines ; the one nearest to D (the a band) being narrower, darker, and with better defined edges than the other ' Pffiifjfir's Archiv, x. 252. T 2 276 THE TISSUES ANU ORGANS OF THE BODY (tlie n band). The centre of the a band corresponds to A 579, of the /j bund to A 553-8 (Gamgee'). As will be seen by looking at fig. 58, a solution of oxyhtemoglobin of concentration greater than 0'65 per cent. and less than 0*85 per cent, gives one thick band overlapping both D and E, and a stronger solution still, only lets the red light through between the C and D lines. A solution which gives the two charactex'istic bands must therefore be a dilute one. But, as before said, we are able with such solutions of Fig. 58.— Graphic representations of tlie amount of absorption of liglit by solution of (I) oxyliKmo- prlobin, (II ) of haemoglobin, of dlflferent strengths. The shading indicates the amount of absorp- tion of the spectrum ; the figures on the right border express percentages (HoUett ). oxyha?moglobin, or defibrinated blood will do equally well, to imitate the reduction of oxyhitmoglobin which occurs in the body. This was first pointed out by Professor Stokes,'^ who employed for the purpose the reducing agent now known as Stokes's reagent. The following are the means by which we can displace the respiratory oxygen in a solution of oxyhaemoglobin : — (1) By boiling it in the Torricellian vacuum of a mercurial air pump. (2) By passing through the solution a neutral gas such as nitrogen, hydrogen, or carbonic acid. (3) By the use of reducing agents. (a) Stokes's reagent : a solution of ferrous sulphate, to which a ' Cramgee, Physiological Chemistry . Where other wave-lengths are given subse- quently, they are taken either from Gamgee's measurements, or from those made by MacIMunn, and pubhshed in McKendi-ick's Physiology. - Stokes, Proc. Roy. Soc. xiii. 357. T.IK BLOOD j>£Q 59_ 1^ Solar spectrum. 2, Spectrum of oxyh.nemoglobin (0-37 p.c. solution). Fir>t baiul, A 5So_ 564 ;' second band, A 5-55-.517. 3, Spectrum of hiemoglobin. Band, A 507-535. 4, Spe.;tiim of CO- hjemoslobin. First band, A 583-564 ; second band, A 547-521. 5, Spectrum of methiewoglob-n (concentrated solution). 6, Spectrum of methiemoglobin (dilute solutionX First baud. A 647- 622 ; second band, A 587-571 ; third band, A 552-532 : fourth band, A 514-490. 7, Speotnim of acid' hiematin (ethereal solution). First band. A 656-615 ; second hand, A 597-577 ; thii-d baud, A 557-529 ; fourth band, A 517-488. 8, Spectrum of alkaline hsematin. Band from A 630-581. 9, Spectrum of hfemochromogen (reduced hjematin). First band. A 569-542 : second band, A 535-504. 10, Spectrum of acid hsematoporphyriu. First baud, A 6 )7-593 ; second baud, A 5''5- 536. n' Spectrum of alkaline hjematoporphyrin. First band, A 633-612 : second band, A 589-561- third band. A 549-529 : fourth band, A 518-488. The above measurements (after ;XIacMunn) a'-e inmillionths of a millimetre. The liquid was examined in a layer one centimetre thick. The edt'es of 111-defiued bands vary a good deal with the concentration of the solutions. 278 TilE Tlr^SUES AND t »KGA>> OF THE 13« )DY little tartaric or citric acid has been added, and then ammonia till the reaction is alkaline. This reagent rapidly darkens in the air, and must be freshly made every time it is used. (6) Instead of ferrous sulphate, stannous chloride may be used in the preparation of the foregoing. This has the advantage of not darkening, as it absorbs oxygen. It however must also be always freshly prepared before using. (c) Ammonium sulphide. This on the whole is the most convenient reagent to use, though it is somewhat slower in its action than the two preceding : a little gentle warmth will liowever hasten its action. Using any of these methods the colour of oxyhemoglobin changes to the purplish tint of htemoglobiu, and by the spectroscope the two bauds are now seen to be replaced by one, called the y band ; this band is not so well defined as either the a or the /3 band. Its position between the D and E lines is denoted in fig. 59 (spectrum 3) ; it is darkest about A 550. On dilution the band fades i^apidly, so that in a solution of such concentration that both bands of oxyhfemoglobin would be quite distinct, the single band of reduced hfemoglobin has disappeared from %"iew. The oxyhfemoglobin bands can be distinguished in a solution Avhich contains only one part of the pigment to 10.000 of water, and even in more dilute solutions which are apparently colourless, the a band is still A'isible. On passing oxygen through a solution of hfemoglobin, or on shaking it up with the air, oxyhsemoglobin showing its two bands, reappears. 2. Methc^moglohin. — This is a compound of haemoglobin with oxygen which can be produced artificiaUy ; it also occurs in the body under certain circumstances, e.g. in certain diseased conditions it occurs in the urine {see Hsemoglobinuria), and after the administration of large doses of potassium or sodium chlorate it occurs in the blood, and death is the ultimate result. It may be derived artificially from a solution of oxyh;emoglobin in the following ways : — (a.) When a solution of oxyha^moglobin is exposed to the air in shallow layers for some time, it becomes acid in reaction, brown in colour, and exhibits the characteristic spectrum of methsemoglobin. (6) On the addition of various oxidising agents the same occurs ; prrtassium permanganate, potassium ferricyanide, nitiite of potassium, nitrite of amyl,' etc., act in this way. Hence the view originally ' Hayem, Comjyt. rend. cii. 698, gives a long list of reagents that act in this way. THE BL0<)1) 279 advanced by Sorljy,' that metha;nioglobin is more highly oxygenated than oxyhivmoglobin ; that it is in fact a per-oxyha?moglobin. (c) Meth.'emoglobin may however be prepared by removing part of the oxygen of oxyhemoglobin by means of the mercurial air pump, or by means of palladium saturated with hydrogen, Hoppe-Seyler,^ who describes the above methods, therefore regards methiemoglobin as a sub-oxyhfemoglobin. Whichever view was held as to its constitution, it was admitted by all that the oxygen of methaemoglobin is more firmly combined than that of oxyhjvmoglobin. Still it can be removed by reducing agents. The oxygen is however not removable by the air pump, nor by a stream of a neutral gas like hydrogen. On adding ammonium sulphide to a solution of methjemoglobin, the first change is to oxyh:emogIobin, and then to reduced ha-moglobin ; these changes can be watched with the spectroscope. More recently, however, Hiifner and Kiilz^ have advanced a third theory concerning the constitution of methfemoglobin, and that is that it contains the same amount of oxygen as oxyhemoglobin, only in a closer state of combination. They are able to make this assertion from actual analyses ;* and these analyses were possible, inasmuch as they succeeded in obtaining pure methemoglobin in a crystalline form. The method of obtaining these crystals is as follows :' — Three or four cubic centimetres of a concentrated solution of ferricyanide of potassium are added to a litre of concentrated solution of hemoglobin. A quarter of a litre of alcohol is added, and the mixture frozen. After one or two days' exposui-e to this low temperature, abundant crystals of a brown colour, which give the absorption spectrum of methemoglobin, are de- posited. They were obtained in this way from the hemoglobin of the dog, pig, and horse, and their form is the same as that of the oxyhemo- globin crystals of the same animals, i.e. rhombic prisms. Gamgee^ had prepared these crystals from dog's blood many years previously, but their true nature was not at that time recognised. His method was much the same as Hiifner's, the chief difierence being that the nitrite of potassium or amyl was employed instead of ferricyanide of potassium. Jaderholm' has also obtained these crystals from dog's blood by the ferricyanide method, and confii'ms Hiifner's statement that they are 1 Sorby, Quart. J. Mic. Science, 1870, p. 400. - Hoppe-Seyler, Zeit. physiol. Chem. ii. 150. 3 Zeit. phtjsioJ. Chemie, vii. * Hiifner and Kiilz employed the spectropliotometric method largely in their work. s G. Hiifner, ' Ueber ki-ystallinisehes Methiimoglobin vom Hunde,' Zeit. physiol. Chem. viii. 366. fi A. Gamgee, ' The action of Nitrites on Blood,' Philos. Trans. 1868, p. .589, et seq. '' Zeitsch. far Biol. xx. 419. Jiiderholm now agrees with Hiifner and Kiilz with regard to the composition of methsemoglobin. 280 THE TISSUE? AND ORGANS OF THE BODY rhombic prisms. He also figures some crystals of methsemoglobin obtained by Hammarsten from the horse by the same method, which were regular six-sided plates, and showed no double refraction if lying flat : they therefore presumably belonged to the hexagonal system : they were more insoluble in water than the crystals of dog s methsemoglobin. When one wishes, however, to obtain a small quantity of crystals for microscopic examination, the following simple method may be employed.' A few c.c. of the defibrinated blood of an animal (rat, guinea-pig, or squirrel) are taken, and an equal number of drops of nitrite of amyl added. The mixture is vigorously shaken for a minute or two. The colour changes to the dark chocolate tint of methtemoglobin, and spectroscopic observation shows the typical absorption bands of that compound. A drop of this liquid is then placed ^ on a slide and covered : in a few minutes crystals form, which oVjservation with the spectroscope shows to be composed of methfemoglobin. The edges of the cover-glass may then be sealed, and the crystals keep unchanged for several months. The cr}-stals obtained from guinea-pig's blood by this process are tetrahedi-a. which differ only in colour and spectroscopic appearances from those of oxyhfemogiobin from the same animal. The crystals obtained from squirrel's blood are perfectly regular hexagonal plates, which remains dark between crossed nicols. The crystals obtained from rat's blood are also perfectly regular hexagonal plates,^ which remain dark between crossed nicols, and which consequently are precisely similar to those of squiiTeFs methfemo- globin. This remarkable fact helps to show that the difference between the oxyhfemoglobin of these two animals cannot be a very deep or essential one. The spectrum of methiemoglobin shows three absorption bands, one in the red alx>ut half way between the C and D lines, and two others between the D and E lines which resemble in position those of oxy- hjemoglobin. but on careful measurement are found to be different.^ A fourth indistinct band in the blue has also been described (see fig. 59, spectra 5 and 6). On adding ammonia to a solution of methsemoglobin, the first two l^ands shift a Kttle towards the violet end of the spectrum ; 1 Halliburton, Quart. Journ. Mic. Science, xxviii. 201. - This must be done immediately after the formation of the chocolate-coloured liquid, as in about a quarter of an hour the whole liquid sets into a gelatinous mass of the same colour, from which no crystals are obtainable. 3 A few triangles and forms intermediate between triangles and regular hexagons are also found. * Araki considers that these bands are due to admixture with oxyhsemoglobin iZeit. physiol. Chem. xiv. 405). TllK IM.ooI) ' 281 this spectrum is sometimes spoken of as that of alkaline methsenio- globin. 3. Carbonic oxide hcemoglohin. — This may be readily prepared l)y passing a stream of carbonic oxide gas ' through blood, or through a solution of oxyha-moglobin. Its colour is a peculiar cherry-red. Its absorption spectrum (see fig. 59, 4) is very much like that of oxyhsemo- globin, but the two bands are slightly nearer the violet end of the spectrum ; the centre of the a band being X 572, of the /3 liand \ 534 to 538 according to concentration (Gamgee). CO-ha?moglolnn forms crystals like those of oxyha?moglobin ; it is remarkable for its stal)ility ; it is not affected by reducing agents like ammonium sulphide, and the carl:)()nic oxide gas can only be driven off by passing tlirough it for ^a long time a stream of air or of a neutral gas, or by a stream of nitric oxide gas which replaces the carbonic oxide and forms nitric oxide hsemoglobin. CO-hjemoglobin also resists putrefaction for a long time (Hoppe-Seyler 2). Carbonic oxide is gi^■en off during the imperfect combustion of carbon, such as occurs in charcoal stoves ; this acts as a powerful poison by combining with the haemoglobin of the blood, and thus inter- fering with normal respiratory processes. The colour of the blood and its resistance to reducing agents in such cases are characteristic. Hoppe-Seyler has, howevei', introduced another test which has been modified by Salkowski ^ as follows : The blood in question is diluted twenty times, and to some of this in a test-tube an equal volume of aqueous soda of specific gravity 1-34 is added. In a few seconds CO blood becomes whitish, then red ; on standing red flocculi separate and finally rise to the surface of a faintly rose-coloured liquid. In normal blood all that is produced by the addition of the alkali is a dirty-brown colouration (h;ematin). Working under Salkowski's direction Kata- yama ^ has discovered a new test which may be briefly stated as follows : The addition of acetic acid and ammonium sulphide (with sulphur in solution) to normal blood jn-oduces a greenisli-gi-ey or reddish green- grey colour ; to CO blood, a beautiful clear rose-red is produced. This solution shows a spectrum which is a double spectrum, indicating that there is in solution CO-ha?moglobin and sulphur-methsemoglobin (Hoppe- Seyler ^), viz. one band between C and D, and two others between D ' The gas may be generated by adding sulphuric acid to oxalic acid or formic acid in a retort, and then applying heat. 2 Hoppe-Seyler, Zeit. ijhysiol. CJtem. ii. 1:31. 5 E. Salkowski, Hid. xii. 227. * Katayama, Virchow's Archiv, 188S, vol. cxiv. p. .53. References will be found in this iiaper to other tests which have been proposed for CO-ha?moglobin. ^ Physiol. Cheni. p. 388. The composition of sulphur methsemoglobin is not known. 282 THE TLSaUES AND ORGANS OF THE BODY and E. The spectrum shown by normal blood after the addition of these reagents is also a double spectrum, viz. of sulphur metha^moglobin and reduced haemoglobin. In other words, in the case of CO-liasmoglobin the colour of that compound completely masks the olive-green tint of sulphur-methsemoglobin, which spectroscopic observation shows to be present. 4. Nitric oxide hcevioglobin. — When ammonia is added to blood, and then a stream of nitric oxide passed through it, this compound is formed (Hermann ') ; it may be obtained in a crystalline form isomor- phous with oxy- and CO-heemoglobin ; it also has a similar spectrum. It is even more stable than CO-hjemoglobin. Other compounds of haemoglobin have been described : one with acetylene (C^Hn), another with hydrocyanic acid (Hoppe-Seyler).^ Dr. Gamgee'' has, how- ever, pointed out the unsatisfactory nature of the evidence upon which the existence of such compounds rests. Recently C. Bohr has advanced the theory that haemoglobin forms a compound with carbonic acid. The importance of this discovery, if confirmed, is very great, and the question will be discussed in the chapter on Respiration (Chapter XIX). Estimation of Hcumoghhin The following methods may be adopted for the quantitative estima- tion of luemoglobin : — • 1. By the amount of iron in the ash. 2. By colorimetric methods. 3. By spectrophotometric methods. 1. A weighed quantity of blood or substance containing haemo- globin is evaporated to dryness, the residue is carefully incinerated at a dull red heat, the ash exhausted with hydrochloric acid to obtain ferric chloride. This is reduced by the action of metallic zinc to ferrous chloride, and the amount of iron in this determined volumetrically with a standard solution of potassium permanganate {see also p. 25). Dry haemoglobin contains 0"42 per cent, of iron. If «i^percentage amount of iron in the specimen under examination, the percentage of hsemo- 1 1 . . , , , . 100 ??i globm m that specimen = -^-r^ • 2. Standard solutions of known strength are prepared from crystals It is formed by passing a stream of sulphuretted hydrogen through a solution of oxy- lipemoglobin. The greenish tint which appears on the surface of corpses a few days after death is due to the develoiament of sulj)liuretted hydrogen, and the consequent formation of sulphur-metliEemoglobin. See also Araki {Zeit. ji^iysiol. Chem. xiv. 412). 1 Hermann, Beichert und Du Bois Beymond's Archiv, 1865, p. 469. - Hoppe-Seyler, Med. Chem. Untersnch. Heft ii. p. 207. •* Gamgee, Physiol. Chemistry, pp. 107-8. i'liK i;l()()J) 283 of oxyhannoglobin. The blood to be investigated is diluted with water until the colour of the standard solution is reached. Knowing tlie amount of blood and the amount of dilution, the percentage of lia-mo- globin is easily ealcuLited (Hoppe-Seyler). The tint of the solutions must be ascertained by examining them in vessels with parallel sides and of the same width. A htematinometer, as such a vessel is called, is usually constructed so that the sides are 1 centimetre apart. Rajewsky ' and Malassez -' recommend the standard solution to be made up of picrocarrainate of ammonia, the tint of which corresponds to that of an oxyhtemogiobin solution of known strength. In Fleischl's ha^mometer a wedge of red-tinted glass forms the standard of comparison ; the wedge is arranged to slide under a hole in a brass plate, the thickness of the glass under observation can thus be varied and adjusted so as to give a red tint equal to that of the blood under examination, which is always diluted to a certain fixed extent. Gowers' htemoglobinometer, like Fleischl's instrument, is designed for clinical use. Tlie apparatus consists of two glass tubes, C and D, of Fig. UU. — Hivmoglobiiiouieter of I)r. Gowers. (Hawkslej-.) the same size. D contains glycerine jelly tinted with carmine to a standard colour, viz. that of normal blood diluted 100 times with distilled water. The finger is pricked and 20 cubic millimetres of blood are measured out by the capillary pipette, B. This is blown out into the tube c, and diluted with distilled water, added drop by drop from the pipette stopper of the bottle, A, until the tint of the diluted blood reaches the standard colour. The tube, c, is graduated into 1 Rajewsky, Pjiiiger's Archiv, xii. 70. - Malassez, Arch, de Physiol. 1877, p. 1. 284 THE TISSUES AND OEGANS OF THE BODY 100 parts. If the tint of the diluted blood is the same as the standard when the tube is filled up to the graduation 100, the quantity of oxy- hemoglobin in the blood is normal. If it has to be diluted more largely, the oxyhi^moglobin is in excess : if to a smaller extent, it is less than normal. If the blood has, for instance, to be diluted up to the graduation 50, the amount of hfemoglobin is only half what it ought to be — 50 per cent, of the normal, and so for other jjei'centages. The instrument only yields approximate results, but is extremely useful in clinical observations.^ 3. The spectrophotometric method for the estimation of coloured solutions has been already described {see p. 50). In connection with the estimation of oxyhfemoglobin it may be added that the region of the spectrum selected for photometi'ic measure- ments is that of the /3 band of absorption. This part of the spectrum in the case of oxyha?moglobin has been found to be that most easily affected by changes in the concentration of the solution through which the light passes. Glazebrook's speetn :»photometer is, in principle, the same as Hiifner's. Light from each of two sources passes first through a Xicol's prism by which it is j)olarised, then through a direct vision prism ; thus, two adjacent supei-posed sj:)ectra are obtained, and these are observed by an eyepiece in which is an analysing Xicol's prism. This eyepiece can be rotated, and the amount of rotation measured l>v a pointer attached to the eyepiece moving over a graduated circle. The nicols are then adjusted in the way already described, so that the two spectra apj^ear of equal brightness. One may then proceed to interpose the coloured solution and measure the angles through which it is necessary to rotate the nicols, or, more simply, in the following way, as suggested by Dr. Sheridan Lea.- On the path of one beam of light is placed a solution of known concentration, and on the path of the other, one of unknown concentration. If the latter is of greater concentration than the first, it may be diluted down till the eftect upon the two spectra is the same ; from the amount of dilution necessary to produce this effect its concentration can be calculated. Or the same effect can be pro- duced by ^'arying the thickness of the layer of tlie fluid under observa- tion ; this latter plan is found to be perfectly fea^^ible, and is applicable also when the concentration of the unknown solution is less than that of the known ; and Dr. Lea has invented an instrument (absorptio- meter) with parallel sides, one of which is movable, and so the thickness of the layer of fluid in it can be varied to kno%\'n extents. Take as an llustration the following example, which reduces to its simplest elements ^ Goweis, Lancet, vol. ii. 1878, p. 8-22. - Sheridan Lea, Jotirn. of Physiol, v. 239. THK ItLOOD 285 the inetl)o > ivom which equation, C the unknown quan- ta o tity is easily calculated. Preyer's method is also a spectrophotometric one. A 0-85 per cent. solution of oxyhsemoglobin (thickness of layer being 1 centimetre) is the most concentrated solution which allows the green light of the spectrum to pass through it (see fig. 58, sign -> «-). Take a known amount of the blood to be investigated and dilute it with water till it just allows the faintest shade of green light to pass through it. If b^ volume of blood taken, and "•= volume of water added, then the per- centajce of oxvh£emoo;lobin in the blood= \ . I append here a table of the results of analysis which I take from Preyer, 'Die Blutkrystalle,' p. 117. 100 tjrams of healthv human blood contain : — Iron Hihinoglohin I Minimum . . 0*048 grm. 11-57 grm. I, Maximum . . 0-057 „ 13-69 „ 0-0508 „ 12-09 0-056 „ 13-45 0-063 „ 15-07 Woman C Minimum Man- Average (11 cases) I Maximum From data given by Malassez it can be calculated that the amount of hfemo»lobin in each human blood corpuscle is approximately 30 billionths of a gramme. Venous and arterial blood contain the same amount of hsemoglobin (Kriiger).' Foetal blood is of lower specific gra-vdty than that of adults, and is especially deficient in hjemoglobin.^ JDetermi nation of the ' Activity of Reduction ' of 0.ryJiamoglohin.^ The time of reduction of oxyhfemoglobin is determined by examining the spectrum of the blood under the thumb nail ; the first band can be always, the second sometimes, distinguished by the direct vision spectroscope. If a ligature 1 Zeit. Biol. sxvi. 452. Kriiger also states that congestion of a part increases the total soUds and ha;moglobin in the blood drawn from it; that the blood of the splenic vein is richer, and of the renal vein is poorer, in solids and pigment than arterial blood. Copeman and Sherrington {Proc. Physiol Soc. 1890, p. -s-iii) have, by a different method, arrived at similar conclusions. - Scherrenziss, Inaug. Diss. Dorpat, 1888. ^ Henoeque, Comptes rendus, ciii. 817. 286 THE TISSUES ANJ) ORGANS OV THE P-oDY is tied round the phalanx, the bands gradually disappear. The time of reduction is the time they take to disap[)ear, and this in a healthy man at rest averages 70 seconds. The quantity of hajmogiobin in the blood is then determined.' A healthy man's blood contains 14 per cent, of oxyh;emoglobin ; from this it is calculated that ()-2 per cent, is reduced per second. This quantity is taken as the unit activity of reduction ; and the activity in other cases = quantity of oxyha^moglobin ^ time of reduction Henocque - has described the variations in the activity of reduction of oxy- haimoglobin in various diseases, and under the influence of various drugs. Tn typhoid fever.^* for instance, periods of high temperatui-e were found to coincide with periods of diminished activity of reduction, and when the temperature was reduced, the activity of reduction increased, and tended to regain its normal value. Composition of Ila^hiofjlohin The following is the percentage composition of haemoglobin as ascertained by various observers.'* Dog Horse Guinca- Pig Squirrel Goo^e ' C.Schmidt Hoppe- Seyler .Jaquet Kossel ZiiioflEsliy Hoppe- Seyler Hoppe- Seyler Hoppe- Seyler c . . . 5415 53-85 53-91 54-87 51-15 54-12 54-09 54-26 H. . . 7-18 T-82 6-62 6-97 676 7-.36 7-39 7-10 N. . . iG-8;j 16-17 15-98 17-31 1 7-94 16-78 16-09 16-21 0. . . 21-24 21-84 22-62 19-73 23-43 20-68 21-44 20-69 s . . . 0-67 0-3it 0-542 0-65 0391 0-58 0-40 0-54 Fe . . 0-43 0 43 0-333 0-47 0-335 048 0-59 0-43 PA- • — — — — — — 0-77 There are thus very considerable disorepancies between the analyses of different observers; we should, however, not be justified in con- cluding from the results of elementary analyses that there are different varieties of hitmoglobin in different animals ; for if the analyses of haemoglobin from the same animal be examined tliere wall be found discrepancies equally as great. A more just conclusion seems to be that the methods we at present adopt are not sufficiently exact to enable us to 2:)repare a pure product. ' Henocque describes a sj)ecial form of litematoscope for the purpose. Dr. Gowers' instrument would do equally well. - CfD/iptes rendus, cvi. 14(!. ^ Henocque and G. Baudouin, Ihid. p. 1245. "• Hoppe-Seyler's analyses will be found in his Physiol. Chonie, p. 377 ; those of C. Schmidt and Kossel I take from a table in McKendrick's Piiijsiologi/, p. 118 ; those of Zinoffsky in Zeit. plujsiol. Cltem. x. 1(5, and of Jaquet, who employed Ziuoffsky's method, in Ihid. xii. 285, xiv. 289. Gschleidlen {Pfiiiger's Arch. xvi. 421) considers that the phosphoric acid of birds' haemoglobin is due to admixture with uuclein. Jaquet is inclined to ccnsider it as an essential part of the hsemoglobin in itself. THK lij.ooi) 287 A temperature sutticient to dry tlie lueinoiflohin thoroui^'lily (120^ C.) causes, for example, a partial disintegration of this unstable organic compound.' We kn«t\v of no ratitjnal formula for haemoglobin, and, in view of the discordance of the above analyses, it would seem rash to calcu- late an empirical one. Preyer's formula is C6()oH,)f,i|N|.,,FeS30|-,j ; Hiifner's, C55oHi,.,oNi4,jFeS40,49 ; ZinofFsky'.s, C7,2H,,3n^'"2i.iFeS.20.,4.v Decomposition of Itr^morjlohin. — On the addition of acid or alkali to a solution of oxyha-moglobin, the colour changes to brown ; this is due to the decomposition of the h;¥moglobin into a proteid, called Globin, and a pigment which contains all tlie iron, called Hccmatin. Con- siderable discnssion has taken place on the question as to whether these two substances are mechanically mixed together, or whether they are chemically combined. It was Lehmann who first brought forward the supposition that hfemoglobin is not a chemical unit, but consists of ha^matin merely mixed with a crystallisaljle proteid. A seeming confirmation of this theory has been more recently advanced by Struve,^ who found that by means of alcoholic ammonia hivmatin can be extracted from the crystals, leaving them colourless. Against this, however, it must be pointed out that alcoholic ammonia is a strong reagent, and is able to effect more than a separation of two substances mechanically mixed ; even alcohol by itself pi'oduces changes in haemoglobin ; the parah?emoglobin crystals of Xencki and Sieber ^ have been shown by Hoppe-Seyler "■ to be a mere coagulation product of oxyha^moglobin brought about by the action of alcohol : these crystals do not show doubL' refraction — that is, they have not the constitutions of true crystals, but are merely proteid masses which, when coagulated, retain the crystalline shape they had previously. Another ground upon which some hold that h?emog!obin is not a chemical unit is the conflicting results of analysis, especially with regard to the quantity of sulphur present. Zinofisky lias, however, pointed out that this is due to bad methods of prepur-ation of the haemoglobin, and by his method he was able to prepare a number of specimens of luemoglobin in which the analysis ga\e concordant results. Zinofisky holds the view, which is now very general, that oxy- h£emoglobin is a chemical unit ; he also states that its molecule yields 1 I have found that, even if the haemoglobin be dried in a ToriueUian vacuum, although a temperature of 40° C. is sufficiently high to drive ofif all watei . f crystallisation, it is also sufficient to cause the formation of hiematin and an insoluble jMoteid. - Struve, Zeit.prakt. Chem. 1884. ^ Xencki and Sieber, Arch, experim. Path. u. Phai-makol. xx. 325. ^ Hoppe-Seyler, Zeitsch. jihysiol. Chem. x. 331. 288 THE TISSUES AND ORGANS OF THE P,(^I)Y on decomposition 1 molecule of lijematin with 34 atoms of carbon, and 2 molecules of globin each with 1 atom of sulphur and 339 of carbon. Glohin. — This proteid is derived from the decomposition of haemo- globin ; this occurs under the influence of heat, when globin is converted into a heat-coagulum ; by the influence of acids or alkalis, when it is converted into acid and alkali-albumin respectively. In all three cases hiematin is simultaneously liberated. On heating a solution of oxy- ha?moglobin a heat-coagulum forms at 68°-70° C. The flocculi are of a brownish colour, as they carry down with them some of the hjematin. Under the influence of acid or alkali both hsematin and the proteid go into solution. The proteid is a globulin ; it is precipitated from its solutions by saturating them with magnesium sulphate or sodium chloride. Gamgee* agrees with Kiihne in considermg that globin wliich was first described by Preyer is probably a mixture of proteids. HiHiiiatin is a brownish pigment which exhibits ditierent absorption spectra, according as to whether it is dissolved in an acid or alkaline medium. It is insoluble in water, alcohol, and ether, easily soluble in caustic alkalis and in alcohol acidulated with sulphuric acid. For spectroscopic examination alkaline hatmatin may be prepared in the following ways : — 1. Add some strong potash or soda to a solution of oxyhaemoglobin or to some diluted detibrinated blood. The rate at which the decom- position takes place varies in different animals ; the same is also the case with acids (see p. 269). 2. Rectified spirit and ammonia, or rectified spirit and soda, may be added to diluted blood, or to a solution of oxyhtemoglobin, and the mixture filtered ; the filtrate shows the spectrum of alkaline haematin. 3. Pure hajmatin may be dissolved in caustic alkali. Alkaline haematin shows one ill-defined absorption band overlappmg d, and extendinff some distance towards the red end of the spectrum {see fig. 59, spectrum 8). For spectroscopic observation acid hcematin may be prepared in the following ways : — • 1. By adding a little glacial acetic acid to diluted blood or solutions of oxylijemoglobin. 2. By shaking up the acid liquid just prepared with ether ; the ethereal extract on standing floats above the watery liquid and con- tains the hfematin dissolved in it. 3. By dissolving pure htematin in acidified .spirit. Acid hfematin 1 Gamgee, Fhijsiol. Chemistrij, p. IIU. THE T.LDOD 289 is sometimes called h?ematoin, or four-banded hivmatin. It shows four bands — one in the red between the C and D lines, but rather nearer the C line than the somewhat similar band of niethsemoglobin ; one narrow and faint band over the D line (this is especially faint when the htematin is prepared by method 3 above) ; and two bands in the green (fig. 59, spectrum 7). Pure hsematin may be prepared in the following ways : — 1. Crystals of haemin (hydrochloride of ha?matin) are dissolved in dilute potash ; this is neutralised with dilute hydrochloric acid, and hfematin is precipitated as a flocculent Vjrown precipitate, which is collected, washed with boiling water, and dried (Hoppe-Seyler '). 2. Blood clot is extracted with rectified spirit containing pure sulphuric acid (1 in 17) ; the solution is filtered, diluted with an equal amount of water, and agitated with chloroform. The chloroform dissolves out the hsematin ; it is washed -with water to remove the acid, and then the chloroform is evaporated ; the haematin remains as a dark brown pigment which dries up to a bluish-black powder (MacMunn 2). The formula for hrtmatin is C34H35X4Fe05, or, perhaps, twice that, CfigH^oX.FeoOio. It forms compounds with hydrochloric, hydrobromic, and hydriodic acids. A compound with potassium cyanide is also described. Hsemochromogen is a reduction product of hsematin. Hsematoporphyrin, hsematolin, and hsematoidin are iron-free pro- ducts of hsematin. These different substances must be taken now one by one, Hctinochromogen. — This substance was called by Stokes reduced hsematin. It may be readily prepared for spectroscopic observation by adding a few drops of Stokes's reagent or ammonium sulphide to a solution of alkaline htematin. Hoppe-Seyler prepares it from reduced haemoglobin in an apparatus from which oxygen is excluded, by the action of alcohol containing sulphuric acid, or caustic potash in solution. This method is far more difficult, and involves the use of special apparatus. Whichever method is adopted, the final result is the same : that is to say, whether we first decompose oxy haemoglobin, and then add a reducing agent ; or whether we first reduce the oxyhsemoglobin, and then decompose it. ^ Hoppe-Seyler, Med. Chem. Untersuch. Heft iv. p. 523. - MacMunn, Journ. of Fhy&iol. vi. 22. 290 THE TISSUES AND ORGANS OF THE BODY On oxidation hsemochromogen yields hpematin. Hoppe-Seyler represents the reaction as follows : — 2(C34H3-N4Fe05)4-Oo=C68H,oN,FeoO,o + 2H20 [hasmochromogen] [hajmatin] In an alkaline solution hfemochromogen is of a purplish colour ; it shows two well-marked bands, one midway between the D and E lines, and the other rather fainter between the E and b lines (see fig. 59, spectrum 9). On vigorously shaking up such a solution with the air, the bands disappear, and the hazy shadow-like absorption band of alkaline hfematin is seen. Hoppe-Seyler protests against the name reduced lijematin being applied to hsemochromogen. Hasmatin is an oxidised product of hsemochromogen, but does not contain so much oxygen as oxyhaemochromogen, which in all probabiUty is the compound that contains the respiratory oxygen in oxyhiumoglobin. Hsematin can be obtained from oxyhfemoglobin by treating it with acids or alkalis, and during this process oxygen is absorbed, probably uniting with the proteid part of the haemoglobin molecule.' Therefore, although hfematin is an oxidation product of haemochromogen, Hoppe-Seyler considers that h;emochromogen is not a reduc- tion product of hfematin. He also considers it probable that h»mochromogen i* a ferro-compound, and hfematin a ferri-compound. Linossier^ has proposed a change in the names of these two substances ; he suggests that hfemochromogen should be called hsematin or reduced hfematin, and that what has hitherto been called hfematin shjuld be named oxyhfematin. But if Hoppe- Sevier's view of the relation between hfematin and hfemochromogen prove correct, this new nomenclature of Linossier's is, of course, inadmissible. Hoppe-Seyler^ has recently succeeded in obtaining hfemochromogen in a crystalline form by heating a solution of haemoglobin with sodium hydrate in the absence of oxygen. By heating a solution of CO-haemoglobin with the same reagent, crystals of CO-hfemochromogen are formed. Linossier had previously obtained the carbonic oxide and nitric oxide compounds of hfemochromogen.* Haemochromogen is thus the atomic group in hfemoglobin which unites with the gases. To represent it in another way : — Hfemoglobin = hfemochromogen + globin (proteid). Oxyhaemoglobin = oxyhfemochromogen + giobin. CO-haemoglobin = CO-ha^mochromogen + globin. Ecemin. — This substance is the hydrochloride of hfematin ; it crystallises readily, and so forms an easily applied test for blood. The crystals^ may be prepared for microscopic examination, by boiling a drop of blood with a drop of glacial acetic acid on a glass slide : on cooling, the crystals which are triclinic plates and prisms of a dark 1 Lebensbaum (Monats. Cliem. viii. 166) finds that oxyhaemoglobin in 01 sulphuric acid solution absorbs I'l per cent, of its weight of oxygen. 2 Comptes rend. civ. 1296. ^ Zeit. physiol. Cliem. xiii. 477. * These show characteristic absorption spectra resembling that of oxyhsenioglobin. 5 Sometimes called after their discoverer, Teiehmann's crystals {Zeit. f. tiat. Med. 1853, vol. iii. p. 375, viii. p. 141). For crystallography see Lagorio, J. Buss. Chem Soc. 1885, p. 35. TIIK J'.Looi) 291 brown colour, often in star-shaped clusters, and with rounded angles (fig. Gl), sepai'ate out. In the case of an old blood stain, when one wishes to apply this test, it is necessaiy to add a small crystal of sodium chloride in addi- tion to the glacial acetic acid. Fn-.^h ^ .. blood contains sufficient sodium j^k"-". ^'' ^^ chloride in itself. ^T^/^'^ ^-^ ^^ "^-f^ On a large scale, htemin may be M \ > ^^ ij *^^ -^^ ^ prepared in the following ways : — -V TT U 1. A solution of hjvmatin in alcohol ^ ^^ ' ^ ^^ ^^ ^ A acidified with sulphuric acid is t "' -i^^. W^ >aj^ -^ i< heated with a solution of sodium ^ ' ^ ^ ^^ J. ''Ja chloride.' 2. Defibrinated blood is '^ ^ T'"- .?^ mixed with a large excess of dilute ' ^ '*' s' ^ '^ sodium chloride (1'5 per cent.) solu- '^ '' ^ -fg^^ ,• „ 1 1 1 J.1 1 Fid. Gl. - Hiemiii crystals maguified (Prej-er). tion ; the corpuscles when they have ' =. v j / subsided are extracted with ether ; the ethereal extract is evaporated to dryness, and the residue heated with glacial acetic acid (Hoppe-Seyler). Ha?min=h*matin-f 2HC1 (Hoppe-Seyler). Hfemin is insoluble in water, ether, chloroform, alcohol, and in cold dilute acetic or hydro- chloric acids. It is soluble in an alcoholic solution of potassium car- bonate, in caustic alkalis, and in boiling acetic and hydrochloric acids. The crystals are decolourised by alcoholic ammonia (Shalfeeff).^ Analogous compounds to hsemin are formed with hydrobromic acid (HBr) and with hydriodie acid (HI). They may be called bromo- hsematin and iodohwmatin respectively, while ha^min may be termed chlorohffimatin. The crystalline form and colour of all three com- pounds are identical (Harris).^ It has been stated that the prepara- tion of iodohpematin crystals is a more delicate test for l)lood stains than that of chlorohfematin crystals (Bufalini).^ Cyan-liamatm. — ' When potassium cyanide is added to an ammoniacal solu- tion of pure hsematin, or to a solution of osyhjemoglobin, a broad band extending from D to E is seen on spectroscopic examination. On adding reducing agents, a spectrum with two well-marked absorption bands is obtained. These optical characters are supposed to depend on the production of a compound which has ' Gamgee, Physiol. Cliem. p. 117. 2 Shalfeeff, Journ. Buss. Chem. Soc. 1885, p. 203. 3 V. D. Harris, Brit. Med. Journ. vol. ii. 1886, p. 103. See also K. Bikfalvi, Che7n. Centr. 1886, p. 499. ■* Bufalini, Arch. Pharm. (3) xxiii. p. 682. The method consists in heating the aqueous extract of the blood stain with a drop of iodine tincture and a little acetic acid on a glass sUde. Crystals form in 1-2 minutes. MacJtIunn obtained a crystalline compound of haematin with sulphuric acid, but the chemical constitution of this substance still remains to be worked out {Journ. Physiol, vi. 24). u 2 292 THE TISSUES AND ORGANS OF THE BODY been designated cyan-hiematin. We are, however, merely acquainted with the spectroscopic characters of the supposed compound.'' Hcumatojiorphyrin. — When ha^matin is heated with fuming hydro- chloric acid to 160° C. the iron is removed from it, as a ferrous salt, and iron-free hajmatin or heematoi^orphyrin is formed. The same result is obtained when hsematin is dissolved in concentrated sulphuric acid, the solution being of a purple-red colour. For spectroscopic purposes only, hsematoporphyrin may be obtained by adding a small quantity of blood or oxyhsemoglobin solution to a large quantity of strong sulphuric acid.^ HfematoporiDhyrin may l)e precipitated from its acid solution by the addition of water. This precipitate is soluble in water, and also in caustic alkalis ; the optical properties of the aqueous solution are the same as those of the acid solution, viz. a broad dark band a little to the right of D, and a narrow fainter band to the left of D (fig. 59, spectrum 10). When hasmatoporphyrin is dissolved in caustic alkalis, it appears to undergo some amount of decomposition (Gamgee) ; the solution has a reddish -brown tint, and the absorption spectrum shows four bands (fig, 59, spectrum 11). Heematoporphyrin is interesting as being not only an artificial product of haemoglobin, but as also occurring in the integument of certain invertebrate animals, viz. starfi.shes, slugs, the common earth worm, and various sponges (MacMunn).^ Polyperythrin (Moseley), a pigment of various actiniae and deep sea polypes, is also probably identical with htematoporphyrin. It is also found in the eggshells of some birds. TJrohoitnato'por'pliyrin is a kind of hiematoporphyrin found in the urine in certain diseases ; it will be fully described under Urine, Hcematoporjihyroidin is a decomposition product of hfiematoporphyrin described by le Nobel.^ The formula given by Hoppe-Seyler "' to hiematoporphyrin is C68HY4N8O12; and its formation from ha?matin may be thus repre- sented {see further p. 294): — C68H7oN8Fe.,0,o + 2H.S0, -f Oo=C,,H,,N,0, , + 2FeS04 [hfeinatin] [sulphuric acid] [liannatopoi'pliyrin] [sulphate of iron] 1 Cyan-hsematin will be found more fully described in Hoppe-Seyler's Med. Chem. Untersuchungen, Heft iv. The above short description I have taken from Gamgee's Fhysiol. Chem. p. 115. ^ Hopjie-Seyler has obtained hsematoporphyrin by the action of nascent hydrogen, and MacMunn by the action of sodium amalgam on hsematin. 3 MacMunn, Quart. Journ. of Mic. Science, 1877; Journal of Physiology, vols. vii. and viii. It may be added that in certain species of actinia3 a pigment very similar to haemochromogen, and convertible into hsematoporphp-in, is found [Phil. Trans. 1885). 4 Che7n. Geniralhl. 1887, p. 538. ^ Hoppe-Seyler has described another iron-free derivative of hasmatin, to which he gives the provisional name of hsematolin {CC8H78N3O7). It differs from heematoporphyrin in being insoluble in sulphuric acid and caustic alkalis [Physiol. Cheinie, p. 397). THE LLOOI) 295 Ilannatoidin. — In old blood clots, such as occur in the brain after cerebral hivmorrhage, in the interior of aneurisms, and in the corpora, lutea of the ovary, small rhorabohedral crystals of a brick-red colour are often found, together with an amorphous deposit' of the same colour {see fig. 62). The name htematoidin was given to this substance by Vir- chow.'^ It is insoluble in water, alcohol, ether, acetic acid, dilute mineral acids and alkalis ; soluble in concentrated acids and caustic alkalis. When treated with fuming nitric acid, the crystals give the same colour reaction as the bile pigment does ^ (Gmelin's reaction). Hsema- toidin is undoubtedly a derivative ©f hiemoglobin, and it is free from iron : so also is bilirubin, the pigment of the bile ; and Salkowski ^ found hsematoidin to be identical chemically with bilirubin. Ber- zelius found in the gall-bladder crystals of bilirubin exactly similar to those of hsematoidin. Preyer, however, states that the two substances differ spectroscopi- cally ; solutions of bilirubin showing 1 1 1 1 • e ^ J • T Fig. C2. — Hismatoidin crystals. no bands, solutions or hfematoidm showing one band between b and F, and a weaker one between F and G. Holm ^ obtained similar results. Thudichum ^ has pointed out that Preyer and Holm mistook the lipochrome (lutein) in the cow's ovary for ha^matoidin, and hence they concluded that it was not identical with bilirubin. Neither htematoidin nor bilirubin shows bands, but both possess a strong absorptive power for the violet end of the spectrum. Hcemosiderhi is the name given by Neumann '^ to a pigment often occurring in extravasations and thrombi with hfematoidin, but differing from it in containing iron. So far, in describing the composition of hi^matin and its derivatives, I have followed Hoppe-Seyler pretty closelj*. More recently, however, the subject of hsematin and its allies has been reinvestigated by Nencki and Sieber,** and the ' The amorphous variety was first described by Eobin, Ann. Chem. Pharm. cxvi. 89. See also Stiideler, Ibid, cxxxii. 328. 2 Virchow's Arch. d. pathol. Anat. u. Physiol, vol. i. (1847), p. 383. ^ JafEe, Arch. f. path. Anat. xxiii. 192. Hoppe-Seyler, Ibid. xxiv. 10, ■• Salkowski, Hoppe-Seyler' s Med. Chem. TJnters. Heft iii. p. 436. 5 Hohii, Juurn.f. pirakt. Chem. c. 142. 6 Thudichum, Proc. Roy. Sac. xvii. 255. Dr. MacMunn kindly furnished me with this and several of the foregoing references. ' Virchow's Archiv, cxi. 25. 8 Nencki and Sieber, Berichfe d. deittsch. chem. Geselhchaft, xvii. 2267, xviii. 392. Monaish. Chem, ix. 115. 294 THE TI88l^ES AND ORGANS OF THE BODY conclusions at which they liave arrived are on many points different from those of Hoppe-Seyler' : to certain of these, however, much importance cannot be attached ; for instance, until we know a rational formula for hiematin it matters but little whether in its empirical formula there are a few atoms more or less of any one element. In certain other matters, for instance, in the composition of hsemin, their views demand confirmation ; I have, therefore, retained the descrip- tion of this substance as usually given, and here add a resume of Nencki and Sieber's work : — They ascribe to haematin the formula C32H3oN4Fe04 ; and the word haemin is applied, not to the hydrochloride of hfematin, but to an anhydride of haematin, of which the formula is Ca^H^oN^FeOa. What is usually called haimin (chlorohsematin) they call hasmin-hydrochloride, of which the formula is C3._,H3„N4Fe03.HCl. This is a crystalline substance, and in its preparation these observers employ amyl alcohol, and a molecule of amyl alcohol of crystallisation is combined with the crystals, the full formula for which would therefore be CT^HadN^FeOj.HCl.CjHijO. This molecule of amyl alcohol can be driven off by heating the crystals to 130° C. HEematoporphyrin may be obtained by the action of concentrated sulphuric acid on either hfematin (Cj^Hg^N^FeO^) or hasmin (C3.^H3oN4Fe03). Its formula is CjoHj^N^O^, and its formation may be represented by this equation : — CsaHa^N.FeO, + H^SO, = Ca^Ha^N.O, + FeSO. + H^O [hrematiii] [sulphuric acid] [liKmatoporphyrin] They subsequently found that when htemin or hajmatin is heated with a saturated solution of hydric bromide in glacial acetic acid it is readily converted into hjematoporphyrin, which shows differences from that obtained in the usual way. It has the fi rmula Ci^HigN.Pj. It is insoluble in water and dilute acetic acid, slightly soluble in ether, amyl alcohol, and chloroform, and readily soluble in alcohol, dilute mineral acids, and solutions of the alkalis. It is reddish-brown in colour, is amorphous, and turns brown and becomes more insoluble at 100° C. Its alkaline solution shows the four bands jireviously described. The hydro- chloride (C,bH,„N.P3.HC1) crystallises in tufts of needles,'-' the sodium salt (C,aH,,NaN.p3 + HoO) in microscopic prisms.^ This substance is considered to be pure htematoporphyrin, and the substance prepared by the action of sulphuric acid on htematin is probably its anhydride (CaoHgoN^Or, = 2C,jH,sN.0g — 2H2O). Hffimatoporphyrin has the same empirical formula as bilirubin, which it resembles in many of its properties. The formation of bilirubin from haematin is represented by Nencki and Sieber by the equation : — Cs^HsjN^O.Fe + 2H2O = C3,H3„N, 0, + Fe [hitmatiii] [bilirubin] When introduced into the living subject htematoporphyrin is partly expelled in the urine, but the greater portion is retained, and is probably utilised in the formation of haemoglobin. Taking a general survey of the .subject, we are able, in spite of contradictory assertions on minor points, to draw a few conclu- ' Hoppe-Seyler has discussed the experiments of Nencki and Sieber, and reaffirmed his own views in the Berichte d. deutsch. chem. GeseUsch. xviii. 601. - Figured in Archiv f. exp. Path. u. Pharmakol. xxiv. plate iv. THE ELOOD 295 sions. There seems to be a group of iron-free derivatives i)f liu'iuatin, and not a single one. Some of these can be produced artificially, such as hivniatoporphyrin with its anhydride, and hivmatolin ; cer- tain others occur in the organism of certain lower animals as such (haematoporphyrin), and certain others are formed during the normal or abnormal disintegration of haemoglobin that occurs in the course of the manufacture of bile and urine pigments (bilirubin, urobilin and uroha^matoporphyrin) ; and lastly one is formed in the disintegration of the blood pigment, that occurs in an old blood clot (hjvmatoidin). In spite, however, of difl'erences between these difierent forms of iron- free haematin (in solubilities, optical characters, &c.), one cannot help being more struck with the resemblances between them. It there- fore appears possible that we may eventually find we are dealing with a number of isomeric or polymeric substances, for in three of them already (bilirubin, ha?matoporphyrin, and ha?matoidin) the same empi- rical formula has been described. TESTS FOR BLOOD In medico-legal cases it is often necessary to ascertain whether or not a red fluid or stain upon clothing is or is not blood. The tests to be applied are microscopic, chemical, and spectroscopic. Microscopic tests. — The corpuscles of the blood should be sought for. If the blood is fairly fresh it is possible to distinguish the human red corpuscles from the red corpuscles of those animals in which they are nucleated, or diflfer from them greatly in size and shape. Exceedingly careful measurements have shown that there are small but very small variations in the diameter of the human red corpuscles and those of the common mammals, but practically it is not possible to discriminate between them. Chemical tests. — The old test with tincture of guaiacum and hydro- gen peroxide, the blood causing the red tincture to become green, is very untrustworthy, as it is also given by many other organic sub- stances, such as potatoes, certain forms of filtering paper, ifcc. &c. The only trustworthy chemical test is the formation of haemin crystals ; if one only has a piece of stained clothing to deal with, this is boiled with glacial acetic acid, and a small crystal of sodium chloride on a slide ; on cooling the crystals foirm as already described. Spectroscopic tests. — If the blood is present in any quantity, the typical bands of oxyhtemoglobin can be readily seen through the spec- troscope ; these give place to the single band of hfemoglobin on the addition of a reducing agent. One must be prepared, however, in the case of old stains for the presence of metha;moglobin, or of haematin. 296 THE TISSUES AXD ORGANS OF THE BODY In such a case, and also when the quantity of hemoglobin is very small, the most readily obtained spectrum is that of hfemochromogen or reduced ha?matin. The stained fabric is extracted vnth a small quantity of water ; a few drops of a reducing agent (freshly prepared sodium hyposulphite is a good one to use),^ and then a few drops of concentrated caustic soda solution to decompose the hsemoglobin ; the spectrum of htemochromogen, or at any rate the best marked band of that substance (the one between D and E), then appears ; the mean wave-length of this band is A 557. This band disappears on heating to 50° C. and reappears on cooHng ; it also disappeai's when the solu- tion is agitated with the air, but the substance so formed is alkaline hsematin, which shows only a faint band, too faint to be seen in such weak solutions as we are considering. In cases where the stain has become insoluble in water, it must be dissolved out with ammonia, and the solution reduced by Stokes's reagent ; the typical band of hpemochromogen is then seen. This test is applicable even in cases where no hoemin crystals are obtainable. In any particular case it is advisable not to rely upon one test only, but to try every available means of detection at one's disposal. 1 Recommended by Linossier, Bull. Soc. CJtim. xlix. 691. 297 CHAPTER XVI THE BLOOD IN DISEASE The common expression ' The blood is the life ' expresses what was till comparatively recent years regarded as true by scientists, namely that the most important of the vital processes take place in that fluid ; the chemical changes grouped together under metabolism, we now know occur not in the blood, but in the tissues generally, the blood forming in great measure a means of putting the other tissues into communication with those parts where nutriment is obtained, or ex- cretions discharged . Not only was this view held of the importance of the blood in the processes of health, but in disease also it was supposed to play an equally leading part. This gave rise to the doctrine of disease called humoral pathology ; and its exponents were called humoralists ; the opponents of this exclusive view of disease arose about the middle of this century, and were dubbed solidists or anti-hu moralists. The great stimulus in starting the opposition to humoral doctrines was no doubt Schwann's great discovery of the important part that the animal cell plays both in health and disease, and the anti-humoralists regarded the life of the organism as the sum of the life of all the constituent cells of its various organs ; similarly too, disordered conditions of the blood were considered to be either secondary to changes in the other tissues, or if primary, that they produced their results by the effects such changes had on the other tissues. In the present day, half a century since the promulgation of the cell theory, pliysicians are now better able to weigh these two counter- doctrines, than was possible in the first flush of a new and brilliant discovery, and their relative importance can be now more fairly estimated. It is now well recognised that all diseases are not morbid conditions of the blood, and that unhealthy blood is often the result of disorders elsewhere ; in the same way, bleeding is not resorted to as a panacea for every ill ; but on the other hand it is also perfectly well recognised that in certain diseases the defect is either in the blood itself, or in the blood-forming organs ; and that in many diseases we have very distinct evidence of the presence of abnormal substances or 298 THE TI8SUES AND OEGANS OF THE BODY poisons in the blood itself ; gout is an instance of this ; but in the early days of anti-humoralism the humoral nature of gout was stoutly denied. In the following brief description of the various altered blood conditions seen in disease, it will be convenient to take first those conditions in which the primary mischief seems to be in the blood itself, and secondly those in which the morbid state of the blood is part of a general pathological condition, or secondary to changes in other organs. THE BLOOD IN AN.EMIA Ansemia is a term which covers a large number of cases in which poorness in one or other, or all the constituents of the blood, is the one constant condition. It may be the result of numerous and very varying conditions ; when the blood-forming tissues are at fault, it may be considered a primary disease of the blood itself ; but there are a large number of other cases in which ansemia is the accompaniment' of a general state of debility or malnutrition, or secondary to chronic affections of other organs. It may be the result of excessive haemorrhage ; the blood then becomes rapidly diluted with lymph, and thus the corpuscles are less numerous than normal until fresh ones are formed to replace those that were lost ; the total of solids of the plasma is also diminished, as the lymph is more watery than normal plasma. Not only hfemorrhage, but other discharges also, produce an anaemic condition, such as the discharges from abscesses, excessive and chronic diarrhoea, discharge of albuminous urine in Brighfs disease, and so forth. On the other hand, the intake of nutriment may be insufficient, and thus the whole body, including the blood, may be wasted. Insufficient food, unsanitary conditions, i.e. insufficient air, light, and exercise, are all potent causes of anaemia. The wasted condition may not however be the result of bad hygiene ; malnutrition may arise from an inability to take food, owing to obstruction in the oesophagus, to disease such as catarrh, or the more serious condition, cancer of the stomach, and to many other morbid states of the alimentary organs. In chronic diseases generally, in chronic lead or mercurial poisoning, there is also associated a marked anaemic condition. Lastly we have those primary conditions of anaemia which are called chlorosis, pernicious anaemia, and leucocythfemia. In anaemia generally it may be stated that the most marked effect is seen in a diminution of the number, size, and colour of the red cor- THK JiLooI) IN" DISKASK 299 pusoles, often ;iu increase actual as well as relative of the white corpuscles, and an increase of water and diminution of the solids of the plasma. The changes in the corjjuscles are investigated clinically by the microscope, the hiemocytometer, and hivmoglobinometer ; while the changes in the plasma require the moi-e complicated methods of analysis, all of which have been described in the foregoing chapter. In chronic amemia the red corpuscles may be diminished to ^, or in extreme cases to ^, of the normal amount. In cases of moderate intensity the amount of ha?moglobin varies between ^ and 5, and in extreme cases to ^ of the normal amount. In all chronic cases the mean diameter of the red corpuscles falls to 7 f^ or even to 6 ^ ; there are also an unusually large number of small red corpuscles (diameter 2"2 — 6 yu), and almost as frequently a certain number of unusually large corpuscles (diameter 10—12 yu). Some of the smaller corpuscles seem to have less consistency than normal, and assume modified, often oval shapes. ^ In anajmic blood the clot shows usually a buffy coat ; this does not seem to be due to the coagulation being very slow, but rather to the subsidence of the red corpuscles being very quick, on account of the low specific gravity of the plasma. Chlorosis is a condition of anfemia which occurs almost exclusively in young women, and is associated with disorder of the menstrual function. There is intense anaemia, falling most especially on the red corpuscles which are few and pale ; this produces a peculiar greenish pallor of the skin, from which the disease derives its name. Chlorosis also is that form of anaemia in which the administration of iron causes the best effects. Often after only a few days the corpuscles are increased, a red colour has returned to the cheeks, and all the other troubles such as palpitation, breathlessness, kc, due to an insufficient amount of haemoglobin, disappear. The following analyses give the condition of the blood before and after the medicinal use of iron in two cases (Andral and Gavarret-). Case Before Iron AVaterin 1,000 parts 866-7 Fibrin . . .3-0 Blood corpuscles . 46"4: Solid residue of serum 83-9 1 Many of the above facts regarding the coi-puscles iu anaemia I have taken from the admirable epitome of Hayem's work given in Gamgee's Fhijsiol. CJiem. p. 148. Hayem, Mecherches sur Vanatomie iiormale et judliologique dii sang, Paris, 1878. Du sang, Paris, 1889. - Andral and Gavan-et, Annales de chimie et tie physique, Ixxv. 225. 1 Case 2 After Iron Before Iron After Iron 818-5 852-8 831-5 2-5 3 5 3-3 95-7 49-7 64-3 83-3 94-0 100-9 300 THE T1^;SL'ES A>T) ORGANS OF THE BODY Considerable doubt has. however, arisen as to whether the iron administered is actually absorbed ; there are many who believe that the iron is absorbed ; on the opposite side, Hamburger among others considers that little or none of the medicinal preparations of iron is absorbed from the alimentary canal, but that iron is absorbed only in the form of organic compounds, such as are formed in the synthetic processes of plant and animal life. The quantity of iron in the whole body is only three grammes, and this quantity is taken many times over during treatment. Bunge' explains the useftdness of iron in chlorosis by its forming iron sulphide in the intestines, removing in this way excess of sulphur from the body : in chlorosis there are ex- cessive fermentation processes in the alimentary canal, and large quantities of sulphuretted hydrogen are foiTued, which destroy the organic compounds of iron that form haemoglobin (haematogen) ; the administration of iron prevents this destruction of ha?matogen. Landwehr- points out that such a theory, however, does not explain the limitation of the disease to the female sex, and the period of early adolescence. He regards the disease as one produced by an excessive development at this period, of substances containing animal gum* necessary for the nourishment of the embryo, and which act injuriously on the ha-moglobin molecule. If this is so, chlorotic people should take little or no carbohydrate food. Landwehr further considers that iron precipitates the gum in the alimentary canal as a jelly-like eoagulum,. and thus excess of gum leaves the body with the faeces, Progres.nve pernicious a/rueinia. — By some this disease has been regarded as simply an advanced form of ordinary ansemia, but its rapid development, and its usually fatal termination, as well as certain other peculiar symptoms (attacks of pyrexia, liability to retinal haemorrhage, kc). place it on a different footing from ordinary anaemia, and most clinical observers recognise it as a distinct disease. The disease was first described by Drs. Wilks and Addison, but since then numerous observers have added greatly to our knowledge of its symptoms and pathology. We have here, however, only to deal with the changes in the blood, and the probable cause of those changes. The changes in the blood have been very thoroughly investigated by Eichhorst,^ and may be summarised as follows : — ■ 1. The coloured corpuscles are diminished in ntmiber, and in the amount of haemoglobin they contain. There is a great increase in the ^ Bunge, Zeit. physiol. Chem. is. 49, - Landwehr, Pfluger's Archiv, si. 21. ^ The qnestion of animal gum and its relation to mucin and similar bodies will be found discussed in detail under the heading Connective Tissues. ■* Die jjrogressive perniziose Aiuimie, Leipzig, 1878. THE JiLooJJ IN DISEASE 301 number of the small red corpuscles, many of which are mis-shapen, and many of which are globular and not discoid. The non-discoid cor- puscles are not, however, constantly present.' Occasionally nucleated coloured corpuscles have been observed (Byrom Braniwell).^ 2. The colourless corpuscles are also few in number. 3. The blood when shed coagulates with difficulty. The question arises, is this disease due to diminished formation of the elements of the blood, or to increased destruction of the same ? In those cases where nucleated red corpuscles have been found, it has been assumed that the red marrow is diseased, as in certain forms of leucocy- thsemia, which will be presently mentioned. But the majority of cases show no disease of the red marrow, and the usual view held is that pernicious ansemia is due to excessive destniction of the cellular elements of the blood. Recent researches by Dr. W. Hunter^ and Dr. Mott * on the pathology of the disease fully confirms this theory ; I quote briefly Hunter's conclusions with regard to the nature of the blood destruction. It is not simply a dissolution of the red corpuscles in the general circulation, such as occurs periodically in paroxysmal hsemoglobinuria, or may be artificially induced by the injection of dis- tilled water or pyrogallic acid into the circulation, Hjemoglobinuria is always absent in pernicious anaemia. In this relation the condition of the liver is of the greatest importance. The condition of the liver is as foUows : (1) It is exceedingly rich in iron ; (2) there is excess of pigment within the liver cells ; and (3) there is fatty degeneration in the central third of each lobule. A condition closely resembling this, though not so marked, is produced by the dinig toluylendiamine. It is therefore assumed that the agent, or agents, which induces the excessive destruction of blood in pernicious antpmia is one whose action on the blood and on the liver cells is the same as that of toluylendiamine. This view is strengthened by the consideration that the form assumed by the haemoglobin after its liberation from the corpuscles is, in cases of pernicious antemia, similar to that assumed by it after poisoning by toluylendiamine. After poisoning by this drug numerous small globules of a yellowish colour occur in the urine, and these exactly resemble the globules of pigment found in the convoluted tubules of the kidney in certain cases of pernicious ana?mia.' The urine contains excess of urobilin (Mott). ' Grainger Stewart, Brit. Med. Journ. vol. i. 1876, p. 40. ^ Edinburgh Med. Journ. xxiii. 408. 5 Hunter Lancet, vol. ii. 1888, p. 634. In this paper will also be found reference to researches of others who have worked at the subject. * Ibid vol. i. 1889, p. 520; vol. i. 1890, p. 287.; ^ Hunter does not however give any proofs that these yellow globules consist of 302 THE TISSUES AND ORGANS OF THE BODY With regard to the precise nature of the poison generated, Hunter suggests it may be of a cadaveric nature, absorbed from the alimentary- tract. The research is, however, specially valuable in fixing the seat of the disintegration of the corpuscles in the portal circulation, and its important annexa, the spleen and liver. {See also Liver, Spleen^ Urine.) Hchwglohinuria. — This is a condition in which the lijemoglobin of the red corpuscles becomes dissolved in blood plasma (ha^moglobina^mia), and passes into the urine, mostly in the condition of methsemoglobin. This condition will be more fully described under Urine. Leucocytltceiaia. — The normal proportion of white to red corpuscles in man is about 1 : 350. This proportion is, howe\er, by no means fixed ; it varies in difterent vessels, at different times of the day, with different ages. There are also certain conditions in wliich the white corpuscles are increased, but still not to such a great extent as to pro- duce the symptoms of what is called leucocythfemia. Thus in many forms of chronic anaemia, the white corpuscles are slightly raised in number absolutely as well as relatively. During pregnancy there is a similar condition, and in many inflammatory affections it occurs also. The term leucocytosis is applied to this condition, whereas the word leucocythjemia is not used until the proportion of white to red reaches 1 : 20. In some cases, however, the proportion may be as high as 1 : 6, or even, it is said, 1:3; and in these cases when the blood is shed it has the appearance of a mixture of blood and pus. The disease is usually accompanied with great hypertrophy of the spleen ; sometimes with a general hypertrophy of the lymphatic glands throughout the body ; it is, however, quite possible to have a very great increase in the size of the lymphatic glands (lymphadenoma) without any leucocythjemia.' There are other cases again in which the red marrow is diseased (myelogenic leucocythjemia), and nucleated red corpuscles like those of the embryo are found in the circulation. It is possible that in certain cases of leucocytha?mia and other forms of intense antemia the affection of the red marrow may be secondary rather than primary. Thus Denys,^ who has investigated the formation of red corpuscles in pigeons, finds that during simple hsemoglobin or are derived from it ; they may indeed be often found in perfectly normal kidneys, and give none of the reactions of hfemoglobin. 1 In one third of the cases of splenic leucoej-thaemia the lymphatic glands are also enlarged ; under the microscope sections of the glands appear nonnal, there is rarely the increase of the interstitial reticulum that occurs in lymphadenoma. Gowers, Eeynolds's System of Medicine, vol. v. p. 238-9. - Denys, La structure de la moelle des OS chez les oiseaux. Travaux du laboratcire d'anatomie pathologique de Vuniversite de Louvain. THE BLOOD JX DISEASE 303 inanition the blood-forming structures degenerate, and are replaced by a mucus-like tissue, and that imperfectly formed or embryonic cor- puscles make their way into the general circulation. Not only is the number of white corpuscles increased, but the red are diminished in number. Elongated, octahedral, colourless crystals have been stated to separate from the blood of leucocytha^mic patients after death by several observers (Charcot, Vulpian, Salkowski, Zenker), and different views have been held as to their nature ; they have been variously considered to consist of pro- teid, of mucin, and of the phosphate of a base with the formula CoHgX (Schreiner '). These fi ^ & crystals are usually spoken of as Charcot's crystals.^ , a They are not, however, peculiar to leucocythsemia ; ly^ {y they have been found also in cases of simple anaemia, and in the sputum of bronchial asthma ^'''aft^r^Stlr; a'fe^w ^Xievden ^\ ''^'^ angles rounded. Xanthine and hypoxanthine occur in greater abundance in leucaemic blood than in normal blood ; these, no doubt, are derived from the white corpuscles, according to Kossel ^ from the nuclei of white cor- puscles. Lactic acid, which has been also described, doubtless owes its origin to the increased number of white corpuscles, which undergo changes resulting in the formation of this acid when the blood is shed. {See footnote 1, p. 261.) Scherer and Gorup-Besanez ^ have described in Uie blood in this ^disease a substance which is soluble in hot water, and sets into a jelly when its solution is cooled, in fact, which behaves like gelatin. Addison's disease. — This is a disease which is associated with great wasting and anaemia ; the skin is deeply bronzed, and in typical cases the suprarenal capsules have been found {j)ost mortem) to be diseased. The relation between bronzing and suprarenal capsular disease is, how- ever, by no means constant, and by some observers the changes occur- ring in the neighbouring semilunar ganglia are regarded as more important than those in the suprarenals. ' Liebig's Annalen, cxciv. 68. 2 A full account with references will be found in Gowers's article iu Beynolds's System just quoted, p. 233. 3 More recently Meissen {Berlin, klin. Wochensch. No. xxii. 1883) has described the same crystals in the expectoration of phthisical and bronchitic patients. * Kossel, Zeitschr. f. phtjsiol. Cliem. vi. 7-22. See also Salomon, Archiv f. Anat. u. Physiol. 1876, p. 762. 5 Maly's Jah7-esbericht, iv. 12G. 304 THE TISSUES AND ORGANS OF THE BODY Nevertheless, it is interesting here to note MacMunn"s' discovery of hjemo- chromogen in the medulla of the suprarenal capsules of mammals. This is partially removed by washing out the blood vessels with salt solution. Hence, and owing also to the fact it is elsewhere excretory, the hamochromogen of the adrenals is probably excretory too. MacMunn further considers that if the adrenals are functionless, as in Addison's disease, the metabolism of haemoglobin (and of allied pigments, to which he has given the name histohaematins) is pre- vented, and the incompletely metabolised pigments circulate in the blood, and lead to staining of the skin and mucous membranes. Myxcedema. — In a few cases of this disease in man, the red cor- puscles, or the ha?moglobin, have been observed to be diminished, but in the greater number of cases, no characteristic changes have been noted by cKnical observers, and in the few instances in which the blood has been more fully examined the only notewortliy alteration seen was the formation of a bufiy coat on the clot. In certain animals the disease can be produced experimentally by removal of the thyroid gland, and disease or atrophy of the thyroid gland is the cause of the disease in the human subject also. Some animals (for instance, pig, donkey), however, do not exhibit the typical symptoms of the disease, and in these the blood and serous etfusions are perfectly normal. But, on the other hand, in monkeys, which exhibit the disease in a very characteristic manner, and show the swelling of the connective tissue which will be fully described with that tissue, it is found that the blood also exhibits certain marked changes, viz. anfemia, slow coagulation with formation of a buffy coat, and presence of small quantities of mucin,- which increase as the disease becomes fully developed. In dogs also, leucocytosis and anaemia follow the extirpation of the thyroid. Enumeration of the corpuscles in the thyroid vein and artery respectively has shown that there is a distinct surplus in the former vessel. One would, therefore, be inclined to conclude that the gland was concerned in the formation of corpuscles; and that this supposition is in part correct, is confirmed by the existence in nodules throughout the gland of a tissue resembling closely that of the spleen, and in some few cases removal of the gland has been followed by enlargement of the spleen. The general alterations throughout the whole body seem, however, to point to the function of the gland as concerned not so much in the elaboration of the corpuscles as of certain constituents of the plasma ; 1 Proc. Roy. Soc. xxxix. 248. ^ Or at least of a substance readily precipitable by acetic acid and insoluble in excess of that reagent. 'I'liK i;l-()()|» in DisKASK :i05 and tlio overijrowtli of the connective tissues, and tlie accumulation of an al)nonnal product in the blood, have led some obsei-vers to suppose that the gland is concerned in the separation of mucin fiom the blood, and then the completion of its metabolism into simpler products. A full account of myxredema, clinicul and experimental, svill be found in the report of a Committee of the Clinical Society, publishelSEASp] BOD zymotic diseases, or acute specific diseases, as they are sometimes called, are produced l>y certain low forms of vegetable life called bacteria, bacilli, micrococci, &c., and their contagiousness consists in the trans- ference of these bacteria or their spores from one person to another. The constant coexistence of a bacterial growth with some of these <1iseases has been proved, but in many other cases the existence of such germs is merely a matter of inference from the resemblance of the disease to other diseases, in which the existence of a specific bacterium has been proved. The various bacterial growths are distinguished from (me another by the shape and size of the bacteria thenjselves, by the way in which they grow upon certain nutritive media (gelatine, agar-agar, blood-serum, and the like), and by the fact that when a pure cultivation is introduced into another animal a certain set of symptoms is invariably produced. In certain acute specific diseases the seat of the bacterial growth is the intestine (cholera,' typhoid fever) ; in others the throat (diphtheria, itc.) ; in others the skin, or subcutaneous tissues (erysipelas, &c.) ; in ■others again, other organs may be the points specially attacked. There are two diseases in which the presence of such germs in the blood has been clearly demonstrated, viz. the spirillum of relapsing fever, and the bacillus anthracis of splenic fever and malignant pustule (AV^oolsorter's disease). Malaria. — The discovery of the bacillus malarije placed ague and the various forms of malarial (intermittent) fever among the acute specific diseases. This was a bacillus found in the blood of malarial patients l)y KleVjs and Crudeli, but upon further investigation it was found that it did not fulfil the ditterent conditions which prove the dependence of a disease upon a micro-organism. These conditions, as laid down by Koch, are as follows : — 1. The micro-organism must always be present in the animal suflfering from the disease in question. 2. The micro-organism must be cultivated for several successive generations in such a manner as to exclude other micro-organisms and other poisons ; and on the introduction of such a pure cultivation into the body of a healthy animal susceptil)le to the disease, it produces the disease in that animal. 3. It is necessary that the second animal so affected should show in its body the same micro-organisms. The bacillus malaria? and several other micro-organisms discovered 1 Owing to the abundant transudations from the ahmentary canal in fholera, the blood becomes relatively rich in solid constituents, and may in severe cases even have a iviscid consistency. 310 THE TISSI'PIS AND ORGANS OF THE BODY in malarial blood from time to time have not been found to fulfil these conditions. Marchiafava and CeJli ^ found that the red blood discs of patients aftected with malaria contain peculiar homogeneous bodies possessed of amoeboid movements. They call them lufmo-jAas- modinm malari(i\ Sometimes these plasniodia include pigment granules assimilated from the pigment of tlie blood discs. Blood containing the plasmodia is capable of producing intermittent fever in man after intravenous injection, and the blood corpuscles of the person so infected again contain the plasmodia. It is not, however,. V)y any means certain that the h;emo-plasmodium is the cause of the disease. The pigment produced apparently from the red discs by these peculiar l)odies is black in colour, and accumulates in and around the smaller vessels of the brain, liver, spleen, and marrow of the bones.^ There is considei-able doubt as to the precise nature of tliis pigment, and its presence in the blood is called melansemia. In the disease known as acute tuberculosis, melan;>:'mia also sometimes occurs. J'hfln.n)<. — This is associated with the presence in the lungs of the tubercle bacillus. The very remarkable statement has been recently made by E. Freund,^ that the tissues, pus, and blood of tuberculous patients contain cellulose. Septicffmia and Pycnuifi .—T\\e. various forms of blood poisoning known by these nan)es are undoubtedly caused by poisons generated during putrefaction, and this is a process which is caused by the activity and growth of micro-organisms. Still, no satisfactory evidence of tlie constant presence of bacteria in the blood has ever been advanced in man. Klein ^ has occasionally found minute bacilli in the blood vessels of the swollen lymphatic glands, and Koch has described a somewhat smaller bacillus in the blood of mice suttering from a special form of septicjemia, and various kinds of micrococci in certain pyjemic processes in mice and rabbits. The question here arises, how do bacteria produce their eftects ? In a few cases, as in those just quoted from Klein, they seem to act mechanically by blocking the vessels and so hindering the circulation through the infected part ; but in by far the greater number of cases of blood poisoning and of acute specific disease they produce a chemical poison, in the same way as yeast produces alcohol and carljonic acid from a solution of sugar, and it is the presence of tliis poison in the l)lood stream tliat causes the widespread general effects tliroughout the 1 Fortschritfe d. Med. Nos. xi. xviii. and xxiv. 1885. 2 Arnstein, VircJiow's Arch. Ixi. 494. 5 E. Freund, Wiener vied. Jahrh. I88(i, p. 335. ■• Micro-organisms and Disease, p. 120. 'I'lIK I;L<»()|) I.N DISEASE 311 body. One of the earliest successful efforts to obtain such a poison was made by Panum and Schmidt (see p. 172). A substance called sepsin (now known to be impure) was separated fi'om putrefying blood, and when injected into the blood stream produced the characteristic symptoms of septict^mia. Since then it has been discovered that during putrefaction processes, substances of the nature of alkaloids, called ptomaines and leucomalnes, are formed. There is no doubt that in many cases it is poisons of this nature produced by bacteria that cause the diseases of which we ha\e been speaking {see more fully Chapters XII and XIII). It must also be remembered that in other cases the poison may be proteid in nature {see Chapter X, Proteids as Poisons, p. 137). For some points respecting haemoglobin crystals in septic diseases see p. 31-5. THE BLOOD IN DISEASES OF VARIOUS ORGANS The changes which occur in the blood secondarily to diseases of other organs have been in part already alluded to. In chronic diseases there is invariably anaemia, and in inflammatory diseases the characteristic changes already described. In some diseases of the heart and also of the lunys there is imperfect aeration of the blood leading to blueness (cyanosis) of the pai-ts most distant from the central circulatory organs. Local patches of cyanosis may occur before the onset of gangrene, and also in certain vasomotor affections (Raynaud's disease). In affections of the liver, in which there is obstruction in the bile ducts so that that secretion cannot get into the intestine, bile pigment and l)ile salts enter the circulation, stain the skin and mucous mem- branes yellow or, in marked cases, brown, and pass into the urine. In some causes jaundice occurs when there is no stoppage in the bile ducts ; this is called non-obstructive jaundice, or sometimes l^lood jaundice. In these cases the bile pigments and not the bile salts occur in the circulation. Boerhaave and Morgagni long ago suggested that the jaundice in these latter cases was the result of suspended secretion, and the consequent accumulation of the elements of the bile in the blood. Bile acids, however, are never found in normal blood, and although many have searched for bile pigments, no satisfactory evidence of tlieir constant presence in the blood has ever been adduced ; the liver, there- fore, is not concerned merely in excreting the elements of the bile from the blood, but it actually forms the acids and the pigment within its own cells. Extirpation of the liver, moreover, never leads to the accumulation of the constituents of the bile in the blood. The ex- planation originally given by Frerichs, and now very generally accepted, of the way in which non-obstructive jaundice is produced is as follows : 812 THE TISSUES AND ORGANS OF THE BODY Under normal circumstances a very little of the bile poured out into the intestine leaves the body with the fieces ; by far the greater amount is resolved into simpler constituents which are absorbed and carried back to the liver to form a fresh supply of bile ; but in certain morbid states the absorbed bile does not undergo the normal metamorphoses, but circulates in the blood staining the tissues. The morbid states that conduce to this result are : — 1. Certain poisons : e.g. those of yellow fever, relapsing fever, pyjemia, &c., snake poison, chloroform. 2. Xervous influences : e.g. sudden fright, ctmcussion of tlie brain, cVrc. 3. A deficient supply of oxygen, as in some cases of pneumonia. 4. An excessive secretion of bile, especially when conjoined with constipation. ^ More recent in\'estigations into the pigments of the urine have shown that all dark brown urines are not necessarily coloured by bilirubin, even although staining of the tissues may be present also. An excess of urobilin in the urine produces a colour very like that of jaundiced urine, but it does not give Gmelin's colour test for the bile pigments, ^^'e have already seen that hfematoidin is produced in extravasations of blooil, and that htematoidin and bilirubin are iden- tical ; moreover, after blood has been extravasated into the tissues in large quantity, the urobilin of the urine is much increased. Urobilin can be artiticially obtained from hfemoglobin, hjematin, and bilirubin by the action of reducing agents (MacMunn ^). Pathological urobilin ditfers somewhat from normal urobilin, but it is a product derived from hsematin and not from the bile pigments."* We have here, then, instances of brown pigments staining the tissues and urine produced in the blood and not derived directly from the bile, and these cases at tirst sight look like jaundice. It is possible, tiiat on further in- vestigation it may be found that certain of the cases described as non-obstructive jaundice are not due to a liver atiiection at all, but that the brown pigment is produced in the stagnant blood of extravasations, or under the influence of certain poisons in the blood stream itself. As illustrating this latter possibility, it may be adduced that injection of the blood of one animal into the vessels of an animal of a different species often produces a breaking up of the blood corpuscles, and the appearance of dark brown pigment staining the tissues and passing into the urine ; the so-callecl jaundice of newly ' The foregoing account of nou-ob=tiuctive jaundice is taken from Dr. Murchison's work on the Diseases of the Liver. - See JIcKendrick's Physiology, p. 131. 5 MacMunn, Proc. Physiol. Soc. 1888, p. vi. THK IJLool) IN DISEASE 813 bom infants also is often ]>rocluced by the forcible expression of the placenta inunediately after delivery ; this sends an abnormal amount of blood into the circulation, and thereby may possibly cause a break- down (if some of its corpuscular elements. The pathology of this disease is, however, at present very obscure. Neumann ' regards it as a true hjtmatogenous jaundice, while Halberstamm - regards it as hepatogenous, finding not only bile pigment but bile acids also in the urine and pericardial fluids. Death from cholct^mia. — Patients suftering from jaundice are often attacked with delirium, coma, convulsions, and indicatiuns of profound prostration (the typhoid state) : in this condition, death may occur. These cases are generally cases of non-()V)structive jaundice, and there is no doubt that some alteration in the blood produces excitation and finally exhaustion of various nerve centres. The symptoms are com- monly attriliuted to poisoning with bile, but Freriehs has repeatedly injected bile into the circulation of dogs without producing ill results, and there is ample proof in cases of obstructive jaundice, that the blood of human beings may be saturated with bile for months, or even years, without cerebral symptoms resulting. Dr. Austin Flint has stated that the poison is cholesterin, one of the constituents of the bile ; but the cases and experiments just mentioned bear just as strongly against this view as against tlie somewhat more vague statement that the bile is the poison. Murchison considers that the cause of death in choljemia is the same as that in ui'temia ; the liver performs a lai'ge amount of the work which is finished in the kidneys, and whenever the liver or kidneys stop work, urea and its antecedents circulate in undue quantity in the blood, and hence the symptoms of poisoning, and, in severe cases, death. One other possibility suggests itself to one, and that is that the cerebral symptoms are not necessarily due to the jaundice, but that both are the results of a poison circulating in the blood ; possibly (in the acute specific diseases and pyaemia) of a nature of an alkaloid or poisonous pi'oteid produced by the A'ital processes of certain micro- organisms. Another disease of the liver which demands special mention is wute yellow atrophy. This disease results in stoppage of the work of the liver owing to rapid fatty degeneration of that organ. There is non- obstructive jaundice, and death occurs after the onset of delirium, convulsions, and deep coma. Very similar symptoms and atrophy of the liver occur in phosphorus poisoning. A distinction between the two disorders is stated to be the occurrence of large quantities of ' Neumann, Virchow's Archiv, cxiv. 3. - Petersburger med. Wochenschr. 1886, No. 10. 314 THE TISSUES AND ORGANS OF THE BODY leucine and tyrosine in the blood and urine in acute yellow atrophy, and the absence of these substances in phosphorus poisoning (see also Liver). Diabetes tneJ/itiis is a disease in which the glycogenic function of the liver is deranged, and of which the consequence is an accumulation of dextrose in the l)l<)od, much of which ])asses into the urine. Normal blood contains about 0"09 per cent, of dextrose (Pavy ^ ), but this may inci'ease to 0"2, 0"3, 0*4, and in severe cases to 0"5 and 0*6 in diabetes. Diabetic coma is a condition somewhat resembling the condition just described, as occurring in cases of non-obstructive jaundice. Some blood- poison circulating in the brain here also causes the condition, and here also the answer to the question — what is the poison ? is somewhat un- satisfactory. It is certainly not the sugar ; the peculiar odour of the breath and urine of these patients has led some to suppose that acetone is the poison, and the condition is spoken of as acetona^mia.- Fetters in fact obtained acetone in the distillate from the urine in such a case. But again large doses of acetone do not produce the symptoms o£ diabetic coma ; and there is no doubt that many patients have the acetone smell, and are far from being comatose : still the prognosis whenever the smell is present is always very grave. ^ Acetone does not in all probability exist free in the blood, but is derived from the splitting up of ethyl diacetic acid, or an allied compound. The formula which would represent its formation in the body is as follows : — C^HgNaO;, + 2HoO= CgHgO + C.HeO + NaHCOg [soilium othyl [acetniie] [alcolnl] [sodium hyilrogreii iliat-etatt.']" carbonate] The urine of diabetics gives a red-brown colour with ferric chloride, which disappears on adding hydrochloric acid. This is a reaction of ethyl diacetic acid, and that this substance is probably what is present, is supported by the fact that Le Nobel ^ has found not only acetone but also alcohol in the expired air of diabetics. Certain facts have, how- 1 Pavy, Croonian Lectures, Roy. Coll. of Physicians, 1878. 2 Eupstein, CentralU. f. d. mad. Wiss. 1874, No. 55. 3 A very complete discussion of this question with notes of some hundreds of cases has been i^ubhshed by v. Jaksch [TJeher Acetonurie, Berhn, 1885: Hirschwald). The question he has investigated specially is not acetone in the blood but in the urine. He states, that diabetics without acetonuria never have diabetic coma. Acetone, however, occurs in the urme in fevers, cancer, starvation, and other conditions ; and according to Ephraira [Inaug. Dissert. Breslau, 1885 : Colm) even in health in small quantity. See also Taniguti [Zeit. lihysiol. Chem. xiv.) 4 Le Nobel, CentralU. f. d. mod. Wissensch. 1884, No. 24. THE ]?].OOT) IN DTSKASK 315 ever, been adduced which tell u^^iinst tliis theory. Tliey will be con- sidered in connection with diabetic urine. A lipjvmic (fat in the blood) condition undoubtedly occurs in many diabetic patients,' but this is not constant, and lij^i'mia may occur in other conditions than diabetes.- In this connection it may also be noted that in diabetes a special form of oxy butyric acid occurs in the urine (see Urine), and by some diabetic coma has been attributed to the formation of the lower fatty acids in the blood (Mayer •'). Bright's diseMtte. — This is the only disease of the kidney that de- mands special mention. In addition to an anaemic condition there is a great increase in the amount of urea in the blood. When the elimina- tion of urea is defective or stops altogether, symptoms of poisoning (con- vulsions, coma, itc.) supervene, and are said to be due to urfemia (urea in the blood) ; but as artificial injection of urea into the circulation produces no ursemic convulsions, we are again met with the difficulty, what is the poison 1 Frerichs' theory that it is ammonium carbonate is now given up as untenable ; and for the present we are obliged to rest content with the vague statement that it is some substance (or sub- stances) constituting an intermediate stage in the formation of urea which produces the symptoms. A ptomaine has been suggested by some ; a proteid poison by others, but these are mere suggestions not supported by evidence. H.^i^MOGLOBIN CRYSTALS IN SEPTIC DISEASES If normal blood is drawn from the finger, placed on a slide, and covered, no formation of crystals appears. If, however, a drop of putrid serum is added, crystals of reduced hsemoglobin appear in twenty-four to forty-eight hours. ■• The blood from cases of septicemia crystallises without the addition of any serum. In cancrum oris, in pyaemia (to a less degree), and in erysipelas (especially if the blood is taken from the red patches) the same is observed. These phenomena are pi-obably due tO' the presence or formation of some ferment produced either by the growth of bacteria, or in leucocythaMnia, when the same crystalline tendency of the blood is present, by the disintegration of animal cells. This ferment produces first a deoxidising action on the oxyhaemoglobin, then its exudation into the serum, and lastly crystallisation (C. J. Bond).'' 1 A number of cases illustrating this will be found in Gamgee's Plujsiol. Cliemistrijy pp. 170-172. See also v. Jaksch, Zeit.f. klin. Med. xi. 307. - I have myself notes of marked lipfemia in one case of Bright's disease where there was no diabetes. ^ Arch.f. exji. Patli. u. Pharmakol. xxi. 119. 4 Copeman considers that this is characteristic of human blood, the blood of other animals yielding oxyhremoglobin crystals {Brit. Med. Joiirn. vol. ii. 1889, p. 190). Plaxton., however, has not succeeded in confirming this statement {Ibid. vol. ii. 1890, p. 113). 5 Lancet, vol. ii. 1887, pp. 509, 557. •316 THE TISSUES AND ORGANS OF THE BODY CHAPTER X\ll THE BLOOB OF lyVEnTKBEATK AXIMALS TxvEETEBRATE animals present in their vascular systems fluids which differ greatly from one another, and from the blood of vertebrates. The lowest groups in the animal kingdom, the Protozoa and. the Coelentera, possess no ccelom or body cavity, and therefore no vascular system ; they obtain food and oxygen direct from the water tliey in- habit ; in the case of the Co?lentera, the water enters the enteric cavity ireely. Many degenerate animals of higher groups, such as the tape- "worm, have also no vascular system. Then there are other groups, such as the echinoderms (which possess the well-known water- vascular system), the acephalous molluscs (lamellibranchiata), and higher in the series the Tunica tes, in which the circulating fluid is principally the sea water or fresh water in which the animal lives ; but it contains dis- solved in it a certain small quantity of organic substances, and in it float a numljer of cells like the white corpuscles of vertebrate blood. Blood of this nature may be called liydrolymplt. Lastly there are certain groups of invertebrates in which the blood is a highly organised fluid, containing in solution much organic matter, and in suspension numerous corpuscles. There is, however, in most cases no distinction between blood and lymph, and hence this variety of invertebrate blood is sometimes called luemolynxph. Worms, most molluscs, and arthropods possess this variety of blood. The hydrolymph of invertebrates discharges only one half of the functions of vertebrate blood, carrying nutriment to the tissues and organs, and removing waste products ; the respiratory function of the blood is not represented, the gaseous exchanges probably occurring directly between the animal's tissues and the medium it inhabits. In hfemolymph on the other hand it is found that there is a nutri- tive and a respiratory function taking place. Ha?moglobin is present in the htvmolymph of many animals of the invertebrate subkingdoms, and in many others it is replaced by other respiratory pigments ; thus there is the pink pigment ha?merythrin, the blue pigment hismocyanin, and the green pigment chlorocruorin . But there is this difference to be noted between the blood of vertebrates and that of invertebrates : TiiK i;i,(>(>i) oi" i.NVKi;rKi;K.\TK anj.mals ;}17 that wliei-eas in the former the respiratory pigment is contained in' special corpuscles (the coloured corpuscles), in the latter the pigment is dissolved in the plasma ; the only corpuscles present being colourless- ones. To this rule there are, however, a few exceptions ; in some eight invertebrates corpuscles coloured by luemoglobin, very like the red discs of mannnals, have been found. In the blood of certain groui)s of animals various other pigments are found (chlorophyll, tetronerythrin, A:c.) which have no respiratory functions. INIany of these various forms of inverteljrate l^lood clot when shed like vertebrate blood does. At one time the clot was considered to be merely a mass of adherent corpuscles, or plasmodium of cells (Geddes') ; l)ut it has since been shown that, in addition to the cells, there is an intercellular substance akin to fibrin which is separated from the plasma ; oi- at least that in many instances this is the case. It is impossible to lay down general laws concerning fluids, which differ so much as do the various forms of blood met with among the invertebrates. It was just stated that in most invertebrate animals tliere is no distinction between blood and lympli. There are, however, certain exceptions to this rule : tliat is to say, there are cases in wliich the fluid in the coelom or body cavitj* is distinct from that in the vessels. The ccelom may be said to contain a fluid com- parable to the lymph of vertebrates, while the vessels contain the blood. The lymph or coelomic fluid never circulates in definite channels or lymphatic vessels as in vertebrates, though the coelom in some cases may become subdivided into- secondary spaces or sinuses. In some cases the distinction between the two fluids is perfectly distinct ; for instance, anyone may, in an earth worm, determine for himself, that a drop of the coelomic fluid is colourless, while the blood is red. In other cases there is much dispute as to whether or no the vessels communicate with the coelom,- and hence doubt has arisen whether the blood and the ccfilomic fluid are or are not identical. It is, however, possible that even if communication does exist, the two fluids might still be different from one another ; for in vertebrates there is a connection between the coelom (pleural and peritoneal cavities) and the blood vascular sy.stem ria the stomata and lymphatic vessels, and yet the lymph and the blood are distinct fluids. The following brief statement of the facts in some of the principal groups is, however, all that we have space for here : — IntheCha;topods there is no doubt that blood and coelomic fluid are distinct : the same may be said for Phoronis, Sipuncuhts, and other gephyrean worms. With regard to the leeches (Hirudines) the vascular sj'stem is in undoubted communi- cation with the coelom ; still there is at least one difference between the fluids in the two cavities : certain large corpuscles found in the sinuses of Clepsine and 1 Geddes, Proc. Boy. Soc. xxx. 252. 2 A concise statement of the best ascertained facts in regard to this question will be found in a paper by A. E. Shipley, Cambridge Philosophical Soc. Proceedings, vi. 213- 220. Tliis paper also enters into a somewhat similar anatomical i^oint, viz. whether the body cavity iuid uephridia in certain groups open the one into the other. •318 THE TISSUES AND ORGANS OF THE I'.ODY Pontobdella are not found in the blood, probably because they are too large to pass through the communicating channels (Bourne).' In the nemertine worms the sinuses appear to differ in origin from those in the leeches, not being ccjelomic l)ut archi-cceloraic (i.e. the form representing the remnants of the archiccel or seo-mentation cavity — Hubrecht'-), and there appears to be no connection between them and the vascular system. Another group separated a long way from these •classes of worms, and in which communications exist between the vascular system and the coelom, is the Echinodermata. Hamann and Koehler showed this first in Spatangids, and Perrier and other French naturalists have shown that the same is true throughout the Echinodermata. It will be now convenient to take up the chief invertebrate pliyla, one Vjy one, and to describe the characters of the ])lood as it occurs in •each. THE BLOOD OF ECHIXODERMS This is of the nature of hydrolymph, i.e. a watery fluid holding in solution saline substances (derived from the sea water) and a v^ery small quantity of albuminous material. In it float numei'ous amoeboid corpuscles. The following is a brief description of the varieties of corpuscles found in the perivisceral fluid of sea urchins and holo- thurians (Geddes^). 1. Large amoeboid cells containing highly refracting sjiherules of a rich mahogany-brown colour. On exposure to the air this brown colour becomes dingy ; V)ut in the vacuum of a mercurial air-pump it rapidly becomes normal again. There is thus considerable probability "that this pigment has a respiratory function. 2. Lemon -yellow amceboid corpuscles are also found in certain sea urchins (Arhoria, Dorocidaris), but are exceedingly al)undant in the perivisceral fluid of the Spatangoidea. 3. In the intestinal vessels of Spatangus amceboid corpuscles, varying much in size and containing variously coloured globules (brown, yellow, purple, green, and blue), are found. Tlie nature of these pig- ments has not been fully worked out, but tliey are probably lipo- chromes. When a drop of the perivisceral fluid is examined microscopically, the cells at first move freely and exhibit amoeboid movements. They soon collect into irregular masses and shoot out long processes which bind the cells together. But the clot is not a mere plasmodiuni ; there is in addition a iiljrin-like material which separates from the plasma. This contracts ' Quart. •/. Microsc. Science, xxiv. 419. - Ihid. xxvi. 417. 5 Geddes in Gamgee's Physiol. Chem. p. V6i. TiiK r.i,t») Extractives . 5-66 1-31 (c) Inorganic salts 8-55 8-78 This table illustrates numerically the various points which have been already mentioned, especially the great diminution in the organic constituents of the lymph as compared with plasma, while the inorganic constituents are approximately the same in the two fluids. Not only are the total inorganic constituents equal in the two fluids, but the same salts are present in approximately equal proportion, sodium chloride in each being the most abundant ; this is illustrated numerically in the next table. 1,000 parts of human plasma contain 1,000 parts of human lymph contain (C. Schmi dt) :— (Hensen and Dahnharc t):- NaCl . 5-54 rNaCl 6-14 Na.,PO, . 0-27 Na.O 0-57 Na^O . 1-53 Soluble K..0 0-49 KCl . 0-36 CO, 0-63 K.,SO, . 0-28 S03.P,0 5 & loss 0-22 Ca3(P0,), . 0-30 / CaO 0-13 Mg3(P0,), . 0-22 MgO ! Fe.03 Insoluble'i p n 1 CO, 0-01 0006 0-118 0-015 ^ MgCO, & loss . 0-021 1 Fano, Da Bois Beymond's Archiv f. Physiol. 1881, p. 277. - C. Schmidt and Lehmann. 3 Hensen and Diihnliardt. The above table gives tlie averages of three analyses. The total solids are rather low in this case ; the average from other cases gives from 30-40 parts per 1,000. l.^'.Ml'll AM) .\I,MKI) \'\. |)S 335 Similar tables might be ([uoted of analyses of tlie lympli from other animals ; the chief points, however, are all sutRcieutly shown by the example given.' The next table illustrates the point already mentioned as to the greater quantity of urea in lympli as compared with the l)lood (Wurtz^). Animal Percentage of Vrea Blood Lymph Cl.ylf Dog Cow Horse Bull 0009 0-019 0-016 0019 0-012 0-021 0-019 0019 ! 1 Observations have been made in dropsical fluids which show ^-hat the relation between the amount of serum-albumin and serum-globulin is very constant in the same person or animal, and is the same approxi- mately in the blood-serum and in the effused fluids, though the total amount of proteids is much less in the effusions than in the serum. If rt=percentage of albumin and 6=percentage of globulin. Then is called the proteid quotient. The proteid quotient is also equal in the same animal in the serum, lymph, and chyle. Salvioli^ has shown this to be the case in dogs. This is a somewhat important point ; it shows that there is no difference in the rate of diifusion of the two proteids in the living animal, for Gottwalt'* found that globulin diffuses more slowly than albumin through dead animal membranes. (Compare p. 15) CHYLE Chyle is the name given to the fluid contents of the intestinal lym- phatics or lacteals during digestion. It may be briefly described as lymph plus certain materials (especially fat) which have passed into these vessels from the intestines. Microscopic examination shows that chyle contains lymph cor- puscles and fat globules in a minute state of subdivision. ' These tables will be found in Hoppe-Seyler's FliijsioJ. Cheinie, pp. 592-3. - Wurtz, Comjjtes rendus, July ia59. I take the table from Gamgee's Phijaioh Cheh p. 224. 2 Salvioli, Du Bois BeymoncVs Archiv f. Physiol. 1881, p. 269. * Zeit. physiol. Chem. iv. 423. 336 THE TISSrP:>J AND ORGANS OF THE BODY The following table, somewhat abbreviated, is taken from Hoppe- Seyler;' the numbers are parts per 1,000. constituents Chyle of Do,^ ^^ ^JJf Chyle of Horse^ Humau Cliyle'' Human Chyle* Water ' 90677 93601 Solids 96-23 63-99 Fibrin Ml — Albumin Sc globulin 2105 45-24 Fat, lecithin, chole- sterin 64-86 6-81 Fattv acids & soaps "l | Other organic sub- - 234 j- 2-91 stances .... J - Mineral salts . . . 7-92 876 1 956-19 43-81 1-27 29-85 0-53 0-28 2-24 7-49 904-8 95-2 1 70-8 9-2 1 10-8 4-4 943 to 958 56 to 41 11 to 13 25 to 27 6-25 The nicst striking fact illustrated by this table is the greater amount of solids in chyle as compared with lymph, and the large percentage of fat. Zawilski found in dogs fed purely on a fatty diet that the chyle micht contain as much as 14-6 per cent, of fat. During the active dio-estion of fat, the blood plasma and serum have a milky appearance produced by the presence in them of excessively minute fat globules. 1,000 parts of the dry residue of the ethereal extract of chyle con- tained :" — F irst Sj}ecimen Second Specimen Cholesterin . 113-2 . . 140-9 Lecithin 75-4 . . 88-4 Olein 381-31811-4 . 430-1/ . 770-7 Palmitin and stearin . The chyle also contains a certain proportion of soaps absorbed from the alimentary canal. This statement was originally made by Hoppe- Seyler, and he still maintains ^ the correctness of his earlier observations, which have been questioned by Lebedeff, Rohrig, and Zawilski. In some new experiments he has found in the serum of the horse, ox, and doo-, a percentage of fatty acids from soaps varying from O-Oo to 0-12. 1 Physiol. Chemie, p. 595. " Ludwig's Arheiten, xi. 147. 3 Analyses by Hoppe-Seyler. ^ Analysis by Sclimidt. 5 Analyses by Rees, Pldl. Trans. 1842, p. 81. The chyle was obtained from a decapitated criminal. 6 Noel Paton. This analysis is not contained in Hoppe-Seyler's table. The chyle was obtained from a patient whose thoracic duct had been ruptured by an operation for tumour in the neck {■Tonrn. of Physiol xi. 109). ^ Hoppe-Seyler, Physiol Chem. p. 597. 8 Hoppe-Seyler, Zeitsch.f physiol Chem. viii. 503. LYMPH AND ALIJKl) I'U'TDS 337 Tnthe chyle' he has found 0*225 per cent, of soaps, and 0723 per cent, of fat. Tlie increased percentage of jn-oteids in tlie cliyle as compared with tlie lymph illustrates the fact that the lacteals an^ not merely concerned in the absorption of fatty, but probably also of albuminous food. In the stomach and intestine the proteids of the food are coiiverted into peptones, substances that diffuse with readiness through living animal membranes ; but no peptones ^ (or proteoses, the intermediate pro- ducts in the formation of peptones) are found in the chyle ; during their passage through the intestinal wall, or immediately on entering the lymph or blood stream, they are reconverted into albumin and globulin. Schmidt-Mulheim ^ tied the thoracic duct in dogs, and found that proteids were still absorbed ; this, however, does not prove that the lacteals are not normally concerned in the absorption of proteid ; it merely shows that animals thus treated can continue to absorb proteid by the other path — the blood vessels. The intestinal lymphatics are thus concerned in the alisorption of fat and of proteids ; they, however, apparently take bvit little part in the absorption of carbohydi'ate food ; the amount of sugar in lymph and chyle is approximately the same as in the blood, and no definite increase occurs when animals are fed on a starchy or saccharine diet (Bernard, v. Mering"*). Probably, as sugar is so easily ditt'usilile, most of it passes into the more quickly circulating blood stream and is carried off, fresh quantities of blood being then available to carry off more. The blood vessels, moreover, lie immediately beneath the epi- thelium, and so the sugar never reaches the more centrally situated lacteals of the villi (Heidenhain ''). By gi'eatly increasing the amount of sugar in the food, however, some does pass into the chyle.*" Quantity of Chyle. — From two cases in which the amount of chyle was measured C. Schmidt concluded that for every kilogram of body weight 0'61 kilo of chj'le was formed in the 24 hours, of which 0-34: comes from the alimentary canal, and the remaining 0"27 consists of the normal lymph. Hoppe-Seyler ' is inclined to think that the proportion derived from the intestine is much smaller. Extidations of CJnjIe.— Owing to the rupture of the lacteals or of the thoraoic 1 Obtained from a case of chylous ascites, i.e. escape of chyle into the peritoneal cavity. 2 This is a statement made by numerous observers ; I have confirmed its accuracy in an examination of cliyle collected from the thoracic duct of two dogs. ^ Du Bois Reymond's Archiv, lb77, p. 549. 4 Ludwig's Arheiten, 1877. Arch. f. Anat. ti. Physiol, Physiol. Ahfh. 1«77, p. 379. ■' Heidenhain, Pfli'iger's Arch, supplemental volume, 1888, p. 71. 6 Ginsberg, Ihid. xliv. 80G. 7 Physiol. Chemie, p. 597. Z 338 THE TISSUES AND ORG.AJSS OF THE BODY duct, or owing to fistulous communications between these parts and other cavities, chyle may pass into the serous cavities, giWng rise to chylous dropsy. For Chvlm-ia i>ee Urine. THE LYMPH IX SEROUS CAYITIES DURING HEALTH The amount of fluid in these ca-vities is in health very smaU ; excess finds its way through the stomata into the lymphatic vessels. The fluid is undoubtedly lymph — that is, dilute blood plasma which has exuded from the blood vessels. In dropsical conditions this fluid is much increased, and our knowledge of its properties is almost entirely derived from a study of dropsical fluids. If excess of fluid accumulates in two of these cavities simultaneously, as, for instance, in the peri- toneum and the pleura, from altei'ations in conditions of vascular pressure (e.g. heart disease), it is found that the composition of the two fluids difiers to a certain extent. On this ground we hold that the normal lymph which moistens the various serous ca^nties probably difiers in those difierent ca^-ities in the same way. The difierences are quantitative only, not qualitative. After death the pericardium, especially in some animals (e.g. the horse), often contains a consider- able quantity of liquid. This accumulation is accounted for by the changes in the circulation immediately preceding death. The liquor pericardii is a liquid which is interesting historically, as so many expeiiments have been made with it in dealing with the investigation of the causes of the coagulation of the blood (see p. 243). The cerebro- spinal cavity is not a serous cavity, and the fluid in it difiers markedly from the lymph of the serous ca\"ities. It will be dealt with sepai-ately. DROPSICAL FLUIDS The modes of causation of dropsy have been already considered (p. 332). These fluids may all be tersely described as hnnph in excess and more watery than usual, except in inflammatory dropsy, where the cells and the solid constituents generally are increased. Nomenclature. — There are certain names given to the various forms of dropsy occurring in difierent situations : — (Edema is the name given to the excessive exudation of fluid into the subcutaneous tissues. Ascites is the name given to a dropsy of the peritoneal cavity. Hydrocele is the name given to a dropsy of the tunica vaginalis, the serous membrane originally part of the peritoneum that surrounds the testicle. LV.MI'H AND ALLIED FLllDS 339 llydrotltorax is the nniiie given to a dropsy of the pleura. Ilydropericnrdinm to that of the pericardium. The names of diseases that are inflammatory in nature terminate in the affix -itis. Thus there is pericarditis, peritonitis, pleuritis (or pleurisy), kc, and in certain stages of all these diseases there is effusion of fluid. Reaction. — This is in all cases alkaline. Colour. — This varies directly with the coltjur of the blood plasma of the patient, and witli the concentration of the ett'used liquid, but there is always a certain amount of the yellowish-green lipochrome (serum-lutein, see p. 253), which can be extracted by means of alcohol. Specific gravity. — This increases j^^^'i pn^'^^t' with the amount of solid constituents. Reuss ^ has examined a large number of these effusions ; he calls the fluids of inflammatory dropsy, exudations, while the dropsical fluids (i.e. diluted lymph) he terms transudations, and the following are the general conclusions he draws with regard to specific gravity. Fluids from cases of peritonitis 1018 or higher \ „ pleuritis 1018 „ _^^^^^^,-^^^^ ,, ,, lunammation lOlo „ i of skin 2 ) „ ,, hydrothorax 1015 or lower] ,, „ ascites .1012 ,, r transudations ,, ,, oedema . 1010 ,, J Coagulation. — Non-inflammatory dropsical fluids do not coagulate spontaneously, or only with exceeding slowness. When mixed with serum, or contaminated with blood, as they are apt to be in the process of tapping, they, however, do coagulate, forming fibrin. The reason why they do not clot is that they contain either very few cellular elements or these may be practically absent. The cause of the coagulation of the blood, we have already seen, is the formation of the fibrin-ferment from white corpuscles and blood tablets. When, therefore, blood or serum or a solution of pure fibrin-ferment or cell- globulin, or of some other active globulin like myosinogen (see Muscle), is added to one of these dropsical fluids, the fibrinogen contained therein is converted into fibrin (see Coagulation of the Blood, p. 2-41). The inflammatory fluids or exudations are, however, different ; they contain abundance of white corpuscles and invariably clot when .shed. ' A Reuss, Deutsches Arch. f. klin. Med. xxviii. 317. HofiEiuaun has made similar observations, Virchoiv's Archiv, Ixxiii. 250. * Such as is obtained by blistering. z 2 340 THE TISSUES AND ORGANS OF THE BODY Sometimes they clot within the serous cavity, the fibrin • sticking to the sides of the membrane. Constituents. — These are the same in kind as those in Ijlood plasma. a. P rote ids. — These are fibrinogen, serum-globulin, and serum- albumin. ^ b. Extractives. — This term is used in the sense explained on p. 251. In some cases cholesterin is found in marked excess. Sugar .seems to be a fairly constant constituent. c. Salts. — These are alike not only in kind but in actual amount to those in the blood. The different dropsical fluids differ from one another in their rich- ness in organic constituents, especially in proteids ; the pleural fluid is richest in these substances, then the peritoneal, and lastly the fluid of subcutanefms oedema. These facts may be illustrated by the following tables : — CompoK'it'Krn, of various droj)sicnl fluuU removed after death from a case of albuminuria ( C Schmidt} ^ : — In jans per 1000 Pleural Fluid Peritoneal Fluid CEdema Flnid 988-70 11-30 H-60 7-70 Comjwsition cf various dropsical fluids removed simultancoushj from a case of albuminv/ria (Hoj}jte-Sei/ler) * : — In parts per 1000 Pleural Fluid Peritoneal Flnid OE'lema Fluid 1 Water SoUds Proteids Extractives and Salts . 957-59 967-68 42-41 .32-32 27-82 1611 14-59 j 16-21 982-17 17-83 3-64 1419 These examples illustrate sufficiently well the fact that it is the amount of proteids that varies in these different fluids, the other constituents being fairly constant. E,une)>erg-5 examined 77 cases of effusions of different kinds ; the amount of total proteids varied from 0-06 to 2-68 per cent. ; while the 1 One often hears these strands of fibrin called lymph in the i)ost-mortem room. - The serum-albumin of these fluids like that of serum can by fractional heat- coagulation be differentiated into thre2 proteids (see p. 247j. 5 Quoted by Hoppe-Seyler, Physiol. Chem. p. 602. ^ Physiol. Cheinie, p. 602. Also in Arch. f. path. Anat. ix. 257. ^ Runeberg, Deutsch. Archiv f. klin. Med. xxxv. 266. LV.AII'H AND ALLIKI) I'H'IDS 341 amount of chlorides, the most aljundunt salts, averaged 1"08, varying only 0"1 per cent, throughout the long series. Runeberg' in three cases (I., II., III.), and my.self in one case (IV.) of heart disease examined dropsical fluids i-emo\'ed simultaneously from different parts. The following numbers give the percentage of proteids in the difterent fluids : — Case I Case H Case in Case IV Fluid from pleura 0-11 Fluid from peri- Fluid from peri- toneum . . . 2-.S toneum . . . 0-12 CEdema fluid . . 0-24 Fluid from peri- i cardium . . . 0*52 | 1-64 0-20 Fluid from pleura . . 1-48 CEdema fluid 0-33 F. Hoftmann ^ examined a series of thirty cases of ascitic fluid as to the relation between the amount of serum-globulin and serum- albumin. The amount of fibrinogen in these fluids is so small, that practically it may be neglected. He found that the fraction serum-albumin i • , • n i .i . • i ±- . • • 1 1 , which IS called tlie proteicl quotient, is very variable, serum-globulin In eleven of the thirty cases he was able to estimate the proteid quotient in the blood-serum of the same patients ; here, also, he found great variations, and the quotient is generally, both in blood and in efiusion, lower than normal.^ The most interesting point, however, is that the proteid quotient is practically the same in the two fluids, blood and effusion, for the same individual. We have seen previously (p. 335) that the proteid-quotient of normal lymph and chyle is equal to that of the blood. This illustrates to us very forcibly the differences between diffusion through a living membrane and through a dead membrane. Senator, A. Schmidt, and Gottwald, using dead mem- branes, found that serum-globulin diffuses with greater difiiculty than serum-albumin ; but here we see that during life they diffuse with equal rapidity. These results have been fully corroborated by Pigeaud,^ and I quote (see next page) the numbers obtained by him in two cases of nephritis. In many cases during the progress of a dropsy it is found necessary to tap the cavity several times ; it is then found that successive 1 Runeberg, Ibid, xxxiv. 1. 2 F. Hoffmann, Arch.f. exper. Path, und Pharm. xvi. 133. ^ It appears doubtful whether we can at present say what the normal proteid- quotient is. It is more probable that it exhibits very considerable variations in health. * J. J. Pigeaud, Over eiivitstoffen in serense vloeistoffen. Doctor. Dissert. Leiden, 1886; see also Maltj's Jahreshericht f. Thierchemie, xvi. 474. 34-2 THE TissrEs and organs of the ];oI)Y Case I Case II F'.uiil Total Proteiil ]>er cent. Proteid Quotie Blood-serum rieural fluirl Ascitic fluid ..... 1 ffidema fluid 5-781 0-900 0-^32 0-775 1-05G 1-142 1-122 1-152 dropsical transudations into the same sac present great constancy of composition. Sometimes difFerenees do occur ; these differences, and also the differences in the proteid contents of the lymph in the various serous sacs, are, no doubt, dependent on alterations and differences in the mechanical conditions of pressure. In successive tappings, however, a difference may arise in another way, viz. a certain amount of inflammation may be set up in the serous membrane itself ; and this may be the result of irritation produced by a sudden removal of the fluid, or, more frequently, it is the result of using imperfectly cleansed instruments for the operation. These diflerent points will be illustrated by further analytical data to be given under the various fluids which we now proceed to take up seriatim. PERITONEAL FLUID This partakes of the general character of dropsical fluids which have just been described. It is an alkaline fluid of a yellowish tint, and is occasionally, even when quite fresh, somewhat opalescent.' It contains few or no cor- puscles, and when removed does not coagulate spontaneously, or only very slowly, the process sometimes lasting several days. Where peritonitis is present, however, colourless corpuscles and epithelium cells of the peritoneum, and, in cases of cancer, cancer-cells ^ also are to be seen in abundance ; the fluid coagulates spontaneously and is much richer in proteid-contents than the fluid of simple dropsy. 1 This opalescence is not removed by filtration, and microscopically no particles are tD be seen to account for it. - H. Quincke, Dentsrh. Arch. f. klin. Med. xxx. 5. LY.MI'JI AND ALLUU) FLl'IDS 343 Some aujilysos have already been given of tlie composition of this fluid ; the following may now be added, as they illustrate more fully certain other points, which may be conveniently stated in the form of propositions, each of which is followed by illustrative analyses. 1. The fluid removed from the peritoneal sac by successive tappings remains fiiirly constant in composition.' Case I Case II- In parts !)«• 1000 1st Tapping 2nil Tapping Cirrhosis of Liver 1st Tai)ping 2ndTapping .^--^h Water Solids Proteids .... Extractives . . . Salts 952-99 47-01 34 90 4-28 7-22 960-49 39-51 29-73 3-75 5-94 984-50 15 50 6-17 1-25 8-46 982-53 983-33 17-47 16-67 7-73 6-11 1-84 3-25 8-13 8-24 2. The amount of proteids is very variable ; the proteid quotient is also variable and apparently does not vary with the cause of the dropsy. This may be illustrated by the following analyses selected from a larger number made by myself. Case Reaction Specific Gravity Total Proteid per cent. 1. Cirrhosis of liver . . 2. Syphilitic disease of liver .... 3. Cirrhosis of liver 4. Non-inflammatory 5. Heart disease 6. Heart disease * . CO y 1010 1012 1012 1016 1016 1018 0-955 0-744 2021 2-235 4-11 4-334 Serum- Serum- Globulin'' Albumin 0-413 0-542 0-252 0-492 1-114 0-907 1-516 0-719 1-48 2-63 2-937 1-397 3. In the same case, however, it is found in successive tappings that the total proteid and the proteid quotient remain very constant. 1 Scherer, quoted from Hoppe-Seyler's Physiol. Chein. p. 602. 2 Hoppe-Seyler, Ibid. p. 603. Other analyses illustrating this point will be also found here. 5 In these cases the amount of fibrinogen is not given, as it was very small, and it is weighed with the serum-globulin. I am indebted to Dr. Sydney Ringer and his house physicians at Univ. Coll. Hosp. for all these various fluids and many others to be described later on in this chapter. See also Brit. Med. Journ. vol. ii. 1890, p. 192. * The amount of proteids in transudation fluids from heart disease is generally greater than in other forms of pressure dropsy. This lends some support to Wooldridge's theory, that the blood is partly at fault in such cases (s(?« footnote, p. 332). 344 THE TISSUES AND ORGANS OF THE BODY Case of BrigliVs Disease complicated with Cirrhosis of the Liver Specific Gravity Total Proteid per cent. 2-037 2-499 2-401 2-152 Serum-Globulin Serum-Albumin First tapping- . . . Second „ ... Third „ ... Fourth „ ... 1014 1015 1015 1015 0-7807 ' 1-2563 0-8960 1-6074 0-572 1-829 0-703' 1-375 4. Even in those cases where, owing to alterations of pressure, the amount of proteid changes, yet the proteid quotient (all>uniin : globu- lin) remains practically unaltered. Case of Cirrhosis of the Lirer in a Boy thirteen years old"^ Date of Tapping Total Proteid per cent. Proteid Quotient July 20, 1885 August 25, 1885 .... September 30, 1885 . November 15, 1885 .... December 27, 1885 . 3-285 0-632 2-368 3-216 2-688 1-483 . 1-573. 1.532 1-525 1-486 5. The total proteids in the fluid in cases of peritonitis is increased as compared with that of simple pressure ascites. Runeberg,^ from the examination of 121 cases, arrives at the following general results : — Percentage of Protoiil 003-0-41 0-37-2-68 0-84-2-3 2-7-3-51 In cases of hydrfemia (including nephritis) the ascitic fluid contain: „ ,, portal obstruction ,, „ „ „ ,, general venous congestion (heart disease) „ „ „ carcinomatous peritonitis „ „ For diagnostic purposes it may roughly be said that a high per- centage of proteid denotes inflammation ; a low percentage of proteid certainly denotes absence of inflammation. The relation of alljumin to globulin (proteid-quotient) is of no diagnostic value, as it varies with the proteid quotient of the blood ; it is therefore merely of theoretical interest. There is generally a small percentage of sugar in the peritoneal fluid as in lymph generally. Many cases of cirrhosis of the liver, how- ever, are often associated with a small amount of diabetes,^ and the ^ In aidition to this, the fibrinogen was in the fourth tapping estimated by adding fibrin-ferment and weighing the fibrin formed ; it amounted to 0-075 per cent. The above analysis is my own. ^ J. J. Pigeaud, Malifs JahreshericM, xvi. 474. ^ Runeberg, Deiifsch. Arch. f. Mill. Med. xxxiv. 1. See also Hofmann {Virchow's Arcli. Ixxiii. 2.50) for a large number of similar analyses. ■* Cobrat, ' De la glycosurie dans les cas'd'obstruction partiolle ou totale de a veine LVMI'll ANIt .\l,I,Il-:i) I'MIDS 345 sugar in tlie ascitic lluid is tlius increased. In one case of syphilitic cirrhosis of tlie liver, in which I examined the ascitic fluid, there was as much as O-l'33 per cent, of sugar present. In a case of cirrhosis recorded by Moscatelli, ' the percentage of sugar in the ascitic fluid was 0*15 ; this fluid also contained a small amount of allantoin. Sugar was, however, in this last case absent from the urine. It is only when the percentage of sugar in blood and lymph exceeds 0*2 per cent, that glycosuria ensues. Chylous ascites. — There have been several cases published of disease affecting the thoracic duct, and causing its rupture ; this leads to the extravasation of the chyle into the peritoneal cavity, and the fluid may be removed by tapping. I here merely quote two cases to illus- trate this : Case I.^ was a case published by Whitla, in which tuber- culous disease led to the rupture of the duct in a boy of 13 ; Case 11.^ is published by J. Strauss, and was a case in which cancerous growths led to the rupture of the mesenteric lacteals. The analysis in the first case was made by Matthew Hay, in the second case by Guinochet. Case I Case IT Constituents Parts per 1000 Fat Proteids Other organic matters .... Mineral salts Loss Total Solids 10-30 28-78 8-02 9-95 59-15 9-48 21-08 11-685 1-595 0-510 43-795 Hay found a small percentage of sugar in the liquid ; Guinochet found neither sugar nor peptone. In some cases of ascites, the fluid though not chylous yet contains a large excess of cholesterin, crystals of which are to be seen floating al^out in the liquid ; in other cases, the peculiar mucin-like substances paralbumin and metalbumin (which are pretty constant constituents of ovarian fluid, and will be described with that fluid) may be found. In still another class of cases of ascites, haemorrhage may occur into the peritoneum, and a liquid more or less stained with blood or altered blood pigment is obtained on paracentesis (tapping). porte,' Lyon. mid. 1875, No. 15. Lepine, Gaz. mid. de Paris, 187C, p. I'io. Quincke, Berl. Uin. Wochenschrift, 1876, No. 38. ' MoscatelH, Zeitscln: f. jihijsiol. Chemie, xiii. 202. - British Medical Journ. vol. i. 1885, p. 1089. ^ Arch, de plnjsiol. xviii. 367. Another case is recorded by Maguire, Brit. Med. Journ. vol. ii. 1886, p. 197. 346 THE TISSUES AND ORGANS OF THE EODY PLEUEAL FLUID The fluid has the same general characteristics as peritoneal fluid. It as a rule contains more proteids than the peritoneal fluid. It does not readily coagulate spontaneously, unless pleurisy be the cause of the exudation. A few more analytical data, in addition to those which have been already given, may be added as illustrations to the following propositions. (1) In hydrothorax, the total percentage of proteid is much lower than in cases of plfoir.isy : the amount of fibrinogen as estimated by the weight of fibrin formed ^ is also less in the fluid of hydrothorax. The following numbers are obtained from analyses of my own :^ Case Specific Gravity Total Proteids per cent. Pi brill Senim- Glohuliu Senim- Albumiu 1. Pleurisy (acute) . 2. Pleurisy (acute) . 3. Pleurisy (acute) . 1023 1020 1020 5-132 3-4371 5-2018 0-016 0-0171 0-1088 8002 1-2406 1-760 2-114 1-1895 3-330 4. Hydrothorax 1015 2-5183 00067 0-6597 1-8519 (Bright's disease) 5. Hydrothorax 1012 1-3242 0-0062 0-4026 0-9154 (Bright's disease) 6. Hvdrothorax 1016 1-482 0-013 ()-779 0-700 (heart disease) (2) In hydrothorax, as in ascites, the liquid removed by successive tappings remains fairly constant in composition. ^ Constituents 1st Tapping 2na Tapping Water 966-24 . Solids 33-76 Organic solids 26-12 Inorganic solids .... 764 i- 963-95 36-05 28-50 7 -.55 Sugar seems to be fairly constantly present in pleui'itic fluid, as in other forms of lymph. In 17 specimens examined by H. Eichhorst^ 10 contained small quantities of sugar. Exceptional forms of pleuritic effusion are sometimes found ; some 1 In the case of the liquid of hydrothorax fibrin may be formed by adding seiiim- or fibrin- fennent to the hquid. The fluid in clirouic pleurisy closely resembles that of hydrothorax (C. Me'hu, Bulletin Med. du Nord, 1872). - The example selected is an analysis by C. Schmidt. It and others will be found on pp. 602-3 of Hoppe-Seyler's Physiol. Cheiuie. ^ Zeitschrift f. Min. Med. iii. 537. lA'.Ml'H AND ALLIED FLUIDS 347 are associated with carcinomatous or sarcomatous tumouis, and the cells characteristic of these growths may be found in the pleural liquid, in addition to the usual leucocytes. In other cases Inemorrhage may occur into the pleura. In one case of ha-mori-hagic pleurisy,' I found a large amount of cholesterin floating about in a crystalline form in the liquid. In another case (not hfemorrhagic) there were large corpus- cles like Gluge's inflammatory corpuscles^ in large numbers, in addi- tion to leucocytes. Cases of chylous pleurisy have also been descril)ed.^ PERICAEDIAL FLUID This fluid is not so often removed from the human subject in cases of disease when it is present in excess, as in other forms of dropsy, because of the greater danger attending the operation. It is stated to contain a lai'ger quantity of fibrinogen than other transudations, and Kiihne found that it contains 0"879 to 2-468 per cent, of proteids. Dr. Friend has made under my superintendence the following analyses of the pericardial fluid of the horse removed after death. 1 In parts per lunu I n ' Water 964-011 957-953 Solids 35-989 42-047 I^oteids 28-611 25-846 Fibrinogen (estimated as fibrin) 0-117 0-260 Serum-globulin ..... 11-069 11-608 Serum-albumin . . . . " . 17-455 13-983 Extractives — 2-432 Salts 7-575 13-769 Specific gravity ..... 1018 1018 ! Reaction ...... alkaline alkaline i It is interesting to note that the pericardial fluid of the tortoise, which I have examined, exhibits precisely the same characters and properties as that of the mammalian animals. Chylous effusions into the pericardium may occur, as in the case of the other serous sacs ; a case is recorded by K. Hasebroek,'* and it may be useful to compai-e the analysis of the fluid he obtained with ' Under Dr. Ringer's charge, Univ. Coll. Hosp. - Corpuscles three or four times the size of white blood corpuscles, containing numerous fat granules. ^ For the analysis of one see Hoppe- Sevier's Physiol. Chem. p. 596. ■* Zeit.jJhijsioh Chem. xii. 289. 348 THE TISSUES AND ORGANS OF THE EODY those of non-chylous pericardial fluid as recorded hy previous observers. The chylous fluid will be seen to contain a greater amount both of proteids and extractives than ordinary pericardial fluid : more than 50 per cent, of the extractives consisted of fat. In parts per 1000 1. Chylo-pericar- .lial Fluid (Hasebroek) 2. (Gorup- Besanez)' 3. CWachsmutlO^ 4. (Hnppe- Seyler)" Water Solids Fibrin .... Proteids .... Extractives . . . Salts 892-782 103-612 73-789 20-481 9-336 955-1 44-9 0-8 24-7 12-7 6-7 962-5 37-5 22-8 961-78 38-22 24-63 HYDROCELE FLUID This fluid with the preceding- (pericardial fluid) is interesting his- torically ; these fluids having been very largely employed in the course of experiments on the coagulation of the blood (see p. 243). It is contained in the tunica vaginalis, originally a part of the peritoneum, and is itself almost exactly like the peritoneal fluid. It resembles the peritoneal fluid and other forms of lymph in reaction, colour, and constituents. It does not as a I'ule clot spontaneously, unless mixed with blood or serum, or containing an excess of leucocytes from inflam- mation. Its specific gravity varies from 1016 to 1022 ; the amount of proteid present also varies very much. The following is the mean of 17 analyses made by Hammarsten -J — Water . 938-85 parts per 1000 Solids . 61-15 Fibrin . . . 0-59 Globulin 13-52 Albumin 35-94 Ether extractives 4-02 Salts . 9-26 NaCl . 619 There are cases of hydrocele which dift'er from the ordinary fluid ; some are viscous from the presence of metalbumin and paralbumin * [see Ovarian Fluid) ; some contain excess of cholesterin ; and others are chylous. The cases of chylous hydrocele are sometimes associated with 1 Lehrhuch, p. 401. ^ Arclt. f. jJafhuI. Anat. vii. 334. ■^ Physiol. Chemie, p. 605. ■* Quoted from Hoppe-Seyler's Physiol. Chemie^ p. 606 (Haramarsten's original paper s in Swedish). Other analyses by Hoppe-Seyler will be found on the same page. 5 E. Devillard, Bull. soc. chiin. xlii. 617. LYMPH AND AIJ.IEI) FH'IDS ;ui) chyluria (cliylous urine) ; and cliyluria is produced by the presence of the hivmatozoon Filaria Sangidnis Ilominis in the blood {see Urine). Lymph tumours and tumours filled with chyle are very common in the scrotum and its neighbourhood in cases of chyluria, and these may discharge their contents from the surface of the skin.' Other cases of chylous hydrocele may however occur, which seem to be produced like chylous ascites by the rupture of lacteal s. I have examined one such case ; it was a iluid oljtained from a case of otherwise ordinary hydrocele, which was shown to the Pathological Society by Mr. S. G. Shattock.'- THE FLUID OF SUBCUTANEOUS (EDEMA This fluid is poorest of all the dropsical fluids in proteid constituents ; otherwise it resembles them very closely. Some analyses have already been given of this fluid, in the com- parisons that we have drawn between it and other eff'usions ; the fol- lowing are some estimations of the proteid constituents which I have made. Case 1, Cardiac dropsy ; fluid from incisions in ankles . . . 2. Cardiac dropsy, another case ; fluid obtained in the same way 3. Blight's disease ; fluid re- moved by Routhey's trocar and cannula from ankles G'-^^i^i- cent. 1012 1013 1009 0-33 0-592 0-6404 Serum- Globulin traces Serum- Albumin 0-0028 traces 0-139 0-453 0-1911 0-4493 In all these cases the fluid which drained away first, coagulated spontaneously on standing ; this was due to a slight admixture with blood. The fluid collected after haemorrhage had ceased did not coagulate spontaneously, but on adding blood or serum to it, a small quantity of fibrin was in all cases obtainable. A. Rosenbach '^ has made a special investigation whether sugar is present or not, and he finds that it is nearly constantly present in cedema fluid in small quantities. Blister jiuid. — This fluid lias the same relation to oedema fluid, as 1 Analyses of such fluids will be found in Hoppe-Seyler's Phijsiol. Clteiit. pp. 008-9. ^ The case was one tapped by Sir Henry Thompson, see Trails. Patli. Sociefi/, XXXV. 250. Mr. Shattock informs me he has since seen a case similar to that which I analysed for him. ^ A. Rosenbach, Breslaiier urztl. Zeit. 1885. No. 5. 350 THE TISSUES AND OKGANS OV THE BODY that of peritonitis or pleuritis to that of simple pressure dropsies into the serous cavities. It contains a large number of leucocytes, coagu- lates spontaneously when drawn, has a higher specific gravity (1018 or more), and a larger percentage of proteids, as is seen by boiling it, when it becomes almost solid from the heat-coaguluni produced. In cases of gout, blister fluid like tlie blood plasma contains excess of urates, and the method of examining it for uric acid, as originally suggested by Garrod, has been already descri})ed (p. 252). THE AQUEOUS HUMOUR The anterior chamber of the eye is essentially a lymph space, and the fluid in it, the aqueous humour, is essentially lymph ; but lymph which contains a very small proportion of proteid constituents. The amount of aqueous humour is directly dependent on the blood pressure (Chavvas).' Lohmeyer^ analysed the aqueous humour of the calf, and the following are his results in parts per 1000 : — Water 986-87 Solids 13-13 Proteids 1'22 Extractives •i-21 Inorganic salts 7-70 (Sodium chloride 0-89) The aqueous humour either does not coagulate spontaneously, or clots very slowly ; it contains in health no formed elements. As in other forms of lymph, however, a clot of filjrin is formed on the addi- tion of serum. Tlie proteids are the same in kind as in blood plasma and lymph generally, viz. fibrinogen, serum-globulin, and serum- albumin.^^ Kuhii * finds among the extractives that a reducing substance like suo-ar is constantly present in the aqueous humour of the ox and rabbit ; the percentage of this substance reckoned as dextrose, present in the aqueous humour of the ox, is 0-03-0-04. This substance is not sugar, as it will not undergo the alcoholic fermentation (Gruenhagen).^ Urea and sarcolactic acid (Gruenhagen) are also present in small quantities. ' Chavvas, Pfli'iger's Archiv, xvi. 143. '■^ See Gorup-Besanez, Lehrhuch, 4th edit. 1878, p. 401. 5 Friend and Halliburton, Brit. Ass. Ecjiorts, 1889, p. 130. 4 Kuhn, Pfliiger's Archiv, xli. 200. ■'■' Gruenhagen, ibid, xliii. 377. LV.Ml'H AND ALLIKI) FLUIDS 351 PERILYMPH AND ENDOLYMPH These fluids of the iuteiuial ear have been exaininetl by Dahnhardt' in fishes. Perilymph contains 2-l-2'2 per cent, of solids ; it is rich in mucin and in sodium chloride. The endolymph is clearer, less viscid ; it contains I'f) per cent, of solids, including a small quantity of mucin. SYNOVIA The fluid in synovial cavities around joints, in bursfe, sheaths of tendons, &c., differs from that in serous cavities (1) in containing a greater proportion of solids ; (2) in containing a slimy mucin-like substance which confers viscosity upon it. The following are the analyses that have been published. In parts per lUOU Calf (Frericbs)- Water i 965-7 Solids I 34-3 Mucin 3-2 Proteids "1 ,(,„ Extractives, fat, &c. J Mineral salts ... 10-6 Synovia of Ox. Mean of Two Analyses (Frerichs)= Synovia fi-om knee of a Man in which there was excess ( Hoppe Seyler)= Human Synovia from t^vo similar cases (Haramarsten)*| Case 1, Chronic Case 2, Acute 1 959-2 928-33 947-19 933-7 40-8 71-67 52-81 66-3 40 6-6 2-7 3-56 26-05 1 51-3 4-47 39-2 4-96 54-21 3-5 10-6 9-3 8-65 8-53 (NaCI6 26) Frerichs found that active exercise diminishes the amount, and increases the concentration of synovia. Some doubt has arisen as to whether the slimy sul)stance present in synovia is really mucin. In Hammarsten's two cases there was no true mucin, but the slimy substance was found to be nucleo-albumin, like that which causes the sliminess of bile ; it contained o per cent, of phosphorus. Landwehr ■' on the other hand maintains that the slimy substance in synovia is true mucin, i.e. a compound (or mixture) of a proteid with a carbohydrate called animal gum. ^ Diihnhardt, Arbeit d. Kieler physioh Inst. p. 103. * Frerichs, R. Wagiier's Handwiirterb, d. Physiol, iii. 463. ^ Hoppe-Seyler, Physiol. Chem. p. 623. In other similar cases Hoppe-Seyler found 10-91 per 1000 of mucin. * Hammarsten, Mah/s Jahreshericht, xii. 484; original paper in Swedish. ^ Landwehr, Pfl'uger's Archiv, xxxix. 193. 352 THE TISSUES AND ORGANS OF THE ]5()1)Y THE FLUID IX OYARIAX CYSTS The fluid contained in ovarian cysts has for its basis a transudation from the blood vessels, as in the different forms of dropsy into serous cavities ; it is, however, generally viscous, from the presence of a mucinoid material, which masks the other proteid constituents of the fluid. Ovarian fluid is alkaline ; it is often coloured with a deep brown pigment, derived in all probability from haemoglobin, and often contains excess of cholesterin, crystals of which are seen floating about. Oei'um ' has examined numerous specimens of fluid obtained from various forms of ovarian cysts, and his general analytical conclusions may be thus summarised : — Colloid Cystomata (24 Cases} | Specific Gravity Total Solids Salts Protei^U. iiicluiling nniciii-like substance, Maximum, In 13 cases 25-75 6-8'7 parts Maximum, 108-32; minimum, 1038 parts per 1000 per 1000 8-8 per 1000. In only three cases Minimum, In 2 cases less .greater than 50 per 1000 ' 1010 than 25, and in Fibrin was formed on mixing | In four cases 1 case more than the fluid with blood. Peptone : only, above 75 per 1000 absent. Mucin-like substance 10.30 always present Paj)illari/ Cystomata. — 2 Cases 10.36 116-1 parts per — 102-67 parts per 1000. In one 1000 case the mucin-like substance was present, in the other ab- sent Hydro^js Follicidi Graajian'i. — 2 Cases 1009 — ! — 1 Fluid clear and water}'. Mucinoid substance absent. Parorarial Cystomata Small in quantity — Clear watery fluid. Mucinoid substance absent. (In one case I had the opportunity of examin- ing I found 1 per cent, of proteids 2) Hydrops Tvia-. — 2 Cases 1008 10-5-11 per 1000 j 6-7 per 1000 ; 1-2 per 1000. No mucinoid sub- ! 1 stance Fluid from Flhro- Cystic Tumour. — 1 Case — ' . — 63-056 per 1000, of which 3-58 per 1000 consisted of fibrin, the remainder serum-globulin and serum-albumin. No mucinoid substance present 1 Oerum, ' Kemiske Studier over Ovariecystevaedsker, &c.' Koebena-\Ti, 1884, Mah/'s .Tnliresbericht, xiv. 459. - Journal of Physiology, v. 163. lA'-MPIl AM) AIJ.IKI) FLUIDS 353 \V(> have here a large number of lluids iVoiu various diseases of the ovary and neighl)ouring organs ; and we see that the chief point of intei'est is tlie constant presence of the niucinoid material in colloid cysts, and this is regarded by ()erum as Ijeing diagnostic of the colloid form of degeneration. It is not present in the normal ovary, nor in cases of hydrops of the graafian follicles. In those cases of ascites where the same material occurs, colloid degeneration is also present. This peculiar ropy, slimy material was by Soberer said to consist of two substances, which he named metalbumin and paralbumin. Both can be precipitated by means of alcohol, and the alcoholic precipitate is easily soluble in water. Metalbumin is the name given to the ropy substance, and paralbumin to the substance which occurs in colourless ovarian fluids which have a gummy consistency. These substances have been investigated by Hammarsten,' whose results are briefly as follows : — MetaJhumin is simply colloid material, i.e. the substance formed in colloid degeneration. It has the physical characters of mucin ; chemically, like mucin, it yields a reducing sugar on Ijoiling with dilute sulphuric acid ; it, however, is not mucin, as it is not precipitable by acetic acid ; the name pseudo-mucin is therefore suggested. Paralbumin is simply pseudo-mucin in loose combination with a proteid. These facts have been fully confirmed by Landwehr ^ ; he regaixls these substances as belonging distinctly to the class of mucins ; they contain a carbohydrate called animal gum, and it is this which is convei'ted into a reducing sugar by the action of dilute sulphuric acid. In one case of ovarian fluid, which I very fully examined,^ the fluid was opalescent but not at all viscous. On adding acetic acid there was an abundant precipitate. On examination, this pi*ecipitate was found to consist of true mucin. THE FLUID IX HYDRONEPHROSIS The ureter of one kidney may become blocked by a stone or new growth ; that kidney becomes functionless, while the other does double work. The functionless kidney becomes more and more filled with urine, and dilates, till ultimately a large sac containing dilute urine is formed, and into it some proteids from the blood also pass. The urinary constituents in time are absorbed. The following details of an analysis (Oerum, loc. cit.) may serve as an example : — ' Mahfs Jalireshericht, xi. 11. Original xiaper in Swedish. ^ A. Landwehr, Zeit. ]}hysiul. Chem. vii. 118. 5 Brit. Med. Joimi. vol. ii. 1890, p. 19G. A A 354 THE TISSUES AND ORGANS OF THE EOl)Y Specitic gravity, 1009. Solids, -lO-Ul per 1000. Proteids, 7-677 ,, „ (serum-globulin and serum -albumin). Salts, 8-654. Urea, uric acid, creatinine, absent. The fluid, in other words, resembles a serous effusion. Cystic degeneration of the kidney may occur from causes which are not known, and consists in the formation of small cysts throughout the kidney substance ; the contents of these cysts are watery, sometimes contain urinary constituents, sometimes are tinged with blood, and sometimes, i.e. when associated with colloid disease, are ^-iscous. THE FLUID IX HYDATID CYSTS The fluid which accumulates in the interior of the cysts formed by the parasite Tcenia echinococcus is colourless, neutral, and sometimes slightly opalescent. Its specitic gravity is 1006-1015 : it contains 1-2 to 1-4 per cent, of solids. The solids may be classifled in the usual way into proteids, which are very scanty, extractives (among which sugar and inosite, and traces of ux'ea, creatine and succinic acid have been described), and salts. Microscopically it is often seen to contain a numljer of booklets from the embryos. If allowed to escape into the peritoneal cavity, it is stated that it sets up peritonitis ; in connection with this point it may ])e noted that Mourson and Schlagdenhauffen ' ha^e found a poisonous ptomaine in the liquid. If the growth has involved organs producing the rupture of blood vessels, or bile vessels, the fluid obtained will be mixed with blood or bile respectively. In one case which I examined the fluid consisted of little else but bile. THE AMNIOTIC FLUID The most probable suggestion as to the origin of the amniotic fluid, during the early months of pregnancy, is that it is simply exuded from the tissues of the foetus. After the formation of the placenta, and chorionic vessels, a transudation of lymph takes place from the maternal vessels into the amniotic cavity."^ In the later months of pregnancy this becomes mixed with foetal urine.^ The composition of the fluid corresponds to its double origin ; it contains, in addition to water, ' Compt. rend. xcv. 791. - Jungbluth, 'Beitrag zur Lelire vom Fruehtwasser,' Inaug. Dissert. Bonn, 1869. 5 Gusserow, Arch. f. GyiK'ik. iii. '268. Prochownik, Tbid. xi. 304. LY.Ml'H AND .\LI,IKI) FLl'IDS 355 proteitls, urea, and tlie salts conimoii to urine and blood. Its quantity varies between one and two pints. In some cases it is abnormally small in quantity, in others (hydramnion) abnormally large. A comparison of normal amnicjtic fluid with that of hydramnion has been made bv Prochownik, wh(j "ives the foUowinj' numbers : — Parts per lOWi -Amniotic Huiil Fluid of Hydraumion Water .... 984B 981-4 Solias .... 1.5-7 18-6 Proteids .... l-!t r>-2 Extractives 8-1 1 '7 Salts 59 5-6 In hydramnion the proteids are thus more abundant than normal. Scherer' and Weyl- found mucin in amniotic fluid. CEEEBEO-SPIXAL FLUID This is the name given to the fluid which is present in the cerebro- spinal cavity, that is, the ventricles of the brain, and the central canal of the spinal cord ; the same fluid is also present outside the spinal cord in the subarachnoid and subdural cavities ; the communication between the fluid outside and inside the central nervous system is by ■means of the foramen of Majendie, a hole which perforates the piece of pia mater which forms part of the roof of the fourth ventricle. By some pre^•ious observers the cerebro -spinal fluid has been regarded simply as an exudation from the blood, and has been classified with the fluids w^hich occur in serous cavities. This is, however, in- correct because : — 1. The arachnoid membrane is neither from the point of view of embryology or structure a serous membrane. 2. The fluid is not a mere lymph moistening the parts already enumerated, but is normally present in sufficient quantity to exercise a considerable amount of pressure. 3. Chemical investigation of the fluid itself show-s that it is very different from the fluids contained in serous membranes, and thus sup- port is lent to the idea originally propounded by C. Schmidt, that the fluid should be classified rather with secretions than with transuda- 'tions. The normal cerebro- spinal fluid is obtained in cases of fracture of • Scherer, Zeit.f. iviss. Zool. i. 89. - Weyl, Arch. f. Anat. u. Physiol. 187G, p. .543. 356 THE TISSl'ES AND ORGANS ()¥ THE T.ODY the base of the skull, where sometimes the fluid escapes by the ear, if the membrana tympani has been also ruptured by the accident. In some cases of congenital deficiency of the vertebral arches (spina bifida), a tumour of the membranes of the spinal cord (meningocele)- projects through the opening, and in this the fluid accumulates, and may be removed by tapping. Tliis fluid may be also regarded (or at least that obtained by the flrst tapping) as normal cerebro-spinal. fluid. In the disease known as hydrocephalus excess of the cerebro-spinal fluid accumulates within the ventricles of the brain, in some cases so- much so that the bi-ain becomes a mere sac suiTounding the fluid. Hydrocephalus may be chronic or acute. Chronic hydrocephalus is due to an accumulation of the normal fluid ; it is often associated with pressure upon, or obstruction in, the veins at the base of the skull, and in these cases, the fluid is therefore cerebro-spinal fluid plus a trans- udation from the blood. In acute cases (tubercular meningitis) the specific gravity rises, and the solid constituents also increase, especially the proteids ; and in such cases the fluid resembles the exudations- which occur in inflammations elsewhere. It is, however, rare for the fluid to become purulent. In animals small quantities of the fluid may be obtained by means- of a small syringe from beneath the dura mater. Necessarily the greater part of our knowledge of the fluid from the central nervous cavity is deriAed from patliological cases where the fluid is in excess. The liquid is always either neutral or faintly alkaline, and has a specific gravity of about 1007-8. Composition. — The following analyses of the fluid from spina bififia were made bj- myself : — ' ! Case 1. Female set. 19 Case 2. Child set. 11 days Case 3. Child at. First Tai>piiig l weeks I In parts per 1000 In parts per 1000 Water 98SJ-75 ! 989-877 Solids 10-2.5 10-I-JH Proteids .... 0-842 l-(;o2 Extractives . . . ,> ,^ ^t . V 0-631 ■.:A Salts 'C ^"•^" \ 7-800 9-fi2t; Foiirtli Tapping In iiarts per 1000 991-658 8 342 0199 3028 5115 The following analyses of the fluid from cases of chronic hydro- 1 'Report of Spina Bifida Couunittee,' vol. xviii. of the Clin. Sue. Trans. Similar analyses will be found in Hoppe-Seyler's Physiol. Chemie, p. 604. LV.MI'll AND ALLIED FLUIDS 357 ceplialus are fi-oin C Schmidt. They show in comparison with spina bitida fluid rather a greater quantity of solids, especially of proteids. lu parts jier 1000 Casel Case 2 1 Cases "Water Solids Proteids and Extractires Salts 1 986-78 13-22 3-74 9-48 984-59 15-41 6-49 8-92 980-77 19-23 11-35 7-88 A tabular statement, such as the preceding, does not show, however, that there is anything characteristic in the fluid. It is when we come to examine the various constituents of the fluid that we find how it •dift'ers from the fluids in serous sacs. These differences consist in the presence of certain peculiar proteids, and of a substance which reduces copper salts like sugar. Proteids. — The proteids in normal cerebro-spiual fluid (removed from cases of meningocele) are as follows : — («) Fibrinogen is absent. There is no heat-coagulum produced by a temperature of 56°C. No fibrin is formed on the addition of seruni or of tibi'in-ferment. (6) All the proteid present is precipitable by saturation with magnesium sulphate. Serum-albumin is therefore absent. Hoppe- Seyler' describes the proteid which is present as a globulin. On re- dissolving the precipitate, however, it is found on heating the solution that a very small heat-coagulum is found at 75°C.,^ but that the remain- ing proteid consists of proteoses or albumoses, i.e. proteids like those -which are formed during digestion, intermediate bodies in the formation of peptone. There, however, appears to be no proteolytic ferment, like pepsin or trypsin, present in the fluid. The most characteristic pro- perties of albumoses are : — i. They are not coagulated by heat. ii. They are precipitated by nitric acid in the cold ; the precipitate disappears on heating, and falls down again on cooling. iii. Like peptones they give a pink colour with copper sulphate and caustic potash ; other proteids give a violet colour. The form of albumose most frequent in cerebro-spinal fluid is prottj- albumose, i.e. one which is precipitable by saturation with sodium chloride or magnesium sulphate. In some few cases deutero-albumose lias been found, i.e. one which is not precipitable by the salts just J Hoppe-Seyler, Physiol. Chemie, p. 608. - This teniperatm-e is the same as that at wliich senini-globulin is coagvdated. The ^obulin present appears to be serum-globuUn. Cell-globuhn (fibrin-fermentj is absent. 358 THE TISSl-ES AND ORGANS OF THE BODY mentioned ; it is, however, precipitated hiy saturation with ammonium sulphate ; and in other cases still fewer in number, true peptone is found, Le. a proteid which is not precipitable by saturation with am- monium sulphate. The existence of albumoses in cerebro-spinal fluid may be shown another way. A large excess of alcohol is added to the fluid : this precipitates all the proteids : and after a few weeks the globulin is rendered insoluble in Avater by the action of the alcohol. The albumoses, however, remain still freely soluble.' In cases of chronic hydrocephalus, the fluid removed by the first tapping has the normal characteristics of cereljro-spinal fluid. The fluid removed by subsequent tappings, however, resembles a dilute transudation from the blood ; and if inflammation supervenes, this be- comes more marked. Albumose can still be shown to be present, but it is obscured by the superabundance of the other proteids. An in- crease in the amount of proteids in successive tappings does not necessarily occur either in spina bifida or hydrocephalus ; but in the latter disease, an increase is very apt to occur, perhaps as a result of irritation, produced Vjy the removal of the fluid ; the quantity does not, however, rise so high as it does in cases of acute, i.e. inflammatory^ hvdrocephalus. At the same time the reducing substance also becomes more abundant. The following case of chronic hydrocephalus in a boy six months old, under the care of Mr. Parker,^ and of which I had the opportunity of examining the fluid in three successive tappings^ illustrates this point very well. Si>ecific Grarity Percentafre of Total Proteiils Kinds of Proteiil present Reducing Substance First Tapping Second ,, Third loot; 1010 1010 0015 006S 0-272 Globulin Proto-albumose Hetero-albumose The same as in the first tappintr Serum-globulin Serum-albumin Trace of albumoses Traces Fairly abundant More abundant 1 I have examined a number of specimens of blood and transudation for albumoses and peptone, but in all cases with a negative result {Proc. Physiol. Soc. 1887, p. xiv. See also J. Physiol, vol. x. p. 232j. * East London Hospital for Children. hVMl'II AM) ALLIED KLriOS 359 In the fluid from a case of acute liydrocephalus,' the following details of the analysis show very well the characteristics of this fluid : — The fluid was clear, alkaline, and straw-coloured ; the colour was extracted by alcohol, and had the normal characteristics of serum- lutein. Proteids : the percentage present was 0-65. The fluid contained a small clot of tibrin. Magnesium sulpliate produced an abundant precipitate of serum-globulin ; serum-albumin remaining in solution. The presence of alljumoses was questionable. Reducing substance : present in small quantities. In other words, the fluid in cases of acute hydrocephalus resembles other inflammatory exudations in the higher percentage of total proteids, and in the presence of fibrinogen, and white blood corpuscles, which caused clotting when the fluid was removed from the body. Reducing substance. — It has long been known that cerebro-spinal fluid contains a substance which reduces copper salts in the same way as sugar does. Bussy^ found it in the fluid obtained from a patient with a fractured cranium, and in the cerebro-spinal fluid of the horse and dog ; but although he considered it was grape sugar that was present, he found he was not able to induce alcoholic fermentation in it. Turner^ discovered the same substance in spina bifida fluid, but he also found that the addition of yeast produced no fermentation ; he concluded that the reduction is brought about, not by a carbohydrate, but by some derivative of albumin. Since then it has been abundantly shown that the substance is not sugar ; it does not reduce bismuth salts ; it does not rotate the plane of polarised light ; it does not form a crystalline compound with phenylhydrazine as sugar does. It is thus not sugar. The sulistance is pyrocatechin (catechol) ; and it may l)e separated in the following way : alcohol is added to precipitate the proteids ; the alcoholic extract is evaporated to dryness, and the residue dissolved in water ; neutral lead acetate is added to the aqueous solution ; a precipitate is formed, this is suspended in distilled water, and the lead separated by a stream of sulphuretted hydrogen, and filtered. The filtrate is shaken with ether ; the ethereal extract on evaporation yields a crystalline deposit which consists of pyrocatechin. This is a substance of which the formula is CgHfjOg ; it belongs to the aromatic group of organic compounds. It is turned green by ferric chloride, brown hj caustic alkalis ; it has a peculiar pungent taste and 1 The Huid was sent to me by Dr. Penrose ; it was removed a few days before death from a boy 5 months old, in the Hospital for Children, Great Ormond Street. - Bulletin cle Vacademie de inedeci>i£, Dec. 1852. s W. Turner, Prac. Boy. Soc. vol. vii. (1854), p. 89. 360 THE TISSUES AND ORGANS OF THE BODY an acid reaction. It is one of the products of decomposition of proteids, and occasionally is found in urine {see p. 77). This substance appears to be a normal constituent of cerebro- spinal fluid, though whether in a combined or an uncombined state is at present douVjtful. Hoppe-Seyler states that the reducing substance only occurs after irritation has been set up by tapping ; but this does not appear to be the case. I have never failed to find it in fluid removed by the first tapping, though it may occasionally be so scanty that the fluid must Ije concentrated before it can be discovered. In subsequent tappings it is always increased in quantity. In one case (first tapping) the quantity (reckoned in tei-ms of dextrose) was 0*002 per cent. ; in another case (fourth tapping) the percentage was 0"165. Salts. — Carl Schmidt remarked that his analyses of the inorganic constituents of cerebro-spinal fluid showed an unusual preponderance of potassium salts. The following are the numbers : Parts per 1000 Casel Hydrocephalus Case 2 Case 3 Hydrocephalus Hydrocephalus 1 K,SO^ . KCl . NaCl . NajPO^ Na.,0 . Ca/POJ., . 0096 2181 4-43H 0-613 1-842 ). 0-307 0-193 1 -48.5 4-101 0-486 2-290 0 362 0-222 0-232 6054 0-115 0-987 0-271 Two of these cases certainly show a remarkably high percentage of potassium chloride ; but subsequent investigators have not found that this is a general rule. Yvon ' gives the following numbers ; the fluid was remo\ ed from a case of hydrocephalus. NaCl 7-098 per 1000 KCl 0-033 J CaO 0-112 , p,o. OoiVd , SO3 . . . traces Iron traces Magnesia 0-238 , P. Midler 2 in another case found that the inorganic salts present were 8-8 per 1000. The most abundant salt present was sodium chloride, and the relation of XaCl to KCl was 21-5 to 1. In my own experiments I sought to obviate error in making obser- 1 Yvon, Journ. (1e pharmacie et de chimie, 4tli series, vol. xxvi. (1H77), p. 240. - F. Miiller, Mittheil. a. d. Wiirzburger med. Klinik, i. 267. LY.Mi'JI AND ALJJEl) i-lAIDS 361 vations on tlie saline constituents of this fluid by avoiding incineration; there is no doubt that some of the salts, especially sodium chloride, pass off with the organic matter during the process of ignition. It is also well to take n large quantity of fluid so that errors may be minimised. The following method maybe recommended for determining the relation of sodium and potassium in organic liquids. The liquid is first evaporated to dryness, and the organic matter is destroyed by heating with fuming nitric acid ; the residue is evaporated to dryness two or thi-ee times on the water-bath with hydrochloric acid, in order to convert all sodium and potassium compounds into chlorides ; phosphates, lime, and magnesia are precipitated by making the liquid just alkaline with baryta water, and the precipitate so formed is filtered oti'. Excess of baryta is then precipitated with ammonium carbonate, and filtered otF ; the residue is evaporated to dryness on a weighed platinum capsule, and the increase in weight gives the total chlorides ; these are dissohed in water, and platinum chloride added ; this precipitates the potassium chloride, and from the weight of the precipitate the potassium chloride can be calculated ; the difference between total chlorides and potassium chloride gives the amount of sodium chloride. The following numbers were obtained in one analysis of hydro- cephalus fluid : 300 c.c. of fluid yielded 2"7825 grammes of chlorides, i.e. 0-927 per cent. The weight of potassium chloride calculated from the weight of the platinum precipitate was 0'0859 gramme, or 0*028 percent. Therefore in 100 parts of chlorides 4*85 consisted of potas- sium chloride and 95-15 of sodium chloride. This is about the same proportion as is present in blood, lymph, and transudations generally. PUS Pus is the creamy fluid which occurs in abscesses. We have already seen how in inflammation the normal transudations from the blood vessels become increased in amount, richer in solids, and in corpuscular elements. The emigration of the white corpuscles may go on to such a great extent that they crowd in great numbei's in the exuded liquid ; in fact, this is the process known as suppuration or j)us formation. The microscopical appearances of pus are as follows : — It is a clear fluid crowded with leucocytes, which have undergone more or less degenerative change ; fat globules are also found which have been liberated from leucocytes that have undergone fatty degeneration and burst. In pus also one usually flnds abundant micro-organisms (micro- cocci and bacteria). The cells of the pus are called pus-corpuscles, the 362 THE TISSUES AND ORGANS OE THE BODY liquid in which they float the pus-seruin. The specific gravity of pus- is 1030-1040, and its reaction is alkaline. Pus corpuscles. — There is n» doubt that these are chiefly white blood corpuscles which have exuded from the vessels ; some of the cells are probably, however, derived from the tissues in which the formation of the abscess is taking place (connective tissue corpuscles). The cells do- not as a rule show any active amoeboid movement. They are spherical, and swollen in many cases with fat globules, their protoplasm having undergone fatty degeneration. In some cases the pus cells have still further disintegrated, and may even have an acid reaction from the formation of sarco-lactic acid. For chemical investigation pus cells may be obtained by mixing pus with an equal volume of dilute sodium sulphate solution (a saturated solution of the salt diluted with nine times its volume of water), and then filtering ; the pus corpuscles remain on the filter, and are washed with some of the same saline solution. The various substances found in the pus cells are like those which we have already described in the white blood corpuscles. The 7iuclei consist of nuclein, a phosphorised albuminoid substance. It may be separated from the investing pi'otoplasm by treating pus with artificial gastric juice ; the nuclei remain undissolved. The nuclein of pus cells was investigated by Miescher, ' and also by Hoppe-Seyler^ whose somewhat conflicting analyses certainly seem to denote that nuclein is not a definite chemical individual. The nuclein of pus cor- puscles in its general characters does not appear to be diflferent from that contained in nuclei elsewhere (p. 202). The 2)rotoplasm consists of proteids chiefly, but it also contains various extractives and a certain small proportion of inorganic salts. The following tabular statement gives the results of Hoppe-Seyler's analyses in two samples of pus-cells. Organic constituents in 100 parts of dried pus-cells : — (1) (2) Proteids 18-762 1 34-257 1 68-585 Nuclein ...... 67-369 Insoluble substances 20 o(J6 1 Lecithin ..... Fats \ 1 14-383 7-564 7-500 Cholesterin ..... 7-400 7-283 Cei'ebrin ..... 5-199 1 10-284 Extractives 4-433 / 1 Miescher, 'Ueber die chemische Zusammensetzung der Eiterzellen,' Hoppe-Seyler's Med. Chem. TJntersuchungen, p. 441. - Hoppe-Seyler, Med.' Chem. Untersucltungen, p. 407. For this reference I am indebted to Gamgee, Plii/siol. Chcin. p. 244. LVMl'li AND ALLIED FLllDS 36^ Inortranic constituents in 100 i)arts cf drierl pus-corpuscles: — NaCl o•^■^o Ca3(r(),), 0-20.-, Mg3(FO,), 01 IH Fe,(PU,)., .... 01 or, PO", . ' 0 91fi Na . 0-068 K ...... . traces Protfids. — My own observations coincide with tlio.se of Miescher as regards the absence of myosin (described by earlier oVjservers). The most abundant proteid is the .same nucleo-albumin already described in the white blood corpuscles ; originally called hyaline substance by Rovida, and also noted by Miescher [see p. 260). With sodium chloride and magnesium sulphate it swells uj? into a slimy mass, and hence one has to use sodium sulphate in separating the pus-corpuscles from the pus- serum. Cell-globulin and cell-albumin are also present in pus-cells as in white blood corpuscles. Fibrin -ferment has been prepared from pus by Rauschenbach.' In addition to these, which are the normal proteid constituents of leucocytes, there are often found in addition considerable quantities of albumoses and peptone produced no doubt during the retro- gressive metamorphosis of the corpuscles. It is very probable that the fever which accompanies suppurati\e processes is often, at any rate in part, produced by the entrance of these substances into the circulation. - The other constituents of pus-cells have been already enumerated, and there is but little to be added concerning them. The large increase of fat and fat-like substances (lecithin, cholesterin, kc.) should be noted ; the fatty degeneration, of which this is an indication, can also be seen by the microscope ; free fatty acids may even be found in old pus, forming crystalline depo.sits. Glycogen can be often demonstrated in pus corpuscles, microchemically by the use of iodine which stains it deep brown (Ranvier^). It has also been separated in considerable quantity from pus-cells (.Salomon^). In containing glycogen, pus-cells resemble white blood corpuscles. 1 Inaug. Dissert. Dorpat, 1883. Mahj's Jalireshericht , xiii. 134. 2 Dr. S. Martin, Brit. Med. Journ. vol. ii. 1890, p. 234. The original statements con- cerning the presence of peptones in pus were made by Eichwald {Vei-handl. d. phijs, med. Gesellsch. Wiirzbm-g, 1864, p. 335), and Hofmeister (Zeit. phijsiol. Chem. ii. 295).. The method adopted by these observers was not, however, perfectly tnistworthy. The only reliable method is that adopted by Martin. The pus was placed under excess of alcohol for many weeks, dried, and extracted with water. Albumoses and peptones alone went into solution, the other proteids of the pus having been coagulated by the alcohol. For an account of the effects of the injection of albmnoses and j)eptones in raising the body temperature, i.e. producing fever, see Ott and Collmar, Journal of Physiology.. viii. 218. 2 Progres med. 1877, p. 422. * Deutscli cmed. Wochenschr. lull, Xo. 35. .364 THE TISSUE.s AND OEGANS OF THE BODY Fus-serum. — This may be separated from the corpuscles by the use of the centrifugal machine ; or it may be obtained diluted with sodium sulphate solution after filtering off the pus corpuscles in the manner already described. The pus-serum is like blood-serum in composition ; it diti'ers from lymph and other forms of exudation which we have considered in con- taining no fibrinogen, and consequently no fibrin is formed when the pus is removed from the body. It is, however, possible that fibrin may be formed within the abscess, and be subsequently dissolved and absorbed; perhaps in some cases this leads to the ' inspissation ' of the pus, as in very severe cases of pericarditis and empyema (purulent pleurisy). The proteids of pus-serum ai'e serum-globulin and serum-albumin ; the extractives and salts are like those in the blood and lymph gene- rally, except that lecithin appears to be more abundant, and leucine and tyrosine have been found (Hop^ae-Seyler). Hoppe-Seylers analysis of pus-serum may be tabulated in the following way : — In parts per 1000 ^1' (2) Water 913-70 905-65 Solids 86-30 94-35 Ori^anic solids .... 78-57 86 58 Proteids ..... 62-23 77-21 Lecithin 1-50 0-56 Other ora:anic matters 14-84 8-81 Inorganic solids .... 7-73 7-77 (NaCl 5-22) (NaCl 5-39) Pigments in pus. — When h;>^morrhage occurs into an abscess the blood pigment more or less altei'ed will be found, and in other cases bile pig- ments have been described. In other cases again, pigments due to the activity of certain micro-organisms are found (chromogenic bacteria) ; thus pyocyanin is a blue or rather violet pigment, produced by the growth of a bacillus. It is soluble in water, alcohol, chloi'oform, and ether, and crystallises from chloroform in prisms or rectangular plates. Pyoxanthose is a yellow pigment similarly produced, and often accom- panies pyocyanin. When the two pigments are together, the pus appears green. Pyoxanthose differs from pyocyanin in its solubilities, and may be separated from the latter body by the use of ether in which it is the more soluble (Fordos,^ Liicke,^ Fitz,-* Kunz,"* Babes-''). 1 Comj)tes rend. vol. li. (1860), p. 215. - Archiv f. Min. Chirurgic, vol. iii. (1862), p. 125. •5 Quart. J. Microsc. Science, Jan. 1880, p. 106. •* Monatsheft f. Cheinie, ix. 361. This paper gives the results of cultivating the bacteria by the most recent methods. 5 Compt. rcJid. Soc. Biol. 1S89, p. 43S. Two other pigments produced by a variety of the bacillus (B. pyocyanicus, /3) are here described ; aromatic bodies are produced as well. 8()5 CHAPTER XIX RESPTliATIOX T\ the foregoing chapters u})on the blood, lyinpli, and similar fluids,, little or no reference has been made to their gaseous constituents. This omission we now proceed to supply, judging it to be more convenient to deal with the blood gases in connection with the function of respira- tion. The respiratory organs consist in air-breatliing animals of the lungs, in aquatic animals of the gills.' The respiratory system also includes the passages by which the air or the water respectively is carried to the lungs or gills, and the musculai- apparatus by means of which the respiratory movements are executed, and these in turn are controlled by a nervous mechanism. The lungs consist essentially of numerous little hollow sacs, in the walls of which is a close plexus of capillary blood vessels. These air cells, or alveoli, communicate with the external air by means of the trachea, bronchi, and bronchial tubes. Inspiration is due to a muscular effort that enlarges the thorax, the closed cavity in which the lungs are situated ; owing to the atmospheric pressure the lungs become distended; the atmospheric air, however, does not actually penetrate beyond the larger bronchial tubes ; the gases which get into the smaller tubes and air cells do so very largely by the process of diff"usion. Expiration is ordinarily brought about by the elastic rebound of the lungs and chest walls, and is only a muscular eftbrt when forced ; but even the most vigorous expiratory ett'ort is unable to expel the alveolar air. The alveolar air and the blood in the pulmonaiy capillaries are separated by the thin capillary wall, and an equally thin epithelium that lines the alveolus. The blood which is venous in the capillaries parts with its excess of carbonic acid and watery vapour to the alveolar air ; this by the process of diffusion, aided by the expiratory eftbrts, passes into the atmosphere. The blood at the same time receives from the alveolar air a supply of oxygen which renders it arterial. 1 Insects jjossess a number of traclieie oi- air-tubes kejit open by a spiral of chitin ; these penetrate to all parts of the body and supply the requisite oxygen. Pulmonary sacs are found in certain groups of invertebrates {r.j. Avachnida) ; gills are present in most aquatic invertebrates. •366 THE TISSUES AND OEGANS OF THE BODY In fish the supply of oxygen is derived from the air dissolved in water. The capillaries of the gills come into close contact with the water, which not only parts with some of its dissolved oxygen, but receives from the venous blood the products of combustion, of which the most important is carbonic acid. In the interchange of gases that occurs in the essential organ of respiration, diffusion plays a certain part, but this is aided by chemical processes ; for instance, the union of oxygen with hfemoglobin. The intake of oxygen and the output of carbonic acid are, however, -only parts of the function known as respiration. The intake of oxygen is the commencement, and the output of carbonic acid is the end, of the series of changes. The intermediate steps take place all over tlie body, •and constitute what is known as tissue-respiration. The compound oxyha?moglobin is only a loose one, and in the tissues it parts with its oxygen supplying them with this element. This oxygen does not necessarily undergo immediate union with carbon to form carbonic acid, and hydi'Ogen to form water, l)ut in many cases, as in muscle, is lield in reserve by the tissue itself. Ultimately, however, the two oxides just mentioned are formed ; they are the chief products of com- bustion. There ure other products, such as the imperfectly oxidised substances (urea, uric acid, tfec.) that pass into the urine. These pro- ducts of combustion pass into the venous blood, and the gaseous pro- ducts, carbonic acid, and a portion of the water in the form of vapour find an outlet by the lungs. Such is a brief account of the vai'ious steps that constitute respira- tion. These we have now to discuss one by one. THE GASES OF RESPIRATION Methods of Investigation The methods, by means of which the changes in the air brought about by respiration can be investigated, are two in number : — 1. An animal is placed in a closed chamber ; the carbonic acid formed is continually removed, and the necessary oxygen supplied in measured quantities. This is the principle of the method of llegnault and Reiset. 2. The animal is j^laced in a chamber through which atmospheric air is passed, and the change in the composition in the air after passing through the chamber is examined. This is the principle of the method of Scharling,' and later of Petteukofer. 1 Ann. Cheiii. Plidrni, xlv. 214. KESl'l RATION 367 The ini'thiul (if Riijnti.aU and Ih-'iset} — The iipjKiratus consists of ;i bell jar, R, in which is placed tlie animal to he experimented on. This is placeil in a cylinder, y// (provided with a thernionmter /), hy which the tem[)erature can be regulated, or which can be employed for calorimetric experiments. A tube, 6, leads into the bell jar R, and through it passes a known volume of oxygen ; to absorb any trace of carbonic acid a vessel containing potash is placed on tlie course of the tube. Two tubes, d and e, lead from R, and are united by caoutchouc tubes with the potash bulbs, which can be raised or depressed alternately by the beam W. In this way they aspirate the air from R, and the increase Pig. 64. —Scheme of the Respiration Apparatus of Reguault and Reiset. R, globe for animal ; jjr, lempiit(il air is tliat wiiich can be forcibly inspired over and above that taken in at a normal respiration. It is equal to 100-130 cubic inches (1500 c.c). Vital capacity is the volume of air which can be forcibly expelled after the deepest possible inspiration. It = reserve air + tidal air + complemental air = 230 cubic inches (3800 c.c). These numbers were obtained by Hutchinson by means of the instrument he invented and called the spirometer, a special form of gasometer adapted for the purpose. The above numbers are averages. Varying conditions which modify the vital capacity are heiyht (one inch in height increasing it by eight cubic inches) ; weight (when the weight exceeds the normal by seven per cent, each kilogramme of increase diminishes the vital capacity by 2-3 cubic inches) ; aye (after 35 it gradually diminishes) ; sex (in a man and woman of the same height the ratio is 10 : 7) ; position and diseases. The frequency of quiet respiration varies with age ; the following average numbers are given by Quetelet ' : in newborn children, 44 ; at the age of five, 26 ; at the age of fifteen to twenty, 20 ; twenty to twenty-five, 18 ; twenty-five to thirty, 16 ; thirty to fifty, 18 respira- tions per minute. Hutchinson gives 16-24 respirations per minute as the average of 2000 observations. cubic metres cubic feet The total air respired in the twenty-four hours =11 ^ 330 litres ,, „ per hour 458 = 13-7 Vierordt gives the following figures : — grammes Oxygen taken in in the twenty-four hours=.744:=516,500 c.c. Carbonic acid given out ,, =900^455,500 c.c. The excess of oxygen absorbed over carbonic acid expired is thus €1,000 cubic centimetres in the day ; most of this combines with hydrogen to form water, and a small quantity is contained in urea, uric Acid, &c. Dumas gives the tf)tal quantity of carbon exhaled in carbonic acid as 8^ oz. in the twenty-four hours ; E. Smith as 7-11 oz. The aqueous vapour averages between 350 and 500 gi^ammes. But this is very variable, the chief factor in the variation being the ' See Ranke, Grundriss d. Ph'jsioh (1. Menschen, p. 333, Leipzig, 1868. B n 2 372 THE TISSUES AND ORPtANS OF THE BODY quantity already present in the inspired ;di- which is dependent on climate, temperature, &c. The effects of varying circumstances on Respiration Many circumstances aftect the respiratory exchanges, particularly with reference to the carbonic acid : such as state of rest or activity, food, day and night, sleep, sex, age, mode of respiration, season of year, alterations of atmospheric pressure, il'C. Atje. — Until the body is fully developed the carbonic acid given off increases with age ; as the bodily energies decay it diminishes. Hence the oxygen absorbed is relatively greater than the carbonic acid given off. The absolute amount of carbonic acid given off is less in children than adults, but in relation to body weight a child gives off twice as much as an adult. The following table is taken from Landois and StirUng's Physiology : — Age In 24 Hours Amount of Carbonic Acid Excreted Amount of Oxygen .\b5orbe 1-2 2'.t-2(; 3-74 1-L> 38-42 l-ol 1-2 40-52 11. 17-33 1-6 2io4 7-50 1-4 31-6 1-26 0-92 34-88 If the expired air be analysed instead of the blood gases, analogous results are obtained. The following example is taken also from S<;zi'lkow's work. Rabbits were the animals emploj'ed : — C.C. in 0 le Miiiuti- I)iu-iiig Resinratorv Quotieut CO.j Expired 0, Absorbed ' I. Repose .... 4-97 12-29 0-404 ' Tetanus .... 13-69 12-11 113 11. Repose .... 7-85 12-76 0-615 Tetanus .... 17-132 1902 0-927 III. Repose .... 6-9!! 17-47 0-400 Tetanus .... r.t-GI 30-35 0-646 The following example is from experiments on human beings : — Discharge of On Deficient Diet On Moderate Diet During Repose CO., .... H.,0 .... Urea . . . Intake of 0., . 695 grms. per diem 26-3 743 1187 1177 26 1042 Experiments on man bj- Yierordt, Speck, and Pettenkoferand Voit, and ou horses by F. Smith,' all gave the same result. In curare poisoning, where the muscles ore inactive, there is much diminished respiratory exchange of ga>es (Zuntz).'- The increase of the gaseous exchanges during forcible respiration is partly explicable by the increase of muscular work. Increased work of the involuntary muscles also produces the same result. The stomach and intestinal tract have been investigated in this direction (v. Mering and Zuntz,' A. Loewy^). Loewy's experiments were carried out both on rabbits and men ; when the activity of the intestinal tract is increased by saline purges, there is a rise in the respiratory gaseons exchanges. No doubt this increased metabolism is due to the activitj- both of the muscular tissue and the glands of the intestine, but probablj- the former is the more important factor concerned. This subject is of practical interest, as, therapeutically, the cures of Carlsbad, Marienbad, &;c., consist in increasing the activity of the alimentary canal by means of saline purgatives. 1 Jotirn. of Physiol, xi. 65. Zuntz and Lehmann {Zeitscli.f. iviss. handwirthscli. 1889 Journ. of Phijisiol. xi. 396) have also made similar experiments with horses. - Pfliiger's Arcliiv, xii. 522. ^ Ihid. XV. 634; xxxii. 173. -* Thid. xliii. 515. 376 THE TISSI-ES .l^D ORGANS OF THE BODY Other forms of activity. — Not only does muscular work increase the amount of gaseous interchange, but all forms of protopla.smic activity act sirailai'ly ; chemical decomiDOsitions are most rapid and extensis-e when an organ is active. Among forms of activity, secretion is the most important after muscular contraction. Nuviber and JDejrtli of Respirations. — The most marked effect of increasing the respiratory movements is not to influence the amount of carbonic acid formed in the body, but to accelerate the removal of that which has been already formed. An increase in the number of respirations (the depth remaining the same), or an increase in their depth (the number remaining the same), causes an increase in the amount of carbonic acid given oif, though with reference to the total amount of gases exchanged it is relatively diminished. This may be illustrated by the following table from Vierordt : — Xo. of Re- spiratious per Minute Tohiine Amount CO, Bepth of Amount CO.. of Air of CO.. per cent. Bespirations of CO, per cent. 12 6,000 c.c. 2.58 c.c = 4-3 per cent. 500 21 C.c. = 43 per cent. ! 24 12,000 „ 420 „ = 3o „ 1000 36 „ =3-6 48 24,000 ,. 744 .. = 31 „ 1500 51 „ =3 4 96 48.000 ,. 1392 ,. — 9-Q W „ 2000 3000 64 ,. =3-2 72 „ =2-4 Corroborative results have been obtained by Voit and Lessen,' Speck,- Berg,-' and Becher.^ Deficiency of Air or of Oxi/yen. Dysjjiuea. Asphyxia — "When a due supply of air is not obtained the oxygen in the arterial blood sinks below the normal, the blood pressure rises, and the respiratory movements become deejaer (dyspnoea or hyperpncea) ; these movements increase until they pass to other muscles, and so a conchtion of general convulsions sets in; this is followed by exhaustion and death : the train of symptoms constituting what is known as asphyxia. A7i increased supply of Air or Oxygen. Apnwa. — After several inspiratory efforts of great force it is easy to hold the breath for a longer time than usual. If air be rapidly pumped into the lungs of one of the lower animals there is no effort made to breathe for the space of some seconds or even minutes. The usual explanation given of these phenomena is as follows : the respiratory centre in the medulla is largely influenced by the quality of the blood sent to it. In ordinary respiration, the normal blood not being fully oxj'genated stimulates it to send out impulses which result in the normal respiratory efforts : too great an amount of carbonic acid in the blood excites it to increased activity (dyspnoea) ; too large a supply of oxygen inhibits its activity altogether (apnoea). This view of the cause of the respiratory movements is, however, not universally accepted. Thus Hoiipe-Seyler^ states that normal arterial blood contains no such reducing sub- stances as have boen considered stimulants of the respiratory centre. He is iiKlir.td to oelieve that the excitation to respiratory activity is to be found in tie ch.mges that occur in the lungs ; these stimulate the terminations of the sensory 1 Zeit. Biol. ii. 244. - Schriften d. Gesellsch. z. Forder. d. Ges. Xaturiciss. Marburg, x. 3. 5 Deutsch. Arch. klin. Med. vi. 291. * Die Kohlensiiurespannung im Bhit, Ziiricli, 18.55. ^ Physiol. Client, p. 544. i;Ksi'ii:.\ri(>N 377 nerves in tlie lunijs, jind by means of rellex action tlio muscular movements of the respiratory nuiscles are l)ronght about. Jlax Marckwald' also adduces many weighty arguments against the generalh" received theory of res))iration. Dj'spncea seems to be undoubtedly caused by excess of carbonic acid in the l^lood, whether this atfects the nerve centre or the nerve terminations. But apnoea, according to Hoppe-Se^der,- is not caused by excess of oxygen in the blood, as normal arterial blood is alreadj- completely or almost completely saturated with oxygen ; from the study of his own experiments and those of other investigators,^ he concludes that it is simply due to fatigue of the respiratory apparatus. PoisoHoiis Gases. — Excess of carbonic acid produces feelings of discomfort (headache, kc.) ; if the excess is very great there is laboured breathing, and ultimately a state of narcosis without convulsions, in which the animal dies. Carbonic oxide is even more deleterious ; it combines with the hfemoglobin, and so prevents the blood, and thus the tissues, from being properly oxygenated {see^. 281). Sulphuretted hydrogen acting as a reducing agent produces similar elfects. Some gases, like chlorine, ammonia, nitrous acid, kc, are irrespirable, producing spasm of the glottis. Nitrous oxide causes narcosis, and is largely used as an aniBsthetic. Ozone, instead of making the blood more arterial, causes it to assume venous characters. in all the vessels; this is perhaps explained by its greater density, interfering with the due excretion of carbonic acid from the blood ; it also causes local initation of the respiratory passages; it slows both heart and respiration (Dewar and McKendrick,^ J. Barlow,^ Filipow"). Hydrogen and marsh gas, if mixed with a sutticient quantity of oxygen, have no effect on respiration. The poisonous effects of res^p'tred air. — Dr. B. W. Eichardson" has made experi- ments on animals in order to investigate the effect ot air that has been breathed previously by other animals. He (inds that after air or oxygen has been once used it is very poisonous, even though all carbonic acid, ammonia, and all appreciable impurities have been removed. He therefore infers that something is removed from the oxygen by the process of respiration ; and that this ' devitalised oxj'gen ' can be revitalised by electrical brush discharges from the positive pole of a frictional machine. As long as oxygen is regarded as an element, it is impossible to accept this explanation. A much more probable explanation is that some impurity is added to the oxygen during the process of respiration, and that this impurity is the poison. What, then, is this impurity ? Jackson** considers it may be carbonic monoxide. What seems to be certain is that it is not carbonic dioxide ; a large admixtitre of pure carbonic acid in the air will not produce the symptoms of poisoning. Men can breathe for two to three hours without marked discomfort air which contains as much as 20 per cent, of carbonic acid. Brown-8equard and d'Arsonval" speak of the poison vaguely as a pulmonary poison, and consider it 1 Innervation of Respirciiio)i, translated byT. A. Haig : Blackie and Son, 1888; Zeit. Biol. xxvi. 259. See also H. Head, Jouni. of Physiol, x. 1. - Physiol. Chem. p. 51V). 5 Rosenthal, Arch. d. Anai. 7(. Physiol. 1864, p. 456 ; 1865, p. li)l. Pfliiger, Pfliiger'.'i Archiu, i. 90. A. Ewald, Ihid. vii. 575; also ' Ueber die Apnoe,' Diss. Bonn, 1873. * Proc. Boy. Soc. 1873-4. •'• Joiirn. Anat. Oct. 1879. '' Pfiiger's Arcliiv, xxxiv. 335. 7 Brit. Med. Joitrnal, vol. ii. l.sdO. CIwiii. News, Iv. 253. « Proc. Physiol. Soc. 1887, p. 31. a Comptes rend. 18H7, 1888, 1HH9. 378 T-HE TISSUES AND 011GAN8 OF THE EODY may be alkaloidal, and that it passes into the expired air from the lungs ; this poison, whatever it is, can be removed by passing the air containing it through tubes containing beads moistened with sulphuric acid. Although the mere presence of 1 per cent, ot pure carVjonic acid in the air has little or no effect, an atmosphere in which the carbonic acid has been raised to this proportion J/y respiration, is highly detrimental ; indeed, air rendered so impure by respiration as to contain even 0-08 per cent, of carbonic acid is very unwholesome. In an hour a man will add about 1 per cent, of the gas to about 70 cubic feet of air ; and if the proportion is kept down to 0-1 per cent., at least 700 cubic feet should be supplied to him every hour, or about 16,800 cubic feet in the 24 hours. Chanr/es in atmospheric pressure} — Gradual diminution of pressure produce .symptoms of asphj-xia ; convulsions, however, are not invariable. A sudden and great diminution of pressure may produce death by the liberation of nitrogen within the blood vessels, and a consequent mechanical interference with the circulation. Increase of pressure up to that of several atmospheres produces symptoms of narcotic poisoning; at a pressure of 20 atmospheres the animals die of asphyxia, as when oxjgen is deficient. The oxidations in the body are at this pressure diminished. Plants, bacteria, Jcc, are similarly killed by too great pressure of oxygen ; and at a high pressure of oxygen even phosphorus will not burn. It is, however, only ^ery great extremes of pressure that affect animals injuriously. As is explained more fully in connection with the subject of the blood gases, very considerable variations of jsressure may take place, especiallj- if gradual, and without any resulting inconvenience to the animal. A mere excess of oxygen in the air breathed has no appreciable influence either on the amount of oxj'gen taken up or carbonic acid given out by the animal.- Marcet ^ states that less air (reduced to 0° C. and 760 m.m.) is taken into the lungs for the formation and emission of a given weight of carbonic acid under lower than under higher atmospheric pressures. THE GASES OF THE BLOOD H. Davy^ was the tir.st to observe that oxygen was evolved on heat- ing the blood. Magnus " made more accurate observations. He found that oxygen could Ije obtained either by passing a stream of hydrogen or carbonic acid through the blood, or by placing blood in the vacuum of an air pump, and that the quantity of oxygen obtained from arterial was greater than that from venous blood. Later Bunsen,'' Lothar Meyer,'^ and later still after the invention of the mercurial air pump Hopije-Seyler, Setschenow and Ludwig, Helmholtz, and Pfliiger worked at the subject. ' Paul Bert, Becherches exjj. stir la j,ression baromeiriqiie, 1.S74. ^ Some recent experiments on this subject by Saint-Martin will be found in the Conipt. rend, xcviii. 2il. 5 Phil. Trans, vol. clxxxi. (1890i, p. 1. * Gilbert's Ann. xii. 593. '" Pocjgendorf's Ann. xl. 583 (1838) ; Ixvi. 177. •^ Bunsen, Gasometrische Methoden, Brauuschweig, 1S57. ' L. Meyer, Die Gase des Blutes, Diss. Gottingeu, 1857. Zcit. rat. Ided. vi;i. -256. EHSl'lliATKiN 379 The general })riiiciples underlying the construction and use of the mercurial air pump have been described in an earlier chapter {sre p. 30). It will be here unnecessary to repeat the principles on which the analysis of the gases is performed {see Chap. IV), and we can pass on now to the results that have been obtained. The following table' gives some numbers illustrative of the results obtained by different observers : — Kind of Bloofl Percentages per Volume' Oxygen Carbonic Acid Nitrogen L. Meyei-^ Dog (from carotid) . 12-18 26-34 3-5 Calf (defibrinated) . 11-5 20 4-4 Setschenow^ Dog (fi-om carotid) . 19-21 40-43 16 Schotter^ „ (from arterv) . 14-22 34-43 1-6-25 „ (from vein) . . o-U. 38-47 1-3-1-6 Sczelkow" ,, (from artery) . 15-22 32-37 1-2-21 „ (from vein) . . 1-6-10 41-52 1-2-1-8 Nawrocki" „ (from carotid) . 10-20 27-45 1-2-2-5 H. Hirschinann" (from carotid. 3 ex- periments) 27, 1(J. 13 24. 40, 37 3, 2, 2 (from femoral in 25, 16, 15 1!). 42. 36 5, 2, 2 the same dogs) Jolyet and Regnard^ have made observations on the blood gases of several aquatic animals, Crustacea and fishes {see also p. 396). Pfliiger,'o whose name is associated with the best known of the many mercurial pumps, from a large number of observations on dogs found in the arterial blood in the mean 58-3 volumes of the mixed gases per 100 vols, of blood ; this was composed of 22-2 vols, oxygen (maximum 2d'4) ; 34-3 vols, carbonic acid ; and 1'8 vols, nitrogen. Pfliiger'i made, like Hirschmann, compai'ative observations on the gases of blood obtained from different arteries (carotid and femoral), and his results were practically the same ; viz. the gases are in the two cases approximately equal, or there may be a slight loss of oxygen in the more distant artery. During the first few minutes after blood is shed, a certain quantity of the oxygen disappears ; in all probability this is stored or used up ^ Shortened from a fuller table given by Hoppe-Seyler, Physiol. Chem. p. 496. - In all cases the volume of gas is measured at 0° C. and 760 m.m. Hg pressure. ' Loc. cit. * Wien. akad. Sitzungsh. xxxvii. 293. s Ihid. xli. 589. b jj,^/. ^Iv. 171. ' Heidenhain, Studien d.physiol. Inst, zu Breslau, Heft ii. 1863, p. 16-2. « Archiv f. Anat. it. Physiol. 1866, p. 50-2. ^ Virchow-Sirsch. Jahresb. 1874, vol. i. p. 201. 10 Centralhl. mcd. Wiss. 1867, p. 724. " Pfiiiger's Archiv, i. 285. 380 THE TISSUES ANlJ ORGANS (W THE EODY by the still living corpuscles. Stroganow ' gives the loss of oxygen that occurs in this way as varying from 1'03 to 1"6 per cent. Schiitz- enberger,"^ by using indigo-white to remove the oxygen from haemo- globin, obtained a result higher by 4—") c.c. per 100 c.c. of blood than by the method of extracting the gas with the mercurial air pump. This diti'erence increases with the length of time employed in the extraction of gas by the pump. Lambling ^ considers that this is in part due to the formation of a small amount of methtemoglobin. Methsemoglobin yields its oxygen to reducing agents like indigo- white, but not to the vacuum of an air pump. Hoppe-Seyler "* .states that the reduction by means of indigo-white is ntjt trustworthy, since it is so powerful. Xot only is oxyhfemoglobin reduced to hfemoglobin, but the reaction goes further, so as to form hfemochromogen. Lambling, however, from numerous experiments considers that Hoppe-Seyler is at fault here ; haemoglobin is formed, and the reaction always .stops short there. The question cannot, however, be regarded as settled. The oxyyen in the blood is not in a state of simple solution. According to Bunsen '' the absorption-coefficient of water for oxygen is O-O-ll ; for nitrogen 0-0203 (0° C, 760 mm. Hg). If the absorption coefficient of blood were equal to that of water, the blood would be able to absorli from the air 0'8G vols, per cent, of oxygen, and 1"60S vols, of nitrogen. The absorption coefficient of blood is a little lower than that of water, and this is smaller still at the temperature of the body (37° C.) than at 0° C, the temperature for which the above numbers are calculated. With regard to tlte nitrogen, the quantity found in the blood is explained by simple solution. But the oxygen is far in excess of what can be accounted for in this way ; the oxygen is in fact nearly all present in the form of oxyha?moglobin ; in venous blood a variable quantity of oxyhsemoglobin is found mixed with haemoglobin. Supposing that 100 c.c. of arterial blood contain 11 grammes of oxy- haimoglobin, this would account for 23'J:3 vols, per cent, of oxygen ; this is rather more than Pfliiger actually found, but all the haemoglobin present is rarely fully saturated with oxygen. The blood-plasma and blood-serum contain only traces of oxygen. In dog's serum Pfliiger'' found 0"2G vols, per cent, of oxygen, 35-26 vols, per cent, of carbonic acid, and 2-24: vols, per cent, of nitrogen. Tlie carbonic acid in the blood is in gi-eat part dissolved in the 1 Pfliicjer's Archiv, xii. 48. - Bull. Soc. Chim. 1873, p. 150. •' Comptes rend. Soc. biol. (2) v. 394, 473. * Phtjsiol. Chew. p. 451. '" Bunsen defined the coefficient of absorption of a fluid for a gas as the vohime of the gas (at O'C. and 760 in.m. barometric pressure) which is taken up by one volume of the fluid. 6 Pfliicjer's Archiv, i. 73. KESI'II^VTION 381 plasiuii, but it is also contained in the corpuscles. The question, in what compounds does it occur ? is a difficult one to answer, since we are not able to separate them out, as we are oxy]iM'iii(>L;lo])iii, tlic conipouiid in which the oxygen is contained.' Al. Schmidt,^ one of the earliest to investigate the matter, ari-ived at the following conclusions : (1) That the corpuscles •* of arterial blood contain a variable amount of carbonic acid ; it may amount, however, to as much as the quantity in the serum ; it is relatively less in venous blood. (2) That the carbonic acid in the corpuscles can be lessened bv shaking blood with oxygen ; this lessening is greater than that of the carbonic acid in the serum. Setschenow found that the carbonic acid in the corpuscles alters with the partial pressure of that gas, and indeed considers that the cor- puscles are the chief source of the carljonic acid given off in the lungs. In the serum the carbonic acid can lie obtained partly by simply placing the fluid in a vacuum, but a larger yield is obtained on the addition of acid. The following are the numbers obtained l)y Pfliiger in two experiments : — I 11 Carbonic acid given off in vnnio 44' 6 vols, per cent. 35-2 -sols, per cent Additional carljonic acid given x , off after addition of phosphoric 4*9 ,, „ 9-3 „ „ acid ' I In the blood itself all the carbonic acid is yielded to a vacuum with- out the addition of acid. From these and other similar experiments, it appears (1) That carbonic acid is present both in red corpuscles and serum. (2) That serum contains the gas in a tii-mer union than the corpuscles. (3) That the red corpuscles, especially if they contain oxyha?mo- globin, act in the same way as an acid, or may give rise to an acid, causing a complete expulsion of all the carbonic acid from the serum. Zuntz ^ compared the action of a vacuum on serum with that on solutions of sodium hydrogen carbonate (NaHCOg), and found that they behaved very similarly. Sertoli ' also believes that the carbonic acid is contained in the serum as a bicarl^onate, and not chiefly in 1 Bohr (Liidivicj's Festschrift, 1887) considers that the carbonic acid is loosely combined with the hsemoglobin itself. See also Jolin, Du Bois ReymoncVs Arch. 1889, p. 26.5. '' Ber. d. sfichs. GeseUsch. d. Wiss. 1867, p. 30. ^ It is difficult to distinguish in this relation between red and white corpuscles Setschenow, however, calculates that in 100 vols, of blood the red corpuscles coHtain 10 and the white 2'.5, vols, of carbonic acid. * Centralhl. f. med. Wiss. 1867, no. 532. 5 Hoppe-Seyler's Med Chem. Unter.i Heft 3 p. 360 (1868). 382 THE TISSrES AND ORCtAXS OF THE BODY union with disodium hydrogen phosphate (NajHPO^), as some earlier investigators stated. He shows very conclusively that the amount of phosphoric acid in the blood, if allowance be made for that contained in lecithin, is quite insufficient for the purpose. Bunge,' however, states that in dogs" blood it is sufficient. One of the most remarkable phenomena in the disengagement of carbonic acid from the blood is the power of the red corpuscles to give off not only the gas they themselves contain, but also to drive off the more firmly combined carbonic acid of the serum. This is not merely due to the phosphoric acid contained in the phosphates of the stromata, nor to the proteid of the stromata. Proteid does drive out carbonic acid in a vacuum from a solution of sodium carbonate, acting in this way like an acid, but the amount driven out is very small ; ^ it appears to be chiefly due to the action of oxyhsemoglobin. Preyer and subse- quently Hoppe-Seyler "^ mixed solutions of pure oxyhjemoglobin and sodium carbonate together, and obtained carbonic acid from the mix- ture in a vacuum. As arterial blood yields its carbonic acid more easily to a vacuum than venous blood, it has been surmised that the arterial blood pigment has more of an acid character than venous blood pigment.* The quantity of oxygen removable from the blood is proportional to its richness in oxyhfemoglobin. ^Mathieu and Urbain ' demonstrated this by successive bleedings from the same dog ; as the number of red corpuscles was thus diminished, the amount of oxygen similarly decreased ; little or no change, however, was observable in the amount of carbonic acid. The amount of oxyha-moglobin, and therefore of oxygen, is less in the blood of cold-blooded than in that of warm- blooded animals ; and is among warm-blooded animals less in the blood of herbivora than in that of the dog.*' 1 Zeit. Biol. xii. 206. - Hoppe-Seyler, Physiol. Chem. p. 503. •"' Physiol. Chem. p. .^0.5. ■* Hoppe-Sevler (Zeit. physiol. Chem. xiii. 4771 draws attention to the fact that the ai-terial blood pigment is not the same thing as oxyha;moglobin, and tliat venous blood pigment differs somewhat from haemoglobin. He suggests the names arterin and phlebin respectively for the arterial and venous pigments as contained in the corpuscles. Arterin is probably a compound of oxyhsemoglobin with lecithin ; phlebin of hsemoglobin with lecithin. The chief differences between the corpuscular pigments and the oxy- hsemoglobin or haemoglobin that can be separated from the corpuscles are : (1) the corpuscular pigments are insoluble, hsemoglobin and oxyhaemoglobin are soluble in the plasma and serum : (2) the corpuscular pigments do not crystallise readily, give off oxygen readily to a vacuum, and decompose hydrogen peroxide readi-y ; haemoglobin and gxyhaemoglobin behave in all these jwints in the opposite manner: (3) the arterial cor- puscular pigment is not altered by a weak solut'on of ferricj-anide of ^ potassium,' whereas oxyhffimoglobin is readily converted into methsemoglobin by such treatment. 5 Arch, de physiol. norm, et path. iv. 14. '■' Bunge, Phystof. Chem. tran?!. by Wooldridge, 1k90, p. 2.56. KKSl'lliATlo.N" sm Changes in the Blood Gases during the circulation The gaseous contents of tlie blo"! underi^o two iinjiortant clianges (luring the course of a complete circulation. The artei-ial blood which leaves the left ventricle becomes venous in the tissues, losing a certain amount of its oxygen, and gaining an increased (juantity of carbonic acid. The venous V)lood in the capillary network of the lungs loses its excess of carbonic acid, and the hannoglobin becomes once more fully, or almost fully, oxygenated. The nitrogen in the blood undergoes no change during the circula- tion if the barometric pressure remains constant ; an increase of atmospheric pressure leads to the solution of a greater amount of nitrogen. An increase of pressure produces a slightly greater absorp- tion of oxygen ; that is, the quantity dissolved in the plasma is increased ; the amount combined with luemoglobin undergoes no alteration with variations of pressure unless the atmospheric pressure be so diminished (to xV^ttV ^^ ^^^ atmosphere) that the point of the dissociation of oxylu^moglobin be reached. The quantity combined with hfemoglobin is by far the most important part of the oxygen in the blood, and that this remains constant under varied barometric relations is a point of great practical interest, as it enables animals to exist even at very great altitudes where the atmospheric pressure is low, and still obtain a normal supply of oxygen. The carbonic acid in the blood undergoes practically no alterations with variations of pressure since the atmospheric air contains a mere trace of that gas. The following numbers selected from Paul Bert's ' experiments illustrate the facts just mentioned : — Pressure Dog 2 Dog 5 0, CO, N., 0, CO, N, 1 atmosphere . 18-3 371 2-2 — — •) 19-1 37-7 30 20-2 871 1-8 •5 ,. ... 20-6 40-.5 6-1 23-7 35-5 6-7 10 ., ... 21i 3 '.-S 11-4 24-7 37-9 9-8 The tension of the Blood Gases In order that we may understand the way in which the changes in the blood gases are brought about, it is necessary to describe the known facts respecting their tension. 1 P. Bert, La pression harometrique, dc. Paris, G. Masson, 1878. Ccmj)t. rend. vols. Ixxiv. and Ixxv. 384 THE TISSUE^ AND ORGANS OF THE BODY The volume of gas aljsorbed by a liquid is indei)endent of the pi'essui'e ; but according to what is known as Boyle's law, the density of a gas, i.e. the number of molecules in a given space, is in proportion to the pressure. Hence, although the volume remains constant, the weight (volume multiplied by the density) of the absorljed gas rises and falls in proportion to its pressure ; this is known as the law of Dalton and Henry. "When two or more gases form an atmosphere above a fluid, the absorption takes place in propoi^tion to the pressure which each of the constituents of the mixture would exercise, if it were alone in the space occupied by the mixture ; this pressure was termed by Bunsen the partial pressure of the gas. Suppose atmospheric air to be under a pressure of 760 m.m. of mercury : the air contains 21 vols, per cent, of oxygen, and "9 vols, per cent, of nitrogen. The partial pressure of the oxvcen='— ~ =159"6 m.m. of mercury; and of the nitrogen 760 X 79 = ' =600--l: m.m. of mercury. The carV)onic acid of the atmo- 100 ^ sphere is present in such traces, that its partial pressure is practically zero. Hence when a liquid like soda water, which is charged with carbonic acid, is exposed to the atmosphere, bubbles of the gas escape fi'om the liquid, until the difference of tension or pressure between the carbonic acid in the water and in the air above it, is balanced ; the gas which comes off from the liquid exercises, as it does so, a certain amount of pressure ; and by the phrase ' tension of a gas in a fluid ' is meant tJie partial pressure in millimetres of mercury, which the gas in question has to exercise \\\ the atmosphere, when no difl'usion between the gas in the fluid and the gas in the atmosphere takes place. If the partial pressure of the gas in the atmosphere increases, a greater weight of the gas is absorbed by the fluid ; if it diminishes, some of the absorbed gas is given oft' from the fluid. We have, however, seen that the formation and dissociation of such compounds as oxyhittmoglobin in the blood is an additional factor to be taken into account, and in point of fact it is found that more oxygen is actually absorbed in the lungs from the alveolar air than can be explained by Dalton's law of pressures. Similarly with regard to the carbonic acid, the compounds which it forms are those which undergo dissociation, and so we have not merely a physical process of gas diffu- sion from blood to air to deal with. It will be here a convenient place to explain what is known as dissociation. Certain compounds of gases are formed only when the partial pressure of the gas is high. When the partial pi'essure of the EESPIRATION 385 gas is (liininished, the coiistituonts of tlie compound are separated. To express it in more familiar language : when the gas is pressed with force against certain sul)staiicos, it refuses altogether to combine with some of them ; with others it forms so fir'rii a combination that it is exceedingly difficult to separate the gas again from them ; and with a third class of substances, those that we are now considering, it forms combinations under protest, as it were, separating from them directly the pressure upon it is reduced sufficiently low. The gas is always tending to separate from the substance with which it is thus loosely combined, and the force which it exercises in this effort is called the tension of dissociation ; when this is greater than the external pressure of the gas, dissociation takes place. Applying this now to the blood, we see that the hfemoglobin of the blood in the pulmonary capillaries finds oxygen in the alveoli, which has a comparatively high partial pressure ; it therefore unites with the oxygen. In the capillaries of the systemic circulation, the oxyhemo- globin comes into relation with tissues poor in oxygen, that is, where the partial pressure of the gas is low ; the oxyluemoglobin is dissociated, the oxygen is supplied to the tissues, and the venous hfemoglobin returns to the lungs for a fresh supply of oxygen. In tlie case of carbonic acid, compounds are formed in the blood of the tissues wh3re the tension of that gas is high ; and in the lungs these undergo dissociation, the gas passing into the alveolar air where the partial pressure or tension of carbonic acid is comparatively low. The diffei-ent conditions under which dissociatioti of oxyhtemoglobin takes place have been recently investigated by Hlifner' and by Brasse.^ Bert showed that dissociation occurs more easily at 40° C. than at temperatures below that point ; Frankel and Geppert obtained similar results, and Hiifner has shown that, besides pressure and temperature, another very important factor is the concentration of the solution of oxyhai'moglobin used ; so that in the body the amount of oxy haemo- globin in the red corpuscles must be taken into account. At high temperatures, as in the blood during fever, the conditions under which oxyhtemoglobin is dissociated are therefore difierent from those which obtain in health. Hiifner calculates that at a height above the sea-level of over 5,500 metres the atmospheric pressure and the partial pressure of oxygen are so low that oxyluemoglobin would be dissociated, and thus such an elevation would be exceedingly perilous to animal life. This coincides very well with the results obtained by actual experience. He suggests, however, that breathing would be still possible at such altitudes, by » Zeit.ijhysiol. Chem. xii. 568; xiii. 285, ^ Qompt. rend. Soc. bioL v. 660. 886 THE TISSUES A^D ORGANS OF THE BODY increasing tlie richness of the blood in haemoglobin ; this may be done by transfusing more blood into the vessels. The researches of L. Brasse relate chiefly to the influence of tem- perature on the dissociation of oxyha^moglobin. He finds that the tension of dissociation for oxyhsemoglobin at 0° C. is nil. The com- pound is thus a stable one at that temperature ; in hibernating animals, the temperature of the body is very low, and the blood is red in the veins as well as in the arteries. The tension of dissociation increases with the temperature, and a mammal dies when the tempera- ture of its blood reaches 45° C. At this temperature, although the tension of dissociation is still lower than the partial pressure of the atmospheric oxygen, it is higher than that of the oxygen in the pulmo- narv alveoli. In birds, on the other hand, where by the arrangement of air sacs the aeration of the blood is very complete, they do not die until their blood reaches the temperature of 50° C. The tension of the gases is thus the sum of the tension of disso- ciation of the oxyhsemoglobin, bicarbonates, and similar compounds, ■with the physical tension of the small amount of the gases dissolved in the blood plasma. The tension (composed of these two factors) of the gases in the blood is not nearly so great as it would be if all the gas in the blood were in a free or uncombined condition. The measure- ment of the tension of the gases in the blood was carried out by Pfliiger,' and his pupils Wolff berg,^ Strassburg,^ and Nussbaum ^ ; the instrument they used is called an aerotonometer.-^ The average results obtained may be thus summarised (Strass- burg) :— Tension of oxygen in arterial blood=29-64 mm. of mercury=3-9 per cent, of an atmosphere. Tension of oxygen in venous blood = 22-04 mm. of mercury=2-9 per cent, of an atmosphere. 1 Pfiilger's Archiv, vi. 43. ^ Ibid. iv. 465 ; vi. 23. 5 Ibid. vi. 65. ■• Ibid. vii. 296. 5 The use of this instrument may be best explained by an example. Suppose that one wished to ascertain the tension of carbonic acid in the blood ; the blood direct from the livinc vessels is introduced into the upper end of a vertical glass tube (kept at a constant temperature by a jacket of water) containing nitrogen and a small known per- centage of carbonic acid. The blood runs down the tube and is at once removed at the lower end, means being provided to prevent air getting to it. If the tension of carbonic acid in the blood is greater than in the mixture in the tube, then the amount of carbonic acid in the tube will be increased after the blood has passed through it ; if the tension in the blood is less, then the amount of carbonic acid in the tube will be found to be diminished. By successive experiments it is found that for a certain percentage the amount of carbonic acid undergoes no change ; this percentage therefore exerts the same tension as the carbonic acid in the blood. Strassbui-g (loc. cit.) gives a figure of the aerotonometer. IM'.SI'IIJATIOX 387 Tension of carltonic Jicid in arleri;il lilood^l'l-l'.^ nun. of mercury = 2"8 per cent, of iin atmosphere. Tension of carbonic acid in venous blood = 41 -01 mm. of mercury =:.'i-4 p^M- cent, of an atmosphere.' The ditferences in the tension of the i,'ases is thus much less than the ditierences in their volume, in tlie two varieties of blood. Let us now compai-e the tension of the blood gases with the partial pressure of the gases in the pulmonary alveoli. Wolffberg obtained the residual or alveolar air from the lungs of dogs by catheterisation, and the following are his mean results : — Tension of oxygen in alveolar air = 27-44 mm. of mercury=3-6 per cent, of an atmosphere. Tension of carbonic acid in alveolar air^27-06 nun. of mercury — 3"56 per cent, of an atmosphere.'-* Now if the line AB in the accompanying diagram represents the alveolar membrane, there is the alveolar air on one side of it, and venous blood on the other ; the tension of tlie two gases in each is represented in the diagram, and the dh-ection in which diffusion takes place is shown by the arrows ; the oxygen passing from alveolar air into the blood, the carbonic acid in the ^-averse direction. Tension A Teii^inn Alveolar air f,A , ~,-_Qr.^ V enous blood 4104; CO., B It has long been felt that the comparatively small differences of partial pres- sure (particularly of oxygen) do not completely explain the very great differences in the volume of the gases in arterial and venous blood, and any account of the gases of the blood would be incomplete without a reference to the ingenious theory recently advanced by Ernst Fleischl v. :Marxow.' The author, after stating the usual theory of respiration and its difficulties, asks how it is that, if the tissues have a greater affinity for oxygen than hicmoglobin, the blood of animals killed by asphyxia still contains a considerable amount of oxyhsemoglobin ; and v. Marxow believes that in the sharp, sudden stroke of the heart's beat he has discovered a physical agency which assists in the work of dissociation ; according to him tbe blood is kept in motion by a series of quick sudden stokes, because for the taking up of the oxygen by the tissues, and the elimination of carbonic acid by the lungs, it is not sufficient that the blood runs steadily through the systemic and pulmonary oapillaiies respectively ; and therefore a short, hard 1 This rose on the coagulation of the blood to 61-79 mm. Hg, = 8-13 per cent, of an atmosphere. - Nussbaum obtained rather a higher number, 29-18 mm. Hg. 3 Die Bedeutitng cles Herzschlages fiir cl. Athmunc/ ; eine neue Theorie der Bespiration, Vienna. I am indebted to Prof. McKendrick's address to the Brit. Med. Assoc. 1888 {Brit. Med. Journ. August, 1688) for the above abstract of v. Marxow's. theory. c c 2 388 THE TISSUES AND ORGANS OF THE BODY stroke is given to it immediately before it enters, and immediately after it has left the lungs ; the systole of the left ventricle assisting in the liberation of the oxygen; of the right ventricle in the liberation of the carbonic acid. That a blow has very considerable power in assisting the liberation of gases can be readily demonstrated with an ordinary liypodermic syringe ; if the piston be pulled up, and water allowed to rush into the vacuum so formed, bubbles of gas will come olf from the water ; but if the handle of the piston first receives a sharp blow from a mallet, the gas bubbles will come off so rapidly that the water froths. Although phj'siologists cannot but treat with the greatest respect the conclu- sions arrived at by so eminent a physicist as FJeischl von Marxow, it must be admitted that there are many difficulties in the way of fully accepting his theory in its entirety. These difficulties are chiefly the two following : — (1) In small mammals the stroke of the heart cannot be nearly so powerful as in large mammals ; but still the same respiratory exchanges go on. (2) In cold-blooded animals there is only one ventricle, and the blood receives only a single blow ; but, nevertheless, on its way from the heart back to the heart again it undergoes two gaseous exchanges, first in the lungs or gills, secondly in the tissues. In spite of these obvious objections, which show that v. Marxow is inclined to exaggerate the importance of the heart beat, it is, however, quite possible that in the warm-blooded animals, where the gaseous exchanges are more extensive than in the cold-blooded animals, the force of the blow given to the blood by the heart may exercise some auxiliary impulse in the liberation of the blood gases. Another attempt to elucidate the perplexing questions involved in the respiratory exchange of gases has been recently made by Christian Bohr. We have already seen that some of the carbonic acid is contained in the red corpuscles, and Bohr considers that it is in actual combination with the hjemoglobin ; he considers that this union is like oxyha^moglobin— a dissociable one — and that dis- sociation takes place in the pulmonary alveoli. If this is really the case, haemo- globin appears to be not only an oxygen carrier but also a carbonic acid carrier. We have, of course, in addition to this, the carbonic acid dissolved in the plasma, in the form of carbonates and bicarbonates. Bohr's theory of the combination which occurs in the red corpuscles appears to me so important and full of interest that I propose here to give a brief resum6 of his paper ' :^ 1 Ludwig's Festschrift, 1887, p. 164. RESPIRATION 389 Setschenow,' and later Zunt'-c,- stated that a solution of hiwmoglobin at the atmospheric; pressure absorbs more carbonic acid than the same volume of water. Bohr's research was devoted to studyinj^ this subject more fully, and to ascertain- ing the relation between the tension of the carbonic acid and tiie amount absorbed per gramme of hemoglobin. A special absori)tiometric method employed was described by him in an earlier paper.* Pure solutions of crystalline h:emo- globin from the dog, and pure carbonic acid, were employed ; these were brought SiO CO wo 740 VcJ(? in contact with one another, the gas being at a known pressure, and the tempei-a- twre kept constant throughout. The amount of gas absorbed was afterwards pumped off and estimated. Some preliminary experiments were made with water ; the result of one of these, in which 41 grammes of water were used, may be repre- sented graphically as in tig. 66. The line which indicates the increase of absorp- tion is constructed from ordinates representing the amount of absorbed gas in grammes, the abscissae the pressure of the gas in mm. of mercury. As is seen, the result is a straight line, the weight of gas absorbed being proportional to its tension (Dalton-Henry law). Experiments were then made with haemoglobin solutions ; the following table represents a portion of one of these : — Pres.«ure of CO, in mm. of Mercury Total quantity of CO. absorbed Physical Absorption ; i.e. the C0„ which would be absorbed by an equal quantity of water Amount of CO, ab- sorbed per 1 gramme of Hjemoglobin Temperature 6-04 11-57 14-62 18-54 24-07 31-98 2-0975 2-8847 3-2295 3-6656 4-1966 4-8548 0-275 0-527 0-666 0844 1-095 1-455 1-269 2-358 2-564 2-822 3-102 3-400 18-2° C 18-4° 18-4° 18-4° 18-4° 18-4° The quantity of carbonic acid absorbed by haemoglobin is thus immensely greater than that explicable on simply physical grounds. The curves in fig. 67 1 Mem. de Vacad. de St. Petersbourg, vol. xxvi. 1879. - Hermann's Handbiich, vol. iy. 2. p. 76. 5 Bohr, Exper. Untersuch. u. d. Sauerstoffsaicfnahme des Blutfarhstoffs, Kopen- hagen, 1885. 390 THE TISSUES AND ORGANS OF THE EODY represent graphically two experiments; the abscissiB represent as before the pressure, the ordinates the quantity of gas absorbed per gramme of hsemoglobin. The curve is very different from the straight line of tig. fifi, and the ascent of the curve is espscially steep at the lower pressures. The upper curve is the represen- tation of an experiment performed with a less concentrated solution of haimo- globin than in the experiment represented by the lower curve. It thus appears that the amount of gas absorbed is less in the more concentrated solution. Con- trasting the curves with those obtained in experiments with other gases (oxygen, carbonic oxide, nitric oxide") which are known to form compounds with haemo- globin, they are found to be different. Hence, if we have to do with a chemical union of carbonic acid and hiemoglobin, the gas is combined differently from that in oxyh „ ... 1-01 25-71 55-50 81-21 2-47 >> Ewald also by the use of Pfliiger's aerotonometer estimated the tension of carbonic acid in four of these fluids ; his results were respectively 7-51, 10-92, 10-73, and 11-5 per cent, of an atmosphere. Thus not onh' the quantity but also the tension of this gas is greater in these transudations than in venous blood. The gases of pus have also been analysed by Ewald ; the following table gives a summary of his results : — Nature of Pus Carbonic Acid Oxygen Nitrogen Removable Removable Total tn vacuo by Acid Empyema, 28 davs standing 7017 1-68 71-85 1-14 10 15-73 2-77 18-50 traces >> tt >j 14-76 ? 1476 ,^ .. 21-46 0-0 21-46 2-9 0-77 Abscess, 21 ,, „ 8 05 0-0 8-05 1-35 0-43 " 7-92 0-0 7-92 — The amount of carbonic acid present increases with the age of the exudation ; the more nearly the purulent exudation approaches pure pus in its characters the smaller is the total carbonic acid, and especially the more firmly combined carbonic acid : indeed, pure pus contains only loosely-combined carbonic acid. It is very probable that the pus corpuscles, in common with the blood corpuscles, possess the power of decomposing sodium carbouate (NaoCOg) and driving off carbonic acid from it. ' Zeit. d. Gesell. cl. Aerzte in Wien, 1859. - Pflnger's Arcliiv, vi. 94. 3 Archiv fur Anat. u. Physiol. 1873, p. 668 ; 1876, Heft iii. 394 THE TISSUES AND ORGANS OF THE BODY CUTAXEOUS RESPIRATION The greater pait of the respirator}- exchange of gases occurs through the thin membrane of the pulmonary alveoli. A certain amount of gaseous exchange occurs also through the thicker mucous membranes of the respiratory tract, and also through the skin. The amount of cutaneous respiration varies in different animals, but is greatest in those in which the epidermis is thinnest, and thus presents the least resistance to the diffusion of gases. In frogs, for instance, where not only is the skin thin, but it has a rich blood supply, Regnanlt and Reiset found that nearly as much oxygen was used and carbonic acid given off after extirpation of the lungs as before that operation. The following are the results of Fabini's ' analyses : — Healthy frogs in the light . . . 632 milligr. of COo per 100 grms. of body weight in the 2i hours Frogs without lungs in the light . .569 „ „ „ Frogs without lungs in the dark . 424 ,, „ " „ The amount of cutaneous respiration in man has been discovered by enclosing a portion of the body, such as a limb, in an air-tight bag, and after a time analysing the gases (oxygen and carbonic acid) contained therein. From this the amount occurring over the whole cutaneous surface can be calculated. In some experiments the whole body was enclosed in an air-tight chamber. The following numbers give the quantity in grammes of carbonic acid which passes out through the whole skin of a man in the 21 hours, according to different observers : Abernethy,- 1-t ; Scharling,^ 32 ; Gerlach,^ 8-5 : Reinhard,* 22 ; Aubert and Lange," 3-8 ; Rohrig,' 14 ; Fabini and Ronchi,^ 68. The last-named observers also found that the quantity of carbonic acid increased with a rise of atmospheric temperature. The amount of carbonic acid excreted by the skin of warm-blooded animals is so small that death which follows varnishing the skin cannot be accounted for by the stoppage of this function. FCETAL RESPIRATION The foetus receives its supply of oxygen from the maternal blood ; the gaseous exchanges occur through the thin walls of the vessels of the placenta ; otlier nutritive materials pass in a similar way from mother to the embryo. The difference between the arterial and venous blood of the foetus is not nearly so marked as in extra-uterine life." The need of the foetus for oxygen is much less than it is after barth, and the amount it receives from the maternal blood is not only ample for its wants, but is sufficient to maintain a condition of apncea. The respiratoi-y changes occurring in hens' eggs during incubation have been ' J. iloleschott, Unters. z. XahrJehre d. Menschen, xii. 100 (1878). 2 Surgical and physiol. Essays, London, 1793-7. 5 J. prakt. Chem. xxxvi. 155. '' Miiller's Arch. 1851, p. 433. * Zeit. Biol. v. 33. ^ Pjiiiger's Arch. vi. 539. - " Deutsche Elinik, 1872, p. 209. * J. Moleschott, Unters. z. Nahrlehre d. Menschen, xii. 100 (1878). ^ The presence and amount of oxyhsemoglobin and haemoglobin in foetal blood has been specially studied by Zweifel (Arch./. Gyndkol. xi. Heft ii. p. 1) and Znntz (Pfliiger's Archie, xiv. 005). EESPIRATION 39J studied by Banmfjiirtner.' The apparatus used was constructed on the principle of that of Kcgnault and Keiset. While the egg is in the condition of rest, no metabolic changes occur ; but wlien incubated, oxygen is absorbed, and carbonic acid given off. In twenty days" incubation ITou'H c.c. of oxygen were absorbed, and 16262 c.c. of carbunic acid given otf ; tlie respiratory quotient was therefore 0-927. RESPIRATION IN FISHES We have hitherto considered respiration in air-breathing animals only ; it is now necessarj' to briefly describe the process as it occurs in fishes and other aquatic animals. Munk- made a number of comparative experiments in which he contrasted what may be called the intensity of respiration ; that is, the amount of oxygen in grammes used per kilogramme of body weight per hour. His results are given in the following table : — Respiratory Quotient Animal Intensity of Respiration V, Cat 1-007 0-77 Dog . 1 183 0-75 Rabbit 0-9 18 0-92 Hen .... 1-800 0-H3 iSmall singing birds . 11-860 0-78 Frog .... 0-084 0-68 Cockchafer^ 1-019 0-81 Man .... 0-417 0-7S Horse 0-563 0-97 Ox ... . 0-552 0-98 Sheep 0-490 0-98 The intensity of respiration is exceptionally high in small birds ; in the frog, which may be taken as an example of a cold-blooded anifnal, it is verj^ low ; the same is true for fishes. This is in consonance with the fact that sea water con- tains onl}^ small quantities of oxygen. The sea water . brought home by the Challenger expedition was analysed by Prof. Dittmar * ; he says : The ocean can nowhere contain more than 15-6 c.c. of nitrogen and 8-18 c.c. of oxygen per litre. The nitrogen never falls below 8-55 c.c. ; but the theoretical minimum of oxygen (4-3 c.c.) is liable to diminution b^' the processes of life and puttefaction ; and as a matter of fact water from a depth of 1 500 fathoms gave 2"o4 c.c, and from a depth of 2875 fathoms 0-6 c.c. per litre, and yet many forrps of life exist at this great depth. Fishes have been dredged from a depth of 2750. fathoms, where the amount of oxygen was probably not so much as 0-06 c.c. per 100 c.c, or 300 times less than that in the arterial blood of a mammal. The amount of oxygen in the blood of a tish is less than in that of a mammal, but it still contains much more oxygen than exists in the same volume of ^ea water. The water is, however, 1 Der Athmungsprocess im Ei, Freiburg, 1861 ; see also Pott, Maly's Jahresb. 1877, p. 328. P finger's Archiv, xxxi. 268. - I. Munk, Physiol, des Menschen mid der Siiugethiere, 1888, p. 82. ^ Insects tliHs take as much oxygen in proportion to their weight as mammals ; this was previously known from the researches of Reguault and Reiset. * Proc. Phil. Soc. Glasgow, xvi. 01. 396 THE TISSUES AND ORGANS OF THE BODY constantly renewed, and the mechanism by which thin sheets of water are pro- pelled over the gills was first fully descriV>ed by Flourens.' Aquatic breathers are not, however, troubled with free carbonic acid. This was shown by the Vhallriujer chemists to be the case, because any carVjonic acid formed is at once absorbed by the excess of alkaline bases present in the water.- There is thus no tension of carbonic acid in the water to prevent or hinder its escape. Oxj'gen also is probably stored in the swimming bladder ; and this presumablj' oxygenates the blood when the fish dives into the almost airless depths of the ocean. Thus Biot' found 70 vols, per cent, of oxygen in the swimming bladders of such fishes, a gas in which a glowing splinter of wood is rekindled. Other observers have, however, shown that in fishes living near the surface of water the quantit}' of oxygen is much less in their swimming bladder. The following analyses have been made : — Oxygen C0„ Isitro^en Carp. . . 10-7 — 7-1 per cent. 5-2 87*7 Cyprinus . 13-2 -M „ 1-4-3-9 80-8-97-5 Margareta Traube ilengarini ^ has shown that the water, that gas soon appears in the swimming whose swimming bladder is a closed one. Humboldt and Provenyal were the first who made quantitative estimations of the respiratory exchanges in fishes ; more complete observations were made by Baumert," and a very exhaustive study of the process in both fresh-water and sea- water fishes and other aquatic animals has been made by Jolyet and Eegnard.'* The following examples may be taken from their tables : — (Humboldt and Provenyal)* (Fr. Schultze)^ if hydrogen is dissolved in bladder even of those fishes Fresh-Water Auimals Temperature of Water Amount of Oxygen taken in per kilog. of body weiglit in one hour Respiratory Quutieut CO3 0^ lu c.c. lu grammes'" Cyprinus tinea .... 14° C. 577 0082 0-66 Murajna anguilla . . . 14^-15° 40O-48-0 0-058-0068 0-6-0-79 Cobitis fossilis .... 17°-22° 8t;-H 0123 0-78 Axolotl 11-5° 45-2 0064 0-56 Astacus fluviatilis . . . 12-5° 38-0 0 054 0-86 Hirudo officinalis . . . 13-5° 14°-15° 230 134-171 0032 0-191-0-244 0-69 0-81-0-86 Sea- Water Animals MuUus Murajna conger .... 1,^°-16° 59-8-75D 0085-0109 0-67-0-72 Pleuronectes solea . . . 14=^ 73'5 01 05 0-81 Cancer 16° 1070 0152 0-85 Homarus 15° 68-0 0-097 0-8 Octopus vulgaris . . . 15°-16° 44- 00628 0-65-0-86 Mytilus edulis" .... 14° 122 0-017 0-76 Asteracanthion rubens . 19° 320 0-045 0-79 1 Memoires d'anatomie et de physiologie cnmparies, Paris, 1844, p. 75. - This is the principle of Fleuss' diving apparatus. 3 Ann. d. Chem. u. Physiol. 1808, iv. 582. * Memoires de phys. et de cliim. de la soc. d'Arcueil. ii. 359. 5 Pjiiiger's Arcliiv, v. 48. ^ Dn Bois Beymnnd's Arch. 1889, p. 54. ^ Chem. JJnters. Hher die Resj). des SchJammpeizgers, Heidelberg, 1852. 8 Maly's Jahresb. 1877, p. 332. ** Weight of shell included in body weight. '^o This table gives what was called the intensity of respiration in the table p. 395. KESI'IKATION 307 It is very necessary to note the temperature of tlie air or water when making observations on cohl-ljlooded animals, since the 'emjieratiire of the animal's body, and therefore its ciiemical activity, rises and falls with that of the surrounding medium. The following example from Julyet and Kcgiiard may be given in illus- tration : — Animal Temijerature of Water Oxygen in c.c. absorbed per hour per kilo of body weight Respiratory Quotient Cyprinus auratus . . . The same animal . , . 2° 10° 30° 14-8 37-8 147-8 0-89 0-96 0-75 The death of fishes when placed in the air appears to be due, not to the drying of the gills, but to the large excess and high partial pressure of oxygen. 398 THE TISSUES AND ORGANS OF THE BODY CHAPTER XX 3IUSCLE INTRODUCTORY '. ' Muscle is a tissue which may be very conveniently considered next to blood, as in many points a resemblance between it and blood is to be noted. Microscopically examined, muscular tissue is found to l)e made up of bundles of fibres, and these fibres which are really elongated cells differ in structure in the voluntary and involuntary muscles. The voluntary muscles, i.e. the skeletal muscles, are composed of transversely striated fibres. The involuntary muscles, with the ex- ception of cardiac muscle, are made up of spindle-shaped fibres, which exhibit no transverse markings, and are often called j:)lain or unstriated muscular fibres. The cardiac muscular fibres are striated, but exhibit certain marked histological differences from the voluntary fibres. Not only do muscular fibres differ histologically, there are also physiological differences, which may be roughly summed up as follows : (1) the latent period of involuntary muscle is much longer, and the contraction slower, than in the case of the voluntary muscles ; (2) the contraction of involuntary muscles is either rhythmical, as in the heart, or has a tendency to become so, as in the uterus, alimentary canal. The contraction, moreover, passes as a wave from one muscular filjre to another, and thus the movement known as peristalsis is produced. What, however, concerns us more especially is not the histology of muscular fibres, nor the physical conditions of their contraction, but the differences in their chemical composition and in their chemical changes. Here, however, we are not able to speak so positively ; nearly all our knowledge of the chemistry of muscle is derived from the study of voluntary muscle ; a much more limited number of observations have been made on the involuntary muscles. Such observations as have been made on the involuntary muscles show that speaking generally they have the same composition as the voluntary muscles, and the changes they undergo during contraction are very similar to those occurring on the contraction of voluntary muscular fibres; the chemical changes, how- ever, like the physical changes during contraction, are not so vigorous. MrscLE 399 Tu our considoration of uiuscular tissue we shall have to study, first, what can be learnt of its chemical structure by means of suuh instruments as the microscope and polariscope ; secondly, the general composition and enumeration of the constituents of tlie tissue ; next it will be necessary to take up certain of these constituents and discuss them at greater length, especially the proteids, the pigments, the ex- tractives, the salts, and the gases of muscle. Lastly, it will be necessary to consider the changes which muscle undergoes when it contracts, and when it dies [rigor mortis), and the influence of muscular activity upon the rest of the body, particularly on the blood which is circulating through the muscles, and on the composition of the expired air and the urine. MICROSCOPIC STUDY OF MUSCULAR FIBRES Voluntary muscular fibres. — These are bound into bundles (fasciculi) by means of areolar tissue. The iibres themselves are cylindrical, or approximately so ; they vary in diameter from yi^ to ^-^ of an inch in the muscles of the trunk and linil)s, and from .^^ to o^-f^Q of an inch in the muscles of the head and face (Kolliker) ; as a rule they are not branched ; in length they do not generally exceed an inch and a half, but in some long muscles, like the sai'torius, they are considerably longer. Each fibre consists of an external sheath that is called the sarcolemma or myoJpmma ; this encloses the contractile substance. The sarcolemma is transparent, elastic, and apparently homogeneous ; it resists the action of acetic acid and of boiling water ; it is, however, by prolonged treatment with these reagents ultimately dissolved. Hence, although it resembles elastin in insolubility, it is not so in- soluble as the elastin of elastic tissue. Beneath the sarcolemma are found a number of oval, flattened nuclei, surrounded by a small amount of granular protoplasm ; these nuclei, derived from the multiplication of the nucleus of the original cell from which the fibre was developed, are rendered evident by treatment with acetic acid ; they consist, as nuclei do generally, of a network of fibres, but the transverse meshes are especially well marked. Very little nuclein is, however, obtainable from muscular tissue [see also p. 204). It is the contrac- tile substance proper within the sarcolemma that has the striated appearance typical of this variety of muscular tissue. At first sight there are alternate layers or discs of light and dark substance ; on closer examination an intermediate dotted stripe is seen in the centre of the light stripe, which has been called a membrane (Krause's membrane), or Dobie's line ; by varying the focus the line appears double. Insects' muscle is very highly developed, and has been largely 400 THE TISSUES AND ORGANS OF THE EODY used for microscopic study ; in this muscle the dai'k stripe appears to consist of a numljer of rods of which the long axis is the same as tliat of the muscular fibres, and the dots of the intermediate stripe appear to be knobs at the extremities of the rods (Schafer). In contraction, the light and dark stripes apparently change places. There is no doubt that the optical conditions vary much with the focus of the microscope, and thus the subjective effects produced by the examination of the tissue are largely accounted for ; a number of theories as to the plan of construction of a muscular fibre have therefore arisen. For a full de- scription and discussion of these various theories the reader is referred to works on histology. What, however, seems to be certain is this : — 1. The contractile portion of the muscular fibre is a semi-fluid material like protoplasm. Kiihne and Eberth observed a minute nematoid worm (JNIyoryctes Weissmanni) moving in the interior of living muscular fibres in the frog, and noticed that the transverse stria" and other markings displaced l>y its movement closed in again behind it, reassuming their foi-mer order and position. 2. The various optical appearances are produced by the existence of two distinct substances in the contractile portion of the muscle. Neither of these has more than a semi-solid consistency ; still, one of them appears to be more solid than the other. The more solid sub- stance is that which forms the structures variously described as rods, knobs, membranes, discs, fibrils, etc., and the less solid material is that in which the more solid structures are suspended. 3. Examined in the dark field of the polarising microscope (see p. 38) the more solid sulistauce remains dark, and is thus isotropous or singly i-efracting ; the less solid substance is bright, that is to say, it is anisotropous or doubly refracting. 4. By certain artificial means, e.g. by weak hydrochloric acid, a muscular fibre can be split into discs ; this separation occurs at the intermediate dotted line. 5. By certain other reagents, e.g. alcohol, a muscular fibre can be split longitudinally into fibrils, indications of which can be seen as faint longitudinal markings in the healthy fil)re. 6. Supposing these two opei-ations to take place simultaneously, a muscle can be finally subdivided into a number of approximately cubical blocks (Bowman's sarcous elements). The most recent investigations on the subject of the structure of the contractile substance of muscular tissue are those in which gold chloride has been chiefly used as a staining reagent (Retzius," Melland,^ Marshall,^ 1 Sitzungsh. d. Wiener Akad. 1881. - Melland, Quart. J. of Microscop. Science, vol. xxiv. p. 371 (1885). 3 Marshall, Ibid. vol. xxvi. (.1887), p. 75 ; ibid. April 1890. MUSCLE 401 van Gehuchten '). These teach that the isotropous material is a network of fine tibrilliv pervading the whole of the contractile substance ; the interstices between the fibrillai are filled up by the less solid, doubly- refracting substance. A typical animal cell is a mass of protoplasm containing a nucleus. It may or may not have a cell wall ; it generally has not. The nucleus consists of a network of nucleoplasmic fibres, and a nuclear matrix, a homogeneous substance that pervades the whole nucleus ; the protoplasm of the cell also contains a network of fine fibrillje, and the unfibrillated stroma in which this fibrillar network is situated is called the enchylema (Carnoy). A muscular fibre is an animal cell ; each one is developed from a typical animal cell ; the fully-formed muscular fibre is, however, an animal cell which has become specialised in certain points both of structure and action ; it possesses, like protoplasm, contractility, but its contractility does not come into play so as to produce movements in all Fifi. 68.— Part of a muscular fibre of Water-beetle. The fibre has been prepare 1 with gold chloride, ami is splitting into discs which show net- works of fine lines. (B. Melland._) Fig. 69.— Living mu;cle of Water-beetle highly mag- nified (E. A. Schiifer). s, sarcolemma : «, dim stripe ; 6, bright stripe ; c, row of dots in bright stripe which seem to be the enlarged ends of rofl- shaped particles d. The transverse filaments connecting these dots are not shown. directions, as in the amoeba or white blood corpuscle, but is limited so as to produce shortening in one direction only ; then in structure it is a cell which has become elongated, and of which the nuclei have in- creased in number and become peripheral in po.sition ; it is a cell with a well-marked cell wall, the sarcolemma ; and, lastly, it is a cell in which the fibrillar network is no longer irregular, but is arranged with 1 Van Gehuchten, Anat. Anzeiger, vol. ii. p. 792 (1887) ; also in La cellule (Louvain), Tol. ii. p. 29.3 (18861 ; vol. v. (1888), p. 247. In the last-mentioned paper, and also in Quain's Anatomy, a bibliography of this subject will be found. D D 402 THE TISSUES AND ORGANS OF THE BODY longitudinal and transverse strands quite regularly, as denoted in fig. 68 ; the intertibrillary substance is the doubly refracting substance. The longitudinal strands extend throughout the length of the fibre, and the cross strands connect these in the centre of what appears in a resting muscle to be the light stripe (Dollies' line). Under polarised light with crossed nicols the fibrillar network is dark, i.e. because it is singly refracting, the enchylema is bright because it is doubly ref I'acting. The question arises, how, then, is the ordinary appeai-ance of the alternate striping of a muscular fibre produced ? No doubt this is an optical efiect ; an oil globule examined in water appears surrounded with a halo of light ; a row of such globules would have a bright line on each side of it ; so the cross strands of the network which are not of equal thickness, but have minute thickenings at the points where the fibres join together, produce a similar effect, and thus the enchylema on each side of the transverse strands appears bright in comparison with the rest. On contraction the longitudinal strands become shorter, and the cross strands thicker, and the granules in the cross strands larger ; hence the cross strands now appear dark, while the rest of the enchylema appears bright in comparison. This is the explanation of the apparent interchange in position of the light and dark strict on muscular contraction. This view of muscular structure and contraction is much simpler than the complicated theories formerly advanced : it brings muscular fibres into the general category of cells, and shows that the optical appearances that vary with the focus of the microscope and the state of contraction of the muscle may all be explained easily on the supposition that the fibrillar network has different optical properties from those of the inter- fibrillar stroma or enchylema which it pervades. Though by means of the microscope and polariscope it is thus possible to distinguish the existence of two substances, it is not possible to say whether the changes that occur on contraction are active in both substances, or whether the movements of one, e.g. the isotropous material, are active, and those of the anisotropous material are merely passive, or vice versa.^ It is also not possible to say whether there is a transference of any material, e.g. water from one to the other during contraction. One must, however, be very careful to recognise that both substances are merely semi-fluid ; there is no justification for supposing that anything in the nature of a solid, firm network pervades the interior of the fibre ; the nematoid worm seen by Kiihne in the interior of a fibre liad no difficulty in progressing in any direction. 1 Eollett {ArcJi. f. niikr. Anat. ISSS, p. 233), for instance, regards the anisotropous material as the actively contractile part of the muscle, and looks upon the network stainable by gold chloride, which Marshall and Melland consider to be the actively con- tractile I avt, as merely interfibrillar material. Haycraft's theory [Brit. Med. Journ. ii. [IbOO] 405) comes into the same category as RoUett's. MU.SCLE 408 In macroscopic as opposed to microscopic chemistry, it is not pos- sible to say whether any one of the constituents of the muscle-phisma corresponds to one or other of the two optically different substances ; but by microchemical methods, the question of the chemical composi- tion of these substances has been the subject of research by several investigators. Briicke ' was the tirst to determine that muscle does contain two substances which act differently on polarised light ; and he assumed that the doubly refi-acting substance is made up of innu- merable positive doubly refracting particles with the properties of uniaxal crystals, to which he gave the name disdiaclasts. Ebner considers that the action of polarised light does not prove that the two substances are chemically different, but merely that there are alternating differences in the elastic tension of different parts of the muscle-substance. Others, again, have supposed that the only difference chemically is a difference of water, the enchylema being the more watery of the two substances ; while others, again, have endeavoured to determine what constituent it is in the muscle-substance that produces the double refraction. Thus O. Nasse^ believes that the anisotropous (doul)ly refracting) substance is myosin ; the precipitate produced by adding alcohol to a saline solution of myosin is thready like fibrin, and, like fibrin, these threads refract light doubly. C. Schipilott* and A. Danilewsky^ find that the more myosin is dissolved out of muscular fibres by saline solution, the less do they refract light doubly ; they consider that the double refraction of muscle is chiefly produced by myosin, but also partly by lecithin. Myosin is converted into acid- albumin or syntonin very easily by the action of hydrochloi'ic acid ; Danilewsky ■* speaks of the substance formed in this way as HCl-myosin ; by neutralising the acid he states that he once more obtains true myosin ; but this is somewhat contradicted by the fact that it no longer doubly refracts light. He has, therefore, advanced the hypo- thesis, that myosin may exist in one of two conditions — doubly refracting myosin and singly refracting myosin. The doubly refracting myosin he also calls crystalloid myosin ; this is the form in wliich myosin exists in the muscle, and is appax-ently the same thing as Briicke's disdiaclasts.' ' Strieker's Handbuch, chap. yi. p. 170. 2 O. Nasse, Zur Anat. u. Physiol, der quergestreiften Muskelsuhstanz, Leipzig, 1882 (Vogel) ; Biolog. Ceniralbl. 1882, ii. No. 10. 3 Catherine Scliipiloff and A. Danilewsky, Zeitschr. f.physiol. Chein. v. 349. These observers also consider that the action of acids and gastric juice in sphtting up muscular fibres into discs is due to the solution of lecithin, which they consider to be especially abundant in the centre of the Ught stripe. This, however, does not appear to me to be proved by their experiments. * Danilewsky, Zeitschr. f. physiol. Chem. v. 158. ^ For Bernstein's views on this subject, see p. 435. D D 2 404 THE TISSL^S AND OEGANS OF THE BODY None of these experiments, however, prove that the isotropous material contains no myosin ; they only show that the anisotropous material contains myosin, or the myosin precursors. Tlte red variety of voluntary iimscidar fibres. — W. Krause ^ was the first to notice that certain muscles in the rabbit (soleus, semi-tendi- nosus, crureus, &c.) were redder in colour than the rest. Similar red muscles have since been described in other mammals and in fishes /"rays). These fibres differ from ordinary voluntary muscular fibres in having a longer latent period and a slower contraction ; they difier histologically in being more distinctly striated longitudinally, in pos- sessing numerous nuclei which are not confined to the sarcolemma, and in the arrangement and size of their capillary blood vessels. Chemically the only important difference is the existence in the interior of the muscular fibres of a larger quantity of haemoglobin than is present in tlie pale muscles. Cardiac muscidar fibres. — These are quadrate cells without sarco- lemma, and with one nucleus in the centre of each. They are branched, and the branches of neighbouring fibres are united by cementing sub- stance which is stained brown by silver nitrate, as is the cementing substance between epithelial cells. The fibres show a well-marked longitudinal striation, an imperfect cross striation, and by polarised light a similarly imperfect fibrillar network is seen to be present throughout the enchylema ; the latter is doubly refracting, as in voluntary muscle. Unstriated or plain muscular fibres. — Voluntary muscular fibres are so much altered from the condition of a primitive cell that the resemblances require to be carefully sought for. Cardiac muscular fibre may be regarded as in an intermediate condition of specialisation, while plain muscular fibres have lost very few of the histological cliaracteristics of primitive cells.^ They are spindle-shaped, or in the blood vessels sometimes have jagged extremities ; each possesses a single nucleus, which is rod-shaped, and has the characteristic structure of nuclei. Each possesses a fine sheath ; each exhibits faint longitu- dinal striation ; and by appropriate reagents the protoplasm can be shown to consist of an enchylema pervaded by a fibrillary network. The fibrils run in a longitudinal direction within the fibre. They never show any double refraction either during life or after death ; perhaps the anisotropous substance is absent ; myosin is, how- ever, present, as we shall see later on. Perhaps, as Hoppe-Seyler^ says, the axes of the particles which 1 Anatomie des Eaninchens, 1863. - The cement substance between these fibres is stained brown by silver nitrate. 5 Physiol. Chemie, p. 669. MUSCLE 405 produce double refraction (Briicke's disdiaclasts) are diflercnlly arranged, so that the liglit passes through their principal axis, and is thus singly refracted. This seems improbable, however, as there is no double refraction in whatever dii'ection the libres are viewed. CHEMICAL COMPOSITION OF MUSCLE A muscle may be considered as composed of two parts, the sujjjxn-t- ing connective tissue often containing fat in small quantities, and the muscular tibres themselves, each of which again consists of two parts, the sarcolemma and the contractile substance which it encloses. The connective tissue of muscle resembles connective tissue else- where ; the gelatin and fat obtained in analyses of muscles are derived from this tissue. The sarcolemma is a substance which resembles elastin very closely in its solubilities.^ The contractile substance is during life of semi-liquid consistency, and contains a lax-ge percentage of proteids and smaller quantities of various extractives and inorganic, salts. By the use of a press, this substance can be squeezed out of perfectly fresh muscles, and it is then called the muscle-plasma. After death muscle-plasma like blood- plasma coagulates (thus causing the stiffening known as rigor mortis). The solid clot corresponding to the fibrin of blood-plasma is called myosin, and the liquid residue is called the muscle-serum. Living muscle has in the resting condition an alkaline reaction ; after extreme activity, and after death, the reaction becomes acid ; this is due to the formation of sarco-lactic acid. There are other changes that occur on contraction and on death of muscle, but the details will be considered later. In round numbers, muscle consists of — 75 per cent, water. 21 per cent, proteids and albuminoids, 25 „ solids. 4 ,, fat, extractives, and salts. The following tables give more accurate data concerning the com- position of muscle : — The percentage of water varies somewhat in different animals : — ^ Birds Amphibians . Fishes . Crab . Pecten (a mollusc) ^ Recent experiments on the solubilities of elastin, sarcolemma, and basement mem- branes have been made by Ewald {Zeit. Biol. xxvi. 1). - Schlossberger, Chemie der Gewehe, Leipzig u. Heidelberg, 1856, p. 1C9; Gorup- Besanez, Lehrbuch, 1878, p. 676; Hoppe-Seyler, Ph-siol. Chemie, p. 636. Man 72- 74 per cent. Ox 77 55 Pig 78 J» Cat 75 5> Pox 74 5) 70-76 pe r cent. 76-80 55 79-82 55 85 55 79^80 ^5 406 THE TISSUES AND ORGANS OF THE BODY In young animals the percentage of water is greater in the muscles tlian in those which are fully grown ; general inanition also increases the amount of water in the muscles. Human muscle (Pectoralis major) gave the following average results : — Water .... 73-5 Solids . . . .26-5 Proteids, including sarcolemma, proteids of connective tissue, vessels, and pigments . . . . .18-02 Cxelatinlp ,, i- x- e i (1-99 -p , rirom the connective tissue ot muscle . . J .^ -,- Fat ) (3-2/ Extractives, creatine, lactic acid, glycogen, tfec. . , 0-22 Inorganic salts . . . . . . . .3-12 Chittenden ' made a similar analysis of the j^lain muscular fibres of Pecten irradians ; he found — Water . . 79-60 to 80-25 Solids . . 20-40 „ 19-75 Proteids . 15-68 „ 15-04 Glycogen . 2-43 „ 1-98 Glycocine . 0-71 „ 0-39 Ethereal extractives . 0-33 „ 0-24 Inorganic salts . 1-26 „ 1-22 The proteids of muscle will be dealt with in the succeeding sections relating to muscle-plasma, and the phenomena of rigor mortw ; subse- quent sections will deal with the pigments, the extractives, the inor- ganic salts, and the gases of muscle. The Muscle-plasma and the Muscle-serum Kiihne ^ was the first to obtain muscle-plasma and to study its reactions ; he used frog's muscle. A stream of 0-5 per cent, salt solution injected through the aorta washed out the blood from the muscles ; these were then removed, cut into small pieces, kneaded with salt solution at 0° C. (to rid them of lymph), frozen, sliced with cooled knives, pounded in cooled mortars, and then subjected to sti-ong pressure at the atmospheric temperature. The muscle thaws at 0° C. and the liquid pressed out has therefore this temperature ; this is filtered and the filtrate is muscle-plasma. It has a syrupy consistency, 1 Ann. Chpm. Pharm. clxxviii. 266. - Kiihne, Lehrlmch der 2)Jiysiol. Chemie, -p. 2T2 ; Untersuchungen iiher das Proto- 2>lus)iui, Leipzig, 1864. MUSCLE 407 and a faintly alkaline reaction. At the teinpcratuie of the air it soon clots, and the clot of myosin contiacts, l)ut not to so great an extent as fibrin does ; the licjuid squeezed out by the contraction of the myosin is called the muscle-serum. Coagulation begins at the points of contact, and is hastened by agitation and by a temperature of about 40° C The muscle-serum contains, according to Kiihne, three proteids : (1) a proteid which coagulates on heating to 45° C. ; (2) an alkali- albumin ; ' (3) an albumin probably identical with serum-albumin. Besides proteids, muscle-serum contains the extractives and salts. Since then it has been shown ^ that the same facts are true for mammalian voluntary muscle ; not only does cold prevent the coagulation of muscle-plasma, but, as in the case of blood-plasma, admixture with solutions of neutral salts (sodium chloride, magnesium sulphate, sodium sulphate, (fee.) also prevents it from undergoing coagulation, Addition of water to the salted muscle-plasma so obtained brings about coagulation ; that is to say, the concentrated saline solution prevents coagulation, but a dilute saline solution has not this inhibitory influence ; this again is exactly similar to what occurs with Ijlood-plasma. The coagulation of the diluted salted muscle-plasma occurs readily at tempei'atures between 30° and 40°, more slowly at lower temperatures, and not at all at 0° C Simultaneously with the production of a clot of myosin, sarco-lactic acid is formed. The similarity between the clotting of blood and of muscle is so great that a similar method of formation is suggested ; just as fibrin is formed from fibrinogen by the action of fibrin-ferment, so may myosin be formed from a precursor (myosinogen) by the action of a similar ferment. This supposition was found to be fully confirmed when put to the test of experiment. Saline extracts of muscle which has undergone rigor mortis resemble salted muscle-plasma very closely ; after dilution they undergo coagulation, which can be described as a re-coagulation of the redissolved myosin ; the process resembles in all particulars the coagu- lation of the muscle-plasma which in the first instance leads to the for- mation of myosin, A saline extract of rigid muscle is, however, acid, and its acidity is increased on re-coagulation, llie properties of the muscle-clot (^myosin). — Myosin may be prepared by allowing muscle-plasma to clot, or on dilution of saline extracts of either absolutely fresh frozen muscle, or of muscle which has undergone rigor. Ammonium chloride solution extracts myosin from muscle in greater quantity than other salts, and then the myosin 1 This ie probably what we shall call later myoglobulin. The various tissue-caseins or alkali-albumins that have been described in tissues are no doubt all globulins. 2 Halliburton, 'On Muscle-plasma,' Journ. of Physiology, viii. 133-202, 408 THE TISSUES AND ORGANS OF THE BODY may be precipitated in a gelatinous form by dialysing away the salt (Kiihne and Chittenden '). Elementary analysis of the myosin so obtained gives the following results : - C, 52-79 ; H, 7*12 ; N, 16-86; S, 1-26 ; O, 22-97. Myosin is precipitated by dro2:)ping a saline solution of it into excess of distilled water. It is readily soluble in 5 to 10 per cent, solutions of sodium chloride and other neutral salts ; it is precipitated from its solutions by saturation with sodium chloride, magnesium sulphate, and ammonium sulphate. These properties clearly place myosin among the globulins. It is very readily soluble in weak hydrochloric acid, forming syntonin or acid-albumin. It is readily digested by gastric juice, forming peptones, the intermediate products being called myosinoses ; it is .still more readily dissolved by pancreatic juice. Myosin, like fibrin, decomposes peroxide of hydrogen. If one takes a perfectly neutral solution of myosin in 5 per cent, sodium chloride solution and then dilutes this with two or three times its volume of water, a forma- tion of a coagulum of myosin takes place, as in the case of muscle- plasma ; that is to say, there is first a jellying throughout the liquid ; the coagulum subsequently contracts and squeezes out a clear liquid ; this occurs most readily at the body temperature, and the addition of myosin ferment hastens the formation of the clot. Thus it appears to be a true ferment coagulation or re-coagulation ; ^ tliis view is supported by the fact that the previously neutral liquid is now acid from the presence of sarco-lactic acid. The liquid squeezed out by the contraction of the clot is free from proteid. We have seen the similarities between the formation of fibrin and that of myosin ; the differences may be summarised as follows : — (1) Fibrin dissolves with difficulty in saline solutions (e.g. 5-10 per cent, sodium chloride) ; the dissolved fibrin has not tlie properties of fibrinogen, and cannot be made to yield fibrin again. Myosin is readily soluble in such saline solutions ; the dissolved myosin has the pro- perties of myosinogen, and on suitable treatment can be reconverted into myosin. (2) The formation of myosin from myosinogen is accompanied by the development of acid, whereas that of fibrin from fibrinogen is not, so far as we know. (3) The formation of myosin from myosinogen is not accompanied by the formation of another globulin, whereas that of fibrin from fibrinogen is (see p. 235). ' Myosin and Myosinoses, Zeit. Biol. vol. xxv. 358. ^ Ibid. ^ Chittenden also regards this as a re-coagulation, Studies from the Lab. of Physiol. Chem. Yale Univ. vol. iii. 1889, p. 116. MUSCLE 409 Myosin when very tlioi-ourtAs after its onset is still a matter of doubt. The usual explanation of the phenomenon is that it is due to putrefaction ; still there are many cases in which rigor passes ort' before putrefaction sets in, and other cases in which rigor persists after the onset of putrefaction. Cossar Ewart ' has shown tliat in fishes there is a persistence of rigidity if putrefaction be prevented and bacteria excluded by a weak solution of mercuric chloiide. I am, however, of opinion that putrefaction will not explain all the facts, but am inclined to think that more probably the disappearance of rigor is 1 Proc. Physiol. Society, 1887, p. xxv. 416 THE TISSUES AND ORGANS OF THE BODY due to the action of an unorganised ferment. We have already seen that muscle contains pepsin, and that it turns acid when it dies. If dead muscle be kept at about the temperature of the body for a feAv hours after death, rigor passes off rapidly, and the muscle is found to contain, not only its normal proteids, but also the products of gastric digestion ; proteoses ' and peptone are found in abundance. Muscle has, in other words, undergone a process of self-digestion. In ordinary circumstances self -digestion does not go so far as this, but only results in the breaking down of myosin into myosinogen, and this would be quite sufficient to cause softening of the muscle. We have already seen how easily the change of myosinogen into myosin, and vice versa, is brought about. The action of pepsin in producing the change is quite analogous to what we have already seen in the case of fibrin ; before the formation of albumoses and peptone sets in, the fibrin is decomposed into soluble globulins (see p. 23.3). This view of the cause of the disappearance of rigor must not, how- ever, be taken as by any means fully proved. Observations are still necessary with regard to the relation between the rapidity of the dis- appearance of rigor and the amount of pepsin in the tissues, which prob- ably varies with the condition of the alimentary canal. Rir/or morti.t in, inrolnntao'y vmscle. — So far as can be stated from actual work done, which is scanty, on this subject, the phenomena of rigor mortis in some invokintary muscles are much the same as in the vohmtary muscle. The heart becomes rapidly rigid, and simultaneously acid from the formation of sarco-lactic acid. Both paramyosinogen and myosinogen are present in the muscle-cells of the heart, and myosin is the result of coagulation. In the stomach and uterus, rigor has been observed, but in other forms of plain muscle it is difficult to recognise and has never been satisfactorily observed. Myosin, i.e. a globulin entering into the condition of a heat-coaaulum at .50° C, has been obtained from all kinds of unstriped muscle. Kossel- examined the proteids present in a muscular tumour of the uterus, and found the proteid which coagulates at 4.5° C. (paramyosinogen) absent. The reaction of unstriped muscle is normally alkaline.^ Lehmann * found small quantities of lactic acid in the muscular substance of the stomach after death. There is, however, no marked change in reaction after death as in striated muscle. Du Bois Keymond ^ observed that the muscles of the stomach and intestines of the bird were, after death, still alkaline. 1 The albumose or proteose already described as myoalbumose does not appear to be a product of digestion, but exists normally in muscle-plasma. 2 Quoted by Hoppe-Seyler, Physiol. Chemie, p. (50it. 5 Bernstein (Kiihne's Lehrbuch, p. 332) found the actively contracting muscles of the anodon acid. * Lehrbuch, iii. 73. 5 Monatsberichte d. Berl. Akad. d. Wissensch. 1859, p. 812. Mrs(],H 417 We have thus sivii that tliL-rc i.s a I'crtain amount of kno\vle(lf,'-o regarding the changes in the proteids that occur wlien muscle dies; but whether any changes take place in the proteids duriny- muscular contraction is quite unknown. The Pigments of Muscles Ifa-moi/lohin. — This is contained in tlie uuscle-plasma of the red muscles of rodents and other animals. Kiihne showed that ha^min- erystjils can be obtained from it. The pale muscles also often contain small «|uantities of ha'inoglobin. In certain gastropod molluscs (Liinnjcus, Paludina) lucmoglobin is absent from the blood, but is present in the muscular fibres of the pharyngeal wall (Lankester). When coagulation of the muscle-plasma takes place, the Iijemo- globin plays no part in the formation of the muscle-clot, but passes into the muscle-serum. Mi/o/iantafiti. — This is one of the most important of a group of pig- ments discovered by MacMunn, and named by him the histohfematins.' These pigments have a wide distribution in the animal kingdom, and often occur in those animals which possess no luemoglobin. They have not, however, been separated in a pure condition, but have only been observed by means of the spectroscope. In so far as conclusions can be drawn from spectroscopic observations, these substances appear to exist in two conditions analogous to those of h;emoglobin and oxyhaimoglobin • that is to say, by means of reducing agents and oxidising agents, different spectroscopic appearances are produced ; in the reduced condition they show well-marked l)ands, and in the oxygenated condition the bands are faint or fade altogether from view. MacMunn considers that these pigments are respiratory pigments, holding the oxygen brought to the tissue by the blood until it is required by the tissue. The spectrum of these substances is somewhat like that of lijemochromogen.- ^Nlyohfematin, the most widespread of the histohsematins, occurs in the muscles of insects, spiders, crustaceans, molluscs, fishes, amphibians, reptiles, birds, and mammals ; in man it has been found in the heart and in the voluntary muscles. The heart-muscles of the pigeon contain the pigment in great abundance, there being little or no hai-moglobin present. If a portion of such a muscle be rendered transparent by glycerine, and then by means of a compressorium it be squeezed out to a great degree of thinness, the bands are readily visible (.<;pp tig. 70). The bands are four in nundjer. ' MacMumi, ' Myohitmatin and the Histoha?niatins,' P/(i7. Trans, of Boijal Societij, Part I. 1886 ; Joiini. of Physiol, viii. No. -2. - Hoppe-Seyler [Zeit. physioJ. Cheni. xiii.l believes niyohsematin is hasmochromo'^eu. E E 418 THE Tissrp:s and ()];t marked buinl : fimrtli ));iiiil, an ill-clefint-il sliiidiiig over tin; 6 line. 2, Absorption spectrum of mollified uiyohfematiu. First band, A 554-5-51.S-5 : second band, A 324-5-519. Avith ether for some days. A yellow lij^ochrome derived from the fat Ijetween the muscular fibres ' passes into solution, and Ijelow this floats a red juice which shows the two bands of modified myohjematin (fig. 70, spectrum 2) ; these resemble those of ha^mochromogen, but are placed rather nearer the a iolet. The Extractives of Muscle The extractives of muscle may Vje divided into two sets: — A. Kitnxj'^n'iiis ' B. Xon-nifro(jenous Creatine i Fats Creatinine j Glycogen Xanthine I Inosite Hypoxanthine I Fermentable sugar Carnine j Lactic acids Uric acid I Urea j Taurine Inosinic acid The method of preparation and chief properties of each of these substances will now be taken up s^'r'udlm. Creatine.— If an aqueous infusion of meat he made, and the proteids ' Hiillilmrbm, Juiini. uf Phijsiol. vii. o'25. .MISCLK 419 precipitated by boiling and tiltei'iug oti" tlie coay;uluiii so formed, the extractives and salts remain in solution. Beef tea is a liquid wliicli contains little more than extractives and salts ; Liebi,g's and other commercial meat extracts are virtually the solid residues obtained on evaporating aijueous infusions (from which the jiroteids have Ijeen separated) to dryness. The following methods of i)rei)ai-atiou may be adopted : — 1. To an aqueous extract of meat (minus its proteids) add baryta to precipitate the phosphates ; filter ; remove excess of baryta from the filtrate by a stream of carbonic acid ; filter off the barium carbonate ; and evaporate the filtrate on the water-bath to the consistency of a thick syrup. Set it aside to cool, and in a few days crystalline deposits of creatine will be found at the bottom of the vessel. These are washed with alcohol and dissolved in hot water. On concenti'ating the aqueous solution crystals once moi'e separate out, which may be still furtliei- purified by recrystallisation (Liebig'). 2. The aqueous extract is precipitated with acetate of lead : filtered ; the filtrate freed from excess of lead by a stream of sulj^huretted hydro- gen, and then filtered and evaporated till crystals appear, which may be purified by recrystallisation (XeuV)auer2). Stadeler^ uses an alcoholic instead of an ai^ueous extract of muscle. 3. Muscle is finely chopped and allowed to stand under ether ; a strongly acid, wateiy fluid in a day or two separates out ; this is red owing to the presence of myoh;ematin ; the ether floats above this watery liquid. On evaporating the latter, crystals of creatine separate out, and may be purified by recrystallisation as before (MacMunn '). Creatine has the formula CjHgNgO.j; this unites with one molecule of water of crystallisation to form transparent, colourless, monoclinic prisms (tig. 34, p. 84). The crystals lose their water of crystallisation at 100° C. They are soluble in w^ater, especially in hot water, and almost insoluble in absolute alcohol and in ether, sparingly soluble in rectified spirit. Creatine forms crystalline compounds with the mineral acids, and Avith meicury (C4H7HgN30^). When creatine is treated with \arious reagents it undergoes a number of different decompositions. The most important of these are the two following, as it is probable that similar changes occur in the body : — (a) C (Hirers ion into creatinine. — When creatine is heated with dilute ' Ann. il. CJiein. u. Pharin. Ixii. -i.")?. - Ibid. cxix. 27. •' J./.jjtak'. Chem. Ixxii. 25(;. J Journ. of Physiol, viii. 58. E K 2 420 THE TISSUES AND ORGANS ()F THE BODY mineral acids, oi- for several days with water, it loses a molecule of water, and creatinine is formed : — ■ C4H.jN30,-HoO=C,H;N30 fiTeatiiie] [creatinine] A similar change occurring in the body no doubt gives rise to the creatinine occurring in the urine. (b) Concersioit info snrcosine and urea. — Creatine seems to replace urea in muscular tissue ; ' the theory that it is a stage in the formation of urea has been advanced because it can be made to yield urea in the laboratory ; its molecule, in fact, contains the .cyanamide radicle (CX.NH.j), which ^//«.s- a molecule of water is equal to urea (COX.,H^). {See further under X'line.) When creatme is boiled with baryta water, the following equation represents the change that occurs : — C4H9X3O., + H20=CONoH, + C3H-NO2 [crtatiiifj [urea] [.■;ai'L'0:5iiie] Synthesis of creatine. — Creatine has been made synthetically, and the following, which is the method adopted, will show what is the con- stitution of its molecule. When methylamine and monochloracetic acid are brought together, the following reaction occurs : — CH31 CH.Cl CH.3N(CH2)H H X+ = , +HC1 H ) CO.OH CO.OH [metbylamine] [monci-cbloracetic [sarcosiiie or acid] tiiethylglycociue] That is to say, sarcosine and hydrochloric acid are formed. Sarcosine is also called methylglycocine, i.e. glycociue (amido-acetic acid), in which one H is replaced by methyl (CH3). On heating sarcosine and cyanamide together, creatine is formed according to the following equation : — C3H-N02 + CN.NH.,=C4H9N30., [sircosiiie] [cyanamiile] [creatine"-] Quantity of creatine in m^iscles. — According to Toit'^ the quantity of creatine is fairly constant in the voluntary muscles ; the quantity is found to vary from 0*2 to 0"3 per cent, in difierent animals. This quantity increases during starvation (Demant"*). Cardiac muscle • Creatine is also fouud sparingly in nervous tissue. - Creatine is thus raethyl-guanidine-acetic acid (Bauniann, Ann. Chem. Phaiin. clxvii. 77). ^ Zeit. f. Biologic, iv. 77. Subsequent analyses by Xeubauer, Hofniann, Creite, and many others have coufirmed Voit's analyses. For references see Hojipe-Seyler's Physiol. Chem. p. 6i'i. * Zeit. physiol. Chem. in. 'i%l. MUSCI.K 421 contains less creatine than the vohmtary muscles (Yoit) ; the same is true for unstriated niusc-le (Lehmann'). Creatinine. -Small quantities of this base are found in muscle;^ it is also a ttonstituent of urine. The strongly alkaline reaction of crea- tinine is said by Salkowski in a recent article ^ to be greatly due to adherent alkaline salts. The com})Ound of creatinine with zinc chlorifle (C4H-N30)2ZnC1.2 has a characteristic crystalline form, and is used as a test for this substance. The statements made as to the relative quantities of creatine and creatinine during the rest and activity of muscle are contradictory ; Voit ' states that creatine is diminislied during activity ; Sarokin' that creatinine increases during tetanus; and Nawrocki^ contradicts these statements. The more recent observations by Monari ^ however confirm the original statement of Sarokin ; on fatigue of muscle, creatinine is produced fi-om the creatine, together with a small quantity of another substance called xanthocreatinine C5H10N4O. Hypoxanthine, Xanthine, and Uric Acid are found in muscles in small quantities. The formulae of these three substances denote tlieir close relation- ship to one another. Hypoxanthine or Sarcine . . . C5H4N4O Xanthine C5H4N4O2 Uric acid C.^H^NjO^ The last-mentioned siib.stance, uric acid, occurs only in the merest traces (Meissner*). With regard to xanthine' and hypoxanthine,'" the following is a .■nummary of the chief facts concerning them : — Preparation. — The mother liquor, from which creatine has been separated, is precipitated with ammonia and silver nitrate ; the precipitate is dissolved in nitric acid of specific gravity 1"1. The liquid is cooled and a compound of liypoxanthine and silver nitrate crj'stallises out. The mother liquor («) is preserved. The silver is removed from the crystals by sulphuretted hydrogen, and the nitrate of hypoxanthine treated with ammonia, and thus crystalline nodules of hypoxanthine are formed. A compound of xanthine and silver nitrate is left in solution in (a) ; it is precipitated by excess of ammonia ; the silver is removed by sulphuretted hydrogen, and the base obtained in white amorphous gi-anules by adding ammonia. * Lehrhuch, iii. 7.3. * This is, however, denied by Neubaner and Nawrocki. ^ Salkowski, Zeit. ]i}ujsioJ. Cliein. xii. 211. * Loc. cit. 5 Vircliow's Archiv, xxvii. 6 Ceidralhlatt, 1865, p. 417. 7 A. Monari, Gazetta, xvii. 367. ^ Zrit.f. rat. Med. xxxi. 144. 9 Found also in urinai-y calculi (Marcet), in guano (Ungei- and Phipson, Chem. Med. vi. 16), and in urine (Bence Jones, Quart. J. Chem. Science, xv. 78; "Weiske, Zeit.f. Biol. ii. 2.54). 1" Found also in spleen (Seherer), in the blood, marrow, and secreting glands. 422 thp: tissues and organs of the bduy Xantldnt Hypoj-anthinr Properties. — It reduces silver salts. It forms compounds with silver. It forms compounds with hydrochloric copper, and platinum. On oxidation acid (hexagonal plates), nitric acid, ice. : with nitric acid it yields xanthine. Its the latter is not rendered purple by nitrate and chlorate are crystalline, ammonia, and so it can be distinguished from uric acid. Avwunt in inusele. -Ov02& (Sche- 002i'-0020 (Neubauer -). Its amoimt rer '). increases in stan ation (Demant). Carnine. — A crystalline base (C,HjX^03 + H„0) ; it was originally found in fairly large quantities (1 per cent.) in American meat extracts (WeideP) ; it has been since found in the flesh of several animals (frog, alligator, ice.) by Kiuken- berg and Wagner.* It is probable that carnine is one of the intermediate prc- flncts between the proteid molecule and the substances of the uric acid group which we have just considered. Om.— (CON.jHj) is probably present in small quantities, but is diflicult to separate from other nitrogenou-s bases. Tavrine. — Found in the muscles of the horse, fishes, and molluscs. In fishes Limpricht * found 106 per cent, of taurin. Glycocine. — Found to the extent of 0-39-0-71 per cent, in the non-striated muscles of molluscs (Chittenden*). Protxc acid (Protsiiure). — An acid formed from the decomposition of proteids, described by Limpricht in the muscles of fishes. This is an acid of doubtful nature. Inosinic acid (C,(,H,,X,0„). — First described by Liebig. It is itself amorphous, but forms crystalline salts with the alkaline metals, with barium, and with calcium. It has since been found and estimated (0005-OOl' per cent.) by Creite." Lecithin and it- decomposition products, such as glycerophosphoric acid, are found in small quantities in muscle, but it is very doubtful whether this substance is a constituent of mu>cular substance pro]>er ; more probably it is derived from the nerve fibres which terminate in the muscle (Hoppe Seyler'). The next class of extractives are those which are non-nitrogenous : these are glycogen, inosite and other carbohydrates, lactic acid, and fat. GlycogBE. — This sub-stance, sometimes called animal starch, has the foiTDula (CgH,,,0,)„. It is present largely in all embryonic tissues;^ 1 Ann. Chem. Pharin. cvii. 314. - Zeit. f. anal. Cheni. vi. 33. ^ Weidel, Ann. Chem. Pharm. clviii. 353. ■* Krukenberg and Wagner, Sitzungsher. d. phijsih.-med. Gesell. zii Wiirzburg, 11*83, No. 4. Krukenberg has also examined the muscles of a large number of fishes and inver- tebrates for the presence or absence of the various extractives enumerated above. His results will be found in Untersuch. des jihysiol. AnstaJt Heidelberg, vol. iv. Heft i.; and in Mahj's Jahreshericht, xi. 340. ^ Ann. Chem. Pharm. cxxvii. 185, cxxxiii. 30(1. ^ Ihid. clxxviii. •26t;. " Zeit. rat. Med. (3) xxxvi. 195. 8 Physiol. Chem. p. 647. ^ Claude Bernard, Comptes rend, xlviii. 673. Cramer iZeif. Biol. xxiv. 67) obtained pm"e glycogen from foetal skin and cartUage. Mist I. K 4-2:5 ill tlie adult it is found chieriy iii tlit- liver, the iiiUftcles, aiul the white 1)1cmk1 corpuscles. Duiing life, a^ in the liver, the muscle glycogen ap|)eai's to l)e converted into sugar, and this change may occur for a certiiiu time after death. Boluu ' considers the change into sugar does not occur till putrefaction sets in, and Demant ^ has shown that the change may l)e gieatly hindered liy the action of weak carbolic acifl In estimating the amount of glycogen in muscle, the tissue should, however, be obtained as soon as possible after death, and immediately lilunged into btiiliiig water to destroy any ferment which converts glycogen into sugar. The glycogen may then be extracted with hot water (Briicke,-^ Xasse^), or with dilute potash (Abeles,' KUlz ^). If a (piantitative analysis is to be made, a weighed quantity of muscle must be taken, tinely divided, and repeatedly extracted until no more glycogen passes into solution. In the case of muscle especially, the dilute alkali effects a much more thorough extraction than hot water (Kiilz). Any proteid that passes into solution is precipitated by potassio-mercuric i(xlide and filtered oft"; the tiJtrate is evaporated to a small bulk and the glycogen precipitated as a white powder by excess of alcohol, or it may be converted into sugar and then estimated polarimetrically. Glycogen forms an opalescent solution in water, and gives, like dextrin, a red- brown colour with iodine. A large number of comparative estimations by weighing and by the polarimeter have been made in Kiilz's laboratory and found to yield very nearly equal results."^ Cramer,'' using KiUzs method, found 1. The glycogen in the two luihes of the body is equal. 2. In the heart, glycogen is unequally distributed in the diflereiit regions, so ditiering from the liver. 3. Ditferent groups of muscles vary in the amount of glycogen they contain, but symmetrical or corresponding muscles contain the same amount. Briicke^ found glycogen in the plain muscle of the stomach; Chittenden '" and Bizio " found it in the plain muscles of gastropods. The amount of glycogen in muscle is variable : the following are the chief facts relating to variations in the amount that is present : — 1. Influence of starvatictn. The nuircle glycogen in warm-blooded animals ' Pfiliger's Archil-, xxiv. Sii. -' Zeit. phij^iol. Cheni. iii. 200. ■"• Brficke, Sitzungsher. d. k. I: Akad. d. Wisscnsch. Wioi, Ixiii. 214. * Xasse, Pfiitger's Archiv, ii. 97. '•> Abeles, Med. Jahrbiichcr. lH77, p. 551. « Kiilz, Zeit. Biol. xxii. 161. ^ Schmelz, Zeit. Biol. xxv. 1«0. « Ibid. xxiv. (!?. ^ Wiener Akad. SitznngHber. vol. Ixiii. 2 Abth. 1871. '0 Chittenden, Ann. Chem. Pharm. clxxviii. 200. 11 Bizio, Atti delV Istitiito Vciiet. di scieiize, vol. xi. (ser. S), 1866. 424 THE TISSUES AND nK(;ANS OF THE I'.oDV disai^pears during inanition luucli more slowly than the liver glycogen ('Wei.'^s,' AldehofI -). Luchsinger' stated that the heart-muscles are richer in glycogen during inanition than those of the limbs, but Aldehoif, who used Kiilz's method of estimating glycogen, and therefore obtained more correct results, was not able to confirm this statement of Luchsinger. 2. Influence of -vrork. Muscular activity lessens the amount of glycogen in a muscle, it being apparently transforiued into sugar (Weiss, Manclie,^ Molinari ^). This is well illustrated by the following table (Manche). Weight of glycogen iu limb at rest AVeiglit of glycogen in o)iiiO:iite limb, whicli \va:i iiiaile to contract from 23-65 minutes Loss of glycogen per cent, in tetanised liml> 1. 0"1277 gi-amme 0114 gramme 12-76 2. 0-2287 „ 0-1942 „ 15-09 3. 0-2267 „ 01017 „ 15-44 In other words, the limbs which wore stimulated to contract lost from 12 to 15 per cent, of their glycogen in an hour. Luchsinger considered that glycogen is not a direct source of energj- in contracting muscle, but this is in no way proved by his researches, for it is doubtful whether he could ever have obtained muscles free from glycogen— as we have already seen the glycogen of muscle disappears very slowly- during inanition. In frogs inanition causes a rapid dis- appejirance of the liver glycogen, but that of the muscles remains practically unaltered (Aldchoff). 3. Effect of removing the liver. Minkowski"^ and Laves" stated that after extirpation of the liver the muscle-glycogen markedly diminishes ; they consider that the muscle-glycogen chiefly originates in the liver. C. Schmelz," using Kiilz's method of estimating glycogen, confirms these results which were arrived at by Briicke's apparentlj- less exact method. Schmelz, however, does not consider the point proved that the liver is the source of the muscle-glj^cogen, for he finds that feeding animals on cane sugar produces no marked increase of the muscle- glycogen either in normal animals or in those from which the liver has been removed. Prausnitz " also considers that the muscles have a glycogenic function quite apart from that of the liver. 4. Effect of cutting the nerve of a muscle. This operation causes an increase in the glycogen of the muscle (Chandelon '"). The following table illustrates the results obtained (Manche) : — Operation of cutting one sciatic nerve. | Increase (per cent.) of muscle-glycogen on licrformeil— ojierated side as compared witli tlie noi-uial T5 days before death 625 2-7 „ „ 2G67 3-20 „ „ 3:i-33 No doubt the intact muscles of the healthy limb continue to contract after the operation, and thus lose a certain amount of glycogen ; in the paralysed muscles on the other hand, the glycogen is allowed to accumulate. 1 Weiss, Sitzungsh. d. k. Akiid. dcr Wi-i'icnsch. Ixiv. 2 Aldeboff, Zeit. Biol. xxv. 137. -* Luchsinger, Dissert. Ziirich, 1875. •« Manche', Zeit. Biol. xxv. 163. •' Molinari, Chem. Ccntralhl. 1889, ii. p. 872. Minkowski, Arcli.f. exj). Path. ii. FJiiinuakul. xxiii. 13i). '' Laves, Inaug. Dissert. Kiinigsberg, 1880. * C Schmelz, Zeit. Biol. xxv. 180. '■> Zeit. Biol. xxvi. 377. ^^ Chandelon, Pfliiger's Archiv, xiii. 02(5. MiscLi-: 425 5. Effect iif i-uttiiig till' tc'iidoii n[ a imisclo. After tenotomy tlie muscle appears to l>c in such a iiatholui^ical condition that glycogen accumulates in it, and does not undergo metabolic changes so reaRff AN.S nF THE BnDY (see p. 409), and the opinion has been advanced that it is formed from proteid, not fi*om carbohydrate. This acid is produced crystallised (Liebig's method, see p. il9j is acidulated with sulphuric acid and extracted with ether. The ether contains the lactic acids in solution : it is evaporated to dryness and the residue dissolved and boiled in water in which carbonate of zinc is suspended. It is then filtered, and the filtrate evaporated to a small bulk. On treating this w ith absolute alcohol, the liquid deposits needles of zinc sarcolactate, the ethene lactate of zinc remainijig in solution. The sarcolactate is filtered off, and the fUtrate evaporated down, when the ethene lactate separates out. ThLs also is filtered off. We thus have zinc sarcolactate on one filter, and zinc ethene lactate on another. The free acid is prepared from each in the same way. The crystals of the zinc salt are dissolved in water : sulphuretted hydrogen is passed through the solution : the zinc sulphide is filtered off; the filtrate is concentrated, shaken with ether, and, on evaporating the ethereal extract to dryness, the free acid is obtained. Propert'ici of the isomeric lactic acids. Sarcolactic acid 1. Dextrorotatory (a = 2. Zinc compound has the formula Zn(C3H-0,)„ -i- 2H.,0. It loses aU its water of crystallisation (129 per cent.) at 100° C. Specific rotation = —7-6°. Very insoluble in alcohoL Soluble in 17o parts of water at 14° C. 3. Calcium compound has the formula 2[Ca(C,H,03)J -1- 9H.^0. Specific rotation = -3-8°. Femientatiou lactic acti !rom milk Etheue lactic acid 1. Optically inactive 1. Optically inactive. 2. Zinc compound has the formula ZnCC^HjOj), 4- 3H.,0. It loses aU its water of crystallisation (1818) at 100° C. Opti- cally inactive. Insoluble in alcohol. Soluble in 58-60 parts of water at I 3. Calcium compound ; has the formula ! Ca(C3HA). -^ 5H,0. Op- i ticaUy inactive. 4. When oxidised with dilute chromic acid, acetic and formic acids are produced. At 100° lactic anhy- dride (CjHj^Oi) and at 140° Lictide (CjH,0„) are formed. 2. Zinc compound has the same formula as the sarcolactate. Optically inactive. More soluble in alcohol. Very soluble in water. 4. Oxidised with dilute chromic acid, ma- lonic acid (C,H,0,) is produced. Lactic anhy- dride and lactide are formed as in the case of the two other acids. .Aiisci.K 427 Quantitij of lactic iicid in miixclc. — Tlii> i> v.dialile ; tlic viirimis analyses that have been made give iuiinliei> \arviiio- tidin dl to 10 per i-ent. (Jacobsen, Takacs, Bolini, Deniant). Fat. A certain (juautity of fat is always present between the muscular fibres ; it is not possiljle to say whether any of the fat obtained from muscle cranes from the muscular substance pi'oper or not. There are two conditions, howe^er, in whicli tat is undoubtedly present : — 1. In the affection known as fatty degeneration, the interior of the sarcoleuina becomes crowded with fat granules and globules ; these tirst obscure and tinally obliteratt' the striations of the contractile substance. It often occurs markedly in the heart ; it may be produced artiticially by certain poisons, especially l)y phosphorus. 2. After death the muscular substance may be replaced by a waxy material, known as adipoce ri\ This occurs especially in corpses buried in damp soil, or in bodies which remain in water some time after death. The length of time after death that these changes occur has been the subject of extended observations, especially at the Paris Morgue ; it is found to occur in the muscles in a definite order, and tlie amount of adipocere present is a very good gauge of the time a body has been dead. ' Adipocere consists chiefly of the calcium soaps of palmitic and stearic acids, and, in some cases, of acid ammonium soaps also.^ Hoppe-Seyler '^ regards the change as a result of a ferment action. The formation of fat from proteids probably occurs under other circumstances ; for instance, fat is formed in the body on an exclu- sively proteid diet. Inorganic Constituents of Muscle The most noteworthy points in the inorganic constituents of muscle are the predominance of potassium over sodium among the bases, and of phosphoric acid among the acids. This appears to be a general rule throughout the animal kingdom. The total amount of ash is from 1 to 1 "5 per cent. The following analyses are hy Bunge ^ : — - ' See more fullj- in works on Forensic iledicine. - Quain, Med. Chir. Trans. Is50, 141. Virtliov,-, Yerhandl. d.phijs. vted. GeseUsch. zii Wihsbiu-g, xol. iii. Vi'etheviW. Joiirii. f. jjnikt. Cheiii. vol. Ixviii. p. 26. K. B. Leli- niann, Bied. Ccntralhl. 1889, p. CO. '' PJiijsiol. Chem. p. 119. ■* Bunge, Zeit. physiol. Client, ix. IHI. Other analyses will be found in Hoppe 428 TlIE TISSUES AND OEGAN.S oi" TJIE BdDY In pai-ts per 1,000^ I. II. KoO .... 4-654 4-160 KagO 0-770 0-811 CaO 0-086 0-072 MgO 0-412 0-381 Fe/J3 , 0-057 .-^ PoO, 4-644 4-58 CI 0-672 0-70 SO3 — 010 The Gases of Muscle The subject of the gaseous constituents of muscle is one at which a large amount of painstaking work has been done. The forms of apparatus used have been already described (p. 31), and we shall now consider the principal results which have been arrived at. In dis- cussing this subject it v.'ill he necessary to draw distinctions between active and resting muscle, and also to consider the gases of tlie blood which is entering and of that which is leaving the muscle. Gases of the muscle itself. — In order to extract the gases from muscle (the muscle in vactw) care must be taken to avoid entanglement with air ; it is necessary to use a form of mercurial aii--pump much like those employed in extracting the gases from the blood : the boiling- Mask is perforated with platinum wires so that, if necessary, the muscle may be excited to contract while mi vacuo, and the froth -chamber is .so arranged that an acid can be made to pass when desired from it to the muscle (fig. 15, p. 32). Muscle which has been freed from Ijlood and removed from the body, then scalded to prevent rigor, and minced, yields a small (juantity of carljonic acid ; this is increased on adding acid, by the liberation of the gas from carbonates. Hermann's ' results were : — Free carbonic acid . Fixed carbonic acid 2-74 per cent. 1-95 Muscles in which rigor mortis has been prevented by freezing and placed in the boiling-flask containing boiled salt .solution give off no gas at 0° C, but above this temperature, especially when rigidity .sets Sevier's Phijslol. Chcm. pp. (J.-JO, O-'Jl. Tliey, however, simply illustrate the same jjoints. ' Hermann (Untersuchunf/e>i ii. d. Stoffirechscl der Miiskeln ausgehend vom Gas- isechsel, Hirschwald, Berlin, 1867). Misci.K 429 ill, tlit-rt' is a discliarge of ,i;as, and, it" put ix- faction Ijf allowed to super- vene, a further discharge. In the portions of gas first set free small (juantities of nitrogen are constantly found, but oxygen is always absent. The following numbers weje obtained Ijy Hermann in one e.xperinient : — Free carbonic acid liberated at 60° C . 11-79 per cent. Fixed carbonic acid, i.e. liberated by acid . 204 „ Nitrogen . . . . . . .1-23 „ Oxygen . 0-0 There is thus a great increase in the amount of carbonic acid as compared with scalded muscle, i.e. muscle in which rigor does not (.ular contraction ou the urine are exceedingly slight ; it is only after prolonged and violent muscular exercise that any change can be percei^ ed at all, and even then it i;> out of all pro- portion to the amount of conti-action. This is in marked contrast to the very great effect that muscular contiuction has on the respiratory excretion {&>->■ pp. 374, 427). It is in the urine that urea, uric acid, and other products of nitrogenous metal^olism or comVjustion leave the body ; and, as we have ali-eady .seen, there is very little change in the chief nitrogenous constituents of muscle, the proteids, on contraction ; 1)utthfr substances which undergo an accelerated chemical change or com- bustion on contraction are the non-nitrogenous constituents. MISCLK 437 Voit ' invcstigateil the t|uestion i}i a dog ; tlif^ animal either went without food or was put on a carefully regulated, fixed diet ; the work (lone was the turning of a treadwlieel, and the urine was carefully collected. A very slight increase in the amount of urea excreted was noted after work, and it was not at all proportional to the amount of work done. The experiments made on hujuan beings fully lorroborate Voit's expei'iments. Of these the experiment of Fick and Wislicenus '^ in the ascent of the Faulhorn has become classical. The following are the chief of the facts they asi-ertained. For seventeen hours before the ascent, during the ascent, and for some hours afterwards no nitrogenous food was taken. The urine was carefully collected and the urea determined in it l)y Xeubauer's method for the periods before, during, and after the exertion. The work done was also estimated. The total nitrogen was determined by combustion with soda-lime. The height of the mountain is 1,956 metres. Fick weighed 66 kilograms, and Wislicenus 76 kilograms. The work done by Fick in raising his body to the top of the mountain =66x1956 = 129,096 kilogram-metres ; similarly, the work done by Wislicenus was 148,656 kilogx^am-metres. This does not take into account any other muscular work, such as the movements of respiration, circulation, movements of the arms and trunk muscles, &c. The nitrogen in the urine (the small quantity which escaped by the sweat and fa-ces being disregarded) during the work and for six hours after was, in the ease of Fick, 5-7 grammes, which corresponds to that obtained from 37' 1 grammes of pro- teid ; and, in the case of Wislicenus, to 5-5 grammes, which corresponds to that obtained from the decomposition of 37 grammes of proteid. Frankland ^ has shown that from the burning of 1 gramme of lean beef a quantity of heat is formed which corresponds to 2161 kilogram- metres, so that the amount of work obtainable from 37-1 grammes of proteid in Fick's case was 37-1x2161 = 80,324 kilogram-metres; in AVislicenus' case 37 x 21 61 =-79,956 kilogram-metres— that is to say, much less than the work actually done. The disproportion is really greater, because the physiological heat-value of proteid is less than its physical heat- value ; proteids do not in the body undergo complete combustion ; the physiological heat- value of proteid is its physical heat- value minus the heat- value of urea. This experiment clearly showed that proteid metabolism will not account for all the work done ; it however does not settle the question ' Untersuch. ii. d. Einjliiiis des Korhmhes, des Kaffcrs mid d,r Miisknlheiregnngrn auf den Stoffwechsel, Munich, 1860. - VierieJjahrrsschrift d. naturf. GeseUsch. in Ziirich, x. IsC"). London, Edin. and Diihlin Phil. Magasine, series 4, vol. xxxi. p. 485. 5 Frankland, Lond. Edin. and DiibJin Philos. Mag. series 4. vol. xxxii. p. 1S7. 438 THE TISSUES AND OKGANS OF THE I'.ODY as to wliether the nitrogen excreted is increased by work ; it so happened in the actual experiment tliat the nitrogen excreted was lessened as compared with the periods before and after the muscular exertion, but the conditions under which the experimenters worked were not sufficiently rigorous to admit of accurate comparisons l^eing drawn. The question as to the influence that work has on the increase or flecrease of the nitrogen excreted has been investigated by Parkes ' on soldiers ; he found a slight increase during woi'k ; by Flint ^ and Pavy * on the pedestrian Weston, who arrived at contradictory results ; and by North,^ who expei'imented on himself. The last-named experiments are by far the most thorough that have been made ; the following is a resume of the methods employed and the results obtained. Each experiiLent lasted nine days ; four days of ordinary occupation, one day's work, and a second i^eriod of four days of ordinary occupation. ' Reserve nitrogen ' was got rid of by thirty-six hours' abstention from food, or by severe labour before the commencement of the experiment. Observations were made twice daily on the pulse, rate of respiration, temperature of body, and body- weiglit. The food, carefully analysed, weighed, and cooked by the experimenter himself, was takeii in four meals, and consisted of bread specially made by Mr. North himself, dried meat-powder, 'desiccated potato,' 'dried julienne,' con- densed milk, cocoa, ' American evaporated apples,' ' Australian beef marrow,' sugar, salt, tartaric acid, and sodium carbonate (for raising tlie bread). None of tliese ai'ticles of food presented any difficulties as regards analysis. The food, of whicli tliere was an unlimited supply, was of constant comjmsltioti, so that for the first time in such experimentation a food, the chemical composition of which was absolutely known, was used. The fieces and urine were collected in specially-prepared bottles, carried in a knapsack during walking, which was the special form of work selected. Tlie nitrogen (estimated by combustion with soda-lime), chlorides, sulphates, and phosphates were estimated both in food and excreta. For full particulars, the original memoir must be consulted, but the following summary of the full tables from one of the experiments will serve as an example to illustrate the general results obtained. The experiment lasted from June 7-15, 1882; June 11 was the day on which work was done. The work was a walk of 32 miles in seven hours, the load carried being 27'75 lbs., and the loss of bod v- weight after the walk. 4 5 lbs. Averages per diem Date Uriue Fa?ces P.O. H.,SO, X P-..0;. N June 7-10 i-ft: 2-75 13-7S 2-17 2-26 „ 11 1-98 3-6.-) IGl.-. — — « 12 1-86 3 02 l(iv!l 2-77 2-57 „ 13-15 1-71 2-74 14-68 2-2.- 2-87 1 Parkes, Proc. Boy. Soc. xi. 339; xvi. 44. - Flint, Journal of An at. and Physiol, xii. 91. ^ Pavy, Lancet, 1876 (numei-ous papers). * North, Journal of Physiol, i. 171. P7-oc. Boy. Soc. xxxix. 443. MISCM-: 439 Dailii hahiiice of Jitijrxia and Excreta. Date N of iagi'stu N (It cxm-ta Juno 7 17fi4 u;-2S 8 :^5-2S Hl'-TC, it 52'.t2 IS 11 „ 10 70-57 r.o-!»o .. 11 88-21 7rt-(;i' o 12 10r,-s.-, !tS-4(> „ 13 128-O0 lie.- lit .. 14 141-14 i:!:!-fis ,. l") 158-78 i-)0-(;.-) il P.,0. I'Xcreted .. intjcsted Difference i-:?r, 2-52 4-51 i»-(;7 H-5!) 7-15 7-:ii 7-4(1 81:5 Total PjO, In excreta daily Difference 3-54 4-08 4-37 1-80 4-75 3-!)4 4-25 3-51 4-50 35-34 34-84 0-50 Per diem Before work After work Difference Nitrogen in excreta . . . . 15-22 \ 17-95 273 P.Os in excreta .... 3-59 4-li) 0-60 H.,SO, in urine .... 2-74 1 2-97 0-23 The weights in the foregoing- tables are expressed in grammes. The results seen are as follows : nitrogen — obvious increase on the day of work continued on the daj's following it. The reserve at the end of the experiment was only 1-54 gramme less than on the day before tlie work : pliosplioric add — the excess of Y.fd-^ excreted over that ingested (0-5 gramme) is probably within the limits of experimental error: sulphnric acid — the increase after the work is undoubted, and proportional to the increase of nitrogenous material excreted ; the amount of sulphates in the food was insignificant, and tliat in tlie urine was therefore derived from proteid metabolisna. These results confirm those of Parkcs, Imt the disturbance produced by very severe labour was much more immediate and of greater intensity than that which Parkes observed, probably because tlie exertion he imposed on the soldiers under observation was inadequate. As in Parkes's experiments, where retention of nitrogen followed the diminution of nitrogen stored in the body, produced by privation of nitrogenous food, so after the disturbance of nutrition produced by severe labour, the immediate effect of which is to diminish the store of nitro- genous material in the system, there follows a corresponding diminution of discharge, the output being less than the intake. This store of nitrogen is more constantly operative than has been hitherto supposed : thus accumulation took place when the daily supply of nitrogen was not more than 17-6 grammes, no extra work being imposed ; this amount cannot be regarded as more than an adequate supply for the normal needs of the bodj'. The retention following starvation or exercise is a mere exaggeration of the normal tendency. Muscular contractic»u thu.s eiilai-ges the total exci-etion of nitroi^en, but the increase is very small and is out of all j^roportion to the work done or the body-weight lost during the exercise. No doubt the nitrogen eliminated is derived ultimately from the muscles ; but, as 440 THE TISSUES AND <,)K(;ANS OE THE JJODY' North's experiments show, it is rather from what may he called reserve- nitrogen that the increased output is derived, not from the muscular nitrogen direct. As Gamgee ' points out, the effete nitrogen may leave the muscle not as urea or any intermediate substance in the formation of urea, such as creatine, but as proteid, and this proteid may be oxidised to form ui-ea somewhere else. ELECTKICAL ORGANS About fifty .species of lislies are believed to possess electrical organs ; the best known of these are the torpedo ray, the common skate, the electric eel (Gymno- tus), and the JMalapterurus. Many interesting physiological observations have been made upon these organs by Du Bois Reymond and by Burdon-Sanderson, Gotch, and Ewart with regard to their histology, development, and electro- motive phenomena.- In Malapterurus the organ appears to be epithelial in origin, but in other cases the organ appears to be analogous to muscle or to be developed from embryonic structures which elsewhere lengthen into muscular fibres ; the nerve terminations, which in muscle form the comparatively small end-plates, in the electric organ form more extensive expansions. But very little chemico-physiological work has been done at this subject ; the observers speak of a mucoid fluid in the spaces between the electrical plates ; and from the torpedo organ Wej-1 ^ has extracted a substance which gives the re- actions of mucin, except that no reducing sugar can ))e obtained from it on treatment with dilute acids; he calls it torjaedo -mucin. A small quantity (if gelatin and of a globulin (coagulated by heat at 5ij°-60°) was also obtained. The heat-coagulation temjierature of the globulin is the same as that of myosinogeii ; ^ and it is also interesting to note, when comparing the organ with muscle, that, like muscle, it becomes acid after death (Boll),'' and much less transparent. Weyl'^ found the percentage of water in the muscles of torpedo to be 77-5, in the electrical organ 89. He was also able to separate a number of organic substances from the electrical organ similar to those occurring in muscle and nerve, such as creatine, xanthine, lecithin, fat, cholesterin, fatty acids, and inosite. Frerichs and Stjideler found urea. In another research' Weyl fdimd that excitation of the organ produced an increased formation of phosphoric acid in it. ' Physiol. CJiem. p. 409. - McKendrick's Physiology., vol. i. chap. xx. 1888, contains a rtsunit of these re- searches, with bibliography. Du Bois Reymond's papers are translated in the Oxford Biological Memoirs, vol. i. 1887. See especially on the Chemical Reaction of the Electrical Organ of Malapterurus, p. 412. (It becomes acid on activity.) ° Weyl, Zeit. x>hysiol. Chem. xi. .525. * Krukenberg ('Weitere Untersuch. zur vevgleicli. Muskelchemie,' Vergleich. 2'hysiol. Studien, 2 Reihe, 1 Abth. pp. 143-147) states, however, that he was unable to obtain myosin from the electrical organ of torpedo. * Arch, f in- Anat. und Physiol. 1873, p. 90. c Moimtsher. d. Jconigl. Akad. d. Wissensch. zn Berlin, April 1881. 7 Du Bois Reymond's Archiv, physiol. Abth. 1884, pp. 31C-324. KlMTIlKLil .M 441 CHAPTFJl XXi ei'ithkljim EpiTiiKLiUM m;iy be detined as a tissue which consists entirely of cells united by a small amount of cementing substance. As a rule an epithelium is spread out to form a membrane, lining a cavity or covering a surface. But in certain cases the tissue is not spread out in this way ; for instance, the liver is an organ which may be said to consist of a mass of epithelial cells, and the vaiious forms of cancel' are also •epithelial growths. Epithelia may be classified from the histological standpoint into those which consist of one layer of cells only, called simple epithelia, and those which consist of more than one layer, which are termed compound. The simple epithelia may be again subdivided into pavement (or endothelium), columnar, cubical, and ciliated, according to the shape of the component cells. The compound epithelia comprise the ti'ansitional epithelium of the bladder and ureters, and the stratified epithelium, such as that lining the mouth, or covering the whole of the external surface of the body, where it is called the epidermis. Separated from these various forms of epithelium on account of their specialised functions, the two following must be mentioned : secreting epithelium, such as occurs in the alveoli of the salivaiy glands, or the uriniferous tubules ; and ner\e-epithelium, the various forms of modified epithelial cells which are connected to the termina- tions of various sensory nerves, and form the receptive end organs for sensations of different kinds ; instances of nerve-epithelium are the rods and cones of the I'etina, the avulitory hair-cells, the olfactorial cells, &c. Very little or nothing is known chemically with regard to a great number of the varieties of epithelium just enumerated. Microscopic research shows that the constituent cells are protoplasmic and contain nuclei, and we conclude that in their essential characteristics the protoplasm of these cells resembles that of other cells which we have better opportunities of examining chemically. With regard to the 442 THE TISSUES AND ORGANS OF THE BODY structure of the nuclei, there is nothing to add to what has already been said relating to cell nuclei generally. On the other hand, various specialised varieties_o£ epithelium differ considerably from ordinary protoplasm. The tegumentary epithelium loses near the surface its protoplasmic character, and the cells become filled with horny material or keratin ; this is exaggerated in certain parts like the nails and hair. Other forms of epithelial growth become calcareous, as in the enamel of the teeth ; and in the invertebrate sub- kingdoms, the exoskeletons and shells are found to be composed of chitin, spongin, conchiolin, and other forms of albuminoid material, more or less permeated with calcareous deposit ; and in a few cases, as in the ascidians, a carbohydrate material akin to cellulose is secreted by the epidei-mal structures. In the various forms of secreting epithelium there are many points of chemical interest to be noted. It will be more con\enient to reserve a detailed study of each until we actually deal with the secretions themselves. At the same time this will afford us an opportunity of glancing at secretion as a whole, the formation of ferments within cells, and the precursors of ferments or zymogens. In connection with nerve-epithelia, the only one which we shall discuss is the retina with its various pigments. PAVEMENT EPITHELIUM (ENDOTHELIUM) This form of epithelium, which consists of a single layer of flattened cells, fitting together like the stones in a mosaic pavement, is found lining the interior of the heart and vessels, and of the serous membranes. In the sei'ous membranes, openings exist between the cells, and from these stomata, as they are termed, capillary lymphatic vessels lead. The pulmonary aheoli are lined by flattened epithelial cells, very like those found in the vascular system ; from the point of view of embryo- logy they are however different, being hypoblastic, while endothelium proper is mesoblastic. These cells are extensible and elastic, and (as is seen in the capillaries, the walls of which consist only of endothe- lium) contractile also. The outlines between these cells can in all cases be rendered visible by staining with silver nitrate. The cement between the cells has the power of forming a compound with this salt, which is reduced by light, and minute granules of metallic silver are thus deposited in it, mark- ing out in black or brown lines the contours of the cells. The cement substance of epithelium is thus similar in this particular to the ground substance of connective tissue, and doubtless both have a similar com- position, consisting chiefly of mucin. Ki'iriiKiJi M 443 COLUMNAR EPITHELIUM This consists of a siiii^le layer of (4oii<,';ited nucleated cells. Such an epitlu'liinn lines the alimentary canal from the cardiac orifice of the stomach downwards, and also most of the ducts of secreting glands. When the column.-ir cells are short, the term cubical epithelium is employed. The border of columnar cells is more strongly refracting than the l)ody of the cell, and though there are no difterences in its resistance to reagents, it no douljt consists of somewhat modified protoplasm. The body of the cell is often vacuolated, and often contains numerous fat globules ; this is especially the case in the columnar epithelium of the small intestine, during the absorption of fat. These fat globules can be identitied by the deep black colour they give with osmic acid (see Absorption). Columnar cells often break down tu form goblet cells, and their more superficial protoplasm is transfoi-med into mucin, the chief con- stituent of mucus. CILIATED EriTHELIUM Ciliated epithelial cells are usually columnar in shape ; the cilia are protoplasmic tapering processes ; in the human subject 4 to 8 /.t in length, but in many invertebrates, like the mussel, they are much larger. Ciliary movement is independent .of the circulatory and ner\ous system, but it is dependent on nutritional changes occurring in the cell with which the cilia are connected, as all movement ceases when they are severed from the cell. The conditions most favourable to ciliary action are a temperature a little aljove that of the body (40° C), and free access of oxygen.' The movement is retarded by cold, liy heat a little over 40° C. (this coagulates the proteids of the proto- plasm of which they are composed) ; weak acids and all strong chemical reagents also kill cilia. Carbonic acid, chloroform and ether stop ciliary action, but the cilia recover when the poisonous vapour is replaced by oxygen. Distilled water acts as a protoplasmic poison here as elsewhere. If cilia are allowed to work after being removed from the body, they will in a varying time get languid and finally stop. If for instance a few bars of the gill of the sea mussel be mounted in a little ' Cilia will, however, like muscle, continue to work some time in an atmosphere containing no oxygen. Their protoplasm, like that of muscle, is able to store up oxygen for future use (Sharpey : sec Quain's Anot. vol. ii. p. 53). 44-1 TIIEjnssUES AND OROANS oF THE F.ODY .sea water, and watched with the microscope, they will probably finally be brought to a standstill in about two hours. This is no doubt a condition resulting from fatigue ; and fatigue in its turn is, as in mus- cular tissue, the result of the accumulation of the products of acti\dty. In the case of muscle, sarcolactic acid appears to be a substance that especially tends to cause fatigue ; probably in the case of cilia, an acid is also produced ; at any rate a dilute alkali will set the cilia going again. A drop of dilute potash (1 partKHO to 1000 of water), passed under the cover-glass, will caitse the cilia in the specimen just mentioned to work vigorously once more. If an acid is produced by the activity of the cilia, the potash no doubt neutralises this, and thus the activity of the cilia, which was hindered by the acid, is restored. The alkali may also act by increasing the amount of imbibition water {.-iee p. 188). In certain particulars, ciliary action resembles amoeboid action : it is for instance accelerated and hindered l)y the same reagents. On the other hand ciliary movement resembles muscular movement : it is not due to contractility occurring in all directions, but, as in muscular movement, in one direction only. Engelmann ' has suggested that the contractile protoplasm is situated chiefly on the concave side of the curved cilium, so that on contraction the cilium will be brought down- wards, and on the contractile motion ceasing, the cilium will be erectef the secretion, by the formation of what are called goblet cells (tii;. 71). The more superficial part of the cell protoplasm undericoes certain changes, which lesult in the formation of a highly refracting globule of mucin : the precursor of mucin within Fig. 71. — (4oliIet t-ell.-. Hi^'Lly iimfniific'l ( Kloin). the cell is called mucinogen : after the mucin is expelled, the basal portion of the cell alone remains. This may once more grow into a normal epithelial cell, and may again undergo this mucoid degeneration. In other cases the mucin is chiefly furnished by certain small racemose glands, situated beneath the general epithelial lining, with its duct opening on the surface. Heie the cells of the acini of the gland undergo, as in the mucous .salivary glands, the .same transforma- tion of the cell protoplasm into mucinogen, and this suspended in an alkaline liquid is expelled as mucin tlirougli the duct upon the surface of the mucous membi-ane. The chief properties of mucin are its stickiness and A-isco.sity and its solubility in dilute alkalis like lime water : from these solutions it is readily precipitated by acetic acid, in excess of which it is insoluble. In composition, it consists of a globulin in combination with a carbo- hydrate called animal gum. By treatment with dilute sulpliuric acid, the animal gum is converted into a sugar, which, like grape suirar, reduces alkaline solutions of cupric hydrate. It is probable that there are several ditierent kinds of mucin, i.e. different proteids combined with animal gum ; that obtained from the snail, for instance, is distingui.shed by Hannnarsten ' into foot mucin (obtained from the foot", and mantle mucin (obtained from the mantle) ; the properties of these two substances are .slightly different from one another, and from the mucin obtained from saliva, mucus, ttc. : and these in turn differ from the mucin found in the ground substance of connective tissue. All mucins, however, are alike in the reactions that Hamniursten, Pfiiigprf Arrhir, xxxvi. 37J5. 446 IHE TISSUES AND nROANS OF THE BODY liave been already mentioned. \ iz. tlieir tenacity, their precipitability by acetic acid, and the fact that a leducing sugar is obtainable from them.' (8ome more particulars concerning mucin will be found under the heading Connective Tissues, p. 476 ; S'i'' also p. 144.) The pseudo-mucin of ovarian fluids differs from true mucin in not being precipitable by acetic acid ; the same is the case with colloid material, formed in colloid degenerations of tumours, and contained within the vesicles of the thyroid Ijody. Pseudomucin and colloid substance are probably identical {see p. 3.53). Xucleo-albumins are like mucin in their physical characters, and in many of their reactions. The slimy material in bile was long mis- taken for mucin ; it, however, is not a compound of a proteid Avith a carbohydrate, but with the phosphorised substance known as nuclein. Such a nucleo-albumin we have already de.scribed as a constituent of the white Ijlood corpuscles ; similar substances occur in the other animal cells : Hammarsten,- for instance, has described one in the cells of the submaxillary gland (which C(mtain however true mucin in addition). 8uch considerations show that all slimy suljstances do not necessarily contain mucin ; and it is especially the nucleo-albumins that must be carefully distinguished from that material. The chief solid constituent of mucus, then, is mucin ; epithelial cells, and debris of such cells, and a few leucocytes are also present, and these are suspended in a liquid which is doubtless a transudation from the blood ; it has an alkaline reaction, and contains a certain small proportion of proteids and extractives, as well as mineral .salts like those in the blood itself. The following table gives a few analyses that have been n)ade <>f iimcus.^ Ti-aclieal Mucus 1 Xasal Mucus Parts per 1000 (Wright) ' ( Berzelius) (Jfasse) Water O.ofrii 9:«-7 95.5-f! Solids 44(1 fi6-3 44-4 Mucin H20 r.3-3 23-7 < )ther or£{anic suV).stances 4-<) 10-4 9-8 Fats — — 2-8 Mineral Salts .... ;V0 -y(y 81 I These are the three characteristics of the inucin-f,Toiip as defined by Hammarsten, Chem. Centralbl. 1884, p. 814. - Hammarsten, Zeit. phijaiol. Chcm. .xii. 163. •" I am indebted to Charles's Physiological and Pathological Cheinistrg, p. 289, for this table. Ei'iTHELir.M 447 Tlu' iiiiu-us of various parts diHers ii little in ai)pearaiice and in ivaction. Charles' de.scril)es the varieties as follows : — Buccal mucus. Ti-ansparent, viscid, alkaline. Stomachal mucus. Thready, greyish, alkaline. /Greyish, viscid, alkaline, rich in fatty Fntestinal mucus. ' particles, and suspended epithelium ( cells. Vesical mucus. Gives a cloudy appearance to urine. Vaginal mucus. Slightly viscid ; acid. Cei'vical nnicus ) Slightly viscid, greyish, tj'ansparcnt, (that of the neck of the uterus) ) alkaline. The amount of mucus normally secreted is small, merely sufficient to lubricate the surface ; in the case of the respiratory cavity, it entangles dust particles ivom the inspired air, and it together with this foreign matter is removed by the activity of the ciliated epithelium. It is stated that the mucus of the alimentary tract may aid digestion. In cases of mild inflammation of the mucous membranes (catarrh), the amount of mucus secreted is inci'eased. In more severe cases, the leucocytes become abundant, and the secretion is called muco-purulent, that is. a mixture of pus with mucus. Sputum consists of the secretion of the mucous membrane of the respiratory tract mixed with a certain amount of saliva and occasionally nasal mucus. The following are some particulars concerning the diffe- rent kinds of sputa in a few important diseases. QtKintifij. — This is very variable, especially in bronchitis. In phthisis it may range from 80-150, in pneumonia from 26-300 grammes per diem. Colour. -In chronic inflammation of the bronchi it may be studded with black particles of carbon, especially in those living in a sooty atmosphere. In acute cases of bronchitis it is yellowish, owing to admixture with pus. In pneumonia the typical sputum is rusty, i.e. brown or yellowish-red, from the presence of altered blood pigment. As hepatisation proceeds, the sputum becomes greyish or purulent. In phthisis the expectoration may be tinged with bright blood. Vixcldity. — The most viscid expectoration is that of pneumonia. The most watery expectoration occurs in the early and late stages of bronchitis. Odour. — In bronchiectasis and gangrene of the lung, the sputum has a putrid (»dour, 1 Charles, loc. cit. p. 288. 448 THE TISSIES AND ORGANS OF THE I'.oDY Quantitutlre Anali/gfs (^Percentages) 1 D isease vv^ater Solids Organic Matters iluciu Proteids Fat Extrac- . , tives ^'" Remarks 1 Bronchitis 97-6-98-3 1-7-2-3 1-17-1-r 0-fi9-l-2 0-08 i 0-53-0-64 Pnenmouia 90-09-93-6 6-3-901 5-.5-.s-35 1-1-1-39 3-09 0-02-0-32 2-8-3-95; 0-66-0-77 Fibrin present in simtum. Sputum very ricli in NaCl, increasiii? as hepatisatiou prcceeds.' Note high iKTcen- tage of pro- teiils ami ex- tractives. riitlii;;!- 1 1 1 94-5 5-5 4-7 l-s-2-4 (1^9-0-39 0-30-0-39 lb-201 1 0-76-O-8 Often contains tissneeleraents of lungs, of wliich ela.stic fibres are most easily recog- nised. It also contains tu- bercle bacilli. SECRETING EPITHELIUM Epithelium is a tis.sue which exhibits varying degrees of vitaUty in different parts according to its function ; thus the outer portions of the epidermis are ahiiost entirely iKtn-protoplasmic, and they undergo few physical and chemical changes of any kind : their function is simply protective. In the secreting glands, on the other hand, the epithelial cells are composed of protoplasm which is the seat of the most active and remarkable chemical operatiuns. the building up of new substances which are di.scharged as a secretion to fulfil important functions elsewhere : or the substances may be simply taken from the circulating fluid by the cells, and poured out from them to form an excretion : that is, these substances are simply gut rid of and discharged from the body by this means. A secreting epithelium may Ije considered as a partition between the blood, or, more properly speaking, the lymph, on the one side, and the lumen of the secreting gland on the other. From the lymph the materials are taken by the seci-eting cells and then worked up into the components of the secretion, and tinally discharged on the other side into the lumen, and thence by the ducts of the secreting gland to their destination. A useful contrast is drawn by Dr. McKendiick - between the activities of three important varieties of organs : — ' The amount of cliloricle> in the urine is correspondingly low. - Physiology, vol. i. pp. 4S4-.". Kl'l'IHHIJlM 449 (1) Muscles. (2) Electrical organs. (3) 8»^ert*ting cells. If a be contraction, b electromotive phenomena, ami c metabolic or chemical changes : in a muscle a is large, b and c relatively small ; in an electrical organ, 7ny, 632,638. G G 2 452 THE TISSUES AND ORGANS OF THE BODY this conversion into keratin is still more marked, and in addition there is a deposit of calcareous salts, especially calcium phosphate. In hairs and feathers also the chief organic constituent is keratin ; cells filled with fat or pigment granules may occur in the medullary portions of the hair. In both hair and feathers silica has been described as a con- stant and important mineral constituent (27-40 per cent, of the ash ; von Bibra) ; iron may also occur. The deeper portions of stratified epithelia, which become horny in their surface layers, remain protoplasmic ; in the skin the protoplasmic layers (Malpighian layer) and the horny layers proper are separated by two thin layers, the stratum granulosum and the stratum lucidum. The granules in the former layer are composed of a su1)Stance which stains deeply with carmine (Langerhans '). It is termed eleidi7i, a,nd is supposed to be an intermediate condition in the replacement of pro- toplasm by keratin. In the cells of the Malpighian layer, granules of a dark pigment called melanin are found ; these are especially abun- dant in the skin of negroes.^ The ceils of the epidermis have a small amount of cementing substance between them, which, like the ground substance of connective tissue, dissolves in weak alkalis : and Ijy such treatment the cells may be separated from one another. Keratin Keratin or horny material belongs to the class of substances that are called albuminoids. It is exceedingly insoluble, and can be freed from other substances by treating cuticle, hair, hoofs, nails, ifcc. with ether, alcohol, water, and dilute acids. A very similar substance called neurokeratin can be obtained from nervous structures, these being, like the epidermis, epiblastic in origin. It is not affected by boiling with water ; but when heated with water in closed vessels to 150°-200° C. it forms a turbid solution. It is not affected by weak acids in the cold, but is dissolved by boiling glacial acetic acid ; it is decompo.sed by boiling with mineral acids, yielding with sulphuric acid products very similar to those obtained from proteids, viz. leucine, tyrosine, aspartic acid, and volatile fatty acids. Like proteids also it gives off when burnt the same peculiar odour. Elementary analyses, from their close resemblance one to another, seem to point to the fact that keratin is a chemical unit, but as in ^ Archiv f. mikr. Anat. laTd. - The dark pigment deposited in the skin in cases of Addison's disease is apparently of tlie same nature. Konitiii fi'Diii Iluir Nails V. I.a.-r Mulder c 50-60 51-00 H G-3G 6-94 N 17-U 17-51 0 20-85 21-75 s 5-00 2-80 KI'IIIIKI-IIM 453 the case of |>roteids we are not iic(iuaiiitt'(l with its lational forniuhi. The following five analyses ' show, liowever, discrepancies in the per- centage of sulphur present ; there is a reniarkahly large percentage of sulphui-, and most of it is very loosely coniljined ^^ no doubt the diflferent methods adopted for the estimation of the sulphur employed Ity the various investigators will sutiiciently account for the different results obtained, sulphur l)einga difficult substance to estimate correctly when occurring in organic compounds. Horn Hi-of Hair Tilaims MuMer r^ >'"'" 'f' "^. Chitteiideu ' 51-03 51-41 49-45 6-80 6-96 6-52 16-24 17-46 16-81 22-51 19-49 23-20 3-42 4-23 4-02 Melanin The term melanin has been applied to a large number of black pigments occurring in the body ; thus we have already noted a black pigment in the blood of persons affected by malaria and other diseases (Melantemia, see p. 310). This, no doubt, is derived from hajmoglobin ; and perha2:)S the other black pigments of the body occurring in the retina, in the skin, and in melanotic sarcomata may ultimately have the same origin. There is no doubt that these pigments are, however, not all identical ; elementary analyses show this ; for instance, iron is present in some, absent in others. In the tissues of the lungs and Vjronchial glands the black pigment that occurs there simply consists of particles of carbon breathed in with the atmospheric air. The black pigment of the skin and of the hair has been examined by Sieber, who made some few percentage estimations of the elementary composition of the substance, but obtained very discordant results. The elements present are carbon, nitrogen, hydrogen, and oxygen.'* It has never been crystallised ; it is soluble in water, alcohol, ether, acids, and strong alkalis ; the brown solution produced by dissolving it in hot potash is decolorised by chlorine water. (The subject of melanin will be more fully dealt with under the Retina (p. 457) and Melanotic Sarcoma, chap, xxiii.). 1 The first four analyses are quoted from Hoppe-Seyler's Physiol. Chem. p. 90. * Hoppe-Seyler, Physiol. Chem. p. 91. ^ Zeit. Biol. xxvi. 291. * See also Hodgkineon and Sorby, Join-n. Chem. Sac. 1S77, p. 427. 464 THE TI.SSUES AND ORGANS OF THE BODY Turacin This is the only one of the many pigments in birds' plumage that has been satisfactorily examined. It is obtained from the touracon, or plantain- eater, of the Gambia. It is of a crimson colour : it is not crv^stalline ; it shows two absorption bands between D and E, and is remarkable as "being one of the few animal compounds that contain copper (Church '). Skeletins The tenii skeletin- has been applied to a number of nitrogenous but sulphur-free substances found in the skeletal tissues of invertebrates. They appear to be intermet. de chimie et de phys. serie 3, tome Ivi. p. 153. Ki'iTiiKi.ir.M 457 called the layer of rods ;iiid cones ; ami it ii;is been satisfactorily proved that it is this layer upon which the images of external objects are focussed by the refractive apjiaratus in front of it. The impressions of light affect tlie rods and cones, and thence they are pi'opagated as uervous impulses via the optic nerve to the brain. External to the layer of the rod.s and cones is a layer of hexagonal epithelium cells con- taining a black pigment. It is these two layers that we have to describe in detail. The retina as a whole gives indications of its twofold structure, nervous and epithelial. Its reaction is stated to be acid ; and, like most animal tissues, it becomes opaque after death. Water dissolves out from it proteids, gelatin, and mucin, the two last-named substances being probably derived from the supporting connective tissue it contains. Alcohol dissolves lecithin from its nerve-fibres and cells. Other reagents are employed to dissolve out other constituents, such as the pigments, from the rods and cones. Cahn ' gives the following quantitative results : — Water . . . . . 86—89 per cent. Solids U- 11 Proteids 2 .... 4— 6 „ Gelatin . . . . . 13 — Iw ,, Cholesterin .... 0-3 — 0-8 ,, Lecithin 1-0-2-9 „ Fat 0-05 -0-5 Salts 0-7 -1-2 INIost of our knowledge of the chemistry of the retina is the result of the labours of Kiihne and his pupils. A resume of the chief facts will be found in Kiihne's article in Herrmann's ' Handbuch der Physiologic' (1879), vol. i. p. 235. The Hexagonal Pigment Cells of the Retina The pigmentary layer of the retina was at one time supposed to be a part of the choroid or vascular coat of the eye, but the facts of embryology have shown that it is in reality part of the retina, and is developed like the rods and cones from the epiblast, whereas the choroid is developed from mesoblastic tissue. The choroid, however, contains branched cells in which is pigment identical with the black pigment of the retina. ^ Hoppe-Seyler's Phijsiol. Chem. p. 699. - Tliree in number — one resembling rajosin, coagulating at 55° C, another like mucin, and a third like serum-albumin. 458 THE TISHUES AND ORGANS OF THE BODY The cells are flattened, six-sided, and form a pavement covering the outer portions of the rods and cones, and sending down long processes between them. Extei-nally the cells consist of a layer of neurokeratin ; internally they are protoplasmic ; in the protoplasm are found one or two nuclei Tig. 73. — Pigmented Epithelium of the Hnman Retina (Max Schultze) highly magnified, a, Cells seen from the outer surface, h, Two cells in profile with fine ofEsets extending inwards, c, A cell still in connection with the outer ends of the rods. and large numbers of black rod-shaped pigment granules. Deposits of a substance called myeloidin by Kiihne, and in some animals of yellow fat-globules, are also found. The black pigment. — Fuscin. — Owing to movements in the cell proto- plasm of the nature of amoeboid movements, the granules of black pigment are differently distributed at different times ; after keeping a frog for several hours in dai'kness, the pigment will be found in the cell bodies, and in the parts of the processes nearest to the cell bodies. But if the frog has been exposed for a similar time to sunlight before death, the pigment granules will be distributed chiefly along the pro- cesses, and a relatively small number remain in the bodies of the cells themselves. In some animals (dog, cat, &c.) much of the retinal epithelium contains no fuscin, but the cells are filled with fine crystals (Max Schultze) ; this forms the tapetum. In some fish, e.g. bream, the tapetum (or pseudo-tapetum) contains guanine, a highly refracting substance ; while in the ox and sheep the tapetum is merely fibrous tissue (Kiihne and SewalP). Fuscin is one of the group of black pigments termed melanins. It has been investigated by Berzelius, who found it contained a small quantity of iron, by Scherer, who found no iron, and also by Rosow and Sieber. The percentage composition obtained by the various observers shows great discrepancies, and this, taking also into account their methods of preparing the pigment, renders it probable that they were not dealing with a pure substance. The failure of some observers, ' Verhandl. dc?- uatin'hist. Vereiiis Hcidelherg, N.S. ii. Heft v. El'lTHELir.M 469 for instance, to obtain evidence of the px*esence of iron was due as Moi'ner ' points out to their having used hydrochloric acid at one stage or other of their operations ; tliis acid dissolves out nine-tenths of the iron from the pigment. May's method - of preparing fuscin is to boil several hundreds of retinH' in alcohol, then in ether, lastly in water ; the residue is subjected to tryptic digestion for twenty-four hours ; the pigment, nuclein, and neurokeratin remain undigested; the tirst-named impurity is dissolved by trituration with alkali, and the last-named must be picked out as well as possible with forceps. Fuscin dissohes by boiling it a long time with concentrated sul- phuric acid, or concentrated caustic alkalis. Like all the other retinal pigments, fuscin is bleached in the air, only very slowly indeed. This is probably due to oxidation. The physiolo.tfical relation of the fuscin-bearing cells with the rods and cones will be dealt with in the consideration of those structures. There is considerable doubt as to whether this pigment is ultimately derived from haemoglobin ; Krukenberg considers it is more closely related to the lipochromes or fatty pigments. It is, however, un- doubtedly nitrogenous. It does not belong to the group of brown pigments, many of which occur in plants called humous substances by Hoppe-Seyler,^ since on fusing with alkali it yields no pyrocatechiu or protocatechnic acid (Hirschfeld ^). {See p. 149.) J/^'^/oiV/i?*.— Myeloidin, or myeloid substance, is not a chemical unit. The term is used as indicating that the cells contain a sub- stance similar to that which forms the white substance of Schwann in nerve-tibres. It is also found in the rods, and will be there more fully dealt with. Yeliow fat-glohules. — These are not present in all animals; they are especially abundant in the retina of the frog. The pigment can be ■extracted by ether, carbon bisulphide, benzene, iVrc. It shows two absorption bands between F and G. The yellow pigment was called lipochrin by Kiihne. It is, however, exceedingly probable that this is the same pigment found generally in adipose tissue ; it belongs to the class of pigments called lipochromes or luteins, and like all these pigments is slowly bleached by sunlight. ^ K. A. H. Momer, Zeit. physiol. Chetn. si. 66-140. In this^paper the references to the writings of the observers mentioned above will be found. - Untersuchungen aus d.physiol. Inst, der Univ. Heidelberg, ii. 324. 3 Zeit. jphysiol. Chem. xiii. 66. * Ihid. xiii. 407. 460 THE TISSUES AND OKGAXS OF THE BODY The Rods and Cones The rods and cones form the nerve-epithelium which receives the impressions of light from without. The accompanying figure shows the general shape and relative size of a rod and a cone. Each consists of two distinct segments, an inner and an outer. The outer or narrower segment is doubly refracting, and is stained darkly by osmic acid, while the inner segment is singly refracting, and stains as protoplasm does with carmine, magenta, Loc. fit. 462 THE TISSUES AND ORGANS OF THE BODY are no well-defined bands, but a general absorption of the central regions of the spectrum. White light bleaches rhodopsin most quickly^ then follows gi'een, blue, and, after an interval, yellow, violet, orange, and red. The sodium tlanie takes about two hours to bleach a frog's, retina, but is more convenient than a red flame, as by light of a red colour it is difficult to detect and avoid blood stains. The intermediate stage of visual yellow is bleached more quickly by rays from the violet end of the spectrum, or it may be that less yellow is produced under the influence of such rays. The rapidity with which ^isual purple fades increases with the temperature up to 76"^ C, at which temperature it disappears instantly even in the dark. Alcohol, ether and chloroform, caustic alkalis and acids destroy the pigment. Putrefaction and tryptic digestion do not. Oxidising agents, such as ozone, liydi'ogen peroxide, osmic acid (the black colour produced with myeloidin having first been destroyed by hydrogen per- oxide, or the myeloidin may be previously removed by ammonium chloride), ferric chloride, potassium chlorate, and iodate have no effect. These reactions show that visual purple is a substance ali'eady highly oxidised. Such reactions, however, are of little interest compared to those produced by the action of light. That the bleaching action of light occurs during life was most conclusively shown by those experiments, in which Kiihne succeeded in obtaining what may be compared ta photographic impressions upon the retina : these were obtaiiied in. rabbits. The animal was first put in darkness by covering its head with a black cloth, it was then exposed to the light of a window, and immediately decapitated, the eyes removed, and the retinal colours fixed by a solution of alum : a small bleached area corresponding in shape to the window, and about a millimetre square, was found on the retina next day. Such optograms may be preserved a long time by drying the retina in vacuo after removal from the alum solution. Regeneration of visual purple. — This is continually taking place during life, and occurs e.specially in the dai'k. This phenomenon appears to be associated with the hexagonal pigment cells which send down their processes between the outer segments of the rods ; if a piece of a fresh retina be lifted from the black pigment cells, and then be exposed to the light, it will become bleached, and if then the retina be placed in darkness the colour will not return as it does in the rest of the retina ; but if the flap be replaced so as to touch the hexagonal cells, regenera- tion of the purple occurs. This function of the hexagonal cells does not seem to depend on the amount of fuscin they contain. It is possible KIMTIIKLU'M 463 that the rods cont;iiu the precursor of visu;il purple, and this is acted upon by some other substaiue from the hexagonal cells ; or it may Ije that the hexagonal cells withdraw the supposed substance from the rods and work it up into visual purple. The subcutaneous injection of pilocarpine causes in the frog (not in the rabbit) a hastening in the regeneration of the visual purple (Dreser). Thi' physiological iises of visual purple. — The rays of light which are focussed on the rods and cones produce in those structures certain Fig. 75.— Diagrams of Absorption Spectra 1, of visual purple ; 2, of visual yellow : 3, of xantho- phane in ether ; 4, of rhodopliane in turpentine ; 5, of chlorophane in ether. This method of representing absorption spectra has been explained in connection with fig. 58, p. 27G. obscure chemical changes which no doubt are very similar to those produced by the action of light upon a sensitive photographic plate. The most tempting hypothesis suggested by the discovery of visual purple was, that that substance is itself the sensitive chemical material, the changes in which are indicated by the changes of colour it 464 THE TISSUES AND (>J{(iANS OF THE BODY undergoes. But further research lias sliown that tliis view canndt be adopted, and that probably the changes in the visual purple are merely accidental accompaniments of other chemical changes that are as yet undiscovered. This conclusion is derived from the consideration that vision occurs in the absence of visual purple altogether ; in birds and reptiles, for instance, it is absent, and in man the part of the eye which is most sensitive to light — the fovea centralis — contains no rods, and therefore no visual purple. It is altogether absent in the bat ; but, on the other hand, it is present in the owl. Both these animals are nocturnal, so the habits of the bat will not explain its exceptional condition. Chromophanes, the pigments of the cones. — In l)irds, reptiles^ and fishes, the inner segment of the cones contains a coloured oil-globule, the colour varying greatly. By using large numbers of birds' eyes Kiihne and Ayres ' succeeded in preparing these coloured fats in large quantities. The retinae were first dehydrated by absolute alcohol and then extracted with ether ; the ethereal solution of the fat was evaporated to dryness and the residue treated with caustic alkali ; the coloured soaps so formed were freed from excess of alkali by washing with water, and were separated by means of their different solubilities in various reagents ; petroleum ether dissolving out a green substance, ether a yellow, and benzene a red material. It was not found possible to obtain the pigments in a state of purity, nor in a crystalline form ; when the pigments were in association with fats or fatty acids instead of soaps they showed no difference in their solubility ; the .soaps can be decomposed by means of glacial acetic acid. The pigments are called chromojihaties — the green one, chl(jrophane ; the yellow one, xan- thophane ; ^ the red one, rhodophane. These pigments belong to the class of pigments called lipochromes, and their spectroscopic appear- ances (fig. 75, spectra 3, 4, and 5) should be compared with those of other lipochromes we have considered before (compare serum-luteiu, p. 254 ; tetronerythrin, p. .325, and lipochrin, p. 459). Chlorophane shows two absorption bands ; xanthophane and rhodophane each show one. The position of these bands shifts a little according to the solvent used, but, as in all other lipochromes, the bands are towards the blue end of the spectrum. Tliere is always, in addition to the bands, a considerable absorption of the violet extremity of the spectrum. Like other lipochromes the chromopiiaiit'S, when evaporated to dryness, give the following colour reactions : — 1 Kiihne and Ayres, Joiirn. Physiol. \. 10!). - Xanthophane is not identical witli lipochrin obtained from the frog's hexagonal epithehum ; the two pigments differ in solubilities and spectroscopic appearances. Ei'JTiiKi-ii .M 465 1. Concentrated suli)hurit^ ucid ; tlie frai^iuonts undcrj^o ericliondrium, as the investing membrane may be called at this stage. The osteoblasts appear first to deposit fibrous structures of a non-calcified nature ; these are termed osteogenic fibres, and are ajiparently collagenous in nature ; they form the intercrossing fibres of Sharpey. In the development of the so-called membrane bones (e.g. the flat bones of the skull, clavicle, &c.) there is no prefigurement of the adult bone in cartilage, but we have merely to deal with a subperiosteal deposition of osteo- genic fibres, and this formation in the case of both varieties of bone is subse- quently calcified by the agency of the osteoblasts which deposit calcareous granules around them till a complete investment is formed. In some animals, like the elasmobranch fishes (rays, sharks, dog-fishes), calci- fied cartilage remains in the adult and is never replaced by subperiosteal bone. In invertebrate animals, bone is not found. Cartilage is occasionally found in invertebrates (Sepia, Limulus, Spirographis, &c.). 9. Dentine. — This substance forms the chief part of teeth ; it is a calcified form of connective tissue ; it is developed by the agency of certain cells called odon- toblasts, and the deposition of calcareous matter is in process of development preceded by the formation of a non-calcified substratum which is termed odontogen. Enamel is a calcified form of epithelium ; it will, however, be convenient to consider it here with the other calcified structures of the body. The scales of fishes may also be convenienth' studied in this connection. In all these hardened tissues the chief salt which is present is calcium phosphate. In such a summary of the characters of the various forms of con- nective tissue as that just given, we see indicated the chief points Avhich it will now be our object to consider more fully from a chemical point of view. We shall have to deal first with the cells of connective tissue, then with the white fibres (collagen and gelatin), then with the elastic fibres (elastin), and lastly with the ground substance (mucin). In connection with adipose tissue we have the important subject fat to consider ; in connection with cartilage, chondrigen, and chondrin ; and in connection with bone and other calcified structures we shall have to take up both the organic and inorganic constituents. Most of the organic constituents found in connective tissue belong to the important but somewdiat heterogeneous group of the albuminoids, a class of substances closely related to the proteids. In studying each of these albuminoids, collagen, gelatin, mucin, elastin, chondrin, tfec. it will be necessary to consider their mode of preparation, their chemical And physical characters, and their physiological meaning and im- portance. 470 THE TISSUES AND OKGAN'S OF THE I'.ODV THE CELLS OF CONNECTIVE TISSUE The cells of connective tissue vary nmch in shape and histological appearances ; some are branched and some not ; some are vacuolated and others tilled to a greater or less extent with fat-globules, this being especially the case in adipose tissue, or in areolar tissue, which is being converted into fatty tissue. The cells also differ functionally ; they are most active in developing bone, and the different activities of osteo- blasts, osteoclasts, and odontoblasts have already been alluded to. The knowledge of the chemical properties of the cells is, however, very limited ; the way in which they are sparsely scattered through the tissue necessarily renders chemical investigation very difficult. The protoplasm of the cells consists chiefly of proteids, and their nuclei of nuclein, or of a mixture of nucleins (phosphorised nitrogenous sub- stances). The cells of the cornea are very contractile, and Kiihne ' surmised that they are closely related in composition to muscular substance. This was confirmed by Bruns,^ who obtained myosin from the cornea, doubtless derived from its corpuscles.'* By some histologists the formation of connective tissue fibres is considered due to a deposition in the ground substance ; this deposition may, however, be influenced by the cells in some way or other ; others consider that the fibres are formed by the direct conversion of the cell protoplasm into fibrous material. The former view is, however, the one generally held."* THE WHITE FIBRES OF CONNECTIVE TISSUE The white fibres consist of a substance called collagen, and this, by boiling or by treatment with acids, is converted into gelatin. Collagen Collagen may be prepared in the following way : finely divided tendons are soaked in water to remove proteid substances, then in 1 Kiihne, TJnters. it. das Protoplasina, p. 123. - Bi'uns, Hoppe-Seyler's Med. Chem. Untersuch. p. 2(>0. '> The following points may here be added concerning the chemistry of the cornea. The eiiithelium is protoplasmic; the posterior homogeneous membrane is elastic in nature. The greater thickness of the cornea is composed of About sixty layers of alternating lanielliB of fibrous tissue (Bowman). This like other fibrous tissue is collagenous in nature, the interfibrillar and intercellular substance being composed of mucin. The anterior homo- geneous lamella is similar in structure. The erroneous idea that the cornea contained chondrin was first fully pointed out by Morochowetz (Verhcmdl. d. naturhist. vied. Vereins Heidelberg, vol. i. part v.) ; 1000 parts of corneal tissue contain 242 of solids, of which 204 consist of collagen, 28 of other organic matters, 10 of ash (His). The sclerotic is also ordinary connective, tissue. In birds, however, it contains bony plates. * See Quain's Anaf. 9tli edit. ii. 70, 71. TJIK CONNKCllVK 'l-ISSTES 471 lime- water to remove tlie interHhrilljir ^lound substance (mucin) ; they are then washed with water, dilute acetic acid, and finally water again. The residue is collagen. By the action of dilute acids or of boiling water collagen is converted into gelatin. Hofmeister ' found that gelatin can be transformed into collagen by heating it to 130° C. By this treatment it loses water, and the reaction may be represented by the t'nllowing formula: — Cl02-"i5i^ 31*^29"" H'20=;C,ooH,.,f)N3iOoH [geliitiii] [colliii-'eii] in other words, collagen is the anhydride of gelatin. As will be seen by the above formula, gelatin, unlike proteids, contains no sulphur. Schiitzenberger, who gives the empirical formula C7,;Hio.iN240.29 to gelatin, also regards the sulphur described by other investigators as being due to admixture with proteid impurities. The chief organic substance in bone (ossein) is identical with collagen. Gelatin rrep(vration. — Gelatin may Ije obtained from the white tiljres of connective tissue either by treatment with dilute acids, by treatment with boiling watei-, or, best of all, by treatment with water in a Papin's digester at the temperature of 110 -120° C. Bones, when similarly treated, also yield gelatin. Hofmeister prepares pure gelatin from commercial gelatin by soaking the latter for several days in distilled water which is frequently changed. The salts pass out by osmosis. The gelatin is then dis- solved in hot distilled water and filtered while hot (by the aid of a hot-water funnel) into 90 per cent, alcohol. As the hot solution falls into the alcohol it is precipitated ; the alcohol is then evaporated otf, and the process may then be repeated, and finally gelatin is obtained so pure that it only contains 0-6 per cent, of ash. This process may very conveniently be employed for the preparation of pure gelatin for cultivations of bacteria. Properties. — Gelatin is insoluble in cold water, but soluble in hot Avater ; the hot aqueous solution sets into a jelly when cold. This property of jellying or yelatinisinf/ is possessed by solutions containing 1 jjer cent, of gelatin or upwards. With every successive solution, however, the power of gelatinising is lessened ; it is entirely destroyed by twenty-four hours' boiling with water ; it is more quickly lost at liigher temperatures and instantly lost at the temperature of 140° C. In order to raise an aqueous solution of gelatin to these temperatures, 1 Hofmeister, Zeit. ijlnjsiol. CJiein. ii. 315. 472 THE TISSUES AND ORGANS OF THE IJODY it is necessary to eui2:)loy either a digester or sealed tubes. Gelatin is insoluble in cold glycerine and soluble in hot glycerine ; such a hot solution gelatinises on cooling (glycerine jelly) like an aqueous solution. Gelatin is insoluble in alcohol, ether, and chloroform. Gelatin is precipitated by saturating its solutions with neutral salts, like magnesium sulj^hate or ammonium sulphate (O. Nasse).^ Tliis is also true for gelatin which has been altered by the action of hot water so as to be no longer or only partially gelatinisable. Aqueous solutions of gelatin are powerfully hevorotatory ; the amount of rotation varies, however, like the power of gelatinisation, according to its treatment with hot water and also according to the temperature. At 30° C. (a)p= —130° (Hoppe-Seyler). Solutions of absolutely pure gelatin transmit a perfectly continuous spectrum as far as wave-length 2024 ; impure solutions and films of gelatin will not transmit rays nearly so far into the violet, and the sensitiveness of photographic plates to the most refrangible rays lies entirely with the character of the gelatin (Hartley ^). Gelatin is not precipitated by acetic acid. Hence it can he readily distinguished from mucin or chondrin. It is not precipitated by acetic acid and ferrocyanide of potassium, by lead acetate, nor by most of the heavy metallic salts that precipitate proteids. Hence it can be readily distinguished from proteids. It gives a violet colour with copper sxdphate and caustic potash like proteids, but it gives only a faint xanthoproteic reaction (Salkowski.^). Gelatin is precipitated by tannic acid and also by mercuric chloride. The compound with tannic acid is an interesting one, as it is the formation of this substance during the process of tanning that converts hides into leather.^ Deririitires of gelatin. — The prolonged action (twenty -four liours) of boiling water, or the shorter action of water heated above the boiling point, destroys the gelatinising power of gelatin. It undergoes a iJrocess of hydrolysis. In peptic digestion a similar change occurs. In i^ancreatic digestion the same j^roducts are formed, but they undergo, as proteid peptones do, a partial decomposition into amido-acids and other simpler substances. The gelatin peptones, as thej' are termed, formed by these ditferent processes are really more akin to the proteoses than to true peptone. They are jjrecipitable by saturation with magnesium * 0. Nasse, Ffli'iger's Archiv, xli. 504. 2 Hartley, Trans. Chem. Soc. 1887, p. 59. ^ E. Salkowski, Zeit. physiol. Chem. xii. '215; Ber. Miti. Wuchcnsdi.. 1885, No. 2. * For particulars of the chemical properties of the compound of tannic acid ancVgelatin, see Bottinger, Annaleii, f. Chem. u. Pharm. ccxliv. 227. Till-; ('(>NNi':("rivH 'I'issues 478 ■sulpliatc or aiiiiiKiiiiiini suliiliatc. liikc the gelatin Iroiii wliicli tlicyare derived they give the xantiio)>niteic reaction very faintly. Hofiueister di.sting'uislie.s two of tliese peptonc-liku substances ; one he terms seml-ghitin is insoluble in 70-80 i)er cent, alcohol, and is precipitated by platinum tetrachloride ; the other, called heml-eolliti, is soluble in 70-80 per cent, alcohol, and is not precipiiable by platinum tetrachloride. The following equation exhibits the rclationslii]) of these substances to collagen : — C,„,H, „N„(),, + :5H,0 = C,,H,,N, A-. + C,,H,„N„(),„ [collagen] [seuii-glutiii] [hemi-colliu] Chemical constitution of gelatin. — The formulaj that have been given to gelatin by different observers are purely empirical ; as in the case of proteids, though there are several theories as to the constitution of the molecule, we are practicallj' entirely ignorant on this point. Strong agents, like sulphuric acid or putrefaction, decompose the substance, forming glycocine, leucine, various fatty acids, carbon dioxide, and ammonia. Schiitzenberger ' heated gelatin in sealed tubes with baryta-water to 200° C. The products he obtained were ammonia, carbonic acid, and oxalic acid, these compounds being in the ratio of the products of decomposition of urea. The other products were amido- acids of the acetic series, the most abundant in the order of their importance being glycocine (amido-acetic acid), alanine (amido- propionic acid), amido-butyric acid, and leucine (amido-caproic acid). The general conclusion is drawn that gelatin, like a proteid, is a compound of urea •with certain amido-acids. It will be noticed in all these decompositions no substances like tyrosine con- taining aromatic radicles are obtained as they are from proteids. THE ELASTIC FIBRES OF CONNECTIVE TISSUE The elastic or yellow fibres, the microscopical characters of wliicli liave been already described, consist chemically of the very insoluble albuminoid which is called elastin. During development, elastic granules ai'e deposited either in rows, which subsequently become fused together end to end to form fibres, or they may be deposited in patches, and by their fusion form an elastic membrane, as in the elastic (fenestrated) membrane of arteries. The elastin granules make their appearance first in the neighbourhood of the cells ; this renders it probable that the deposition of the granules is influenced by the cells, "but it does not prove that they are formed by a direct conversion of the cell-protoplasm. Elastin Preparation. — As usually described elastin is an albuminoid sub- stance wdiich, like gelatin, is free from sulphur, but which offers great resistance to the action of reagents. The method of preparing this ^ Coniptea rcndus, cii. 1296. For some work on the decomposition of gelatin see also Buchner and Curtius, Berichte d. cheni. Gesellsch. Berlin, xix. boO. 474 THE TLSSUf:s AND ORGANS OF THE JiODV material from tissues which contain a large (quantity of it (such as the ligamentum nuclue of the ox, horse, or giraffe), consists in treating the finely divided ligaments successively with reagents in which it is. insoluble, and in which adherent collagenous and proteid suljstances- readily dissolve. The ligaments are treated first with l)(jiling water, then with 1 per cent, potassium hydroxide solution, then in 10 per cent, acetic acid, then in ^ per cent, hydrochloric nc'u], and lastly with alcohol and ether. This method of purification takes several days,, large excess of each of the reagents mentioned must be employed, and each fluid must be changed two or three times. Composition. — By this means a substance free from sulphur is. obtained. Chittenden and Hart ' in some of their preparations of elastin omitted the extraction with potash, and in these a small per- centage of sulphur (0'3 per cent ) was found ; it is, however, doubtful whether this is really in the elastin molecule or in proteid matter which is present as an impurity. The following table sliows tlie results in percentages of elementary analj'sis by different observers : — Muller--= Tilanus ^ Horbaczewski * Chittenden and Hart Carbon . . . 55-09-55-72 Hydrogen . . .7-11- 767 Nitrogen . . . : 15-71-16-52 Oxygen . . . 207 -21-15 54 90-55-65 7-25- 7-41 17-52-17-74 19-50-20-33 54-32 6-99 16-75 21-94 54-24 7-27 16-70 21-69 The .higher content of carbon in the preparations of Miiller and Tilanus is doubtless due to the presence of more or less fat, not conipletel}' extracted with ether. Properties. — Elastin is not soluble in any liquid that does not decompose it. It is soluble in hot concentrated caustic potash, in cold concentrated sulphuric acid, and also in cold concentrated nitric acid. When boiled with sulphuric acid elastin yields leucine, but no tyrosine. When digested with pepsin or trypsin, elastin is gradually but slowly dissolved. The older writers looked upon it as being almost insoluble in the digestive juices. Kiihne and Ewald "' appear to be the first who obtained a solution of elastin by digestive agents, and these 1 Chittenden and Hart, Zcit. Biol. xxv. 3(>H ; Sfialirs fruiii tlic Lab. of Physiol. Chem. Yale University, iii. 19. ^ Midler, Zcitsch.f. rat. Med. 3rd Heries, vol. x. part ii. 3 Tilanus, Goriijj-Brsauez' Pliysiol. Chem. 3'® Aufl. p. 14H. * Horbaczewski, Zeif. j^hysiol. Chem. vi. 330. ^ Ewald and Kiihne, Die Verdamiiig ah Jtistol. Methode. THE CONNECTIVK I'lSSlKS 475 observers noticed that pepsin w;is more uctive than trypsin. More recently Etzinger, ' Horbaczewski,* and Moiochowetz •* have shown that finely divided liganientum nuclue, or powdered puritied elastin, ai-e fairly S(.)luble in artitieial digestive juices, and Horbaczewski was able to verify this with natural gastric juice obtained fi-om a man with a gastric fistula. Horbaczewski iiaiuod tlio two products of dioestion hcnii-clastin and elastin- peptone. Cliittenden and Hart/ who repeated these experiments, using the methods adopted by Kiihne and Chittenden in the examination of the digestion products of proteids, found that both these substances are analogous rather to the proteoses, the intermediate stages in tlie formation of peptone, than to true peptone ; they are both, for instance, precipitable from their solutions by satura- tion with ammonium sulphate, while true peptone is not. They have named the two products of digestion of elastin, proto-elastose (this corresponds to Horbac- zewski's hemi-elastin) and deutero-elastose (this corresponds to Horbaczewski's elastin-peptone) ; the former is precipitable by saturation with sodium chloride, the latter is not. Both are precipitable by saturation with ammonium sulphate, and both give the xanthoproteic and biuret tests. The names just mentioned are analogous to the names of the albumoses, the first cleavage products in the digestion of albumin. These same substances can be obtained from elastin by the action of trypsin, or by the prolonged action of acidulated water at 100° C, THE GROUND SUBSTANCE OF CONNECTIVE TISSUE The ground substance of connective tissue, like the cement-substance of epithelium, has the power of forming a compound with silver salts, which becomes reduced in the light, and consequently brown or black from the deposit of metallic silver in it. This property is of great value to the histologist, as a means of demonstrating the spaces in the ground substance in which the cells lie. These spaces, which are connected to one another, form a branching network of irregular canals {Saft Kandlclien) in which lymph circulates. The chief constituent of the ground substance is mucin. This is readily soluble in lime-water or other weak alkalis, and so the various tissue elements of fibrous tissue and other forms of connective tissue fall apart when they are treated for about twenty-four hours with lime- water, owing to the solution of mucin. The other organic constituent of the ground substance is a proteid ; this occurs in small quantities ; it belongs to the class of proteids (which are insoluble in distilled water, soluble in dilute saline solutions, and insoluble in saturated .saline solutions) called globulins. It is coagulated by heating its solution to 75° C. Mucin is obtained in greater abundance from endjryonic connective ' Etzinger, Zeif. Biol. x. 84. - Loc. cit. ^ Morochowetz, Mahj's Jahrshcricht, 1S8(>, p. 271. '' Loc. cit. 476 THE TISSUES AND ORfrANS OF THE BODY tissues than from those of the adult ; in the fully formed comiective tissues the ground substance is very laigely replaced by fibrous (colla- genous) material. In the vitreous humour ' and Whartonian jelly of the umbilical cord, in which the fibrous and cellular elements of con- nective tissues are reduced to a minimum, mucin can be obtained in abundance. Mucin Mucin is a substance which has a slimy consistency, and of which there are several varieties. It is found, not only in the ground substance of connectiv^e tissue and the cementing substance between epithelial cells, but, as we have already seen, in many epithelial structures {see Mucus, p. 444). We shall also have to consider it in a few secretions, such as the saliva. It must be carefully distinguished from certain nucleo-albumins which have similar physical characters, such a.s, for instance, the so-called mucin of the bile. Mucin, again, is an ingre- dient of the tissues of certain invertebrates, and the mucin obtained from the snail has been studied by Eichwald and Hammarsten. Elementary analysis has shown that mucin from different sources differs in composition very much. Many of the minor reactions of the substance also vary, and it is now pretty generally granted that different mucins differ from one another in the nature of the proteid which is combined with a carbohydrate radicle to form the mucin molecule. The name given by Landwehr - to the carbohydrate Avhich may be obtained from the various forms of mucin is animal gum. Hammarsten ^ gives the following three properties as characteristic of a mucin : — 1. Its viscidity and stickiness. 2. Its solubility in dilute alkalis ; it is precipitable from such solutions by acetic acid, being insoluble in excess of that reagent. 3. AYhen heated with dilute sulphuric acid, it yields a reducing sugar. Connective-Tissue Mucin Preparation. — The different methods that have been adopted for the preparation of mucin from connective tissue are all essentially the same, though they differ in detail ( Rollett,^ Lcebisch ■^). The tissue is finely minced, washed with water, and then extracted for twenty-four to 1 Gorup-Besanez {Phi/siol. Chem.) gives the foUowing analysis of the vitreous humour by Lohmeyer : water in 1000 parts, 986-4 ; membranes 0-21 ; proteids and mucin 1"36 ; fats 0-016; extractives (urea &c.) 3'2 ; sodium chloride 7"76; other mineral matters I'Oo. - Landwehr, Zeif. physiol. Chem. vols. vi. vii. viii. ix. 3 Chem. Centralhl. 1884, p. 814. « Rollett, Strieker's Handbuch, i. 72. ^ Lcebisch, Zeit. j^hysiol. Chem. x. 40. THE COXNKCTJVK TlSSlES 477 forty-eight hours with a very large excess of lime-water, or baryta-water (lihited to five times its bulk with distilled water. The extract is then precipitiited with excess of acetic acid, the precipitate is allowed to stand a few hours ; in this time it collects into large flocculi or stringy masses if a large quantity of mucin is present, as when one is dealing^ with the vitreous humour. The substance is collected and may be purified by redissolving it in lime-water, filtering, and reprecipitating it from the filtrate by acetic acid. Some recommend that the tissue should be first placed in alcohc^l for a week or two to coagulate the proteids that are present. This^ however, is quite unnecessary ; for if any proteid is dissolved by the lime-water it is precipitated, as alkali-albumin always is, by the acid, but is readily soluble in excess. The spirit has the disadvantage of rendering the pieces of tissue hard, and so they cannot be permeated easily by the lime-water ; ultimately, also, it renders mucin insoluble. Instead of using lime-water or baryta- water, other weak alkalis^ like a 1 per cent, solution of sodium carbonate, may be employed ; or even distilled water, no doubt in virtue of the alkaline salts in the- tissue, will extract a consideraljle quantity of mucin. Estimation of mucin. — The amount of tissue taken is weighed ia the first instance ; it is extracted with lime-water repeatedly till no more mucin goes into solution ; and the mucin precipitated from this by acetic acid is collected on a weighed filter and washed with 2 per cent, acetic acid, distilled water, alcohol, and ether ; it is finally dried,, weighed, and incinerated, the amount of ash being deducted. The- percentage of mucin can be thus calculated. In view of the alleged increase of mucin in a disease known as myxoedema, it is important to- have certain data concerning the amount present in normal tissues. Although the above method cannot claim to be absolutely accurate, it gives very good comparative results. On p. 478 is the result of a number of analyses made l»y Dr Stevenson and myself.^ In all the cases there enumerated the percentages refer to the organ as weighed in the fresh condition. The following numbers represent the averages obtained in normal tissues. The details are given in the tables on the next page. Peroeutage of mucin Skin (children) 0-766 Skin (adults) 0-385 Connective tissues. ..... 0-521 Parotid | Heart tendons / traces 1 Clin. Soc. Transactions, vol. xxi. supplement, report of Myxoedema Committee. 478 THE TISSUES AND ORGANS OF THE BODY Skin Sex of Age of Part from wliicli the Percentage of patient patient skin was taken mucin present M. Stillborn Leg 0-96 M. 7 weeks ryajinia Abdomen 1-02 F. 2 years Syphilis 0-74 M. 6 „ Gang-rene of lung 0-72 F. 9 „ Nephritis 0-39 M. 20 „ Tuberculosis 0-42 F. 40 ., Carcinoma 0-29 M. 53 ., — 0-11 M. 56 ,, Aortic disease 0-64 M. 63 „ — 0-264 F. 67 „ Pneumonia 0-59 Other Tissues Case Tissue m Percentage of Mucin Analyst Bright 's disease Connective tissue fro Stevenson thigh (?) 0-5 (' Clin. Soc. Trans.' XV. 94) Case of accident a. Connective tissue from thigh 0-5 ' h. Tendo Achillis 0-77 , Ditto e. d. Tendons of breast Pericardial fat ( Traces — Heart tendons Doubtful Dr. Stevenson (^Itdd. xxi. suppl.) Female Tcndo Achillis 0-539 •\ (Bright's disease) Male Tendo Achillis 0-298 Halliburton (septicfemia) - — Parotid Trace The high percentage of mucin in the incompletely developed con- nective tissue of young children is due to the greater quantity of ground substance present there. In cases in which fluids are to be analysed for mucin, the precipitate produced by the addition of acetic acid may be collected and weighed, or alcohol may be added to the fluid ; this precipitates both mucin and proteids ; the former may then be dissolved out from this precipitate by means of baryta-water or lime-water, and precipitated therefrom by acetic acid. Lcebisch states that mucin has an acid reaction, and that tlie amount present may be measured by the deci'ease of alkalinity of an alkaline solution employed to dissolve it. Properties and reactions of connective-tissue mucin. — Mucin is a slimy, glutinous substance insoluble in water and in alcohol. It is IIIK CONNKC'l'lVK '1'ISSUE.S 479 soluble in weak iilkaliiic solutions, such as liinc-vvator, from which it is easily precipital)lo hy acetic acid, and is not soluble in excess of that n applied by some authors to the fats is wholly incorrect ; a hydrocarbon is a compound like marsh gas ^C^i) or olefiant gas (CjH^ consisting of hydrogen and carbon only. In spermaceti the fats are not glycerides, but derivatives of cetyl alcohol, C'leHrs.OH, the chief being cetyl palmitate. In Chinese wax (produced by the Coccus ceriferun) and in bees' wax there are also no glycerides. The wax is a C H-- mixture of ceryl-cerotate p'TT^'f-. - O, free cerotic acid, CirH^O, smd melicyl palmitate, a derivative of melicjl alcohol, C5,>EIei.H0 (Schorlemmer, Org. Chem. p. 174). THE COXNECTIVK TISSl'KS 491 From methyl alcohol, CH3.HO, formic acid, CHO.HO, is obtained „ ethyi „ C2H,.,. HO acetic „ C.HsO.HO ,, propyl „ C^H^.HO propionic „ C3H5O.HO „ „ butyl „ C.Hg.HO butyric „ C4H7O.HO „ amyl „ C5H, ,. HO valeric „ C^HgO.HO „ and so on (see p. 65). Or in general terms : From the alcohol with formula C'uHgn+i-HO, the acid with formula CnHon-iO.HO is obtained. This is the series of acids known as the fatty acid series ; the sixteenth member in the series has the formula C15H31O.OH, and is called ^^aZmi^ic acid ; the eighteenth has the formula C,8H350.0H, and is called stearic acid. Each acid, as will be seen, consists of a radicle, CnHon-iO, united to hydroxyl ; and it is these radicles that unite with glycerin to form fats. Oleic acid, however, is not a member of the fatty acid series proper, but belongs to a somewhat similar series of acids known as the acrylic series, of which the general formula is C'nHan-sO.OH, It is the eigh- teenth term in the series, and its formula is CigHgjO.OH (see p. 69). Glycerin, or glycerol, is a triatomic alcohol, C3Hg(OH)3, i.e. three atoms of hydroxyl united to the radicle glyceryl, C^Hg ; or three atoms of water in which half the hydrogen is replaced by the triatomic radicle, CgHg. The hydrogen in the hydroxyl atoms is replaceable by other organic radicles. As an example take the radicle of acetic acid, acetyl (C2H3O). The following formuke represent the derivatives (ethers) that can be obtained by replacing one, two, or all three hydroxyl hydrogen atoms in this way: — (OH (OH I (OH .O.CoHgO C3H5-OH C3H5 OH C3H5 O.CHgO C3Hs.'O.C2H30 iOH 'O.C2H3O iO.C2H30 (O.C2H3O [glycerol] [ruonoacetin] [tliaceiiu] [trlacetin] Triacetin is the type of a neutral fat ; stearin, palmitin, and olein ought more properly to be called tristearin, tripalmitin, and triolein respectively. Each consists of glycerol in which the three atoms of hydrogen in the hydroxyl are replaced by radicles of the acid. The following formulae represent their constitution : — Acid I Radicle Formula of glycernl Palmitic acid, CjgHjiO.OH Palmitvl,C,6H3,0 — Palmitin, C3H,(O.C„H3,0)3 Stearic acid, C^Hj^O.OH StearAl. C„H.^O C3H,(OH)3 Stearin,C3Hs(O.C„H350)3 Oleic acid, C,8H330.0H ' Olevl, C,sH330. — i Olein, C3H5(O.C,8H330)3 492 THE TISSUES AND ORGANS OF THE BODY Decomjwsition products of the fats.— The fats aplit up into the substances which we have seen go to build them up. Under the influence of superheated steam, and in the body under the influence of certain ferments (for instance, the fat-splitting ferment steapsin of the pancreatic juice), a fat combines with water, and splits into glycerol and the fatty acid. Take, as an example, tripalmitin or palmitin, as it is more generally called. The following equation repre- sents what occui's : — C3H5(O.C,6H3,0)3 + :3H,0 = C3H,(OH)3 + 3C,6H3,O.OH [palmitin] [glycerol] [i)iilmitic acid] In the process of saponilication, we have much the same sort of reaction ; the final products are, however, glycerol and the palmitate of the base employed. As an instance take what occurs when tripalmitin is heated with potassium hydroxide ; mutatis mutandis similar reactions occur with other fats and other bases ; the equation representing the reaction is as follows : — C3H5(OC,6H3,0)3 + 3KHO=C3H5(OH3)+3C,6H3iO.OK [lialmltiii] [glycerol] [potassium palmitate] When fats decompose, certain volatile acids are liberated, and these it is which give the characteristic smell to rancid fats. When strongly heated, fats give ofi" a characteristic penetrating odour. This is due to the formation of acrolein (CaHjO, the aldehyde of allyl alcohol, C3H5OII) from the glycerin. Glycerin is obtained commercially from fats by decomposing them with superheated steam. It may also be obtained by precipitating the fatty acids as insoluble soaps from a solution of fat by litharge and water ; glycerin remains in solution in the water, and may be freed from lead by a stream of sulphviretted hydrogen. BONE Bone, like all the other tissues of the body, consists of water, organic substances, and mineral salts ; it difiiers from most of the other tissues in the large amount of mineial matter present. The mean percentage of water as estimated by Volkmann is 48 ; Lukjanow ' gives approximately the same num]:)er (46-7 per cent.) ; this is a mean of twenty determinations of pigeons' bones. The general composition 1 Lukjanow, Zeit. jjhijsiol. Chcm. xiii. 339. Aeby {Centralbl. f. d. vied. Wissensch. 1871, No. 14) considers that 11-12 per cent, of the water present is in a state of loose chemical combination, analogous to water of crystallisation. TIIK CONNKC'TIVK TISSUKS 493 of uorni.il uiidrietl bone without the separation of marrow or blood is given by Hoppe-Seyler thus : — Water .... 50-00 per cent. Fat .... 15-75 Ossein . . . .11-40 „ Bone earth . . .21-85 It may be said roughly that two-thirds of the solids present in bone consist of inorganic matter and one-thiixl of organic substances. Zalesky's analyses are as follows : — Human bone Bone of ox Bone of guinea-pig- Organic constituents . . 34'56 32-02 34-70 Inorganic „ . . 65-44 67-98 65-30 When a bone is soaked in acid (5 per cent, hydrochloric acid, or a saturated solution of picric acid, c^'c), it is but little altered in appear- ance, but it is soft and flexible and has lost two-thirds of its weight ; the inorganic salts have been dissolved out by the acid. The opposite process, the destruction of the organic matter, may be accomplished by heating the bone to a white heat ; the organic matter is thus burnt away, and the bone then appears somewhat whiter than normal, and has lost one third of its weight. The organic constitueiits of bone consist of — a. Ossein. — This is the most abundant of the organic matters in bone. It is identical with collagen {see p. 471). By boiling with water it is converted into gelatin. b. Mastifi. — This is present in small quantities only. Some of the perforating fibres and a thin membrane lining the Haversian canals, lacunar, and canaliculi form the source of this substance in bone.' c. Proteids and nuclein — from the cells. d. Fat. — This is always present in small quantities, even after the I'emoval of all connective tissue and marrow. The inorganic constituents of bone are— a. Calcium phosphate — Ca;j(PO^)._,. This is the most abundant of the mineral matters present in bone. b. Calcium carbonate — CaCO,. c. Calcium chloride — CaCL. d. Calcium fluoride- — CaFL^. e. Magnesium phosphate — Mg (P04)2. /. Small quantities of sulphates and chlorides. ' This membrane lining the Haversian canals was sui^posed by Brijsicke to be composed of keratin; but H. E. Smith (Zeit. Biol. xix. 469) has conclusively shown that this is not the case. 494 THE TISSUES AND ORGANS OF THE BODY Numerous analyses of the bones of difterent animals are given in full in Hoppe-Seyler's 'Physiol. Chemie ' (p. 105).' The total amount and relative proportion of the inorganic constituents is, however, very constant in different animals, and the average from the analyses of Heintz, Recklinghausen, and Zalesky (quoted by Hoppe-Seyler) is as follows : — (The numbers represent percentages of the total ash) Ca PO4 CO, Fl Mg CI 38-49 5446 6-24 1-28 0-44 0-19 Prom his own numbers Zalesky has calculated the probable com- position of the mineral constituents of bone. Calcium phosphate . ... . . . 83"889 ,, carbonate ....... 13"032 Calcium in combination with tiuorine, chlorine, &c. . 0*350 Fluorine 0-229 Chlorine 0-183 Hoppe-Seyler believes that the characteristic inorganic ingredient of bone, dentine, and enamel is one which has the same constitution as the mineral apatite. The formula for apatite is : Ca,„Fl,(PO,)„. In another variety chlorine takes the place of the fluorine : Ca,„CL(PO,V Very small quantities of these compounds, however, occvir in bone ; the chief compound is one built on the same plan, in which the radicle CO3 takes the place of the FL, or Cl„ : Ca,„C03(P0,)„. In other words, if such a compound exists, it is a combination of three mole- cules of calcium phosphate with one of calcium carbonate : 3Ca3(PO,), + CaC03 = Ca,„C03(P0,)«. During the deposition of eartliy matter in tissues like bone and shell the deposit occurs, not in crystals, but in the form of globules and granules. In 1857 George Rainey showed that certain crystalline substances when deposited in viscovTs solutions assume globular and cell-like forms.- These globular bodies arc 1 A large number of other analyses will be found in Gamgee's Physiol. CJieiii. jip. 278-280, quoted from Fremy, Aim. de Chim. et de Physique (S), xliii. 47-107. The general result is approximately the same as that given above. In contrast with what is found in true bone, the analysis of the calcified cartilage of the ray may be given : ash per cent. 30'00 ; calcium phosphate 27'7 ; magnesium phosphate trace ; calcium carbonate 4-3. Fossil bones also analysed by Fremy show a smaller percentage of organic matter than recent bones ; they yield gelatin on boiling. - Rainey, Quart. Journ. Micros. Science, 1858. See also Ord, 0« tJic Injiuence of Colloids on Crystalline Form, London, 1879. I'llK CnNNKCTlVK TISSUES 405 termed calcospha^ritcs by Hinting. Onl has shown also how in urine the presence of albumin .and other colloid substances influences the crystalline form of urinary sediments, causing the anj^les of the crystals to be rounded, the molecules arranging themselves not in straight lines, but with a curvilinear disposition. DENTINE, ENAMEL, AND OTHER CALCAREOUS AND SKELETAL STRUCTURES Denthie consists, like Ijone, of water (10 per cent.) and .solids (90 per cent.). The solids are organic and inorganic. The organic solids are rather less abundant than in bone ; they consist of collagen and elastin ; the latter is derived from the lining of the dentinal tubules. The inorganic solids are like those in bone. From Aeby's analyses, Hoppe-Seyler calculates that the solid matter of dentine is composed of the following constituents : — Ca,oC03(PO,),; 72-06 per cent. MgH(PO,) 0-75 „ Organic substances .... 27 '70 ,, Enamel. — This is the hardest tissue in the body ; in the adult it contains 95-97 per cent, of mineral matter, in the infant 77-84 per cent. Hoppe-Seyler's quantitative analyses give the following mean result : — Ca,oC03(PO,)c 96-00 per cent. MgHP04 105 „ Organic substances .... 3-60 „ The inorganic matter thus resembles that in bone and dentine. The organic matter does not yield gelatin ; this is interesting in view of the fact that enamel is not of a connective-tissue origin, but is epithelial (epiblastic). CruMa 'petrosa, or cement. — This is simply bone both from a histo- logical and chemical point of view. Scales of fisliex. — The scales differ in structure in different groups of fishes : in the Elasmobranchs they are composed of true dentine; the Ganoid scales are covered with a brightly polished plate of enamel ; this is very rarely found in the Teleostean fishes, in which the scales are bony ; the Dipnoi have horny scales. PearU from oysters were analysed and found to consist of calcium carbonate 91-72, animal matter 5-94, and water 2-23 per cent. They are not soluble in vinegar unless pulverised (Harley).' 7>>rfOT««-«7«"ZZ.— The shield of the tortoise is firmly fixed to the skeleton: it consists of a layer of epidermis or tortoise-shell composed of homy matter or keratin and a layer of bone beneath. ' Troc. Roy. Soc. xliii. 461. 496 THE TISSUES AND ORGANS OF THE V.oDY The exo-skeleton of the armadillo is t-omposed of bony plates. Egg-shelU {see Eggs). Sliells of invert chr ate h (.see p. 454). Otoliths. — These concretions, formed in various parts of the auditor}' organs of all animals, consist chiefly of calcium carbonate in a crystalline form ; the crystals are imbedded in mucus.' PJileholitlm. — FhleV)oliths or venous calculi have a tendency to form in veins in which, from dilatation of the coats, the circulation is abnormally slow, as in the veins of the prostate and bladder, and in varicose veins anywhere. They com- mence, no doubt, as deposits of fibrin, and to this the less soluble salts of the blood adhere, chiefly phosphate of cal- cium, and in less quantity the sulphate.'? of calcium and potassium. Calcareous deposits in atheromatous arteries have a similar composition. Fig. 77. — Crystals of Calcium Carljonate from an i, - 7 rri -ii. j.- i otolith, consisting of small thick columnar Uram-sand. — Iha gntty particles crystals, combinations of rhombohedra, ami found in the pineal body and in the hexagonal prisms. ■,.-,, -, ,. choroid plexuses are composed of earthy matter (phosphate and carbonate of lime, with a little phosphate of magnesia and ammonia) mixed with organic matter. This substance is not a product of disease, but is present at all ages, and even in the foetus. Its amount increases with age.- The corpora amylacea found in the follicles of the pineal gland and pituitary body are coloured brown with iodine, and blue with iodine and sulphuric acid. They are non-nitrogenous, but as they do not yield sugar on treatment with boiling dilute sulphuric acid they are probably not carbohydrate in nature.' A colloid sub- stance like that in the thyroid vesicles is sometimes found in the alveoli of the anterior lobe of the pituitary Fig. 78.— Corpora amylacea , -, from human liraiii. DOCiy. THE FAT OF BONE MAEROW C. Eylert * described in ox-bone marrow a new fatty acid melting at 72-5° C, of the formula C„iH.^.,0.,, which he called medullic acid. As nothing further was discovered as to the properties and salts of this acid, P. Mohr^ reinvestigated the matter. The fatty acids were separated in the usual way, and the hypothetical acid was found to be nothing but stearic acid ; the acids in the marrow fat being present in the following proportions : palmitic acid, 22 ; stearic acid, 10; and oleic acid, 63 per cent. 1 Dahnhardt, ' Endolymphe und Perilymphe,' Arbeiten d. Eielerphysiol. Instif. p. 186. Barruel, 1838, quoted by Dahnhardt. - Quain's Anat. ii. 327. 3 Hoppe-Seyler, Fliijuiol. Chem. p. C89. ■i Wittstein's ViertelJaJirsdirift f. j'rakf. Pharin. ix. 330. ^ Zeit. Physiol. Chem. xiv. (Ib90) 390. 497 CHAPTER XXIII THE COXXECTIVE TISSUES IN DISEASE INTRODUCTORY TiiK diseases in which tlie connective tissues are involved are nume- rous, but our knowledge of pathological chemistry in this direction is limited. In actual post-mortem experience chemical methods are compara- tively seldom resorted to, as a naked eye or microscopical examination of the organs gives the observer, as a rule, sufficiently complete infor- mation of the morbid condition present. Many of the morbid conditions affecting connective tissue differ from the normal condition in degree rather than in kind. Thus there may be excess of white fibres, producing what is known as fibroid degeneration, cirrhosis, or sclerosis ; or excess of fat may occur as in general obesity ; in this condition widespread fatty degeneration of heart fibres, kidney, liver, itc. may occur in association with increase in the amount of adipose tissue. In another class of cases hypertrophy may be not geiieral, but localised, forming what is known as a tumour ; thus there are bony tumours (exostoses), cartilaginous tumours (en- chrondromata), fatty tumours (lipomata), tumours comjDOsed of jelly- like connective tissue, as in certain forms of nasal polypi, and so forth. Tumours of this kind are composed of tissue, showing practically no difference from that normally present in the body, and when removed show little or no tendency to recur. There are other new growths of connective-tissue origin which are malignant ; these constitute the numerous class of the sarcomata. A sarcoma, speaking roughly, is com- posed of embryonic connective tissue in which the cellular elements are especially numerous and active ; and malignancy runs parallel to the activity and rate of growth of these cells. One especially malignant form of sarcoma is that known as melanotic sarcoma. The pigment melanin separated from the tumour has been the subject of several chemical investigations, a brief resume of which will be given. Another disease which will demand special notice is that known as myxcedema ; and in connection with bone diseases we shall have to K K 498 THE TISSUES and organs of Till-: F.ODY consider the chief alterations that occur in rick' is, osteomalacia, caries, and necrosis. Joint diseases are exceedingly numerous. Those of a simple inflammatory nature are accompanied by incre.i e of the synovial fluid, the composition of which we have already consiilpred (see p. 351). The more severe the inflannnation, the greater is tlif percentage of organic solids in the etl'used fluid, and it may even becmiie purulent. In other diseases of joints the cartilage and bone ma} iindergo various patho- logical changes, and with age hyaline cartila-< niay become calcified, or even ossified. The so-called loose cartilaut -; that are met with in diflTerent articulations consist generally >ver, it has been .stated that they sometimes consist of lumps of coaguhitod blood. A disease which affects cartilage is gout, and we shall have to describe briefly the crystalline deposit of uratf- lound in articular car- tilage and other situations in this disease. In the condition known as dropsy the coHiiertive tissues become infiltrated with watery lymph (cedema). Areolar tissue is the most extensively distributed of the tissues, and it is. moreover, continuous throughout the body, and from one region ii; inay be traced without interruption into any other, however distant ; rims it is that dropsical fluid, air, blood, or ui-ine effused into the aii ular tissues, and even pus, may spi-ead far from the spot where they w ere first introduced or deposited. The composition of the fluid of .--i!l)Cutaneous cedema will be found on p. 349. Diseases of marrow might justly Ije includi i! in diseases of connec- tive tissue ; the chief known facts concern] nu these have, however, been already descriljed in connection with ili' formation of blood- corpuscles (p. 302), A rough sketch like the foregoing of the nidihid conditions in which the connective tissues are either primarily m secondarily involved is sufficient to indicate the great variety of diseased processes that may occur, and the few points that we have now tn take up a little more in detail are those in which chemical resean-h has been instrumental in adding to our knowledge of the pathology of such conditions. TlIK C'()NNHC11\K IISSIKS 1\ J)1SK.\SE 499 THE PIGMENT.^ OF :\rELAXOTIC SARCOMATA Tlie name mi'lanin has been hitliei-to used for tlie pigments occur- ring in the eye, hair, skin, in pathological new growths, and also for the decomposition products of chromogens in the urine. We have already considered the bhick pigments of the eye and skin ; the follow- ing account of the pigment of melanotic tumours is an abstract of a papei- on the subject by Prof. Morner, of Stockholm.' This pigment was first investigated by Heintz, who found that it was soluble in alkalis with ditticulty and that it contained no iron. An elementary analysis gave the following figures : C, 53-4 ; H, 4*02 ; N, 7-10. Dress-er made a similar investigation, and found in the pigment a small quantity < )f iron. Berdez and Nencki named the pigment phymatorusin ; they found it to be insoluble in water, alcohol, and ether ; easily soluble in solutions of fixed alkalis or their carbonates, and in ammonia ; from such solutions it was precipitable by acids, but was somewhat soluble in excess. The preparation contained c^arbon, hydrogen, oxygen, nitro- gen, and sulphur (in large amount 10"67 per cent.), but no iron, phosphorus, or chlorine. In horses they found in melanotic tumours a pigment with somewhat different })roperties, which they called hippo- melanin.2 In the urine of patients suffering from melanotic sarcoma a dark pigment has been found ; this, according to some, is an excess of the ordinaiT urine pigment, and, according to others, is the same pigment that occvirs in the tumour. It is turned dark brown by the oxidising action of nitric acid, or sometimes by mere exposure to the air. Again, in other cases of these tumours, particles of Vjrown pigment are found in the blood, the corpuscles having the normal shape and colour ; similar granules have been occasionally described in the urine and the urinary passages. Morner's research was undertaken in order to clear up, if possible, some of these doubtful points ; the material was supplied from a case of which full clinical and post-mortem records will be found in the paper already referred to. During life the urine showed the peculiar colouration just mentioned ; after death the tumour itself was investigated. Its situation was the shoulder, but secondary growths occurred elsewhere. The blood, except for a low percentage of haemo- globin, was normal. The pigment did not give any absorption bands when examined with the spectroscope, but produced a general dimming, ' Zeit. physiol. Chem. xi. 66-140. Nencki criticised some of Miirner's statements in Arch.f. exp. Pathol, und Pharmakol. xxiv. 27. The difficulties raised by Nencki were fully explained in a subsequent paper by Morner, Zeit. physiol. Chem. xii. 229. ' For further particulars see Arch. f. exp. Path, unci Pharmakol. xxiv. 17 ; also Chem. Centralbl. 1888, p. 587. K K 2 500 THE TISSUES AND ORGANS oF THE BODY especially near the violet end. By the spectrophotometer the extinction coefficients in different parts of the spectrum were determined. The- pigment was also subjected to elementary analysis. It was found to contain iron, which was estimated spectrophotometrically as well as by the usual methods ; the spectrophotometric method consists in con- verting the iron of the ash into ferric thiocyanate, and comparing its extinction coefficients ^vith those obtained from a solution of ferric chloride of known strength similarly treated. The failure of some previous observers to obtain proof of the presence of iron is accounted for by their having used hydrochloric acid in the preparation of the pigment. It is found that this acid dissolves out nine-tenths of the iron from the pigment. Baryta-water caused a precipitate in the urine, which cariied down with it some of the pigment ; in the filtrate the remainder was precipi- tated by lead acetate. For the methods which were adopted for sepa- rating the pigment from these precipitates and from the tumour the original paper must be consulted. The pigment obtained from all these sources was a brownish amorphous powder when dry. It was partly soluble in acetic acid, and partly insoluble. The following table represents the percentage composition and the relative absorption for the region of wave-length=562, for these different preparations : — Percentages Variety of Pijrmeut Absorption C H X s Fe A. Pigment insoluble in acetic acid :^ 1. From the tumour . 55-72 6-00 12-30 7-97 0-072 0-00038 2. From the urine . . 5576 5-95 12-27 8-65 0-22 0-0003i B. Pigment soluble in acetic acid: — 1. From the tumour . — — - 5-90 0-21 0-00094 2. From the urine . . 58-07 803 11-08 4-75 0-20 0-00085 i Although from paucity of materials the analyses are incomplete, the general conclusion seems to be that either two pigments are present, or are produced from a mother substance by the action of the acid. The high percentage of sulphur in the one insoluble in acetic acid, agrees with what Berdez and Nencki found in phymatorusin. An important point brought out is the identity of the tumour pigment with that in the urine ; it is probably brought to the urine by the blood, in which feebly alkaline liquid it is slightly soluble. It gives a very different spectrophotometric chart from ordinary urobilin. THE CONNKCTIVK TlSSKKS IN DISKASK 501 Braudl and Pfeitier ' have more recently made a similar investi^'u- tion, and have obtained corroborative results. They consider with Miimer, and in oppositi(m to Nencki, that melanin originates from hivmoglobin. Melanuria. — This subject (melanin in the uiine) has been also investigated by v. Jaksch.^ He finds that the best reagent for detecting melanin or its precursor, melanogen, in the urine, in cases of melanotic sarcomata, is a very dilute solutioia of ferric chloride, which gives a black precipitate. These urines also give very markedly the Berlin blue reaction in adding a cyanide, and an alkali, and subse- quently an acid. Melanin itself when separated out from the urine does not give the reaction ; but it is apparently due to some other substance excreted simultaneously : this substance, whatever it is, appears to be present in traces even in normal urine, and is especially abundant in those urines which yield a large amount of indigo. MYXCEDEMA ^ Myxoedema is a well-defined disease, which affects women much more frequently than men, and the subjects are, for the most part, of middle age. In women there appears to be no constant relation be- tween the myxoedematous condition and disease of the generative organs ; '* but pathological and clinical observations both indicate in a most decisive way that the one condition in all cases is a destructive change of the thyroid gland, a delicate fibrous tissue being substituted for the proper glandular structvire. Myxcsdema is practically the same disease as that named sporadic cretinism when affecting children ; it is also identical with the condi- tion known as cachexia strumipriva, which occurs after removal of the thyroid gland in surgical operations, and lastly it can be artificially produced in certain animals by removal of the thyroid gland. Affections of movement, speech, sensation, and intellect form a large part of the symptoms of the disease ; the most marked morbid condition is an increased bulk of the body, which is due to hypertrophy of the subcutaneous tissues. Interstitial development of fibrous tissue is much less frequently observed in the viscera, and the appear- 1 Zeit. Biol. xxvi. 348. ^ Zgif_ 2)}iijsiol. Chem. xiii. ^ The following account of myxoedema is taken from the report of a committee of the Clinical Society, Clin. Soc. Trans, supplement to vol. xxi. * This is a somewhat noteworthy point, in view of the exaggerated importance attached by Landwehr to the co-relation between activity of the female generative organs and an increased formation of Kubstances like mucin, which contain animal gum. See p. 480. 502 THE TISSUES AND ORGANS OF THE P.OI)Y ance presented by the new tissue is suggestive of an irritative or inflammatory process. In the early stages of this new growth, the microscope reveals an open-textured appearance, probably due to the excess of ground sub- stance, such as is generally met with in young connective tissue ; later this tissue is very generally replaced by fat. Dr. Ord,' in de- scribing the typical appearances in a case in which death occurred before replacement by adipose tissue had taken place, says : ' The skin did not show the condition of ordinary cedema ; during life it was tough and resistant,' and after death remained firm on section without exudation of fluid from the cut sui'faces : this taken with the micro- scopic appearances indicates that whatever material filled the unusual interval between the bundles of white fibres had something of a gelatinous consistence.' The name myxcedema was originally given to this disease on the grounds that one of its most pronounced symptoms was this peculiar oedematous condition, which was not the result of a watery dropsy, but of a swelling of the subcutaneous and other connective tissues, due to an excess of a mucin-yielding intercellular substance. In the disease artificially produced in animals a similar condition was found to be present. The analyses of the amount of mucin present have been made by various observers, who have all adopted, with slight variations, the method which has already been described when we were dealing with the amount of mucin in normal connective tissues (p. 477). Mucin in inyxcedematous tissues {man). — It will be first con- venient to recapitulate the chief numerical results obtained with regard to the quantity of mucin present in normal tissues : — Percentage of muciu Skin (infants) ..... 0*766 Skin (adults) 0-385 Connective tissues . . . . 0'521 Parotid ...... traces The first analyses of mucin in myxoedematous tissues wei-e made by Dr. T. Cranstoun Charles in the case published by Dr. Ord in the ' Medico-Chirurgical Transactions,' Ixi. Dr. Charles states (p. 62) that the skin of the feet yielded fifty times more mucin than the skin of healthy people, or those suffering from ordinary a?dema. In this case the patient was a female, set. sixty, and death occurred after the disease had lasted ten years, the patient being still in the swollen coiidition. » lirjwrf, p. 184. THE ("(|\NK(T1VK I'lSSlKS IN DlSKASl'] 503 Siucf tluMi a lUiiiiln r of investigations have Iteen made in siniilai- cases, and the ohsel•ve^■^ have gixcn tlicii- results more acciu'ately tlian in tlie somewhat loose statement made l»v' Charles. The results may he lonveniently drawn up in tabular form : — Duration (,'■■11 litiiiii at time of Analysis percentages of mucin ' Ciise of ileatli "^U g-C Otlicr Analyst ilisease Skin c ^ = £ eonncctive ^- — c r. tissues f° g § 1 and fat 1. Male (Et 4G 4vrs.(?) Yorv fat "T"" 1-9 0-4 Dr. Stevenson 'J. Feiuale „ G2 ■ - - Wasted traces — — „ 3. .. ,, 67 Very fat 0-012 — 1-72 traces „ 1. ,. ., 56 32 years I'l-.il.ablv iio wastiiiar 0-3O-0-38 — Dr. Bernays •''. .. 1, 58 3 „ S\Milleii : uuK'li fat 0-36 -0-81 — — ,, i;_ __ ^, 41 16 „ Much fat : possibly 0-17 — 1-91 — ,, -ome wasting 7. „ „ 44 4 „ Very fat 0-72 — 1-09 O-UC-0-20 „ K. Male 73 S wollen 1-43 — — W. D. Halli- '•>■ « 1. 52 IJ year Very fat 0-38 — — 1 — burton l'»- 47 5 year» Swol'en 0-37 — — — . ., 11. — 5 „ Swollen 0-237 1 " In several cases other organs, like the brain, liver, and spleen, were examined ; but the results are of little importance, because we have lu) analyses of the amount of mucin in these organs when in the normal condition ; in organs containing many cellular elements, nuclein and mucin would no doubt Ije weighed together. In Case 8, how^ever, the percentage of mucin in the following glands may be stated : — Parotid gland 0-188 .... 0-159 0-185 Submaxillary gland Pancreas In some few cases the blood, and fluids in the serous cavities, which were often increased in amount, were examined. The genei'al conclusions are as follows : — Skin. — This tissue was examined in most of the cases, and in ten the analysts stated their i^esults numerically. The lowest percentage of mucin obtained was O-Ol'i, the highest 0-81. In two cases there was an increase of mucin, the percentages being 0-81 and 0-72, the average amount in normal adult human skin being 0-385 per cent. The average of the ten analyses gives a number (0-374 per cent.), which is approximately the same as in normal skin. Connective tissues. — The tendo Achillis was examined in one case, and the percentage of mucin oljtained was 1-42. The cardiac tendons ■were examined in four cases, and in all there was an increase of mucin. 504 THE TISSUES AND ORGANS OF THE BODY the average number obtained from the four analyses being l-Go per cent. The average percentage in normal tendinous tissues is 0'521. Other organs. — -The spleen Avas examined in two cases, and in one gave a large percentage of mucin (2-21). The lungs were examined in the same two cases, and in one case there was a high percentage of mucin (0-72). The liver and brain were examined twice, and the intestine, submaxillary gland, and pancreas have each been examined once ; they gave low percentages of mucin, but the amount present in these tissues, when normal, has not been investigated. The parotid, which normally contains only a trace of mucin, gave in the one case in which it was examined a comparatively high percentage of mucin (0-188) ; this was even higher than the percentage obtained from the sub- maxillary gland of the same patient (0-159). Fluids. — The blood has been examined in one case, but no mucin was discoverable in it. It, however, showed very imperfect coagula- tion, a point ill which it resembled the Ijlood of animals in which the disease had l^een artificially produced. The pericardial, peritoneal, and cerebro- spinal fluids have each been examined in two cases, the pleuritic fluid in one, but in all no mucin was found. There has been no record of mucin havnig been found in the urine. It is seen from the foregoing summary of the results of analysis that the increase of mucin, as found by Dr. Charles, has not been found to anything like so great an extent in subsequent cases ; nor is the increase so marked as in the experiments on animals. In certain cases this is accounted for hj the fact that the patients have not died while in the typical swollen stage, but in the subsequent atrophic period of the disease ; and in other cases the subcutaneous connective tissue has become replaced by fat, and in other cases still the analyses are, to a great extent, vitiated by the keeping of the specimens for long periods under alcohol before analysis. In the case of animals it is easier to avoid all such sources of error. It is imjDortant to remember that the source of mucin in the body is twofold : — 1. It results from the degeneration of the protoplasm of epithelium cells, as in the goblet cells of mucous membranes, and the cells of the acini of mucous glands like the submaxillary. In the myxcedema of human beings this source of mucin has not been to any gi-eat extent investigated. The most important analysis bearing on this point is the one analysis of the parotid gland which has been made, and which showed a distinct increase of mucin. This is interesting in connection with Horsley's experiments on monkeys, in which it was shown that in the myxcedema artificially produced in them, the cells of the parotid, THE C'DNNKCTIVK TlSSlKS IN DISEASE 505 which noinially secrete cleur saliva, secrete a viscid saliva ; by the microscope the cells were found swollen by niucinogen, and by chemical analysis mucin was found tu be greatly increased in amount. 2. It forms a constituent part of the ground substance or stroma of connective tissue in which the colls and fibres are embedded. This substance is chemically a muco-albuminous material. One of its con- stituents is mucin, the other a proteid of the globulin class, which in its reactions resembles the seium-globulin or paraglobulin of the blood. In the chemical investigation of myxttdematous tissues only one of these constituents, viz. the mucin, has been estimated. In new and loose connective tissues the ground substance is present in greater amount than when the librous (collagenous) material replaces it in a later stage. This is illustrated by the fact already noted of the higher percentage of mucin in the skin of infants as compared with that of adults. In myxcedema it seems that the swelling is at a certain stage due to the increase of this ground substance, and hence the increased percentage of mucin ; but at later stages, when white fibres or fat- cells have permeated it, the increase of mucin is not so marked. Myxcedema in Animals The second part of this question deals with the results of the chemical investigation of the tissues of animals. The animals were those in which the thyroid gland had been removed. Some comparative analyses were also performed with the tissues of animals in which the thyroid gland had not been removed. In these experiments the operations were perftjrmed by Prof. Horsley at the Brown Institution, and the chemical analyses by myself. The question which has been the chief subject of chemical inves- tigation is the percentage of mucin in the tissues and fluids of the body. The first series of analyses made were those which have already been published in Horsley's Brown Lectures (' Brit. Med. Journal,' vol. i. 1885, p. 211). In the tabular form in which they then appeared they illustrate very forcibly the fact that the percentage of mucin is increased in the tissues after thyroidectomy. The increase is not only marked in the connective tissues, but also in the salivary glands. The pi'esence of mucin in large amount in the parotid which normally contains none is especially noteworthy. This chemical result was confirmed by microscopical examination, the cells of the acini simu- lating those of a mucous gland like the submaxillary. Another im- portant fact is the presence of mucin in the blood in increasing amount as the myxoedematous condition of the animal becomes fully developed {nee p. 304^). It will be noted that the normal per- 506 THE TISSUES AND (HJOANS OF THE BODY centage of mucin in the skin of monkeys is less than in man. The table, with the additional cases that have been examined since, is as follows : — In some of the above animals the ))lood ^\■as further examined. In monkeys Nos. 1, 3, f), and 10 clotting of the blood took j^lace very slowly, leading to the formation of a well-marked bufty coat, whereas in normal monkeys the blood coagulates quickly without the formation of a bufFy coat. The percentage of proteids in the serum was found to be approximately the same in both normal and myxoeclematous monkeys (about 4 to 5 per cent.), the serum-globulin and serum-albu- min being present in about equal amount. In one case the tempera- ture of heat-coagulation of the serum-albumin was rather different from the normal. The urine of monkey Ko. 3 contained a small amount of mucin.'^ The tissues of a few other animals (jiig and donkey), from which the thyroid had been removed, showed no increase of mucin, and the animals themselves showed no typical symptoms of myxcedema, in the same way that monkeys do. The case of a sheep in which myxcedema occurred after removal of the thyroid is regai'ded by Horsley and Ord as of great interest ; the chemical part of the report in this case ran as follows : — The blood of the animal was examined twenty-seven days after the 1 Monkey No. 21 was not myxoedematous, being kept at a high temperature, and the percentage of mucin in its tissues is seen to be ai^proximatelj- nonnal. - The urine in the other cases was not examined. THE CONNF.CTIVK 'IISSIKS IN DISK ASK 507 operation. Xo mucin was fountl in it, and the proteids of tlie scrum were both qualitatively and quantitatively normal (serum-globulin 'i-iS'i, serum-albumin 4'13 per cent.) The animal was killed nearly two years afterwards, it having developed myxi'oportion of grey to white matter in the human brain has been the subject of investigations by Bourgoin (Mecherches chiniiques sur le cerveau, Paris, ISGO), and by De Eegibus (Maly's Jahrcsh. xiv. 346). Both find the grey matter is the more abundant. De BegibuB gives these numbers : for every gramme of grey matter there is 073 gr. of white. THE NKRVOUS SYSIKM 515 lujule by dilli'it'iit ol)ser\t'i'.s of the reaction (jf nervous tissue vary considerably ; they, however, all agree in the fact that nervous struc- tures after death are acid. The difficulty in ascertaining the re;iction -during life is very great, as the nervous structures are permeated by jdkaline bk)od and lymph. The nervous tissues, moreover, of all tissues ^ire those that undergo degenerative processes most readily when their normal blood supply is cut off ; so that any attempt to obtain nerve free from blood would not give us living, healthy nerve any longer. Moreover the mere application of reagents in order to test the reaction is generally sufficient to kill the nerve. The experiments that have been published on this subject are as follows : — Heidenhain' and Gschleidlen^ state that the normal reaction of the «,xis-cylinder is alkaline ; on death, or after long-continued activity, the I'eaction becomes acid. The grey matter of the brain is acid even dui'ing life. Tlie acid is probably lactic acid. The sympathetic ganglia are neutral or weakly alkaline. O. LangendorfF^ used the frog in his experiments, as in the cold- blooded animals post-mm'tem changes occur less readily than in the warm-blooded animals. He found that the central nervous sy,stem is alkaline in this animal dui'ing life, but rapidly becomes acid after •exposure or death. He also made similar experiments on rabbits and guinea-pigs, and found that there also the reaction of the brain was alkaline — in newly born animals so strongly, that even after death they did not become acid. On stoppage of the blood-stream, acid accumulates in the bi-ain ; but on allowing the blood once more to flow, the accumulated acid is washed away. J. Moleschott and A. Battistini^ found the brain, spinal cord, and sciatic nerves acid, the grey matter Ijeing more strongly acid than the white ; on activity the acidity increased, especially in the grey matter. These observers used a very •dilute solution of potash and phenolphthalein as indicator. In my own •experiments, I have never failed to find an alkaline reaction in fresh brain, cord, or nerve ; they, however, rapidly become acid, as a rule, iifter death. All observers, however, agree on the most important fact, that acidity, whether present initially or not, increases on activity and on Zeif. physiol. Chem. i. 3'SO. 518 THE TISSn:s AND OKGAXS OF THE EDDY The grey matter is stated by Schlossberger to be richer in total ash, but poorer in phosphates, than the white matter ; Petrowsky, on the other hand, obtained more phosphoric acid from grey than from white matter. Tlie following table gives some of the typical quantitative analyses that have been made of the propoi'tion in which the principal solid constituents occur in dijfferent nervous structures : — Portion of nervous svstem Leci- Cliolesterin! r^ .k_:„ Xetirokera- Other thin anrl fat tin *'''^'"'' matters I Grey matter of ox ' bram(PetrowskT) 55'37 "^liite matter of ox brain (ihid.) . . 2472 Spinal cord (Mole- "' schott) .... 23-8 75-1 1-1 17-24 18-68 0-53 GTl I'^o 9-90 .)1-91 9-.55 3-34 0-57 Human sciatic ners-e | " (Josephine Cheva- i I lier') 36-8 32-57 12-22 11-.30 3-07 4-0 — After having looked at the nervous tissues as a whole, and before going on to describe in detail the principal organic substances con- tained in them, it will be next convenient to take the individual histological elements and the facts we know respecting their chemical composition. Here we have, to a large extent, to rely upon the methods of micro-chemistry, which almost necessarily afford us limited infor- mation. Nerve-cells. — These cells vary much in size and shape in different parts of the central nervous system ; the body of the cell is proto- plasmic, and therefore chiefly proteid in nature. In this way the high percentage of proteids in grey mater Ls accounted for. In many nerve-cells masses of a greyish pigment are often present ; this pigment does not seem to have been specially investigated, but is no doubt ultiuiately derived from hfemoglobin like the other pigments of the body. The nerve-cells of the ganglia of the worm Aphrodite aculeata are tinged red. This is due to the presence of haemoglobin. - From this fact, and from the fact also of the greater vascularity of grey as compared with white matter, we may assume, as Gamgee says, that respiratory exchanges go on more actively in nerve-cells than in nerve-fibres. Nerve-cells have always a well-marked nucUvs. The substance of 1 ZeU.physioh Chem. x. 97. * Gamgee, Physiol. Chem. p. 420. THE NERVOUS SYSTEM 519 which it is composed, uppears to I)e one of the class of phosphorised .•ill)uiniiioi(l.s known as nucleins ; v. Jaksch ' separated it from the brain, and Geoghegan ^ estimated that the amount present in that organ was 0"14 per cent. Elementary analysis shows very marke24 THE TISSUES AND ORGANS OF THE BODY THE PHOSPHOKISED CONSTITUENTS OF NERVOUS TISSUE 111 tlie year 1865 Liebreich' sepHifitecl from the brain a material he "termed jrrotagon ; he further found tliat ^^■hen decomposed by baryta- water it yielded two acids — stearic acid and glycero-phosphoric acid — and a base called choline. Hoppe-Seyler, and Diaconow ^ workin<^ under Huppe-Seyler's direc- tion, denied the existence of this substance protagon, and considered that it was a mere mechanical mixture of a phosphorised fat called lecithin, with a nitrogenous non-phosphorised substance called cerebrin. Lecithin yields the same three decomposition j^roducts that were ob- tained from protagon by Liebreich. Diaconf)w's elementary analyses were, however, far from convincing. The subject in this country was taken up by Gamgee and Blanken- horn -^ ; and the result of their work has been that Liebreich's discovery has been fully verified. They showed that protagon is a perfectly definite crystalline substance of constant elementary composition. They also showed that even prolonged treatment with alcohol and ether ■will not extract lecithin from protagon, as alleged by Diaconow. When protagon is digested with alkalis it yields the same decom- position products as lecithin does. Bavinistark ' lias since this fully confirmed Gamgee's work. An elaborate research by Thudichum'' has led him to the conclusion that there are three groups of phosphorised substances in the brain, which he terms keiDhalines (very soluble in ether), myelines (far less soluble in ether), and the lecithines (characterised by their extreme instability). In each of these groups there are several members, the •empirical formuhe of which ha^•e been calculated. Though somewhat indefinite, Thudiehum's researches demand a passing notice in this short historical sketch of the chief steps by which our knowledge on this subject has been attained. We can now pass on to a more detailed consideration of lecithin, protagon, and their products of decoiiipositit)n. ' Liebreich, AniidJcii dcr CJiciii. ii. FJiariii. cxxxiv. '2it. - Diaconow, Coitralhl. f. <1. mod. Wissensch. 18C8, p. il7. ■" Gamgee and Blankenhorn, Jourii. of Physiol, ii. 11:5. Fioin this paper and from Dr. Gamgee's account of his work in his PhijisioJ. Clirm. 427 H scq.ihe above description eing the base winch is united to the distearyl-glycero-phosphoric acid. He states in favour of this view that, on shaking an ethereal solution of lecithin with sulphuric acid, the products of the reaction were choline sulphate and distearyl-glycero-phosphoric acid. The following graphic formula will therefore represent Diaconow's view of the constitution of lecithin : — i lO-N(CH3),,-C,H,OH. Strecker •' on the other liand, considers that lecithin is an ether- like combination, the choline being united to the acid by means of the 1 The term iieurine s sometimes Ubetl synoiijinously with choline ifscc, 'however, p. 179). -' Ccntralbl.f. d. med. Wiss. ISOs. ^ Annaleii Chcm. Pharin. 18G8, cxlviii. p. 77. TIIK NKRVorS SYSTEM 529 oxygen of the hydroxyl ; and iciapliically tlie fonuula foi- lecitliin would tlierefore be ((C,.H3,(),)-- In favour of this latter view Hundeshagen has stated that the clioline salt of distearyl-glycero-phosphoric acid prepared synthetically has none of the properties of lecithin. E. Gilson ' reinvestigated the action of weak sulphuric acid on lecithin. He found that the products of the action were small quantities of glycero-phosphoric acid, another phosphorus-containing compound (? distearyl-glycero-phosphoric acid) in still smaller quantities, and free phosphoric acid ; the last named is the most abundant. These results certainly negative Diaconow's theory that choline plays the part of a base in a combination resembling a salt, and we must therefore draw the conclusion that Streaker's is the more correct view to take of the nature of lecithin. Having now seen the way in which lecithin is built up, it is easy to understand how it is we obtain on its decomposition stearic acid, glycero-phosphoric acid, and choline. The following equation represents what occurs on boiling lecithin with alkaline solutions : — C44H90NPO9 + 3H20=2Ci8H360, -h C3H9PO, -f C.H.^NO., [leoitliiu] [water] [stearic acid] [glycero-phosphoric [choline] acid] The acids in the above equation further unite with the alkaline base used to form salts. Lecithin should more properly be called distearyl-lecithin ; other lecithins probably exist in which palmityl, oleyl, or other fatty-acid radicles take the place of the stearyl in the lecithin we have been considering. The following points may be here added with regard to the chief products of decomposition of lecithin. Stearic acid has been already considered (p. 491). GIi/cern-j)7to.y)horic acid. — This may not only be obtained by the decomposi- tion of lecithin or protagon, but may be prepared s^iithetically from phosphoric acid and glycerin. It is a syrupy liquid of a sweet-acid taste : its salts, except the lead salt, are soluble in water, and all are insoluble in alcohol. The salts that have been particularly studied are those of barium (C3H.P>aP0J, of calcium (C3HjCaPO,;.H..O and CjHjCaPO^.CjHgPOJ, of zinc (CjH.ZnPOJ, and of lead (CjH.PbPOJ. Clioline. — This base was first obtained by Strecker- from bile, and named by 1 Zeit. phijsiol. CJiem. xii. 585. * Strecker, Annalen Chem.Ph arm. Ixxii. 77. M M 530 THE TISSUES AND OEGANS OF THE BODY him choline. It may be obtained by decomposing lecithin or protagon by boiling it with baryta-water. The liquid is then filtered, treated with carbonic acid, boiled, filtered, and the filtrate concentrated. A syrupy residue so obtained is extracted with absolute alcohol ; the solution is then filtered, faintly acidified with hydrochloric acid, and platinum chloride added. A yellow precipitate con- sisting of the double salt of choline and platinixm chloride is obtained, and choline is obtained free from the metal by the method already described in the case of lecithin {see p. 527). Choline may also be prepared syntheticall}' from trimethylamine, ethene- oxide. and water [N(CH,)3 + (CH,),0 + H,,0 = C.H,3N0J ; or the hydrochloride may be obtained by heating together ethene-chlorhydrin and trimethylamine [C1.CH„.CH2.0H + N(CH3)3 = N(CH3)3(C.,H^.0H)C1] (Win-tz >). Choline is a sjTupy liquid, markedly alkaline in reaction, soluble in alcohol and in ether. It forms compounds with hydrochloric, carbonic, and sulphuric acids ; its hydrochloride forms compounds with gold chloride and platinum chloride, of which the formulfe are respectively (C^Hi^NOCl) + AuClg and (G,H, jNOCl)^ + PtCl ,. Choline can be always identified by adding to the substance suspected to contain it gold or platinum chloride, and then estimating the amount of the metal present in the compound so formed. The gold compound of choline contains 44'4 per cent, of gold, the platinum compound 31'9 per cent, of platinum. When heated, choline splits u]^ into the diatomic alcohol glycol and tri- methylamine : — ^^^' { OH.NCCH^), = ^'-'"^ 1 OH + ^'(CH^X [choline] [gb'^'ol] [trimetliylamiiie] Physiological hnportance of clioline. — Choline is of great interest and im- portance, as it is a type of the alkaloids known as ptomaines and leucomaines. These are alkaloids which are formed either after death (ptomaines), as a result of putrefactive changes, or during life (leucomaines), as a result of metabolic processes. Two of the most constantly present are choline and the allied alkaloid neurine {see p. 179). Another important alkaloid obtained from poisonous mush- rooms, and called muscarine, can be obtained from choline by oxidation. "We thus are able to prepare synthetically many of the important organic principles l)roduced in nature by the decomposition of proteids and other complex sub- stances. In Chap. XIII an account of the animal alkaloids will be found. Choline in small doses produces p\Texia.- In larger doses it produces symp- toms resembling those of curare poisoning — that is, parah'sis due to poisoning of the motor end-plates. Xeurine in its physiological action resembles the closely related alkaloid choline, but the former is the more powerful of the two. Botli, like muscarine, are antagonistic to atropine with regard to their action on the heart and glandular sj'stem.' Fate of lecithin in the hody. — Lecithin is largely contained in certain forms of food. In the intestine, pancreatic juice splits it up as it does the simpler fats into its constituents (Bokay^); the fatty acid behaves like that obtained from adipose tissue, being partly saponified, partly absorbed as such, and fm-ther ^ Wurtz, Annal. Chem. Pharm. Sup. vi. 116 and 197. - Ott and Colbnar, Journ. Fhysiol. viii. 218. 3 Cervello, Arch. ital. hiol. vii. 232; Chem. Centralbl. 1887, p. 1150. * Bokay, Zeit. physioh Chem. i. 1.57. TJIJ-; .\"KI{\'()IS SYSTEM 531 oxiiliseil ill tlu' body to form Ctaibonic acid and water. Glycero-pliosplioric auid is also probably absorbed as such, or as a gh'cero-pbospbate. Sotnischewsky ' found it unaltered in the urine. When mixed with putrefactive organisms it is not decomposed into gaseous comixMinds. Choline, on the other hand, when mixed with mud containing putrefactive organisms is split up into carbonic acid, methane or marsh gas (CH,), and ammonia." No doubt a similar decom- position is i)roduced by the bacteria of the intestine, and so tlie poisonous action of clioline is obviated. We are still ignorant of what hajipens to the lecithin or protagon of the brain. A supposed increase of the output of phosphates during mental activity has never been fully proved (Hojipe-Seyler'-'). CHOLESTEPJX This sul)stauce i.s contained, not only in nervou.s tissue, Ijut also in blood-coi"puscles, in bile, and elsewliere. In nervous tissue it appears to be an especially abundant constituent of myelin or tlic white sub- stance of the medullary sheath. Preparation from brain or spina/ cord. - The tissue is first dehydrated by cold alcohol, then finely divided and extracted with boiling alcohol. The alcoholic solution is filtered hot, and cooled. A deposit occurs •which consists of protagon and other phosphoi'ised constituents, cerebrin, and cholesterin. From it the cholesterin is dissolved out by ether, and the ether distilled off. To get rid of adlierent traces of lecithin, the residue is heated for an hour with alcoholic potash : this decomposes the lecithin, and the residue obtained by evaporating to dryness is dis- solved in a mixture of alcohol and ether ; from this solution cholesterin crystallises out as its solvents evaporate off. Cholesterin is obtained readily from gall- stones by simply extracting them with boiling alcohol, and treating with alcoholic potash to free it from extraneous matter. Froperfies of cholesterin. — It is freely soluble in hot or cold ether, glycex'in, petro- leum, benzol, and solutions of bile salts, in hot alcohol and in chloroform. From anhy- drous ether or chlorofoim it separates in the form of needles containing no water of ciystallisation ; from alcohol or ether containing water it separates in the form of rhombic tables (C26H44O-I-H2O) ; these are easily recognised under the microscope (see fig. 79). Dry cholesterin melts at 1-1:5°, distils 171 vacuo at 3G0° C. Its Fig. 79.— Crystii's of Cholesterin. 1 Sotnischewsky, Zvit. phijsiul. Chciit. iv. 215. - K. Hasebroek, Ibid. xii. 148. Fhijsiul. Chciii. ]>. ()8H. M JI 2 532 THE TISSUES AND ORGANS OF THE IIODV specific rotatory power (a)p= — 31-6°. Its specific i^ivnitv is 1-046. It may be recognised by the following colour tests : — • (1) With iodine and concentrated sulphuric acid the crystals give a play of blue, red, and green. (2) Heated with sulphuric acid and water ("> : 1) the edges of the crystals turn red. These two tests can be watched under the micrr)- scope. (3) A solution of cholesterin in chloroform shaken with an equal amount of concentrated sulphuric acid turns red, and ultimately purple, the subjacent acid acquiring a green flviorescence (Salknwski). Cliemical constitution and derivativps. Cholesterin is a monatomic alcohol, CogH^g) ^ Hi' It forms a compound with bromine (C26H^40.Br2) ; the compounds of the radicle cholesteryl (C2(;H4 3) that have been examined are the chloride (C2GH43CI) and the amide (CoeH^gNHj). Cholesterin yields, on treatment with hot nitric acid, cholesteric acid, CnHniOfl (Witthaus), and on oxidation, by means of cln^omic acid, it yields oxycholic acid, C24H40OC. By oxidation with potassium per- manganate three acids are obtained, viz. ^-cholesteric,C2(jH4./34, oxy- cholesteric, C26H42O5, and dioxycholesteric, C2r,H4 20(i (LatschinofF). Physiological importance of chol ester im.. — Cholesterin is very widely- distributed in the body (nervous system, blood-coi'puscles, yolk of Qgg, semen, spleen, milk, bile, fjeces, &c.) It occurs in excess in certain pathological conditions, e.g. in gall-stones, in atheromatous, cancerous, and tubercular deposits ; in cataract and in certain de- generative diseases of the retina glistening crystals of cholesterin may be often seen with the ophthalmoscope ; crystals of cholesterin are- often found floating about in ovarian fluids, less frequently in ascitic and pleuritic fluids. It is present, to a large extent, in the seeds- and oils of certain plants — cereals and pulses ttc. A substance very like cholesterin (isocholesterin) was prepai'ed by Schultze from sheep's wool ; the vegetable cholesterins have been named para- cholesterin by Reinke and Rodewald,' and phytosterin by Hesse.^ These differently named compounds differ slightly in melting point, specific rotatory power, &c. but they are apparently all isomeric. ' The mode of origin of cholesterin in the body has not been clearly made out. "Whether it is formed in the tissues generally, in the blood,, or in the liver is not known, nor has it been determined conclusively that it is derived from albuminous or nervous matter. It is alsa I Journ. Chenu Soc. Abstracts, 1881, p. 753. - Annalen Chem. Pharm. cxcii. 179. See also Jacobson's paper on vegetable fats,. Zeit-physiol. Chem. xiii. 32. THE NEliVOrs SYSTEM 533 ■doubtful if we (.•an regard it as a waste substance of no use in tlic body, as its pi-esence in tlie blood-cori)UScles, in nervous matter, in the egg, and in vegetable grains points to a possible function of a liistogenetic ■or tissue-forming character' (McKendrick).' Deteniiint(tk>/i of Cholestcrhi, Lecithin, and Fats {Jlojijx'-Si-i/lcr) The method with slight variations is applicable to estimations of cholesterin and lecithin whenever they occur in blood, brain, &c. A known volume of the liquid (20 to 50 c.c.) or a known weight of the solid is treated with large excess of absolute alcohol ; the insoluble residue is again extracted and washed with alcohol, and finally extracted with a mixture of alcohol and ether. The mixed extracts and washings are evajjorated to dryness on the water-bath. The residue is dissolved in ether, again evaporated to dryness, and weighed. The combined weight of cholesterin, lecithin, cerebrin, and fats is thus obtained. The residue is then treated with alcoholic potash, and heated on the water-bath till all the alcohol is driven off. The residue contains caustic potash, cholesterin, and the products of decomposition of fats (glycerin and soaps), and of lecithin (choline, glycero-phosphoricacid, &c.)- To this water is added, and ether is agitated repeatedlj'^ with the mixture. The ethereal solution is evaporated to dryness and weighed ; this gives the weight of the cholesterin. The cholesterin having been removed by the ether, the watery solution is evaporated to dryness and fused with sodium hydrate and pure nitre. The fused mass is dissolved in water, and to it an excess of nitric acid and then ammonium molybdate added ; the mixture is allowed to stand twelve hours, the precipitate is dissolved in ammonia, and the phosphates again precipitated by magnesia mixture. This pi'ecipitate is washed, dried, ignited, and weighed as magnesium pyrophosphate, 100 parts of which correspond to 764-5 pai-ts of lecithin. The weights of cholesterin and lecithin having been thus obtained, the weight of the fats and cerebrin is obtained by difference. The cerebrin is subsequently estimated in another portion of brain substance. THE CEREBRINS These form a group of ill-detined, nitrogenous substances existing in the white substance of nervous tissue, and it is said also in yolk of ■egg, pus-corpuscles and spleen. - Miiller^ obtained cerebrin by rubbing brain up with baryta- water so as to form a milky fluid ; this is boiled and the resulting coagulum is extracted with boiling alcohol ; on cooling, the alcoholic solution deposits cereljrin and cholesterin. The latter is removed by ether, and "the foi'mer is purified by repeated crystallisation from boiling alcoliol. 1 Physiolugij, i. 147. - Hoppe-Seyler's Phijsiul. Chem. pp. T'iO, 788. The cerebriu in the spleen is doubt- less obtained fi-om the white corpuscles contained in that organ. •> Miiller, Aiutaleii Chem. Pharin. ciii. 131; cv. 301. 534 THE TISSUES AXD ORGANS OF THE BODY According to Miiller, its foniiula is Ci^HajXOg: according to Parous,' CgoH, 50^2^1 5- Parous also obtained two other similar substances with different formul*. Adopting a slightly ditfereut moilns operandiy Geoghegan- obtained a substance with the formula 0.5711,10X2025. Thudichum-^ obtained three nitrogenous substances, which may be classified as cerebrins, which he has named cerebrin (C34HggN208), phrenosine (CgjHgyXOg), and kerasene (C^gHgiNOg). Gamgee^ found that whilst protagon cannot be separated by the action of solvents into lecithin, and a non-phosphorised substance cerebrin, yet such non-pliosphorised substances do exist by its side in the brain, and one which he tenns pseudo-cerebiiu (C^jH^oNO^) can be obtained from protagon by the action of caustic baryta. The above facts show that there are probably several cerebrins, but that our present knowledge of these non-phosphorised, nitrogenous constituents of the brain is most incomplete. The cerebrins are like mucin in being nitrogenous gluco.sides ; when boiled with acids they yield a laevorotatory, unfermentable .sugar (Liebreich,^ Diaconow. Otto,' Geoghegan,^ Thudichum"). Tliis sugar is galactose (Thierfelder,* Brown, and ^lonis''). ' Parens, Joitrn. f. prcM. Chem. exxxii. 310. - Geoghegan, Zeit. phyaiol. Cliein. iii. 332. ■' Loc. cit. * Virchow's Arcli. xxxix. 183. ° Ihid. xli. 272. ^ Geoghegan stated that the substance whicli reduced alkaJine solutions of cupric salts had the formula CtiB-^O^ ; he named it ceti/Ud. By fusing it with a caustic alkali,, palmitic acid was obtained. There is, however, no doubt that cetj-hd was a mixture of a sugar vrith other decomposition products of cerebrin. " Journ. f. pralii. Chcm. xxv. 28. * Zeit. physioh Chcm. xiv. 20'.). ^ Proc. Chem. Soc. London, 1889, p. 107- 535 CHAPTEK XX\' THE ORGAXS OF Till-: BODY Thk animal IkkIv is built of a luiinlter of constituent part.s called organs. Each organ has a .special function. The functions of diflerent organs are, however, interrelated more or less closely. Those of which the functions are more closely connected to one another are grouped together into sets of organs or systems. We have thus the circulatory^ respiratory, alimentary, and other systems. The organs, moreover, are built up of certain elementary textures or tissues, and in the preceding chapters we ha^•e been dealing with the chemical physiology of these tissues. In considei'ing the chemistry of the organs, we shall tind each to consist of several of the tissues, and therefore containing the substances found in those constituent tissues. Many of the organs we have now to consider may therefore be dismissed in a few words ; others, such as the liver, will demand more detailed study ; and others again which form secretions will be only studied in part. In this chapter the chemical constituents found in the organs themselves will alone be considered ; the secretions, they form can be more conveniently studied in relation to alimentation, nutrition, and excretion. Relation of water and solich in various or gams. — The following analyses, most of them by Oidtniann,' of some of the organs which we shall con.sider give the relative normal amount of water, organic matter, and mineral matter in each : — Organs Water organic substances Inorganic substances Liver (child) . . ; . . Liver (old woman) . . . Spleen Thvmus (dog) Thvmus (calf) .... Thyroid (child) . . . . Thyroid (old woman) . . Suprarenal body .... Kidney (child) " . . . . Kidney (old woman) . . Lung Testis (Jliescher) . . . 74-14 24-78 80-68 18-65 70-77 28 HO 80-7 19-2 77 21 77-2 22-8 82-2 17-6 80-08 19-88 77-82 21-47 8109 17-92 79-G 19-8 1-07 0-71 0-5-0-9 0-2 20 0-5 0-9 009 0-71 01 0-G 75-0 25-0 1 PreisscJrrift Wiirzburg, 1858. 536 THE TISSl-ES A>'D ORGANS OF THE P>ODY The relation of water to solids was determined in a large number of the organs and tissues of twenty normal pigeons by S. M. Lukjanow.' These were compared with similar observations on twenty pigeons from which food and water had been ■withheld for some time. The chief conclusions to be drawn from the exhaustive tables of results are as follows : — Organs and tissues of the starving animals only showed important ■changes in the relation of solid to water when the total body-weight was diminished by 34 per cent, and the animal had taken no solid or liquid food for 13-3 hours. The relation in some organs (heart, kidneys, thorax muscles, alimentary tract, blood, brain, and lungs) undergoes little or no change ; in others (thigh muscles, bones) the water is increased ; while in a third category (spleen, pancreas, liver) the water is diminished. Sex and initial weight are apparently factors that have no influence. The follo%\'ing table gives the average percentages of water and solids in the organs as found by Lukjanow (see also p. 58) : — Blood Brain I-i-' ^ Spleen Kid- neys Heart Thigh Lungs mus- ; cles Thigh boue Normal — Water. . . 77-07 80-16 72-95 74-27 75-29 76-53 78-90 77-41 77-14 78-08 74-86 46-48 Solids . . . 23-93 19-84 2705 25-73 24-71 23-47 21-10 22-59 22-86 21-92 25-14 33-52 In inanitiou — Water. . . 77-44 1 79-78 73-25 7219 74-08 76-21 78-22 77-55 77-05 77-67 76-57 51-52 Solids . . . 22-56 20-22 26-75 27-81 25-92 23-79 21-78 22-45 22-95 22-33 ' 23-43 ' 48-48 THE LIVER The liver may be considered as a mass of epithelial cells prevaded in all directions by blood-vessels and bile-vessels. In most forms of ■epithelial tissue, the constituent cells are spread out in layers to form a membranous investment or lining of some organ. But in the liver the cells are collected together into lobules, the whole being bound together by means of connective tissue. The liver is one of the largest masses of cells in the body, and is larger in proportion in the embryo than in the adult ; these cells perform many important functions. They are formed from the same embryonic layer, the hypoblast, as that from which the cells that line the alimentary canal are formed, and the function of the liver is intimately connected with the processes of alimentation. > Zeit. phijsiol. Chciit. xiii. 339. THE LlVF.Ii 537 The livei- receives a supply of artt'iial blood by the hepatic artery. This appears to l>e concerned chiefly in supplying the supporting connective tissue of the organ. The chief supply of blood to the liver is venous blood ; this conies via the portal vein, formed by the union of the mesenteric and splenic veins ; the portal vein breaks up into capillaries after the manner of an artery ; the blood leaves the liver by means of the hepatic veins, which open into the vena cava inferior. During digestion the portal vein carries to the liver certain products of digestion absorbed from the alimentary canal : this is taken from the blood by the liver-cells, and stored up there chiefly as glycogen. This is again given out as necessity arises, probably in the form of a soluble sugar, and leaves the liver by the hepatic vein. The storage capacity of the liver led Claude Bernard to compai-e its function with that of "the tuber of a potato plant ; the tuber stores up carbohydrate in the form of starch, receiving it in a soluble form from the leaves, where it is formed, and giving it out again as a soluble carbohydrate. The important secretion called bile is also formed by the liver-cells. This will be considered in connection with digestion. The liver-cells have lastly a most important action in producing urea -and uric acid, and other products of nitrogenous metabolism, wdiich ultimately pass into the urine, and will be considered in connection with that secretion. Chemical Composition of the Liver Substance The fresh liver is alkaline in reaction, but after death soon becomes acid, and this, as in so many cases, is due to the development of sarco- lactic acid. The number of organic substances occurring in the liver is very numerous. There are proteids and nuclein contained in the protoplasm and nucleus respectively of the hepatic cells themselves ; there are substances, like glycogen, sugar, and fat, which are stored up by the liver-cells, or produced from stored-up substances ; there are certain •constituents, such as gelatin and mucin, derived from the connective- tissue framework ; blood and bile may als(j he found if means have not been taken to remove these previously. There are also extractive matters like xanthine, hypoxanthine,'and uric acid ; and lastly a certain small proportion of inorganic constituents. The proportion of water present is roughly the same as in muscular tissue, viz. 75 per cent. The following numl:)ers are given by v. Bibra : ^ 1 V. Bibra, Cliemi>>che Fragmente Hber die Leber, 1849. 538 THE TISSUES AND ORCtANS OF THE r.OI)\ Water Insoluble tissues Proteifls Gelatin Extractives . Fats 7G-17 9-4-1: 2-40 3-37 2-40 2-oO Oidtmann ' found 1-1 per cent, of inorganic material of whicb potassium phosphate, as in most solid organs of the body, was the most abundant. His numbei's ai*e as follows : — Potash, 25-17 ; soda, 14"17 ; lime, 362 ; magnesia, 0-19 ; iron oxide, 2*75 ; phosphoi'ic acid, 43-37 ; sulphuric acid, 0-91 ; silicic acid, 0-27 ; chlorine, 2-5 ; traces of manganese, lead, and copper. Calcareous deposits, consisting chiefly of calcium phosphate and carbonate, may occasionally be found in the liver. The Proteids of the Liver-cells This has been the subject i>f an interesting research by P. Plosz.^ Fresh liver substance was found to be alkaline, after death it became first neutral and then acid. The liver at the same time became harder and less transpai-ent, and these changes are all attributed to a condition resembling the rigor mortis of muscle. The liver was rapidly washed free from blood and bile by means of a stream of ice-cold salt solution (0-75 percent, sodium chloride), cut into small pieces by means of cooled knives, frozen, and the pieces subjected to pressure. As the pieces thawed, an alkaline juice was expressed from them, which may be termed liver-plasma, as it appeared to be analogous with Kiihne's muscle- plasma [see p. 406). Here, however, resemblance ceased, as clotting never occurred in the plasma ; myosin is therefore not present in liver-plasma. The liver-plasma contained in solution a proteid coagulating at a temperature of 45'^ C. (in this it resemljles muscle- plasma) and a nucleo-albumin : in the cells from which the juice had been expressed, a globulin which is more difficult of solution than the two just mentioned. The liver-plasma also contained glycogen and small quantities of sugar. A more thorough investigation of the proteids was made by extracting the proteids from the liver- cells by means of saline solutions ; 0-75 per cent, sodium chloride, 10 per cent, sodium chloride, and solutions of sodium sulphate and other salts were employed to dissolve the proteids. The proteids present were : — 1. A proteid coagulating at 45° C. ; wholly soluble on gastric- digestion. 1 Oidtmann, 'Die anorgunischen Bestancltheile der Leber,' Premr/;///y, Wiirzburg, 185H. - Pfliiger's Archiv, vii. 371. THE LIVKK 539' '2. A nuclei i-;illjiuiiiii coagulating at 70° C, yielding an insolul^le residue of nuclein on gastric digestion. .'?. A globujin coagulating at 7->° C. This was most readily extracted with a 10 per cent, sodium chloride solution. It also was wholly digested by gastric juice. 4. Alkali-all lumin. ."). The nuclei contained nuclein. I have repeated Plosz' e.xperiments with certain slight variations, and find that saline solutions extract the following pioteids from the liver-cells : — ' 1. A globulin coagulating at 45° C. 2. A globulin coagulating at 56° C. 3. A globulin coagulating at 70° to 75° C. 4. An albumin coagulating at 70° to 73° C. No. 1 is probably identical with what I have termed cell-gloV)ulin a (see p. 260). No. 2 resembles myosinogen in its coagulation-temperature, Init like Plosz I have failed to find any further e^'idence of myosin. T should propose the name hepato-globulin for this substance. No. 3 is cell-globulin (see p. 260). No. 4 is cell-albumin, but is present in the merest traces, and may be practically absent in many cases. I have failed hitherto to obtain any evidence of nucleo-albumins. I am inclined to regard the hardening that occurs in the liver after death, and which is very slight, as not being comparable to the I'iffor mortis of muscle, but is more probably due to the solidification of the fat in the cells, which during life is liquid. It is, however, possible, as P16sz suggests, that if coagulation does occur in the cells with the formation of a myosin-like clot, this takes place so rapidly that our present methods do not ena1)le us to separate its precursoi- from the cells.. The Glycogen of the Liver The glycogen of the li^er-cells can be frequently demonsti-ated in^ them micro- chemically by means of iodine; glycogen is stained a reddish-brown colour by this reagent ; it occurs in globules or in irregular amorphous masses within the cells (Heidenhain ^), and when abundant reduces the protoplasm of the cell to the condition of an open network which becomes Aery distinct after solution of the glycogen (fig. 80). Preparation of glycogen from tlip liver. — A rabbit is killed three or ' Proc. Physiol. Soc. 1H90, p. 9. ' Heideiihiiiii, Hermann's Haiulhuch, 1880. )40 THE TISSUES AND ORGANS OF THE BODY four hours aftei- ;i hearty meal of carrots. The blood is washed from the liver Ijy passing a stream of salt solution through the vessels, a cannula is inserted into the portal vein for this purpose ; another cannula is placed in the vena cava inferior and the mixture of blood and saline solution which comes from the liver can be collected. The iirst portiijns that come through contain sugar. When the organ is rendered colourless, and the salt solution that leaves the liver is no longer deeply tinged with blood, the liver is removed and plunged into boiling water acidulated with a little acetic acid. A certain amount of glycogen is in this way extracted, and the proteids of the liver-cells are coagulated. Any ferment too which may be present, and which i Fig. so. — Hepatic Cells from the Liver of a Dog, founeeii hours after a full meal (Heidenhain) : «. with glycogenic deposit ; 6 and c, after its solution. In c the network wliich remains is finer than in b, and impai-ts a somewhat gi-aniilar appearance to the cells. The external layer of the protoplasm contains no glycogen. would convert the glycogen into sugar, is destroyed. The pieces of liver thus scalded are then thoroughly extracted with boiling water, and tiltered off. The extract is very opalescent, and contains a trace of proteid, which may be precipitated b}' means of a little hydrochloric acid and potassio-mercuric iodide, and tiltered off. The filtrate is con- •centrated, and excess of rectified spirit added. This precipitates the glycogen as a white amorphous powder, which is collected, washed with ether and absolute alcohol, and dried (Briicke '). Kiilz - recommends a dilute solution of potash instead of water for •extracting the glycogen from the liver. If a quantitative estimation is to be made, a weighed quantity of liA'cr must be taken in the first instance, and it must be repeatedly extracted until no more glycogen passes into solution. The dried glycogen obtained from all the extracts mixed together is also weighed ; or the glycogen may be converted into sugar by boiling with sulphuric ^cid and then estimated polarimeti'ically ( Kiilz), or by means of Fehling's solution. In the liver the glycogen is equally distributed throughout ; it is ^ Briicke, Sitzungsher. der Wiener Akad. Ixiii. 214. - Kiilz, Zeit. Biol. xxii. 161. TIIK lAVVAl 541 therefore only necessaiy to use ;i small poitiou of the liv«M- foi- quiintitative estimations (Cramer '). Glyco(i;en is also found in muscles (see p. 422), in many fo'tal tissues,, in white blood-corpuscles, and in numerous invertebrate animals. Variations in the aimnotl of .V^yco/r/p/i in the hrer. In 1843 CL Bernard - recocfnised that in the liver was a source of i(rape-su«ifai-, and that the suyar in the blood did not directly depend on the intake of carbolivdr.ites in the alinicntary canal ; he found, foi- instance, that sugar occurred in the blood when the animal was fed on a purely proteid diet. Bernard found sugar in the liver of all animals which were in a healthy condition. The next important step in the investi- gation of this subject was in the year 1856, when Bernard •' and Hansen ^ independently of each other prepared from the liver a carbo- hydrate which like starch formed an opalescent solution with water, and was convertible into sugar by saliva or other diastatic ferments.. This is the substance we now know as glycogen. Briicke,' Abeles,^ and Kiilz ^ have since that time improved on the original methods, adopted for its preparation. Bernard stated that the foetal liver contains little or no glycogen, and considered that the placenta, which contains glycogen, takes the place of the liver as a source of sugar in intra-uterine life. Hoppe-Seyler,*" however, finds that the fcetal liver like most other fcetal tissues, including the placenta, contains abundance of glycogen, and in new-born dogs. Demant^ found the glycogen to be more abundant than in adult animals. Salomon "^ has made similar observations on still-born children. During inanition the glycogen t»f the liver disappeai-s ; none can be found in the livers of rabbits after six to nine days' abstinence from food; other animals have also been investigated with similar results (Luch- singer," Weiss,'- Kiilz,'^ AldehofF,'^ and others). The hepatic glycogen seems to disappear more quickly than the muscle-glycogen. During hibernation the liver continues to be rich in glycogen (Aeby, ''^ Yoit '*^). 1 Cramer, Ihid. xxiv. 67. Cramer's careful experiments eorrectefl the statement, , originally made by v. Wittich [Centralhl. vied. Wiss. 1875, No. S), that different parts of the liver contain different percentages of glycogen. 2 Bernard, NouvelJe fonction du foie, 1853; Arch, gtneralcs dc wed. Oct. 1848. •* Bernard, Gaz. vitd. de Paris, No. 13, 1857; Comjit. rend. xliv. 578. * Hensen, Verhandl. d.plujs.mcd. GeseUsch. zii Wiirzhurg, July 185(i, vol. vii.p. 219; Arch. path. Anat. xi. 395. ^ Xjoc. cit. 6 Abeles, Wien. ined. JahrhUcher, 1877, p. 551. '' Loc. cit. * Hoppe-Seyler, Physiol. Chew. p. 708. 9 Demant, Zcit. j'h/jsiol. Chcin. xi. 142. 1*^ Salomon, Centralil. med. Wiss. 1874, No. 47. 11 Pfiiif/cr's Archie, xviii. 472. '- Weiss, Wicn. Akad. Sif.viiiirjsber. Ixvii. . 1' Kiilz, Sit.~iatgsb. d. Marburgrr GcsrU. 1870, No. 5. 14 Aldehoff, Zeit. Biol. xxv. 137. 15 Aeby, Arch. cxp. Path. n. Pluirtii. iii. 184. "• Yoit, Zcit. Bivl. xii. 209. 54-2 THE TLSSL-ES AND ORGANS OF THE ];<>I)V A few lioufs' active exercise causes a greatei- refluction of tlie hepatic glycogen than days of starvation (Kiilz'); muscular exercise also reduces the amount of glycogen in muscle (st-e p. 424). Strychnine poisoning has the same result, even when it does not produce con- ■vidsions ; and, strangely enough, curare, which abolishes muscular move- ment, has the same effiect ; in both cases the animals become at the same time diabetic (Demant -). In fever, the liver of human beings is found pout morteni to be almost free from glycogen and sugar (Bernard). In diabetes the glycogenic function of the liver is deranged, so that an increased (quantity of sugar enters the circulation. In poisoning by arsenic and phosphorus, the glycogen of the liver is diminished. These drugs produce a fatty degeneration of the liver- cells. In other forms of fatty liver the glycogen is also diminished in -quantity, or it may be absent. It has been found experimentally in animals that ligature of the bile-ducts, no doubt by interfering with the normal metabolism in the liver-cells, also causes a diminution of the hepatic glycogen (v. Wittich). Fommtion of glycogen in the liver. — Bernard supposed that pioteid food was the source of the hepatic glycogen ; Pavy, on the other hand, considered that it was fi'om carbohydrate food alone that glycogen was derived : the usual view accepted at the present time is that both varieties of food may act as sources of glycogen. The glycogen is most abundant after carbohydrate food, but it also occurs in the liver of flesh-feeding animals, and in animals kept exclusively on a proteid diet. Glvcogen is not fonned from fat. The following are the chief experimental facts bearing on this subject : — Grape sugar, starch, dextrin, cane sugar, inulin, fruit .sugar, milk sugar, and lichenin given as food increa.se the amount of glycogen in the Hver (Dock,-^ Luchsinger,* Frerich.s,'' Kiilz, Bernard, Tscherinoff,'' Pink,' Komanos,"^ Salomon, v. Mering^). Gum ai-abic, inosite, mannite, •erythrite, and quercite have no such effect (Salomon, Kiilz, v. Meiing, Luchsinger). Fats and soaps are also inactive (Bernard, Tschei-moff, McDonnel,'" Xuchsinger, Kiilz). 1 Kiilz, Pfliiger's Arch. xxiv. - Demant, Zeit. phijsiol. Chem. x. 442. 3 Dock, Pfliiger's Arelnv. v. .571. * Luchsinger, Ihul. viii. 289. 5 Frerichs, Dm. Wiirzburg, 1876. ® Tscherinoff, Wien. Akad. Sitzungsh. li. (2), 412. ' Pink, Diss. Konigsberg, 1874. * Komanos, Diss. Strasbiirg, 1875. 9 V. Mering. Pfiigers Archiv, xiv. 274. '" McDonnel, Conqit. rend. Ix. 693. ■I'liK i,i\Ki{ ri43 Gelatin increases the amount <>f i,f]yc()!?en in the liver (Bcrnunl, Salomon, Luehsinger). Pi-oteid foixls also increase the amount of glycogen in the liver (Bernard, Finn,' v. Mering, Xaunyn '•'). Some observers have failed to make out any marked increase (Dock, Weiss, Luchsinger). Glyceiin undoubtedly causes an increase of the liver glycogen (Weiss, Luchsinger, .Salomon). Anunonium carbonate increases the glycogen also (Rohmann ^), and certani amido-compounds (asparagine, glycocine) act similarly (Rohmann'). As sodium carbonate does not act in this way, it is suggested that ammonium carbonate does not exert its influence by relit into glycogen on the one hand, and a nitrogenous product, such as urea, on the other. Forster ' <»btained an increase in the glycogen of tlie liv'er by in- jecting a concentrated solution of sugar into the portal vein, and at the same time the urea in the urine was increased. Luchsinger ^ found that, by passing a stream of arterial blood containing grape sugar in solution through a liver just removed fi'om the body, glycogen continued to be formed, and Seegen and Ki'atschmer '' found that a calf's liver after removal from the body continued to foi-m glycogen even though no blood Mas passed through it. Prausiiitz,"* from experiments in feeding hens on cane sugar, con- cludes that the quantities of glycogen in the whole body, in the liver, and in the muscles run closely parallel to one another : the maximum of glycogeii-formation, as evidenced Ity the quantity found after death, occurs twenty hours after feeding. This is somewhat later than is stated by previous observers, and is certainly not coincident with the maximum of bile-formation. From such an enumeration of the substances that have been found to cause an increase of the liver glycogen, one would be justitied in at once concluding that the building up of glycogen in the liver is by no means a simple process. The following suggesti\e remarks bearing on this subject are taken from a paper by E. Pfliiger ^ on synthetical proce.sses in the animal oi'ganism. ' Finn, Wii izbitrger Verhandl. d.phijs. meil. Ges. X.F. xi. No. 192. - Naunyn, Arch.f. exp. Path. u. Pliarm. iii. 85. 5 Rohmann, Centralhl. f. klin. Med. 1884, No. 35. * Rulimann. FffUgcr's Arch, xxxix. 21. ^ Zeit. Biol. xi. olo. 6 X)/.s.s. Ziirich, 187-5, p. G2. ' Pfluger's Archiv, xxii. 33. ® Zeit. Biol. xxvi. 371. ^ Pfliiger's Archiv, xlii. 144. 544 THE TISSTES AND ORGANS OF THE P.ODY ' A living liver free from glycogen will again form that substance not only from carbohydrates, but from gelatin, proteid, or from glycerin. V. Mering ' fed dogs on phloridzin, whereby they became diabetic, and in a few days all carbohydrate materials in the body had been discharged as sugar. If now the same drug were given to the same animals after a few days' interval during which they had no food, they once more became intensely diabetic, and the quantity of sugar passed was so enormous, that it cannot be supposed to have come from the drug itself. It must therefore have been formed from the proteid substances in the animals'' tissues. One explanation of the way in which glycogen is formed after- the administration of glycerin is the well-known ' economy theory '; ^ another is that glycerin and similar substances act as stimuli to the activity of the liver-cells. It certainly cannot be supposed that glycogen is directly formed from the substance administered, or at least not in^ all cases ; for instance, from ammonium carbonate. ' The question then arises as to the genetic relationsliip existing between glycogen and albumin. Experiments outside the body on the decomposition products of proteids have in no case yielded a carbo- hydrate, and not only that, but proteids never yield any of the- commoner decomposition products of carbohydrates such as mucic acid^ tartaric acid, etc. Still we have tlie formation of glycogen taking place- in the liver, when no food but albuminous food is taken. ' The following genei-al considerations will, however, lead to a better understanding of the suljjeet. The chemical differences between animal and vegetable cells are not so great as was at one time supposed ; their chemical composition, so far as it is known, is the same ; all living cells breathe oxygen and produce carbonic anhydride, water, and amido- compounds. Synthetic processes are more highly developed in chloro- phyll-holding plants, but they also occur in animal cells. As instances of synthetic processes occurring in animal cells, the formation of hippuric acid from glycocine and benzoic acid, or of an ethereal sulphate from phenol and sulphuric acid, may be taken. A special kind of synthesis must moreover occur in the i-etrogressive metamorphoses of proteids that lead to the formation of uric acid and urea. In albumin itself, and in the derivatives of albumin obtained in the laboratory like indole and leucine, the number of carbon atoms is much greater than that of nitrogen atoms, but in many of the products of metamorphoses, in the body, the nitrogen and carbon atoms are nearly equal in number, or, as in the cases of urea and guanidine, the nitrogen atoms are the- ' Verltandl. VI. Congresses in never Med. Wiesbaden, 1887. - This theory may be briefly stated thus: the glj-cerin is used in combustion instead' of glycogen, so allowing the latter body to accumulate. THE LIVER 545 more lunnerous. The iinpU. '11 IK I,1\KK 547 if the livei* be removed from a healthy animal with sufficient rapidity :after death, and l)<)iled or scalded so as to kill a supposed ferment, no sugar was obtainable from it, and that durin<,' life the blood in the hepatic vein contained no more sugar that the portal blood. Pavy,' however, subsequently admitted that the noi-nial liver contains 0*2 to 0-6 part per 1000 of sugar. The large quantity of sugar found in a liver after death was attributed to the action of a fei-ment which was considered to be formed in the Ijlood from the solution of the blood- corpuscles.2 This fei-ment Avas considered to be a diastatic ferment, •i.e. one which like ptyalin and diastase converts starch and glycogen into sugar. The quantity of sugar in the hepatic vein as compared with that in the portal vein is a very important point to settle in connection with this question, and this apparently simple investigation has been the subject of very contradictory statements. v. Mering ■' found less sugar in the blood of the hepatic than in that of the portal vein ; Bleile,^ like Bernard, found more; and Abeles,' like Pavy, found about "the same amount in the two varieties of blood. Prof. M. Foster, speaking on the subject in his 'Text Book of Physiology,' ^ says: 'In "view of this conflicting evidence we shall not go far wrong in assuming that Bernard's view is not as yet clearly disproved. The quantitative determination of sugar in the blood is open to many sources of error. When the quantity of blood which is continually flowing through the liver is taken into consideration it is obvious that an amount of sugar ■which in the specimen of blood taken for examination fell within the limits of errors of observation might, when multiplied by the whole •quantity of blood and by the number of times it passed through the liver in a certain time, reach dimensions quite suflScient to account for the conversion into sugar of the whole of the glycogen present in the liver at a given time.' Others, puzzled by Pavy's researches on the very slight increase or absence of any increase of sugar in the hepatic blood, considered that the glycogen of the liver is converted, not into sugar at all, but into fat. This view was chiefly based on the fact that carbohydrate food may be in some way or other a source of the fat of the body. In view of recent researches, however, this theory of the fate of the liver glycogen is unnecessary. Among recent investigators, Seegen has done most to add to our knowledge on the subject. Many of his conclusions have not, however, ' Croonian Lectures on Diabetes, 1878. - Tiegel, Pfliiger's Ajxhiv, vi. 24'J. ' V. Mering, Arch. f. Anat. a. Pliijsiol. 1877 ; Flujslol. Ahtli. p. 412. * Bleile, Ibid. 1879, p. 75. ^ Abeles, Wien. nied. Jahrbilcher, 1875, vol. iii. '' Fifth edition, p. 726. K K2 548 THE TISSTES AND OEGANS OF THE BODY met with genei-al acceptance. It will be com eiiieiit to discuss these- researches, not in chronological order, but under the following heads: — ■ Tlte 2^&^'centage of siu/ar in diffemd kinds of blood. — From a large number of experiments Seegen' gives the following averages : — Normal amount of sugar in cardiac and arterial blood 01 — 0"15 p.c. „ „ portal blood 0'119 „ „ ,, hepatic blood 0'23 ,, The following four experiments'- illustrate the same fact, viz, that hepatic blood contains, roughly speaking, twice as much sugar as portal blood : — Percentage of sugar Portal blood Hepatic blond 1. 0-101 0-258 2. 0-090 0-175 3. 0-107 0-209 4. 0-120 0-287 What kind of siKjar is j^i'esfnt in the blood. — Although the sugar formed from glycogen by diastatic ferments is maltose, that found in the blood leaving the liver is dextrose (Kiilz, Seegen,"^ Eves'*). .Chittenden and Lambert^ speak of the sugar as a mixture of maltose and dextrose, but Seegen has shown that this is prol)ably due to the fact of small quantities of dextrin and cane-sugar, which are partly absorbed as such from the alimentary canal, having been reckoned as maltose. Formation of sugar in excised livers. — The liver continues to form sugar after death, and according to Seegen*^ the glycogen does not diminish in a corresponding ratio. He concludes, therefore, that the glycogen does not furnish the sugar, but fulfils some other, at present unknown, function. If pieces of excised liver be placed in contact with solution of peptone, sugar is produced, and the same occurs with fat. He therefore concludes that the liver forms its sugar from proteid and from fat. This conclusion is, as Hoppe-Seyler" states, not to be accepted without fuller inquiry. Seegen does not show, for instance, that the peptone diminishes in proportion as the sugar increases. An equally possible explanation is that the peptone merely stimulates the- activity of whatever agent it is that produces sugar in the liver after 1 Bied. CentralU. 1H84, p. 747. - Seegen, Pfliiger's Archiv, xli. 52G.. s Ihid. xl. 48. 4 Jonrn. of Physiol, v. 350'. * Studies from Lah. PJii/siol. Chcm. Yale Univ. 1885. ^ Seegen and Kratschnier, Pfliiger's Archiv, xxii. 8; xxxvii. 348; xxxix. 121. " Physiol. Chcm. p. 717. 'UK l.lVl'.h' 549 •death ; or a tliinl possible explanation and a very probable one, as it was on carnivorous animals (dogs) upon which Jiiost t)f the experinienLs were performed is that glycogen may Ije an intei-mediate product in the formation of sugar from proteids. Tlu^ figures obtained by other analysts are entirely conti-ary to Seegen's on this point. Thus Chitten- den and Lambert, Bohm and Hoffmann,' H. CJirard,' Abeles^ and Panornow,'' and A. Dastre •" have all arrived at the same conclusion, viz. that as the sugar increases, the glycogen diminishes. I quote the following table from H. Girard's analyses, which brings out this point very well : — - Auimal 1 10 mimites after death 1 24 hours after death 48 hours after death Sugar Grlycogen Sugar Glycogen Sugar Glycogen ' Dogl 2 Cat 1 » 2 Rabbit 1 „ 2 0-55 0-74 0 48 0-62 0-75 0-65 2-12 4-05 5-88 4 96 9-56 10-25 1-80 300 2-95 315 3-58 4-12 0-76 1-50 3-20 2-08 6-35 6-24 1 1-75 ' 312 306 3-48 3-85 j 4-20 0-75 1-38 , 2-88 1-87 4-28 505 The ferment theory of the change of glycogen into sugar. — These -changes just described that occur after death are no doubt indications of what is always occurring during life ; in the normal process of metabolism, glycogen is formed from something else on the one hand, and given out as sugar on the other by the same cells. After death, as the liver-cells retain a certain amount of vitality, the process still ■continues.^ There is no necessity to assume the action of any sf)ecial ferment developed after death to account for the phenomenon observed, and in fact Seegen and Eves have both shown that no such ferment can be extracted from the liver in larger quantity than from any other tissue of the body. We have, for instance, already seen that such a fei'ment can be obtained from muscle (p. 412), and it seems that diastatic activity is present in all living proteids. The diastatic fer- iment which is obtainable is, however, not derived from the blood, but 1 Pflilger'H Archie, xxiii. 205. ''' Ibid. xli. 294. ^ Wie»ier vied. Jahrb. 1887, p. 383. * Polish paper; Abstract in Mahj's Jahrrsh. xvii. 304. ^ Arch, dc Phijsiol. (4), i. B9. * In favour of this view it may be stated that the greatest formation of sugar tliat -occurs in the excised liver takes place within an hour or two after its removal. That the liver retains its vitality for this length of time is supi^orted by the fact that if a stream of blood be passed through an excised liver, it continues to form bile for a couple of hours iSchmulewitsch, Bcr. d. S(ichs. Akad. d. Wiss. 18C8) 550 THE TISSUES AND oROANS OF THE JloDV from the liver-cells (v. Wittich'), and it moreover does not convert glycogen into dextrose, but into maltose. In a recent I'esearch on the influence of glycerin on the liver, W. B. Ransom 2 finds that the administration of glycerin to I'abbits prevents the glycosuria that usually follows injury to a certain spot of the medulla oblongata ; and also that it prevents the post-mortem change of glycogen into sugar ; he therefore concludes that the glycerin checks the glycosuria by inhibiting the formation of sugar in the liver-cells, and that in this way the accumulation of glycogen in the liver is fully explained. Fat in the liver-cells. — The normal liver contains about 2 to 3 per cent, of fat ; in acute yellow atrophy the percentage may rise to 7*6y and in fatty degenei-ation to 19-5. Fatty degeneration occurs in many wasting diseases, such as phthisis, as a result of chronic alcoholic poisoning, and also as a result of poisoning by phosphorus, arsenic, and antimony.^ Grohe and Mosler ^ state that in the duchy of Brunswick the peasants give to the geese when producing the famous fatty livers a small quantity of white oxide of antimony every day. The fat in the liver-cells can be readily seen by the microscope in the form of minute globules. These are especially abundant after a meal, particularly after a fatty meal. These globules are seen in greatest number in the so-called portal zone of a hepatic lobule ; that is, in the outer region of the lobule, which is the part to which the portal capillaries are first distributed. In fatty degeneration it is generally noticeable that the morbid process commences in the same region. The amounts of glycogen and fat in the healthy liver run parallel with one another. In hunger both disappear, on feeding they reappear ; and just as glycogen may under certain circumstances be formed from substances other than carbohydrates, so fats seem also to be formed from substances like proteids other than fats. Fat like glycogen is here a result of the metabolic activity of the liver protoplasm. Other organic constituents of the liver-cells. — Urea, uric acid (especially in birds), xanthine, and hyjDOxanthine are found in the liver. -^ These substances are instances of the destructive metabolism of proteids ; the liver is considered generally to be the organ where a very large amount of the organic bodies of the urine are formed. Thei^e pass into the lilood-stream and leave the body by the kidney secretion. 1 Pjiiiger's Arch. vii. -is. - Jour)i. of Physiol, viii. !)'J. •'• A recent paper on the relation of fat in the liver to various toxic agents will be found by Chittenden and Blake, Studies from Lab. Physiol. Chetu. Yale Univ. iii. 106 See also Salkowski, Viixhoiv's Archiv, xxxiv. 78. * See "Wood's Thei-ajteutics, p. 161. * Sclierer, Ann. Chein. PJtarin. cvii. 314; Cloctta, Ibid, xcvii. 289. 'I'lii': i,i\'Ki{ 551 AVlieii tliis fii notion of the liver-cells is considered in addition to those already enumerated, we see the vast importance in analytical and especially in synthetical processes that is possessed Ijy tin; mass of protoplasm we call the liver. Dnicine and tyrosine do not normally occur. They are, however, found in the li\er of eases of acute yellow atnjphy, and in cases of phosphorus 2)oisoning.' Various (tther substances ha\e been desciibed by various observers, but do not appear to be constantly present ; such as guanine, inosite, scyllite,^ cystin (in a pathological case),^ sarco-lactic acid (probably formed after death). ^ A suhst-Ance, jeroriii, containing phosphorus (C,05Hij(gN;,SP3O4Q) has recently been separated from the liver by Drechsel.^ In its properties it somewhat resembles lecithin ; it however like sugar, but unlike lecithin, reduces Fehling's solution. According to Baldi,^ it occurs also in many other organs .spleen, muscle, braiii, Arc. The so-called waxy or amyloid substance replaces the px'otoplasm of the liver- cells in the condition known as waxy degeneration. In this condition large quantities of cholesterin are also sometimes found in the liver.'* The inorganic constituents of the liver. — These have been already enumerated ; it is now necessary to add a few words respecting one of the most interesting of these, viz. iron. Iron in the liver. — This has been the subject of a special research by S. S. Zaleski.^ The liver was tirst thoroughly freed from blood by a stream of 2"5 per cent, cane-sugar solution. The quantity of iron in the blood-free liver was found to vary between wide limits, but it was constantly found in organic combinations in the liver-cells, especially with nuclein ; and one of the iron-nuclein compounds named hepatin was isolated. The iron in these compounds is present in two, probably in three states of oxidation, in the ferrous, ferric, and ferroso-ferric states. Of all the macro-chemical reactions for detecting iron the most delicate were found to be (1) that with potassium thiocyanate and hydrochloric acid, (2) that with potassium ferro- or ferri-cyanide and ' Sotnitschewskj', Zeit. j'hysiol. Cliem. iii. 391. * Frerichs and Stiideler, Mitth. d. Zi'tricher naturf. Gesellsch. 1855. ^ Hoppe-Seyler, Physiol. Chcmie, p. 718. * Minkowski {Arch. ejcp. Path. ii. Pharm. xxi. 14); Marcuse [Pfliit/cr's Archiv, xxxix. 425) and Nebelthau [Zeit. Biol. xxv. 123) found that after extii-pation of the Hver lactic acid appeared in the urine. * Journ. prakt. Chem. xxxiii. 425. * Du Bois Retjxwnd's Archiv, supplement, 1887, p. 100. ^ Zeit. phijsiol. Chem. x. i53; xiv. 274. This subject was first brought into promi- nence by the researches of Quincke {Deutscli. Arch.f. kl'i)i. Med. xxv. 5(57 ; xxvii. 202 xxxiii. 23). See also Peters [Ibid, xxxii. 182). 552 THE TISSUES AND ORGANS OF THE P.()I)Y hydrochloric acid. The green or blue colour produced by the latter test was found to be best adapted for microscopical investigation. After the administration of iron compounds the metal collects in the liver, and Zaleski ' concludes that iron is excreted by the liver, not by the intestinal glands as other heavy metals are. There can, however, be no doubt that the iron-containing pigment present normally in liver- cells is derived from the blood-corpuscles. The great increase of the iron in the liver in cases of pernicious anaemia has been already alluded to (see p. 301) ; a certain amount of blood is always being destroyed in the liver in health, and this is increased in the disease just mentioned (Hunter,'-^ Mott,^ Delepine "*). Dr. Mott has recently recorded three cases which confirm this view of the pathology of the disease. The portal zones of the lobules were so crowded with albumino-ferruginous compounds that the sections when stained with potassium ferrocyanide and hydrochloric acid appeared as if injected. The urine as in most cases (Fagge) was highly coloured. Mott's view of the decomposition that occurs is that the haemoglobin is acted on by the liver-cells to form urobilin or an allied pigment that appears in the urine, and the iron is left behind and accumulates as the disease progresses. In two of the cases the amount of iron found in the liver by analysis was very large, while that in the spleen was not greater than normal. In the third case the iron was increased in both organs. Hunter * has also recorded a similar case, except that he found pathological urobilin instead of normal urobilin in the urine. Bemmelen '' from his own researches and from those of Stahel ^ and Graanboom ^ concludes that the normal percentage of iron in the liver is O'l. In a case of leucaemia this was reduced to O'Ol. It is present in greater proportions in the liver of new-born animals, and probably acts as a storehouse of iron subsequently used in the formation of blood-corpuscles (Bunge). Delepine belicAes that this function per- sists throughout life. Certain pathological conditions of the liver. — It has been necessary to allude to several pathological conditions like fatty degeneration, diabetes, itc. in the foregoing paragraphs. I have here merely to add a table which collects together various quantitative analyses that have » Ckem. CentralU. 1888, p. 759. 2 Lancet, ii. 1888, pp. 555, 608, 654. Full references to previous workers will be found in these papers. s Lancet, vol. i. 1889, p. 520 ; vol. i. 1890, p. 287 ; Practitioner, Aug. 1890. * Practitioner, August 1890. 5 xHij^ September 1889. * Zeit. physiol. Chem. vii. 497. ^ MaJij's Jahrcsh. xi. 427. * Ibid. p. 429. See also Lapique, Compt. rend. Soc. Biol. 1889. ■IILE Si'LKKN 553 v. Bibra FolwarczMV " l'iitt\- 1 ,„ , . , livei- . iyP''"i'l (tubercle) I f*'^'^'- Dia- betes Embolism of liei>ati(' artery Water . . . . | 71*0 Soluble proteids 1-3 Gelatin. ... 44 Extractives . . 2(; Fat 17-4 Insoluble tissues ;>-l Salts .... — 751 2-6 40 4-5 3-3 |lO-2 75-3 6-7 1-1 2-2 1-9 11-7 0-9 80-7 21 11 :?■(; 2-4 8-9 Oit Frericlis Oiiltmaiin Fatty liver Cirrhotic liver Sypliilitic liver in new-born child 73 0 80-2 82-5 ! 3G 3-5 1 17-2 |ll-5 2-2 >16-5 40 :5-6 } 9-1 ! been made, and for which I am indebted to Charles' 'Physiological Ohemistry.' ' Thi.s table is chiefly interesting as showing the great increase of fat in fatty livers, and of gelatin due to the overgrowth of connective tissue in a cirrhotic liver. In a liver that had undergone acute atrophy Rohmann ^ found albumose and peptone, sarco-lactic acid and a mixture of amido-acids, alanine, leucine, and tyrosine being the most abundant. The latter were absent from the urine, which contained, however, excess of aromatic oxy-acids. THE SPLEEN ' The spleen is invested with a fibrous and muscular capsule, and this again has a covering derived from the serous membrane. The capsule sends fibrous bands or traheculce into the oi^gan, and these join with similar trabecule which pass in at the hilus with the blood-vessels. In the interstices of the framework so formed lies a puljyy substance containing blood, and therefore of a red colour, within which are seen small whitish specks, the Malpigliian corpuscles. These are composed of lymphoid tissue which is gathered into masses which envelop the smaller arteries, while the pulp which everywhere surrounds them is composed of a close network of flattened and branched cells like connective-tissue corpuscles. Coursing through the pulp and com- municating with its interstices are capillaries connected with the terminations of the arteries : in other parts venous channels arise from the pulp and bring the blood which has passetl into its interstices from the arterial capillaries towards the larger veins of the organ which run in the trabeculse and are by them conducted to the hilus.' ' The cellular elements of the pulp are of three kinds, viz. peculiar large amoeboid cells called splenic cells, lymph-corpuscles, and the l)ranched cells which form the sponge-work. The first named are ' P. 35.5. - Berlin, klin. Wocli. 1888, Nos. 43 and 44. 554 THE TISSUES AND ORGANS OF THE BODY frequently found to contain coloured blood-corpuscles iu their interior in various stages of transformation into pigment.' The foregoing brief account of the histology of the spleen taken verbati'iii from Schjifer's 'Essentials' ' shows us the number of microscopic elements with which we have to deal, and thus the large numVjer of chemical substances obtainable from the spleen is fully accounted for. Chemical composition of the spleen. —Oidtmann- states tliat the perceutHge Cloi-tta, Scherer. " Frerichs and Stadeler, Mitth. Ziiricher Jiaturf. Gesell. 1855. 8 Hoppe-Seyler. ^ Baldi, Du Bois Beymond's Arch. supp. 1887, p. 100.. ^'^ Verhcoidl. Wiirzburger phys. med. Gesell. ii. '6'1'i. '* Zeit. phyaiol. Chem. xi. 41. ^- Quoted by Hoppe-Seylei-, Physiol. Chem. p. 720. THE I.V.Ml'llAriC (il.AM)S 655- cumpiunds containing iron which have been described as artificial or post-morteui (.leconiposition pruilucts of hiemoglobin. Lapique states that the spleen of young animals contains less iron than that of adults, which is tht^ opposite to what is the case in the liver. Functions of the spleen. — The lymphoid tissue is no doubt a place for the manufacture of white blood-corpuscles. With regard to the red corpuscles, some hold that they are destroyed, others that they are formed, and others again that both processes may occur in the spleen. The splenic cells are also believed to liberate hannoglobin from. ' eftete ' corpuscles, which, passing to the liver, is there transformed into, bile-pigment. This is erroneous, but the question will be considered, again in connection with the bile. 8chitf and Herzen ' supposed that the spleen also manufactures the pancreatic ferment. Hosier has sho^vn that this is, however, probably not the case. The spleen has been removed from healthy animals (Galen) and also from the human subject without any bad results following. In cei'tain animals, e.g. the dog, the operation has been followed by hypertrophy of other ha^mopoietic tissues (lymphatic glands and red marrow) ; but in certain other animals, e.g. the rabbit, this does not appear to be the case.^ In the disease known as splenic leucocythjemia, in which the spleen is hypertrophied, there is a great increase of the white coi'puscles of the blood (see p. 302). In this disease Charcot's crystals {see p. 303) are also found in the splenic pulp. In progressive pernicious anjvmia the destruction of blood in the liver and also in the spleen is much increased, and so an increased quantity of iron is found in those organs (see pp. 301, 552). The administration of toluylenediamine produces similar results to those observed in pernicious ansemia (Engel and Kiener,^ Hunter). In attacks of ague the spleen becomes enlarged, and this is apparently connected with increase of uric acid in the urine. After many attacks the spleen becomes permanently enlarged and hard from the overgrowth of connective tissue. LYMPHATIC GLANDS These structures are composed of lymphoid tissue with an invest- ing capsule and trabecular of fibrous tissue. The connective-tissue structures yield the same chemical materials as this tissue does in general, especially gelatin and mucin. The 1 Yircliow, Hirscli, Med. Jahresb. IbTO, i. 100 (_origiiuil paper in Italian). See also. A. Herzen, Pfiiiger's Archiv, xxx. 295 and 308. - Tizzoni, Internat. Monatsschri/t fiir A)taf. uiul Plnjsiol. ix. 143. •" Coni])t. rend, cv.^465. 556 THE TISSUES AND ORGANS OF THE BODY lymph-cells are simply white blood-corpuscles, the chemistry of which has been already described (p. 258). In a lymphatic gland about two-thirds are water, the remainder solids. The gland is alkaline during life, and turns acid after death. The acid present is sarco-lactic acid (Hirschler '). In the overgrowth of lymphoid tissue that occurs in scrofula and tubercle, there is a great tendency for the new tissue to undergo degenerative changes, caseation and softening, leading to the formation of cavities and abscesses. In the condition of hyperti'ophy known as lymphadenoma, this tendency is absent. THYMUS This body is also lymphoid tissue, and contains the same sub- : stances as the lymphatic glands. Its cells, like those of the lymphatic glahds, have been already -described in connection with the blood (p. 258). The so-called ' concentric corpuscles,' which are peculiar to the thymus, do not seem to yield any special chemical substance. Towards puberty the thymus undergoes fatty degeneration, and is a mei'e mass of adipose tissue in the adult. The presence of extractives like xanthine, hypoxanthine, &c. has been noted by Scherer, Gorup-Besanez, Frerichs, Stadeler, &c. whose wi-itings have already been I'eferred to. In fact these substances -appear to be constantly present in all structures rich in cellular •elements. Schindler - has estimated these nitrogenous bases quantitatively in the thymus of the calf, with the following results : — Percentage in Adenine Hypoxanthine Guanine ' Xanthine ' Fresh tissue . . . Dry tissue .... 0-179 ' 00023 1-919 0-218 00075 0-038 0071 0360 The high percentage of adenine (a base derived from nuclein ; see p. 203) is especially noteworthy. Like all the other organs also that we have examined, the reaction, alkaline during life, becomes rapidly acid after death. This acid is .sarco-lactic acid (Moscatelli "'). ^ Zeit. phijsiol. Chem. xi. 41. * Schindler, Zeit. jihysiol. Chem. xiii. 438. ^ Zeii. phijsioh Chem. xii. 410. THK TJlYKUil) AND SC I'JiAHENAI. IJnltV 557' THYKOID This is also a cellular oi) mixed with small quantities of organic substances, like mucin and albumin. In tubercle and phthisis generally, the chemical composition of the lung differs with the very various physical conditions that may be present, such as consolidation, fibroid overgrowth, softening, breaking down, calcification of tubercular deposit, ttc. The term caseation as applied to a certain stage in the breaking down of a tubercle (which in origin is a mass of lymphoid tissue) is one derived from the cheesy a ppearance of the deposit ; there is no proof that any substance of the nature of casein is formed. It appears to be a stage in the fatty degeneration of the cells. The presence of the tuljercle bacillus in cases of phthisis is constant. The very remarkable statement has been made by E. Freund,' that the tissues, blood, and pus of tuberculous patients contain cellulose, and apparently the amount of cellulose stated to be present is greater than would be accounted for by the presence of cellulose in the cell-walls of the bacilli themselves. In pneumonia, the alveoli become filled with proliferated cells, and lymph exuded from the blood-vessels ; the lymph coagulates, and thus the lung tissue is solidified, producing the condition known as hepatisation. Budde - attributes the coagulation that occui's to the presence of a large excess of very active fibrin-ferment in the tissues in this disease. When speaking of the occurrence of intravascular coagulation (p. 305), the fact was mentioned that solution of the clot often takes place with great rapidity ; a vessel that is hard to the touch like whip-cord, from the presence of a clot within it, may in a few hours become perfectly pervious. The same holds with regard to the lung in pneumonia. Every clinical observer is familar with the rapid re-solution that occurs in cases of recovery from pneumonia. The functions of the lung in respiration (Chap. XIX), and the composition and variations in the sputa in different conditions (p. 447), have been already considered. (For Charcot's Crystals sre pp. :30.'{, 563.) TESTIS The observations that have been made on the testis, and its secre- tion, the semen, are mostly of a fragmentary nature. A large pro- portion of the chemical constituents of the organ is composed of proteids, or substances closely allied to proteids, of which the most 1 E. Fieuml, Wieiiei- med. Jahrb. 188G, p. 33.5. - Budde, Ueber das Fiirinferment, Wiiizburg, 1889. O O 662 THE TISSUES AND ORGANS OF THE BODY important is nuclein. In addition a large number of extractives, both nitrogenous and non-nitrogenous, have been found. The following is a brief resume of the chief observations that have been made. Sertoli ^ found that a watery extract of the fresh organ had an alkaline reaction ; Treskin ^ found it had an acid reaction. The acidity is probably due, however, as in so many oi-gans, to the commencement of post-mortem changes of the nature of putrefaction, and this view of the case is supported l)y the fact that Treskin found leucine and tyrosine to be present. The proteids present are serum-albumin and a globulin precipitable by saturation with sodium chloride (SertoH). Nuclein is present in abundance. The extractives present are leucine and tyrosine (probably produced by post-mortem changes), lecithin, cholesterin, and fat (Treskin) ; creatine (Schottin ^), inosite (Schottin, Kiilz *) ; and, in a case of diabetes, glycogen (Grohe '') ; adenine, xanthine, hypoxanthine, and guanine (Schindler ^). The salts present aj^pear to be chiefly chlorides of sodium and potassium (Treskin). The greater number of the above observations have been made on the testes of the lower animals, bull, dog, &c. Semen. — This is the secx'etion of the testis, generally mixed with the secretion of the prostate. It is a whitish, Wscid fluid, containing innumerable spermatozoa, wliich originate from the cells of the tubules of the testis. While alive the tail of the spermatozoon exhibits lashing move- ments, akin to those of a cilium, by means of which locomotion is iiccomplished. This power is retained for hours, or even days, in the alkaUne fluids of the body, but it is destroyed by weak acids, and by nil strong reagents like alcohol, chloroform, strong alkalis, &c. The movement is stojjped by cooling to 0° C, and also by a temperature over 53° C. The latter temperature appears to coagulate the proto- plasm and quite kills the spermatozoon. The chief chemical constituent of the spermatozoa is nuclein (Miescher') ; this forms an external coating to the head, and within it ' Sertoli, Gazz. inecl. veterinaria, anno ii. Milano, 1872; Hopiie-Seyler's Physiol. C'hciii. p. 773. - Treskin. Fflugefs Archiv, v. 122. •' Schottin, Hoppe-Seyler's Physiol. Chem. p. 773. -* Kiilz, Sifztingsh. d. Gcsellsch. zu Beford. d. Nafiiriciss. zu Marhurg, 1870, Xo. 4. ■' Grohe, W. Kiiliiie, ArcJi. f. jjathol. Anat. xxxii. '' Schindler, Zeit.physiol. Chein. xiii. 438. '• Vcrh:iiuU. d. naturforsch. Gcsellsch. in Basel, vi. 138. TIIK TESTIS 563 are proteid matters. Miescher ascribes the formula C.29H4,jN9P30o2 to the nuclein obtained from the semen of the bull. In containing no sulphur this nuclein differs from that obtained from pus-corpuscles. By mixing semen with 10 to 15 per cent, sodium chloride solution, the outer portion of the spermatozoa swells, and thus a slimy, jelly-like mass is obtained. The nuclein of the outer covering of the spermatozoa does not appear to be in combination with a proteid, but with a Ijase called protamine, to which Piccard,' from an examination of its platinum compound, has ascribed the formula C,rjH3oN902. Another organic substance akin to a proteid was described in spermatozoa by Miescher ; it was found to contain 4 per cent, of sulphur. Next to nuclein and proteids the chief organic substance present in spermatozoa appeal's to be lecithin (Diaconow ^). Cholesterin and fat are also fairly abundant. Miescher gives the following percentages for the spermatozoa of the salmon : — Xuclein . 48-68 Protamine . 26-76 Proteids . 10-32 Lecithin . 7-47 Cholesterin . . 2-24 Fat . 4-53 In addition to these, small quantities of the other extractives already mentioned as being obtainable from the testis are present. The crystals generally described as Charcot's crystals (p. 303) are said to form in human semen on evaporation (Bottcher ^). Schreiner'* considers these crystals to consist of the phosphate of a base of which the foimula is CgHgN, and to which he gave the name spermine. This substance appears to be identical with the base called ethylenimine, which can be prepared artificially from ethylenediamine hydrochloride.' The name spermatin has been given to the mucin-like substance in semen (Yauquelin, Kolliker) (see p. 145). The prostatic secretion. — This secretion is slimy, opalescent, and 1 Ber. d. deiitscli. cliem. Gesellsch. vii. 1714. - Diaconow, Hoppe-Seyler's Med. Chem. Unters. ii. 221 ; iii. 405. ^ Arch. f. ijathol. Anaf. xxxii. 525. * Liehig's Annalen, cxciv. 68. Li the account of the crystals on ^i. 303 the formula is incorrectly given. 5 Laclenhurg and Abel, Ber. d. deutsch. chcm. GescUscli. xxi. 758. O O 2 564 THE TISSl'ES AND OEGANS OF THE BODY in man and the dog of a neuti'al or alkaline reaction. According to Buxmann ' its composition is — Water . . . . . .98-0 per cent. Proteids .... 0-4:5 to 092 Salts 1-0 „ The most abundant salt present is sodium chlorifle ; potassium salts, sulphates, and phosphates also occur. Prostatic calculi, of which the most important constituent is calcium phosphate, may occur (Paulizky,- Iversen^). OVAEY The connective-tissue element of this organ is veiy large and yields chiefly gelatin and mucin. Proteids and nuclein are derived principally from the ova and other cells present. The corpora lutea are composed of cells, and are coloured by a yellow pigment called lutein. This was first described by Thudichum ; * this observer was also the first to point out that this pigment is distinct from hsematoidin or bihrubin (a derivative of haemoglobin), which is often also present.. Lutein is one of the class of pigments known as lipocliromes, and other members of the .same group occur in the blood (p. 254), egg-yolk, retina (p. 464), adipose tissue, lirc. Lutein shows two absoi-ption bands, one well marked between b and F, but nearer the latter ; the other less well defined between F and O. (For Ovarian Cysts see p. 352.) THE EYE The outer coat of the eye, the sclerotic, with the cornea, which is continuous with it, has been described with the connective tissues. The middle coat, the choroid, is the vascular coat of the eye : the connec- tive tissue corpuscles are, however, pigmented : the same is true for the cells of the iris ; the pigment present is probably the same as fuscin contained in the hexagonal pigment-ceUs of the retina. The retina has already been described in the chapter on epithelium (p. 458). The aqueous humour is lymph, and has been descril^ed under that headincf (p. 350) ; the vitreous humour is jelly-like connective tissue, and will be found described under that heading (p. 467). The crystal- line lens is the only part of the eye that still demands description.. I Buxmann, Beiirdge zur Kennfniss des Proatatasaftes. Diss. Giessen, 1864. - Paulizky, Diss. Berlin, IS."*?. ^ Iversen, Maly's Jahresh. 1874, p. 358. * Thudichum, Centralbl. med. Wiss. vol. vii. 18C9, p. 1. THE KYE 565 The lens.— The crystalliue lens of tlie eye is composed of many layere of fibres whicli are in origin elongated epithelial cells. It is enclosed by a capsule which is homogeneous, and resembles elastin in its insolubility. The specific gravity increases from the centre outwards from 119+ to^l076 (Chevenix ') ; the refractive index also increases in the same way. The reaction of the lens is alkaline. The following are the results of analyses made by Laptschinsky : — ^ Water 6.3-.50 Solids ...... .36 -50 Proteids 34-93 Lecithin 0-23 Cholesterin 0-22 Fats 0-29 Salts 0-82 The proteid present is a globulin ; albumin is absent. The name globulin was first given to this proteid, and afterwards extended to include other proteids which form the well-defined class we call globulins. The name crystaUin was then given to this particular globuUn by Berzelius. Hoppe-Seyler describes crystaUin as very like vitellin in its properties. It coagulates on heating it to 70° C Laptschinsky speaks of this proteid as being fibrino-plastic ; Kiihne says this is not the case. Cataract. — This may be due to the formation of vacuoles in the lens-fibres in cases of diabetes ; this is the condition produced in frogs by injecting sugar into the circulation. The opacity produced in the lens after death is caused in a similar way, and is not due to the coagulation of proteid, such as occurs in the rigor mortis of muscles. Ordinary senile cataract is, however, a fatty degeneration, and the ■opacity appears to be chiefly due to the deposit in a crystalline form of cholesterin in the lens ; at the same time the proteids are diminished in quantity. Cahn^ gives the following percentages of dry residue in a case of cataract : — Proteids 8.5-37 Cholesterin Lecithin Fat Salts 4-.55 0-803 1-19 3-86 Calcareous salts are said to be occasionally deposited in the lens. 1 Kiiline, Lehrbuch, p. 404. - Laptschiusky, Pjf tiger's Archiv, xiii. 631. ' Hoppe- Sevier's Physiol. Chem. p. 692. 566 THE Tissn:s and okgax^; of the body THE EAE The pinna consists of elastic tissue (p. 473) covered by skin. The external auflitory meatus is composed of hyaline car-tilage (p. 481). The composition of peiilymph and endolymph (p. 351), and otoliths (p. 4:96) has alx-eady been described. Beyond this there is as yet no chemical knowledge of this organ. THE SEIX The epidermis is a stratified epithelium ; its surface layers are homy in nature. This is especially mai'ked in nails, hoofs, horns, and hairs. (For Keratin, or horny material, see p. 452.) The deeper layers of the epidermis (Malpighian layer) are proto- plasmic. The ti'ue skin is composed of fibrous tissue {sei^- p. 467). The secretions of the .skin ^-ill be taken later with other secre- tions of the body. la ichthyosis there is a great increase in the horny layer of the epide^mi^ : there is also found a quantity of fat, cholesterin, and hippm-ic acid. In peUugru fat and cholesterin are also found, with a little leucine and tvrosine. In both diseases the ash of the skin contains ruucli silica. PAET IV ALIMENTATION 5GU CHAPTER XXVI FOOD Foods are the substances which are required for the nutrition of the body. It has been calculated that a man of average weight loses about 1000 grammes of matter daily ; this passes out in the expired air, the urine, sweat, fa?ces, and other excretions ; the substances that pass out are comparatively simple bodies, like water, carbonic acid, and urea, formed in the chemical decompositions always going on in living matter. Food is necessary to replace this waste if the body-weight is to remain constant ; but the substances taken in in the form of food undergo many changes befoi'e they ultimately become a constituent part of the body. The changes are partly of a physical nature, such as mastication in the mouth, a thorough mixing by the peristalsis of the stomach, and solution in the watery secretions of different parts of the alimentary tract ; these same secretions also fulfil a much more im- portant function, namely, that of causing chemical changes in the food, converting insoluble into soluble, indiffusible into diffusible substances. These two sets of changes, physical and chemical, constitute what is called digestion. Absorption follows digestion ; that is, the products of digestion pass through the walls of the alimentary canal into the blood or lymph circulating there. The blood- or lymph-stream carries the absorbed products to the tissues, which take them and make them part of themselves : this is assimilation. In some cases, however, assimilation may not occur immediately, but an organ like the liver may intercept such an absorbed material as sugar, and store it (in the form of glycogen), giving it out by degrees as it is wanted. Before we can study the chemical processes concerned in digestion, absorption, assimilation, and nutrition, it is necessary that we should be acquainted with the raw materials, the foods, on which the digestive juices act. THE PROXIMATE PRINCIPLES OF FOOD If we examine the food-stuffs, such as milk, eggs, meat, and vegetables, we find that they are mixtures of various inorganic and organic materials, which are named proximate principles ; and, more- 570 ALIMENTATION over, the chief proximate principles of food are in the main the same- as the chief proximate principles of the body they ai'e destined to build up. They may be classified as follows : — I Wafer. Inorganic Salts, e.g. chloi-ides, phosphates, carbon- I ates of sodium, jDotassium, calcium, &c. ,Proteids, e g. albumin, myosin, casein, ikc. \ Albuminoids, e.g. gelatin, chondrin, nuclein, A:c. Simpler nitrogenous bodies, lecithin, creatine, &c. \ Iron-containing comjjounds.^ Nitrogenous Organic Non -nitroo'enous / Fats, e.g. cream, fats of adipose tissue, I Garboliydrates, e.g. sugar, starch, &.c. Simpler oy-ganic bodies, e.g. alcohol^ vegetable acids and salts, etc. Liebig inaccurately divided the organic foods into assimilable or plastic (proteids) and combustible or respiratory (fats and carbo- hydrates) ; we, however, now know that all varieties of ffiod are both assimilable and respiratory. Water. — It is recognised as a matter of every-day knowdedge that a good water supply is essential to the health and well-being of the community. The water used for drinking must be clear, odourless, and colourless. It must not be contaminated with sewage, nor with the pathogenic bacteria apt to occur in sewage. For the purposes of safety it should be filtei-ed through an efficient filter, or, better still, boiled before it is consumed. Distilled water is insipid ; rain water has the same disadvantage ; the softness of water, i.e. its freedom from salts, deprives it of its pleasant taste. The salts of sj)i'ing or river water vary immensely according to the rock through which the river or spring passes. ^ When the salt (magnesium sulphate, iron salts, &c.), or gas (carbonic acid, sulphuretted hydrogen, (fee.) contained in any special spring, is accentuated either in qvuxntity or quality, the water is- often found to be useful as a therapeutic agent. Pure water alone, without any specially dissolved salt, is also a most useful addition to the physician's stock of remedies. The subjects of water analysis, of 1 Tliere is some doubt whether inorganic compounds of iron are absorbed (see p. 300) ; iron-contaming foods are therefore chissified with the organic proximate principles. ^ A good drinking water shoukl not contain more than twentj- degrees vi hardness, i.e. twenty parts of Hme in 100,000 of water. ro(ii) 571 water as a therapeutic- ayent, and of mineral waters are obviously too lari^e to be treated of in a work on physiology. Water is taken into the body, not only as water pure and simple, l)ut all other forms (if food, and especially bev'erages, are composed of water mixed with something else. Even the solid foods contain large pi'oportions of water ; meat, for instance, contains about 7"), bread 37,^ milk 86, eggs 74, potatoes 7o per cent, of water. Inorganic salts. — The importance of these agents in nutrition has. been already dwelt on ; they are daily excreted in certain amounts and must be daily replaced by the same or approximately the same amount ; and the enumeration of the salts of the body (p. 60) is also an enumeration of the salts of the food. Sodium salts, especially the chloride, are essential. About twenty grammes of sodium chloride is in the mean taken per diem, partly in the articles of diet themselves, but mostly in a separate foi*m as a condiment. This salt doubtless supplies the chlorine for the acid of the gastric juice. Potassium salts are found more abundantly in muscle, nerve, and other solid structures of the body, and are especially contained in meaty foods and in potatoes. Calcium salts, particularly the phosphate and carl)onate, are more especially necessary for Ijone and tooth, but are also universally dis- tributed in the tissues, though in smaller proportions ; these salts are chiefly derived from milk, eggs, cei"eals, and other vegetables. Iron is found not only in hjemoglobin, but also in the organs, such as liver and spleen, and in most of the fluids of the body, in milk, in eggs, and in many vegetable foods. Probably the iron absorbed from the alimentary canal is furnished wholly by organic compounds of iron formed either during plant life or during the life of other animals, being there again ultimately derived from plants. Bunge ' terms these organic com- pounds of iron licematot/ens. The following table compiled by Beaunis - gives the percentage composition of the ash of various foods : — Food Potash Lime Magnesia Soda XaCl Iron oxide 1\0,-, SO3 Silica Milk . . . 23 17 2 7 4-7 0-47 28 005 0-06 Muscle . . 39 1-8 3-8 4-8 l-y 1 46 0-3 — Brain. . . 84 0-7 1-2 10-4 4-7 _ 48 0-75 0-4 "White of egg 27 3 2-7 12 39 0-5 3 1-7 03 Yolk of egg 11 13 2-2 1 9 2 (JO — 0-6 Wheat . . 27 2 6-6 0-4 — 1-3 62 — — Barley . . 20 1-6 7 — — 2 38 — 29 Potatoes 51 :} 13 — 2-4 — 12 6-5 7 Lentils . . 35 G 2-4 13 4-6 2 36 — — 1 Bunge's Physiol. CJiem. transl. by Wooldvidge, 1890, p. 100. Hwmatogen in most cases appears to be a compouncl of iron and nuclein. - Physiol, hnmaine, i. 621. .572 .ILIMEXTATION Proteids. — These form the most abundant source of nitrogen to' the Ijody, and they are found in hirger quantity in animal than in vegetable foods. The chief animal proteids used in food are the myosin of flesh, the casein and albumin of milk, the proteids of egg, and of blood. The chief vegetable proteids are glutin, vegetable myosin, and other vege- table globulins. Gelatin has also a certain nutritive value, but an animal fed on gelatin, to the total exclusion of proteids, wastes. Carhuliydrates, on the other hand, are derived chiefly from vegetable foods ; the most important are starch, cane sugar, and grape sugar. The carbohydrates found in animal food are lactose in milk, glycogen in liver and muscle, and inosite in muscle, and other organs. Cellulose, gums, and mucilages are of little or no use in nutrition. Fats. — The fat of adipose tissue consists of olein, stearin, and palmitin. The fat of milk contains certain lower glycerides in addition. The vegetable oils consist chiefly of olein and palmitin. Vegetable acids and salts of those acids. — Oxalic, tartaric, citi'ic, and malic are the most important of this group. In the body they are con- verted into carbonates. THE PRINCIPAL FOOD- STUFFS We do not actually use as foods the various organic proximate principles in the pure condition ; it is necessary that in a suitable diet these should be mixed in certain proportions, and in nature we find them already mixed for us. In mUk and in eggs, which form the exclusive food-stufi" of young animals, all varieties of proximate principles are present and mixed in suitable proportions ; hence these are spoken of as perfect foods. Eggs, though a perfect food for the developing bird, do not form a perfect food for mammals, as they contain too little cai'bohydrate. In most vegetable foods carbohydi-ates are present in excess, while in most animal foods the proteids are pre- dommant ; hence in a suitable diet these should be mixed in proper proportions. The food-stuffs we shall consider fully are milk, eggs, meat, bread, various flours, and seeds of plants used as food, and in conclusion certain accessories of food, such as alcoholic beverages and other stimulants and condiments. The table on the next page gives at a glance the percentage composition of the principal food-stuffs.' Milk Milk is a secretion which is characteristic of mammals. The acini •of the mammary glands are during periods of non-lactation lined by flattened epithelium. During lactation, which begins after the birth of ' From McKendvick's Phijfiiologij, ii. !). MILK 51S Foixl-stnffs 1 Water I'roteids Starch Suj;iir Fat Salts Bread . . . 37 8 47 3 1 2 Wheat flour . . 15 11 66 4-2 2 1-7 Oatmeal . . . 15 12-6 58 5-4 56 3 Rico . . . . la fi 79 0-4 0-7 0-5 Poas . . . . 15 23 55 2 2 2 Potatoes , . . 75 2 18 3 0-2 0-7 Milk . . . . 8G 4 — 5 4 0-8 Cheese . . . 37 33 — — 24 5 Lean beef . . 72 19 — — 3 5 Fat beef . . . 51 14 — — 29 4 Mutton . . . 72 IS — . — 5 5 Veal . . . . 63 1() — 16 4 ■\Vhite fisli . . 1 78 18 — — 3 1 Salmon . . . 77 16 — _ 5-5 1-5 Egg . . . . 74 14 — — 10-5 1-5 Butter. . . . 15 — — — 8-3 2 Oats . . . . 12 10 57 11-2 (cellulose) — 3 Hay . . . . 13 9 41 27 — 7 Rye straw . . 14 4 35 40 — 6 Red clover . . 78 3-5 8 8 — 2 the offspring, the cells are larger and are continually undergoing a fatty degeneration. They disintegrate, and the fat-globules so liberated float in a clear liquid which is also secreted by the cells from the lymph circulating in the gland. The mammary glands of male animals are inactive; there have, however, been exceptional cases in the human species, as well as among lower animals, in which a secretion has been obtained from the mammae of the male.^ The mammary glands of new-born animals of both sexes secrete a small quantity of milk for a few days ; it is popularly termed ' witches' milk.' It does not differ qualitatively from the milk of the adult female, but it does differ quantitatively ; but the few analyses that have been made show great variations in the amounts of the different con- stituents present (see Tables of Analyses). The mammary glands themselves, apart from their secretion, do not seem to have been chemically investigated in a thorough manner. Their protoplasm, nuclei, and interstitial connective tissue have doubt- less a similar chemical structure to that] of these same constituents in other situations. Bert^ found in the secreting glands a substance con- 1 Schlossberger {A7i7t. Chem. Pharm. li. 431) analysed a specimen from a lie-goat; lie found the milk was alkaline and contained 9-6' per cent, of proteids and insoluble salts. 2'65 per cent, butter, and 2'G per cent, lactose and soluble salts. - Gaz. hebdom. 1879, No. 2. o74 ALniENTATK tX Tertible by boiling with water or dilute acids into a sugar ; and- Landwehr' regards this substance as animal gum, which he here con- sidei's to be the mother-substance of milk sugar. The object which milk is intended to fulfil is that of supplying a food to the growing ofispiing. In the case of many animals domesti- cated by man (cow, goat, ass, ttc.) the milk is collected and applied to other uses, namely, the feeding of man himself. Milk is, moreover, a perfect food : it contains members of all classes of proximate principles; its proteids are casein and an albumin ; its carbohydrate is termed lactose or milk sugar ; the fats constitute what we call butter ; and the salts are chiefly phosphates and chlorides. Microscopical appearances of railh. — The microscope reveals the fact that milk consists of two parts : a clear fluid, which may be called the milk-plasma, and a number of minute particles floaticg in it : the o-reater number of these are minute oil-globules, varying in size from 0-001 5 to O'OOo millimetre in diameter:- the great majority are nearer the lower limit than the higher. In addition there are minute particles of casein and nuclein suspended in the fluid. -^ The milk which is secreted for the first few days after parturition, is somewhat e^ imperfectly formed, and differs quantitatively from , ' true milk. This is indicated microscopically by the a presence of cells from the acini of the gland con- m^) ^^0) taining fat-globules wliich they have not yet liberated d ® by cUsintegrating. Tliese cells are tenned colostrum ^'5ita^ls^o"'^IcoTol" coi'pusdes,'- and the mUk of the first few days of rTcoioSS^TS;"]: lactation is caUed colostrum, cies -nith fine and ^,l\\k \s, One of the most perfect emulsions ; the coai^e fat-gloliules re- ^ ' spectiveir; c, ; liad previously recognised that this substance is not glycogen. - Fleischniann, Das Molltereiivesen, Braunschwieg, 1876-9, p. 206. ^ Kehrer, Arch.f. GijnceJ:oTogie, ii. 1. ^ Strieker iWien. Akad. Sitzungsber. liii. Feb. 1, 1866) states that they show amoeboid anovements when placed on a warm stage at 40° C. 3IJLK 575 -ii sholl of casein. Asi-liorsou • sliowcd that with an artiiicial oiiiulsiou of oil in an alkaline albunnnous iluid the oil (h'ops became coated with ]»roteid ; and (duiiieke- demonstrated the same fact, in whicli mucilage was used instead of albumin ; each oil droplet had a gummy envelope. Casein certainly is not in solution in the milk-plasma or only in small quantities; if milk be Hltered under pressui'e through a porous cell, the filtrate is free, not only from fat-globules, but from casein also.-* The different methods employed for precipitating casein cause also a pre- cipitation of the fat with it. In spite of these facts, however, Hoppe- Seyler ' i-egards it as improbable that the greater part of the casein is present in the form of casings for the globules ; he obtained the same amount of casein from cream as from portions of milk below the creain. The part of the casein around the globules is therefore, if present, imponderable. Hoppe-Seyler also states that it is not so difficult, as stated by the earlier observers, to remove the fat by simply extractini;- with ether ; after the fat has been thus removed the fluid is still cloudy, but this is from the presence of the particles of casein and nuclein which vwere described by Kehrer. Reaction. — This is nearly always alkaline. Milk turns readily acid or sour as the result of fermentative changes, part of its lactose l)eing transformed into lactic acid. In carnivora the milk is acid ; in herbivora an amphoteric reaction is often observed, the acid sodium phosphate in it (H.^NaPO^) turning neutral litmus paper red, and the alkaline sodium phosphate (NajHPO^) turning it blue. Specific gravity.- Thi^ is usually ascertained with the hydrometer. That of normal cow's milk varies from 1028 to 1034; when the milk is skimmed the siDecific gravity rises owing to the removal of the light constituent, the fat, to 1033 to 1037. This, fraudulent milk -vendors correct by the addition of water. Tables of specific gravities are published for the guidance of analysts, which indicate the purity of milk w^hen skimmed and unskimmed, these numbers varying with the amount of water added. Amount secreted. — The quantity of milk secreted by a woman is about 700 to 800 c.c daily, sometimes as much as a litre, and sometimes even more when the mother is suckling two or three children simul- taneously. A good cow secretes six to seven litres daily. Qnavtitativp coi)ipo>K2 Solid.^ .... 11-418 Proteids . . . 3-691 Fat 3-532 Lactose .... 4298 .Salts 0169 90-581 Ti.ly 86-271 86-32 to 88-79 87-24 9-419 l3-72;i 11-21 „ 13-68 12-75 2-911 2-950 1-68 „ 3-15 1-90 3-345 5-370 2-59 „ 5-39 ' 4-32 3-154 5136 5-79 „ 6-61 5-97 0-194 0-223 0-23 „ 0-34 0 28 On account of the difficulty in separating casein and alljumin in human milk, they are given together in the above table. Other observers have, however, determined these two proteids separately ; thus Tolmatscheff ^ found 1*28 of casein and 0-34 of albumin per cent.; Makris^ found 1-8 to 4-8 of casein, and O'T to 1-7 of albumin per cent. The gases of human milk have not been investigated. Vernois and Becquerel ' have in^•estigated the influence of age on the quantitative composition of human milk, but with no specially interesting results. The same authors state that menstruation lessens the amount of lactose, but increases that of fat and casein. The milk of blondes contains less casein and sugar, but more fat, than that of brunettes (L'Heritier *") ; YernoLs and Becquerel agree with L'Heritier only so far as the ca.seiu is concerned : while Tolmat- scheff could find no constant difference at all. It is indeed probable than other causes than complexion were at play in the cases observed by the French chemists just mentioned. 4. Artificial human milk. — Many recipes have Vjeen given to enable mothers who cannot suckle their children to prepai-e from cow's milk a milk like their own. Cow's milk is poorer in sugar, but richer in casein and butter, than human milk. Frankland gives the following table contrasting the milk of woman, ass, and cow : — Casein Butter Lactose Salts ^'ornan Ass Cow 2-7 1-7 4-2 3-5 1-3 3-8 5-0 4-0 3-5 0-2 0-5 0-7 I Malifis Jahresb. 1874, p. 168. 3 Med. chem. Unters. ii. 272. ^ Compt. rend, xxxvi. 188. * Traiti de chimie pathol. Paris, 1842, p. 683. - Dissertation. Erlaugeu, 1877. * Dissert. Strasburg, 1876, p. 31. P P 578 AIJMENTATKIN The following are the principal recipes ior preparing artificial human milk : — ' («) Cow's milk . . . 600 grammes Water .... Cream . Lactose .... Calcium phosphate (h) Heat half a pint of skimmed cow's milk to 35° ; add rennet. After ten to fifteen minutes, break up the curd finely, strain the whey off, and boil it, adding 110 grains of lactose; Sti-ain again, and add it to two-thirds of a pint of fresh cow's milk and then two teaspoonfuls of cream. This should be freshly made every twelve hours (Frankland). 5. Cot&s milk. — The following analyses of the milk and colostrum of the cow have been made : — 339-5 „ 13 15 1-5 ,, (Coulier) Constituents Colostrum ° ' Milk ' Milk* Water 78-7 per cent. 84-28 per cent. 85 to 86 per cent. Solids 21-3 15-72 14 „ 15 „ Casein 7-3 „ 3-57 3 „ 4 ., Albumin 7-5 0-75 0-3 „ 0-5 „ Fat 40 e pi-ecipitated from milk by the addition of acids, or by saturation with neutral salts, like sodium chloride or magnesium sulphate. In Ijoth methods the fat is en- tangled with the precipitate, and, when the saturation method is used, the adherent fat renders the precipitate so light that it floats (jn the surface of the concentrated saline solution. I have obtained caseinogen most satisfactorily by combining the two methods. Milk is first saturated with magnesium sulphate ; the precipitate of caseinogen and fat is collected on a filter and washed from milk-serum by a saturated solution of the same salt. Distilled water is then added to the precipitate on the filter ; this, in \ irtue of the salt adhering to the precipitate, dissolves •out the caseinogen, and the fat is left on the filter, while the solution of caseinogen in dilute magnesium sulphate solution passes through the filter and is collected. From this solution the caseinogen is precipitated by means of excess of acetic acid ; it is collected, thoroughly washed, dissolved in dilute alkali such as lime-water, and purified by repeated precipitation with acid and re-solution in alkali. If the caseinogen has been washed completely free from all calcium phosphate (a long and difficult process), the addition of rennet to the solution causes no forn)ation of casein ; but rennet plus calcium phosphate will produce almost immediate clotting at 40° C. ; the > Mahfs Jahresh. 1874, p. 135. * Jourii. of the American Chcni. Soc. May 1888, p. 15. 5 Text-book, 5th edit. p. 419. 582 ALniENTATlDN addition of a little calcium chloride is also beneficial (Hammarsten '). Hamniarsten himself does not use the word caseinogen, but speaks of both casein and its precursor as casein. A method introduced by him to show that casein precipitated by acid from milk (caseinogen) will undergo reiuiet coagulation (formation of true casein) is as follows ; The caseinogen precipitated by acetic acid is well washed with distilled water until free from salts ; it is mixed with powdered calcium carbonate and lime-water added till the pap-like mass so formed is just alkaline ; a few drops of 0"5 per cent, phosphoric acid is added (to form calcium phosphate with the lime water), and a drop of rennet. The previously semi-fluid mixture now sets into a firm jelly-like mass. Caseinogen, usually .spoken of as casein, is often comjDared to alkali- albumin. The latter, however, does not clot with rennet and is, unlike caseinogen, readily soluble in acids. Both are alike in precipitability by neutral salts, and in the fact that neither is coagulated on heating its neutral solution. Caseinogen is like a globulin in the way it behaves to neutral salts. A solution of a globulin, however, coagulates when heated. A solution of caseinogen, such as that in dilute magnesium sulphate or sodium chloride solution, becomes a little cloudy at 70°, but this disappears when the solution is cooled if the heating has not been continued too long, but there is never any apjDearance of a fiocculent precipitate. Caseinogen as analysed by Chittenden- has the following percentage comjjosi- tion : C, 53-3; H, 7-07: N, 15-91; S, 0-82; 0,22-04. Danilewsky^ has asserted that it is a mixture of two proteids — caseo-protalbin, partly soluble, and caseo- albumin, insoluble in hot 50 i^er cent, alcohol. Hammarsten ■■ has shown that this peculiar behaviour of Danilewsky's preparations is due to their containing calcium phosphiite ; and this impurity depends on the use of hydrochloric acid as a i^re- cipitant, as this acid does not favour the removal of the salt as well as acetic acid. Both Hammarsten and C'hitten den's analyses favour the view that caseinogen is a single proteid. Chittenden has also studied the products of digestion of caseinogen and casein, and finds that peptones are ultimately formed; certain intermediate bodies resembling the albumoses are also formed, and termed caseoses. Proto-caseose, hetero-caseose (produced only in small amounts), and deutero caseose can be separated, and correspond to the albumoses with similar • names. An insoluble semi-gelatinous substance separates in the first stages of gastric digestion ; it is only ver^' slowly changed into soluble bodies, and is termed casein-dyspeptone.'' Sebelien," who has also prepared pure casein-peptone, states ^ The calciiun salt must be a soluble one ; other alkaline earths may be substituted for lime (Lundberg, Mali/s Jahresb. 1876, p. 11; Ringer, Joiirn. of PJiijsiol. 1890). - Studies from Lab. j'hyHiol. Clion. Yule Univ.W. ISt!. ^ Zeit. jjltysiol. CJiem. vii. 4S3. * Tbid. vii. 2-27. 5 Chittenden, Studies from Lab. 2>Jnjsiol. Chcni. Yale Univ. iii. CO. 6 Bied. Centralhl. 1889, p. 717 MILK 58a that it is optically inacti\e. AH other inoteidfi, so far as is at present known, are licvorotatory. The question whether the easeinogen of milk is in suspension or sohition, or both, has been already disriissed (p. 57o). Casfiiti. — This name should be lestricted to the proteid foi'ined by the action of leniiet or reuiiet-Hke ferments from the caseinogen of milk. It is more insoluble than caseinogen in dilute alkalis. The casein of human milk, unlike that of the cow, separates in fine flocculi ; when dried the powder fonned is more yellowish tliau that from cow's milk. This corresponds to certain differences that have been described in the caseinogens. The caseinogen of human milk is more difficult to pre- cipitjite by acetic or caibonic acid, and more readily precipitated by magnesium sulphate than that of cow's milk. There are also stated to be differences in elementary composition.' Casein is the chief cons.tituent of cheese. There have been various explanations advanced to account for the action of lime-salts in favouring the coagulation of milk by rennet. Hammarsten is not inclined to believe that the lime combines with the caseinogen, but that the ferment produces the change in the caseinogen, and that the casein so formed will not separate out unless the calcium-salt is present. _ Green *_ suggests that there is some definite relationship between the ferment and the salt, resembling that which exists between pepsin and hydrochloric acid, and that the ferment cannot act without the presence of its inorganic ally. Ringer^ finds that casein dissolved in lime-water separates out as a curd on the addition of calcium chloride ; this curd is more soluble in cold than hot water, so resembling other lime-salts. Whether, however, casein normally formed by rennet is nothing more than a caseate of lime must be for the present regarded as uncertain. Lactalhumin. — After the precipitation of caseinogen by magnesium sulphate, this proteid is left in solution. It can be incompletely precipitated from this solution by saturation with sodium sulphate. It coagulates between 70° and 80° ; in cow's milk, which I have specially examined, at 77° C. It is not separable, like serum- albumin, into several proteids by fractional heat-coagulation. It moreover is coagulated by heat very slowly. The solution must be kept some hours at 77° before the proteid is entirely precipitated. Its specific rotatoiy power ^ is (a)^ = — .36°. Its elementary analysis gives the following percentages : C, 52-19 ; H, 7'18 ; X, 1.5-77 ; 8, 1*73 ; O, 23-13 ; it thus differs from serum-albumin in specific rotatory power, in its high percentage of sulphur, and in its solubilities. The scum which forms on the top of milk when it is boiled is probably in part produced by the coagulation of the lactalbumin by heat ; this carries to the surface a little caseinogen and fat. If the scum of boiled milk ' Bredert and Schriiter, Centralhh f. Agricultur-Cliemie, 1888. - Journ. of Physiol, viii. 371. ^ Proc. Physiol. Soc. IsiiU, p. iv. * J. Sebelien, Mahfs Jahresh. xv. 184. 584 ALIMEXTATinX be removed, another f(jrms, and this may be repeated many times in succession. The contact with the air thus appears to be of influence in causing the solidification which results in the fonnation of the scum ; it may be because evaporation is more rapid from the surface exposed to the atmosphere. The boiling of milk before it is used as food is advantageous in two ways : (1) All germs of disease are destroyed; (2) the gastric juice, in -s-irtue of its rennet, causes a flocculent, not a bulky precipitate.' These advantages quite outweigh any slight diffi- culty of digestiliility which is alleged to occur (Raudnitz).""^ LoxtofjIohuHn. — Sebelien states that after removal of the caseinogen by saturating milk with soflium chloride an additional precipitate is obtained by saturating the filtrate with magnesium sulphate : this precipitate he considers to con.sist of a globulin which he calls lacto- cflobulin. There is no doubt Sebelien has here fallen into an error ; for double saturation with the two salts just mentioned will precipitate albumins (see p. 246), and he has mistaken the precipitate of lactalbumin so produced for a globulin. A solution of the precipitate produced by saturating milk with magnesium sulphate never coagulates on boiling in the specimens I have examined ; globulins are therefore absent, though doubtless a globulin is present, as Sebelien states, in colostrum. WJo'y-profetd {Molk'-n-Protfin) (Hammarsten).— Rennet, according to Hammarsten, splits caseinogen into two proteids : one is the insoluble casein, the other a soluble proteid found in the whey, and equivalent to the lacto-protein of other investigators. Lacto-protein Ls stated by some investigators to be a peptone- like substance ; and peptones and peptone- like substances are altogether absent both in fresh milk and in the whey of fresh milk. I have examined whey repeatedly and failed to find any peptone or proteose in it. On saturating it with magnesium sulphate a proteid is precipitated, and this appears to be Hammarsten's whey- proteid ; its solutions do not coagulate on heating, and it differs from caseinogen in not being convertible into casein by means of rennet. I -should suggest that caseinogen and whey-pioteid should lie included in a new class of proteids intermediate between globulins and albuminates. Lacto-proteiti, jyroteoses, peptones. — The separation of proteoses and peptones from other proteids has only been possible since the introduction of ammonium sulphate as a reagent. The older method of estimating peptones (with which proteoses were confounded) was to acidify and heat, filter off the coagulated proteids, and the proteid left in solution was called peptone ; this was precipitated by tannin and 1 The reason that boiled milk will either not curdle at all, or more slowly than fresh milk, is that by boiling, a part of the dissolved calcium salt is precipitated as tricalcium phospiiate. 2 Raudnitz, Zeit. jjhysiol. Chem. xiv. 1. MILK 585 weighed. Tn reality this so-called peptone consists of the primary proteoses (proto- and lietero-pioteose) formed by the hydrating action of the acidified hot water. This applies not only to milk, but to other fluids also. Milk, howevei", is a ,i,daring example of how this n)istaken method has led to mistaken results. Thus Struve ' and Schmidt- Miilheim ^ describe peptone in milk ; DogieP calls it lacto-protein, and ■J. Schmidt ^ speaks of it as hemi-albumose. Milk and whey undei- no circumstances contain true peptone. After saturation with ammonium sulphate and filtering, the filtrate is always free from proteids. Proteoses, such as the albumoses, may be identified by placing a liquid containing a mixture of proteids under alcohol for many months. All proteids but peptones and proteoses are by this means rendered insoluble ; water, however, extracts peptones and proteoses from the precipitate, as these are not coagulated by alcohol. This solution is then saturated with anmionium sulphate ; the precipitate, if any occurs, consists of proteoses ; the proteid in the filtrate, if any is present, is peptone. By this method of testing, fresh milk and whey from fresh milk are found to be free from proteoses. Sour milk or whey from sour milk contains a good quantity of primary proteoses. Koumiss and the similar substance kephir also contain abundance of proteoses, and, according to some, peptones also.'^ The so-called ' long-milk ' of Upper Scandinavia also contains peptones (Sebelien). Nudein is found in small quantities in milk. The difierence between this and the true nuclein of nuclei has been already pointed out (p. 203). The iron in milk is combined with nuclein (Bunge). Th,' Fntx of MUk The chemical composition of the fat of milk is very like that of adipose tissue (p. 4S7), with small quantities of the triglycerides of bu- tyric, caproic, caprylic, caprinic, myristic, and arachic acids in addition.^ Milk contains also small quantities of lecithin, cholesterin, and a yellow lipochrome. 1 J.praTxt. Chem. 1884, p. 73. - Pfliiger's ArcJiiv, 188-2, p. '2M7. 5 Zeit.phi/sioL Chem. iHSo. p. (iO'2. * Diss. Moskau, 1882. * The above observations on the proteoses and peptones of milk have been published by Neunieister {Zeit. Biol. xxiv. 271) and Sebelien {Zeit. physiol. Chem. xiii. 135). I have independently made identical observations {Journal of Physiology, 1890). The subject of koumiss and kephir has quite a literature of its own. Full references will be found in the last ten or twelve volumes of Mali/'s Jahresberichf. ''■ Crrunzweig, Ann. Chem. Pharm. clxii. 215. E. Wein, Dissert. Erlangen, lH7(i. Chevreul, Becherches siir les corps-gras, Paris, 1822. Lerch, Ann. Chem. Pliarm. xlix, -212. Heintz, Ibid. Ixxxviii. 300. 586 ALIMENTATION Cream. — This is simply the upper layers of milk allowed to stand, in whiclv therefore, the fat -globules are more numerous than in ordinary milk ; the amount of fat in cream varies from 14 to 44 per cent. Butter. — The fat -globules are broken up by mechanical agitation ; the strokes of the chiirn must not, however, exceed thirty or forty per minute. About one- third of the original fat is left in the butter-milk. Butter contains small quantities of caseinogen and lactose, in addition to fat : sodium chloride is added. The fats of cow's butter consist of 68 per cent, of palmitin and stearin (solid fats), 30 per cent, of olein, and the remaining 2 per cent, of the specific butter fats (Bromeis).' In the winter time the sohd fats are said to be increased (Fleischmann). The butter from human milk is richer in fluid fats than that made from cow's milk (Hoppe-Seyler).- By exposure to the air, butter becomes rancid ; this is partly due to a breaking up of the higher fats, and the production of lower fatty acids — formic, acetic, butyric, valerianic, Sec. — partly to the formation of acrolein from glycerine, and partly, and according to Hagemann chiefly, to the formation of lactic acid from the lactose mixed with the butter. Artificial Butter. Margarine. — The best forms of imitation butter are made from beef fat freed from the greater part of its stearin, and mixed with milk, colouring, and flavouring reagents. The worse forms of imitation butter are made from lard, tallow, olive oil, rape seed oil, ^c. A good margarine contains 80 to 90 per cent, of fat and 5 to 6 per cent, of casein, salts, and pigment. Though not so assimilable as the butter from milk, it is a cheap and wholesome food. MUk Sugar or Lactose The characters of milk sugar whicli have been ah'eacly described in the chapter on Carbohydrates (p. 102) are the same whether it is derived from the milk of the woman, cnw, goat, and of all other animals from wliich it has been separated. The formation of lactic acid from lactose by the activity of certain bacterial growths gives rise to the souring that occurs in stale milk. Hoppe-Seyler ^ has supposed from the rapid appearance of lactic acid in some cases, that the milk already contains a lactic acid enzyme, when it leaves the mammary glands. Lactose by inverting ferments is changed into dextrose and galactose ; these undergo on the addition of yeast the alcoholic fermentation, and so koumiss is prepared. Extractives of Milk There are a number of organic substances dissolved in the milk- plasma which may be called extractives, and of which a mere enumera- tion will suffice. The caseinogen may be precipitated by acid or by rennet ; this carries the fat down with it, and the two together are then 1 Ann. Chem. Pharm. xlii. 46. - Physiol. Chem. p. 727. ^ Arch. j)athol. Aunt. xvii. 417. MILK 587 tiltcred otr. The filtrate contains all)iuuin, lactose, salts, and these extractives. Among the latter, Ritthausen ' separated a second carbohydrate (^f doubtful nature. Bechanip ' obtained traces of alcohol, and acetic acid ('0-021 to 0*2 grannne to the litre) from cow's milk ; SCO c.c. of asses' milk yielded 30 c.c. of a distillate containing 3'") pei- cent, of alcohol and 0-036 per cent, of acetic acid. The koumfss of Russia is made by allowing mare's milk to undergo the alcoholic fermentation. A similar substance, kephir, is made by adding tlie so'-called kephir- grains to milk; the kephir-grains are masses of fungi and bacteria. Kephir contains less alcohol (0-5 to 1 per cent.) than koumiss (1 to 2 per cent.). Small quantities of lactic acid can generally be obtained from the freshest milk (Hoppe-Seyler ^). Traces of urea have been described by several obsei-vers '^ ; Commaille "' found a trace of creatinine, and Musso '"' of a sulpho-cyanide. The Salts at Milk This subject has been worked at quantitatively by Bunge,' and with the f ollowino- results : - Hiuiiau milk I II K.,0 0-78 0-71 lsa.A) 0-23 026 CaO 0-33 0-34 MgO 0-06 0-06 Fe..O, 0003 0-006 P.A 0-47 0-47 CI 0-43 0-44 Total ash per 1000 . 2-22 2-18 Dog's milk Cow's milk Horse-s milk I II 1-41 1-68 1-76 1-04 0-80 0-69 1-11 0-14 4-53 4-28 1-59 123 o-i;t 0-21 0-21 012 0-02 001 0-003 0-015 4-93 4-(i7 1-97 1-31 1-62 1-8 1-69 0-31 3-15 12-96 7-97 4-17 The chief acid present throughout is phosphoric acid ; the chief base in human milk is potash ; but this in the other animals in the list is second to lime ; the lime in dog's milk is especially high. In connection with the quantity of iron in the milk, Bunge ^ has made the interesting observation that although the other mineral constituents of milk are present in the same proportion as they are contained in 1 .Joiirn.f.])rakt. Chem. N.F. xv. o4s. '- Compt. rend. Ixxvi. 654, H3G. ^ Arch.f.pathol. Anat. xvii. 43o. * Pieai-d, These, Strasburg, 1856; Lefort, CuDipt. renil. Ixii. 190; and others. * Commaille, quoted in Hoppe-Seyler's Physiol. Chem. p. 723. " Mahfs Jahresb. 1877, p. 168. T Diss. Dorpat, 1874. 8 Zeit. physiol. Chem. xiii. 399. 588 ALi:\IENTATIO^ ^he foetal tissues, the quantity of iron in the milk is very much less. "This is illustrated by the following analyses : — A hundred parts by weight of asli contain — K2O NaaO CaO MgO Fe^Og P2O5 CI Oxygen equivalent of the CI 111 new-born (lop 11-42 In (log's milk 14-98 . 10-64 8-80 29-52 27-24 1-82 1-54 0-72 0-12 . 39-42 34-22 8-35 16-90 101-89 103-80 1-88 3-81 loo -00 100-00 The milk ash is rather richer in potash and poorer in soda than that of the new-born dog ; this is easily explained by the fact that in the young animal the potash-rich muscle is increasing, and the soda-rich cartilage is diminishing. The higher percentage of chlorine is also explicable, as the chlorides not only serve to build up tissues, but also act largely as solvents in removing the end-products of metabolism through the kidneys. But the percentage of iron in the milk is only •one-sixth of that in the foetal tissues. The explanation appears to be that the foetus obtains the greater part of its supply of iron before birth through the placental circulation, and stores it in the liver {see p. 552). Bunge has published analyses that show that a kilogramme of body-weight contains less and less iron as the young animal grows. Iron appears to pass to the child by the placenta rather than by the milk, because of the difficulties of absorbing iron by the alimentary canal, and the danger that hfvmatogenous compounds may there become the prey of bacteria. Bunge regards it as probable that the large ^amount of iron which passes to the foetus is not all derived from the mother's food during the relatively short period of pregnancy, but that a storage of iron occurs in the maternal organs even before the first conception, and this may explain the occurrence of chlorosis at the age of puberty. Preservation of Milk ;Milk may be preserved for a short time by boiling and tightly corking the A'essel in which it is contained. Antiseptics, of which tlie most commonly used are boroglyceride and boracic .^cid, may be added. -MILK 589* Tlie milk may be concentrated at a low tcinptrature and then presened in- hermetically sealed tins. Most condensfd inilhs have an antiseptic added to them, of which the most commonly employed is cane sugar. Frozen milk should be thoroughly tliawed and well shaken before it is used as food, as the ice first formed carries a large percentage of the casein and cream to the surface.' Cheese Cheese is an important product of milk. The cheeses made iir various parts differ according to the amount of cream mixed with the milk, and thus their percentage of fat varies. The essential con- stituent of cheese is the curd which is thrown down by rennet. All cheeses in addition contain a small admixture of lactose and a variable amount of salts. During ripening the fats and proteids both undergo decomposition, and thus free fatty acids are generally present. The following table ^ gives the percentage compo.sition of some common cheeses : — Cheese Nitrogenojis principles Fats Salts Water 1 Cheshire . . . . '■■ S6-14 25-48 4-78 30 39 1 Grruv^re . . . . . ! 35-10 28-0 4-79 32-05 ; Roquefort . . . . 32-95 32-31 4-45 26-53 ' Cheddar . . . . . 28-4 311 4-5 360 , Camera bert . . . . 18-rt 21 4-7 1 51-9 ' The Chnnges jyrodiiced in Milk hy Disease It is a matter of every-day experience that the milk of a strong, healthy woman is more nourishing to the infant than that of weakly or sickly women. This has been .supported by analyse^.^ Filhol and Joly ' described a very abnormal case in which casein was altogether wanting. Bile-pigments and salts were described in milk in a case of jaundice by Frank,'' but in similar ca.ses .subsequent observers^ have failed to find them. Certain drugs given to the mother pass into the milk, e.g. iodine, mercury (when given in large doses), arsenic, antimony, lead, zinc, and bismuth. Opium and morphia have never with certainty been found in the milk, but the milk of a mother dosed \vith opium is very fatal to the child. ' Kaiser and Sclunieder, Bied. Ceyitralhl. 1887, p. "iiiT. - Charles, Physiol. Chemistry, p. 27. ' Decaisne, Gaz. mid. 1871, p. 317 ; Vernois and Becquerel, Loc. cit. * Gorup-Besanez, Lehrbuch, p. 438. s £)iss. Giessen, 187',>. . '■ V. Jaksch, Frager med. Wochenschr. 1880, No. 9. 590 ALniENTATION The milk of animals, especially of the cow, is so important a food that it is essential it should be derived from healthy, well-fed speci- mens. In cases of cattle plague the milk is found to contain blood.' The milk in cases of pearl disease should also be avoided, as the danger of infection fi'om the presence of the bacillus of tubercle is greatly to .be feared.^ The milk from cases of foot and mouth disease and all ■ affections of the teats of cattle is also injurious. Milk no doubt often acts as a carrier of infection ; in certain cases it has been supposed that scarlet fever may be transmitted by its means ; hence the pro- phylactic measure of boiling the milk befoi'e it is used. Concretions -consisting chiefly of calcium carbonate Avith small quantities of phos- phate and fat are occasionally met with in the teats of cattle.^ Blue milk "* owes its colour, according to Fiirstenberg, to triphenyl- rosanilin ; it is doubtless produced by a bacterium and is said to produce diarrhiva. A purple-red micrococcus {M. prodigiosus) quickly grows in milk allowed to stand. Another bacterium (j5. synxanthuvi) causes a yellow colour. JMWt Annlf/ns The estimation of the specific gravity of milk is important, as it furnishes a •guide to the most frequent adulteration milk undergoes, namely, admixture with water, usually after removal of the cream. The estimations of specific gravity, total water, solids, inorganic and organic, may be carried out by the jsrocesses already described in Chapter II. The total solids should not be less than 11-5 per cent. The microscopic examination of the milk should be then carried out most care- fully. Starch grains, globules of other oils, and many other adulterations may be thus detected. Quantitative estimation of the fat. — (1) An approximate estimation may be made by the thickness of the layer of cream. If milk be allowed to stand in a ■graduated vessel the cream should occupy 10 to lo per cent, of the column. (2) To 20 c.c. of milk add 20 c.c. of a- 10 per cent, solution of potassium hydrate, and 100 c.c. of ether; shake the mixture vigorously; pour off the ether from the sur- face, and add more ether to the milk several times in succession until a fresh portion of ether shaken with the alkalised milk extracts no more fat. Mix together the ethereal extracts, and evaporate the ether on a water-bath in a weighed capsule; dry at 110'' and weigh agcain ; the increase of weight is the amount of fat in the 20 c.c. of milk. The normal minimum for fats in cow's milk is 2-.5. (3) Many optical methods have been described, the opacity of the milk •depending on the number of globules present. In Donne's galactoscope a candle * Hussoii, Compt. rcml. Ixxiii. V6'M. - For analyses are Storch, Miihfs JnJircah. xiv. 170. •^ Fiirstenberg, Die Milchdriiseii dcr Kali. Leipzig, 1H()8. •* For recent .observations on this subject see Eeiset, Co))i2}t. 7'cnd. xcvi. 682, 745. light is (>xninino(l tlinniyli n v;ni;il>lc Iciii^tli of a (Column of milk until the li<;lit, is occhided. Bv coiiipiirisnii willi a standard tlii^ number of globukiS is calculated from the lengtli of the cohiniii. In Vnn^crs method small measured portions of milk ai'e adtled to 100 c.c. of water until a portion of tlie mixture examined in a vessel with jiarallel walls is n])a(|ue to light. (4) Soxhlet's ' method is one in wliich caleidations are made from the sjiecilic griuity of the milk before and after removal of the fat. EstiviatUw of the proteids. — A large number of methods for (estimating tlie ]>roteids of milk have been devised from time to time. They consist essentially of the following: The casein is estimated by weighin|j either the curd produced by rennet, or the i)reeipitate ])roduced bj' acetic ^id, after all fat has been removed from it by thorough I'xtraction with ether. The albumin is estimated by weighing the precipitate ])roduced by boiling after the removal of the casein or caseinogen. I have carefully examined the various methods that have been proposed, and arrived at the conclusion that J. Sebelien's - plan is the best. Tn outline it is as follows : The proteids are estimated, not by weighing them, but by estimating the nitrogen in the precipitates produced by various reagents ; for this purpose admixture with fat does not matter ; Kjeldahl's process of nitrogen estimation renders this method less formidable than at first sight it appears to be. The nitrogen nmltiplied by 6'37 gives the amount of proteid. (1) Total nitrogen estimated in a known volume of milk. (2) Nitrogen estimated in the precipitate produced by adding tannic acid to milk. This multiplied by (vST gives the total proteid. (1) mimiti (2) is the non-proteid nitrogen : this is more abundant in colostrum than in milk. (3) Nitrogen estimated in the precipitate produced by saturation with magnesium sulphate (casein + globulin^). (4) Casein-nitrogen estimated in the precipitate produced by adding acetic acid to milk (approximate). (5) Globulin-nitrogen : two estimations ; maximal obtained by the dilference ■ between (H) and (4) ; minimal estimated in the precipitate produced by mag-nesium sulphate after separating the casein by saturating with sodium chloride. This is the only pare of the process I regard as fallacious, for reasons already stated (p. 584). (6) Albumin-nitrogen estimated in the precipitate produced by adding tannic acid to the filtrate after removal of the caseinogen by saturation with magnesium sulphate and filtering. Estimation, of the sugar. — A precipitate of caseinogen and fat is produced by adding acetic acid to a known volume of milk, and filtered off ; the precii)itate is wa.shed with water, and the washings added to the first filtrate. This is then boiled with dilute sulphuric acid for half an hour, and the amount of dextrose so formed, estimated in it by means either of Fehling's solution or by the polarimeter. Lactose may also be directly estimated by titrating the whey y;/?/.* the washings of the curd (produci'il by rennet) with Fehling's solution : 10 c.c. of this solution is decomposed bj- 0-0()76 gramme of lactose. Or the lactose may be' estimated by the polarimeter without first converting it into dextrose ; (o)d = -f- 59-3. 1 Zeit. (7. huxhr. Vereins 7)i Biiijer>i, 1882, p. 18. See also Egger, Zeif. Biol. xvii. 110 ; Schmiiger, J. f. Landw. xxix. 129. . -' Zeit. pJiysiol. Chew. xiii. 135. ■* As, already shown fji. 583), globulin is absent from milk, though present in colostrum. 592 ALIMENTATION Uterine Milk This is ;i creamy alkaline secretion of the uterine glands ; it is especially abundant in ruminants. Its specific gravity is 1033 to 1040; it becomes acid quickly and coagulates. Microscopic investigation shows the presence of fat- globules, epithelial cells, nuclei, and di'lmn of cells. The uterine milk of the cow has the following percentage composition: Water, 87-9; fat, 1-23; proteids and cells, 10'56; salts, 0-37. This secretion is doubtless nutritive to the embryo in the early stages of development. Secretion of the Crop of Illrds Although milk is peculiar to mammals, John Hunter made the very remarkable observation that the mucous membrane of the crop of certain birds (doves) secretes a fluid very like milk for the first few days after the chick emerges from the shell. CI. Bernard ' compares this to milk, and believes that the birds feed their young with it during the first few days of their life. Leconte analysed this remarkable secretion, and found in it casein and salts 23-23, fat 10-4:7, and water f)()-30 per cent. At other times the crop secretes a weakly alkaline fluid which has no nutritive or fermentative action. Eggs The ova of mammals are small and contain only a small amount of food material for the nourishment of the growing and dividing proto- plasm ; the close connection between fcetus and mother enables the former to obtain its nutriment from the latter. But in animals whose development ab ova occurs outside the body of the mother, the neces- sity of a large store of food material is obvious ; hence in oviparous- animals the eggs are large, this increase of size being entirely due to- the store of food material. In vertebrates the food material is at first continuous with the embryonic tissues ; later it is placed in the yolk- sac which is attached to the primitive alimentary canal. As in the case of milk, the food thus provided for the developing oSspring is diverted by man from its natural uses and employed by him as a food for himself ; like milk, also, it is a highly nutritious food. The eggs of birds, and in this country especially the eggs of hens and ducks, are those particularly selected as a food-stufi". It will be, however, con venient to deal here, not only with hens' eggs, but to treat the subject from a more general standpoint. Egg-sliells consist in birds and some amphil)ia of a highly resistant keratinous material infiltrated with calcium carbonate and traces of magnesium carbonate and calcium phosphate.'^ The envelopes of the eggs of frogs and fishes are transparent and mucilaginous in consistency, ' Lei;ons sia- les propriites physiol. dcs liqiiidcs dc I'org Paris, lS5i», ii. '232. - Analyses are published by Hilger {Her. deatsch. Ckein. Gesellsch 1873 p. 165),, Wicke (Ann. Chem. Pharm. xcvii. 350; cxxv. 7»), Bruimerstiidt (ibid. xcv. 376). E(i(;.s 693 beiiii( composed almost entirely of mucin.' In insects the egg-shells are chitinous. Egg-shells are formed in vertebi'atf^s not by the ovary, but by tlu^ walls of the passages by which they pass to the exterior. 31any eggs are coloured green, blue, red, lirown, and so forth. The function of the pigment appears to be protective. The green and blue pigments are stated by Liebermann ^ to l)e derivatives of the bile- pigment. The chief calcareous constituent of the egg-shells of Ijirds is calcium carbonate. Dana and Buchanan state that the calcium car- bonate exci-eted by polyps is absorbed as sulphate and converted first into sulphide and then into carbonate. Irvine and Hims Wood- head^ therefore experimented with fowls, and fed them only on <;alcium sulphate ; the birds, however, continued to lay eggs with normal shells. These observers also showed that fowls are unable to store up in their gizzard more lime as carbonate than is sufficient for the formation of the shells of two or three eggs, and that if lime be not procui'able, either they will lay soft eggs or will cease to lay. White of pgg. — This is situated between the shell and the ovum •proper or yolk ; it forms an additional protection to the ovum, and is gradually absorbed by the yolk as development progresses. It is semi-fluid in consistency and pervade F.,Or, per cent.; Voit). Smoked meat. — The outer surface is hardened by the coagulation of the outer layer, certain nuitters in the smoke (creosote, &c.) acting as antiseptics. Soups. — These contain the extractives of meat, a small proportion of the pro- teids, and the principal part of the gelatin. The gelatin is usually increased by adding bones and fibrous tissues to the stock. The presence of gelatin causes the soup when cold to gelatinise. Beef-tea is an extract nuule by gradually and gently warming lean beef in water. Its stimulating effect is due to the extractives creatine, xanthine, hypo- xanthine, lactic acid, and salts. It is nutritious only to a slight extent, as it con- tains mere traces of proteids, gelatin, and fats. Hebig's Extract, and many other meat extracts now made, are concentrated beef -teas. Liebig's Extract contains 78 per cent, of solids, of which 61 are organic (extractives) and 17 inorganic. Its good effect in the sick-room is due to the extractives, which are stimulating, and to the salts, which are also stimulat- ing ; a simple solution of potassium phosphate is very refreshing. Vegetable Foods The chief distinction between animal and vegetable cells is the presence in the latter of an excessive amount of carbohydrate material, including an investing wall of cellulose ; and this replaces to a large extent the original protoplasm (proteid) of the cells. Vegetable foods, then, speaking generally, are rich in starch, sugar, and cellulose, and comparatively poor in albuminous substances. The cellulose is indi- gestible or almost so ; hence the fjeces, which consist of undigested residues, are larger in volume in herbivora than in carnivora. Vegetable proteid is, so far as analysis goes, practically the same as animal pi'oteld. For some reason not yet understood, it is, however, not so easily digested ; hence, even if a vegetable food contains as much nitrogen per cent, as an animal food, for purposes of nutrition it contains less.^ The varieties and properties of the vegetable proteids are described in Chapter X (p. 131). In the ash of vegetables the salts of potassium and magnesium ai-e, as a rule, more abundant than those of sodium and calcium. Cereals. — The average percentage composition of the cereals is given in the following table (Munk) : — 1 The investigations of Rutgers (Zeit. Biol. xxiv. 251) seem to point to the fact that this is due rather to the admixture of vegetable proteids with indigestible substances than to any peculiarity in the proteids themselves. 598 ALIMENTATION Coustituents ^Vlleat Rye Barley Oats Rice Mai/.e 1 Millet Water .... 13-6 151 13-8 12-4 131 131 110 Albumin . . . 12-4 115 ll-l 10-4 7-9 9-9 10-8 Fat \i 1-8 2*2 5-2 0-9 4-6 5-5 Carbohj-drates . 67-9 67-8 649 57-8 765 68-4 66-8 Cellulose . . . 2-.5 20 6-3 11-2 ()-6 2-5 2-6 Ash .... 1-8 I., 2-7 .0 1-0 1-5 2-4 Flour is made from cereals and from other seeds by removing the husk and grinding the remainder. The best wheat flour is made from the white interior of the wheat grains, and contains the greater pro- portion of the starcli of the grain, and most of the proteid. Whole flour is made from the whole grain minus the husk, and thus con- tains not only the white interior, l)ut also the harfler and browner outer portion of the grain. This outer region ctjntains a somewhat larger proportion of the proteids of the grain. Whole flour thus contains 1 to 2 per cent, more proteid than the best white flour ; but it has the disadvantage of being less readily digested. Brown flour con- tains a certain amount of liran (the coating of the grains) in addition ; it is still less digestible, but is useful as a mild laxative, the insoluble cellulose mechanically irritating the intestinal walls as it passes along. The best flour contains, or should contain, little or no sugar. The presence of sugar indicates that germination has commenced in the grains. In the manufacture of malt from barley, this is purposely allowed to go on. Wheat flour when mixed with water f < )nns dougli, a sticky, adhesive mass. Tliis is due to the ft)rmation of gluten ; and tlie forms of grain which ai'e poor in gluten cannot be made into dough or bread (oats, rice, ttc). Gluten does not exist in the flour as such, but is formed on the addition of water from the pre-existing globulins (Martin ; see more fully p. 135). Bread is made by cooking the dough of wheat flour mixed with yeast, salt, and flavouring materials. The yeast acting at the com- mencement of the baking, when the temperature of the oven is little above that of the body, forms sugar and dextrin from the starch, and then the alcoholic fermentation occurs. The bubbles of carbonic acid burrowing passages through the bread make it light and spongy. This sponginess enables the digestive juices subsequently to soak into it readily, and aSect all parts of it. In the later stages of baking, the gas and alcohol are expelled from the bread, the yeast is killed; and a crust forms from the drying of the outer portions of the mass of dough. Other methods have been, or are, adojitecl for making dough light ; the VEGETAKIANIS.M 599 leaven of the ;iucients was a piece of putrid dough ; baking powders are mixtures containing sodium bicarbonate, from which the carbonic acid is driven oti' during baking. White bread contains in 100 parts, 7 of proteid, 55 of carbohydrates (starch, dextrin, and sugar, the two last more abundant than in the flour), 1 of fat, '2 of salts, and the rest water. An adult would require daily about I'G kilo, of In-ead to supply him with the recjuisite amount of proteid ; this would, however, contain an overdose of carbohydrate. Lfgumhwus plants.-- -The meal t»f peas, beans, and lentils are rich in proteids, and are used by vegetarians as substitutes for meat. Potatoes are chiefly starchy. The percentage composition of the foods just mentioned is given in the following table : — Coustitiient.s Lentils Peas Beans Potatoes 7G-0 Water 12 o 14-H 14-8 Proteids .... 24-8 22-6 23-7 2-0 Fat 1-S) 1-7 1-6 0-2 Carbohydrates . . -A-8 53-2 49-3 20-0 Cellulose .... ■M\ O'O 7'5 0-7 Ash 2-4 2-7 _ 31 l-O It has been calculated that 4-5 kik>grammes of potatoes would be necessary daily to supply an adult with the requisite amount of proteid ; a, bulk far too great for an ordinary alimentary canal to manipulate. In fact, unless a vegetarian diet is supplemented by some concentrated form of proteid, like milk, eggs, or cheese, it will be found impracti- cable, and the person who takes it will waste. Fermentative changes occurring in the alimentary canal, and giving rise to gases (carbonic acid, marsh gas, etc.) from the starch and cellulose, give rise to flatu- lence, which is a most serious drawback to vegetarianism. • Rice has not this particular disadvantage to such a marked extent. It is well known that the inhabitants of certain countries (e.g. the coolies of India) are able to sub-sist on a smaller quantity of nitrogenous food than the average European. Recent experiments have shown that even Europeans can train themselves, gradually, to maintain bodily equilibrium for short periods on less than the fifteen grammes of nitrogen daily which lias been hitherto supposed to be iiecessary.^ The disad- vantages of pure vegetarianism resulting from overloading the stomach and flatulence remain, however, unaltered. Looking at the table just given, it is seen that beans contain rather more nitrogenous material than beef ; but experiments on man have shown that beans are a most 1 Sep. Rutgers, Zcif. Biol. xxiv. Sol. - F. Hirschfeld, Pfiiiger\s Archiv, xli. .533. 600 ALIMENTATION unsuitable form oi. iood when taken exclusively. It cannot be too often repeated that the digestibility of a food, as well as its percentage com- position, must be taken into account in estimating its nutritive value. Prausnitz' ' experiments with beans gave the following results : The faeces contained 18*3 per cent, of the food weighed as dry material, and 30*3 per cent, of the nitrogen undigested. Beans thus compare most unfavourably with bread, lentils, and other forms uf vegetable food. Green vegetables. — These ai-e taken as a palatable adjunct to other foods, rather than for their nutritive properties. Their ^^otassium salts are, howevei-, abundant. Cabbage, turnips, and asparagus contain 80 to 92 water, 1 to 2 proteid, 2 to 4 carbohydrates, and 1 to 1-5 cellulose per cent. The percentage composition of the green foods of herbivora have been already given (p. 573), and the small amount of nutriment they contain accounts for the large meals made by, and vast capacity of the alimentary canal of these animals. Accessories to Food Alcohol.— ^moXl quantities of the alcohol taken leave the body by the breath and urine as such ; the greater amount is decomposed into simpler products (acetic, oxalic, carbonic acids, and water) ; the forma- tion of these must give rise to a certain amount of bodily heat. It has been calculated that a man can burn off in his body two ounces of absolute alcohol daily. Alcohol is thus within narrow limits a food^ It, however, lessens proteid metabolism by about 6 per cent., and thus ultimately leads to a diminution of the heat produced in the body. It is, moreover, a very uneconomical food ; much more nutriment would have been obtainable from the barley or the grapes from which it was made. The value of alcohol used within moderate limits is not as a food, but as a stimulant, not only to digestion, but to the heart and brain. 2 The percentage of alcohol in liquors is as follows : Spirits, 50 to 60 or 65 ; port and sherry, 16 to 25 ; clarets and champagne, 5 to 13 ; porter and Bass' l^eer, 8 to 10 ; light beers 2 to 5. Various liquors are differently coloured and flavoured ; some, like hocks, are acid ; others are sweet from the presence of sugars and glycerine ; port abounds in tannin, sherry and brandy in various ethers and alcohols ; some, like champagne, are sparkling from excess of carbonic acid. Malt liquors contain bitter and other principles from the hop. Condimeyits, like mustard, pepper, ginger, cui'ry powder, are stomachic stimulants. Their abuse is followed by dyspeptic troubles. 1 Zeit. BioJ. xxvi. 227. 2 The reader interested in the subject of alcohol in diet should i-ead Chapter VITI of Bunge's Physiol. Chem. (transl. bj' Wooldridge). ALCOHOL, TKA, AND COKKKK 601 Tea, coffee, and cocoa. — These are stiinuhiuts chiefly to the iiin-vous system. Tea, coffee, mate (Paraguay), guarana (Brazil), cola nut (Cen- tral Africa), bush tea (South Africa), and a few other plants used in various countries, all owe their chief property to an alkaloid called theine or caffeine (CyH,oN^02 + H20) ; cocoa to the closely related alkaloid, theobromine (C7HgN402) ; coca to cocaine. These alkaloids are all poisonous, and, used in excess, even in the form of infusions of tea and coffee, produce ovei'-excitement, loss of digestive power, and other disorders well known to the practical physician. Coffee differs- from tea in being rich in aromatic matters ; tea contains a bitter principle, tannin ; to avoid the injurious solution of too much tannin,, tea should only be allowed to infuse (draw) for a few minutes. Cocoa is a valuable food in addition to its stimulating properties, containing about 50 pel" cent, of fat and 12 per cent, of proteid. I have not atten)i)ted to give references to the vast amount of literature published on the subject of alcoboL A few of the more recent papers on the subject will be found referred to in the chajjter on alcohol in Bunge's book. I am indebted to the same book for the following references to papers on the constitution and physiological action of caffeine and allied alkaloids. Caffeine is trimethylxanthine (i.e. xanthine with three methyl groups introduced into its molecule), and can be prepared artificiall)'' (E. Fischer, Licl)'ig's Annalen, ccxv, 253). Theobromine (the alkaloid contained in cocoa, and mixed with caffeine in guarana) is dimethylxanthine. Mono-methylxanthine is at present unknown. The physiological action of this series of substances has been studied by Filehne {Du liois Reymoncrs Arcldr, 1886, p. 72), and Kobert {Arch. f. exj). Path. u. Pharm. xv. 22). 602 A LI. MENTATION CHAPTER XXYIT DIET A HEALTHY and suitfible diet must possess the following characters : — 1. It must contain the proper amount and proportion of the various proximate principles -proteids, fats, carbohydrates, salts, and water. 2. It must be adapted to the climate, age, and sex of the individual, and to the amount of work done by him. 3. The food must not only contain the necessary amount of elements, but these must be present in a digestible form. The subject of diet is necessarily related to that of excretion. The object of the food is to repair tlie waste of the body ; the amount of waste or loss must be known before the amount necessary for repair can be ascertained. The varying relations betAveen income and expenditure, and the balancing of the two sides of the sheet, cannot here be conveniently studied in detail, but after our consideration of the ui'ine and other excretions, we shall be then better able to con- sider the exchange of material or metabolism of the body in relation to nutrition. For the present we must content ourselves with stating very briefly the principles on which diets have been constructed, and this we can do most i-eadily undei- the tln-ee heads enumerated above. The relation between the proximate principles in a diet. — We have seen that a proteid contains carbon, hydrogen, oxygen, nitrogen, and sulphur, and it might be said that proteid j^l'is water and mineral matter would supply a man with all the materials he wants, and the question will be asked, what is the use of the fats and carbohydrates, which only contain carbon, hydrogen, and oxygen ? If we examine the materials that leave the body, we shall obtain an answer to this question. A man doing a moderate amount of work will eliminate, chiefly by the lungs in the form of carbonic acid, from 250 to 280 grammes of carbon per dieia. During the same time he will eliminate, chiefly in the fonn of urea in the urine, about fifteen to eighteen grammes of nitrogen. ' In order to repair this loss, the daily food should contain, roughly, 1 In addition about six grmnuies of hydro^jen, and 700 grammes of oxygen, and thirty grammes of salts are parted with, but the dietetic value of a food depends chiefly on the amoiuit of carbon and nitrogen it contains. 1)1 KT 003 the sanio ijuantities of carbon and iiiti(>i;i'n, and the I'clation between carbon and nitrogen should be 250 to 1'), or 16-6 to 1. The propor- tion of nitrogen to carbon in })r(>teid, is, liowever, ^)'^ to 15, or 3-5 to 1. Hence if a person lives entirely on proteid food, his diet will be incorrect in one of two ways : if the amount taken is adjusted to give the right weight of carbon, the nitrogen will be much too high ; or if the amount taken is adjusted to give tlie right weight of nitrogen, the carbon will be much too low. In the first case, when the amount taken brings the quantity of carbon to the correct level, the food would be a bulky one, in fact so bulky as to be impracticable ; 250 grammes of car- bon would mean 500 grammes of proteid, and this would be obtainable in two to three kilogrammes (five to six pounds) of beef. This quantity of beef would contain much more nitrogen than the body has lost, and much more than the kidneys can excrete. In carnivorous animals, the capacity of the body for producing urea is greater than in man ; but in man the accumulation of nitrogen and increased woi'k of the kidneys, which are doing their best to get rid of the nitrogen, lead to ailments of which gout, obesity, and Bright's disease are the most common. If, on the other hand, the quantity of proteid takeii be kept down, so as to balance the daily loss of nitrogen, the result is that too little carbon is taken in to repair the lai-ge output, and the body conse- •quently wastes. Thus a purely proteid diet, though practicable for a short time, is impossible if the" body is to be maintained for long in an approximately healthy condition, not to say -a condition of equilibrium. In the practical construction of a suitable diet, what is first done is to keep the quantity of proteid food at such a level as to replace the amount of nitrogen lost, and secondly to supplement this with the carbonaceous but non-nitrogenous foods, so bringing up the quantity of carbon to the requisite standard. The non-nitrogenous foods are on this account sometimes called j)roteid- sparing foods. The same term is applied to gelatin, which within certain limits may be mixed with proteid, and so helps to supply the necessary nitrogen. The non -nitrogenous foods are the fats and the carbohydrates ; in the latter the hydrogen is already fully oxidised, and only the carbon is available for combustion ; in the fats both carbon and hydrogen can undei'go oxidation. There has been considerable discussion as to whether both fats and carbohydrates are essential, and as to how far one can replace the other. We shall not here enter into considerations of a theoretical nature, because tlie explanations they offer are not at all .satisfactory ; food and diet are subjects which, above all others, are 604 .\LJMKN'J.\'IHiN intensely ]jrHctical ; no doubt there are Siitisfactoiy reasons for every- thing relating to diet, but in many cases they have still to be discovered. Practically it is found that animals thiive best on diets whicli supply thern witli the bulk of their carbon, fiorn both fat and carbohydrate ; the diets which inf-n constructed froni experience long before they had even heard of metabolism contained both fat and carbohydrate ; the foods "which nature has pro\'ided for growing animals, in the shape of milk and eggs, contain also both fat and carbohydrate. Moleschott ' fixes the following daily diet foi- a man performing a moderate amount of work : — X. in frmtiiitK^ C, in vTaaimc-i 120 grammf>. of proteid (4-232 oz. avoird.) 18-88 64-18 90 „ fat (3174 „ ) — 70-20 330 „ carbr.hydrate (11-G4 oz. avoird.) — 146-82 Total . . . \^-HH 28r20 The total nitrogen and carbon are thus approximately equal to the total daily loss of the same two elements. Prof. V. Ranke '^ has performed many experiments on him.self, and his table of an adequate diet closely resembles ISIoleschott's ; it is as. follows : — K. in ffranime* C. in grammeit 100 grammes of proteid 1.5 -.5 53-0 100 „ fat — 79-0 250 „ car);oliyd i-ate — 93-0 Total i.v.-, 225-0 A diet consisting of fat or cai-bohydrate, alone or combined, is free- from nitrogen, and obviously incompatible with life for more than a short periwl. A diet consisting chiefly oi carbohydrate.s, as in a vege- tarian diet, has disadvantages which are just the opposite as regards the carbon and nitrogen to those already fully explained in connection with a diet consi.sting exclusively of proteids. Ranke and Moleschott in their experiments did not feed on proxi- mate principles, as the aVjove tables would seem to imply, but u.sed the food-stuffs of every-day life, of which the percentage composition was known. Ranke's diet, for instance, con.sisted of meat and bread, with small quantities of potato, butter, and egg. So is it in the construc- tion of diets nowadays. A table is consulted in which the amounts of the proximate principles in the chief fofxls are given (p. 573) ; from this the amount of such foods required to yield the necessary amount of these proximate principles can be calculated. The following tables- ' See Pavy'H Food and Dicteticn. - TJii Boia Reynumd's Archiv, 1862, p. :-ilt, and numerouK other papers. DIET 605 of the ratio of nitrogen and c'arl)oii in various foods, tlie amount of such focids necessary for the 120 grammes of proteid, and 420 grammes of non-proteid material on the l)asis of Moleschott's diet will be also found useful : — ' I'ouil N c Fooil Oysters . . N 1 1 ^ Food N C Beef with- 21 7-2 Oatmeal . . . 1-9 44 out bone . 3 11 Cheshire Potatoes . . 0-3 11 Roust V)eef . 3-5 17-7 cheese . 41 41 Dried figs . . 1 34 Salt cod-fish 5 16 : Beans . . 4-5 42 , Infusion of 3 ioz. yardines in 1 Peas . . . 3-6 44 coffee . . . 1 !) ' oil . . . 6 29 ! Flour . . 1-6 88-5 Infusion of 308i Salt herrings HI 23 1 Barley . . 1-9 40 grains tea . . 0-2 2 1 Eggs . . . 1-9 13-5 Rice . . . 1-8 41 Chocolate, 3^ oz. 1-5 58 Cow's milk . 1 0-6 6 Fresh butter . . 0-6 83 1 1 Food Weight of tlie food whicli ooiitains 120 grammes of proteid 420 grammes of iiou-proteiounds ; 1 foot-pound=0"13.'< kilogramme-metre ; 1 kilogramme-nietre=0'00328 foot ton ; 1 calorie=42o*5 gramme-metres : =0'4:25 kilogramme-metre ; 1 oz. avoird. = 2''"*"35 grammes. The loss of heat and aotion is replaced by the combustion of fresh material, which ultimately comes from the food. Tables are con- structed which give the heat value of food suVjstances when burnt out- side the bofly. and their nutritive Aalue as a source of energy within the body is deduced from these. It must, however, be clearly under- stood that no combustion occui's in the food in the alimentarj' canal, none in the blood on its way to the tissues, but it is only after assimi- lation, that is, after it has Ijecome a part of the living tissues them- selves, that it is oxidised and gives rise to heat and motion. In some cases, especially that of fats and carbohydrates, the amount of heat produced when they are burnt outside the body and estimated by calorimetric processes, is the same as that produced in the interior of the body after the carbon of these substances has become the carbon of the living cells. But in other instances the physiological heat-value is a different thing from the physical heat- value ; this is the case with substances like proteids which are only incompletely burnt in the body. Frankland estimated that a gramme of dry proteid when bunit in the calorimeter yielded 4998 calories, or heat-units ( = 2124 kilo, metres of work;. In the Vjody, however, 1 gramme of proteid yields one-third of a gramme of urea. The heat- value of 1 gramme of urea is 220^ ; one-third of this ( = 735) deducted from 4998 gives us 4263, the physiological heat-value of 1 gramme of proteid, which, expresse^50 which is more than sufficient to supply the energy expended ; we must,, however, remember that food materials are in the lirst place not wholly digested, in the second not completely oxidised in the body. Variations in diet necessary in relation to work, sex, climate,. &C. — Work. — A study of prison dietaries, of military dietaries, and so- forth, shows that the greater the expenditure of energy, the greater is the amount of food necessary. Muscular work falls especially on the non-nitrogenous, not on the proteids of the muscle (see p. 43G) ; hence 1 Philosophical Mac/, xxxii. 608 ALIMENTATION the increase of proteid food actually necessary during labour is probably smaller than in the annexed table : — • Ouuces 0 Dynamic value in Proteids Fats Carbo- )iydrates Salts Total foot-tons Subsistence diet 2-230 0-84 11-690 _ 14-760 2453 Soldiers during peace 4-215 1-397 18-690 0-714 25-016 4026 Soldiers in the field . 5-410 2410 17-920 0-680 26-420 4458 Koyal Engineers 5-080 2-910 22-220 0-930 31140 5232 Navvy .... 5-640 2-340 20-410 ■ — 28-390 4849 English sailors . 5-000 2-370 14-390 — 21-760 3911 Prisoner under 7 davs 1-800 0-480 10-712 — 12-992 — „ 21 „ 2-448 0-608 14-792 — 17-848 — „ with hard labour . 4-075 1-557 18-806 1-963 26-401 4072 „ with light labour . 3-508 0-315 16-727 1-715 22-265 3577 „ with industrial em- plovment 3-710 1-562 17-310 1-616 24198 3787 Prisoner with penal servi- tude .... 3-784 1-580 10-864 0-972 26-200 4193 Prisoner undergoing punishment . 1-296 0-256 8160 0-368 10-080 1541 Af/e and sex. — -Young animals require more food in proportion to -their weight than adults, because they are growing in addition to maintaining metabolic processes. Aged persons requii-e less food than those of middle age, and -women less than men. The average minimum diets for different ages are thus given, in grammes : — - 1 1 Proteid Fat Carbohydrate i Child under 1^ year „ from 6 to 15 years . Adult man (moderate work) . „ woman ..... Old man „ woman 20 to 36 70 to 80 118 92 100 SO 1 80 to 45 ! 60 to 90 37 to 50 1 850 56 ' 500 44 400 68 366 50 260 1 Climate. — Cold increases the appetite, increases the loss of heat, increases the desire and necessity for food with high heat- value ; the fats have the highest heat-value of the proximate principles. Fat is •even better than carbohydrates. Dr. McKendrick puts the matter thus : the dihitation of its Ijlood-vessels, and thtMV, is also an increase in tlie How of saliva. Further ohservation soon showed, however, that the increased How of seci'etion is not due to the increased vascularity of the secreting organ, for the tliree following reasons : — (t. The pressure in the duct is often higher, sonn'tinies twice as high as the pressure in the artei'ies. h. Tf the experiment be performed on the head of a recently decapitjited rabbit, stimulation of the nerve still produces a flow of saliva ; and here there cannot be any interference from alterations of blood-pressure. c. By the use of small doses of the alkaloid ati-opine, the two kinds of fibres contained in the nerve can be differentiated one from the other ; the di-ug produces paralysis of the secretory fibres, but it has no effect on the vaso-dilatator fibres ; excitation of the nerve produces under these circumstances a dilatation of the blood-vessels of the gland, but no increased flow of saliva. The next question which ai'ises is this, Admitting the existence ■of seci'etory nerves, is there any histological evidence that nerve-fibres terminate in secretory cells 1 The answer to this is an unsatisfactory one ; the nerve terminations are probably connected with the cells, but the exact method of connection has not at present been ascertained. Pfliiger made observations in which he described the direct connection of nerve-fibres with the nuclei of the salivary cells, but his assertions Slave never been corroborated. The submaxillary gland is the salivary gland in wdiich the nervous mechanism of secretion has been most fully woi'ked out. We will therefore consider this gland first. It is supplied by two nerves, the chorda tympani, and by branches of the cervdcal sympathetic, which -enter the gland with its artery, and supplies vaso-constrictor fibres to it. We have already seen that the chorda tympani supplies the gland with secretory fibres, and its vessels with vaso-dilatator fibres ; the sympathetic supplies the vessels with vaso-constrictor fibres. Has it any secretory fibres ? Such a question can only be answered by means of experiment — the experiment of stimulating the cervical sympathetic nerve. When this is done not only are the vessels constricted, but in the dog a slight flow of saliva results, which is remarkably \ iscid, of higher specific gravity and richer in corpuscles than is the chorda saliva. In different animals the results varies ; thus in the rabbit, both chorda and sympathetic saliva are free from mucin, but the latter ■contains moi'e proteids : in the cat, chorda saliA-a is more viscid than 618 AI^nrENTATJO.V sympathetic saliva ; but in all these animals the symimthetic saliva is smaller in quantity than the chorda saliva, and in all of them the blood-vessels are constricted. To explain this difference between the action of the two nerves of the gland, Heidenhain ' has advanced the theory that the cells of a secreting gland are supplied by two kinds of nerves ; the one, trophic, exciting chemical processes in their protoplasm ; the otlier, secretory, having to do with the separation of the secreted products. In all cells, gland-cells among the number, two processes ax'e continually occurring : one the building up of their subst-ance and contents (anabolism), the other the bi'eaking down of the same (katabolism). That each of these processes is governed by a special nerve-filament was an ingenious si^eculation, which it turns out, on further investigation, is supported in several ways. The existence of the two kinds of fibres, and theii* admixture in various proportions with one another, and with vaso-motor fil)res, will explain very largely the result of stimulation of the nerves we have mentioned ; to take the case of the dog's submaxillary again, the chorda contains many secretory fibres and few trophic fibres ; hence the secretion which follows its stimulation is copious and watery. The sympathetic, on the other hand, contains few secretory and many troj)hic fibres ; hence the secretion which follows its stimulation is scanty and viscid. Bayliss and Bradford - have confirmed the jirobable existence of Heidenhain 's two sets of fibres by demonstrating that the electx'ical changes in the glands are of the opposite kind on stimulation of the two nerves ; and that atropine destroys the chorda variation (hilus positive to surface of gland), but only slightly lessens the sympathetic variation (hilus negative to surface). Langley, however, considers that the existence of more than one kind of secretory fibre is very doubtful ; and he shows, too, that this assertion is not irreconcilable with the conclusions of Bayliss and Bradford. The reasons for Langley's conclusions are entered into fully in the papers quoted below,'' and briefly they are these : - (1) The plienomena of atropine-poisoning give no indication of the existence of more than one kind of secretory nerve-fibre. By the use of very small doses of atropine, administered successively, all varieties of secretory nerve-fibre are equally and simultaneously paralysed. (2) Exjjeriments on the submaxillary, in which the two nerves, supplying the gland are alternately stimulated, also tend to throw doubt on the existence of two varieties of nerve-fibre. The sym- I Hermann's Handhiicli, 1880, vol. v. ' - Proc. Roy. Soc. xl. 203. 3 Joiirii. PJii/sioIofjij, ix. .">."); x. '291. 8AT-IV.\ C.l!) j>atlietif saliva is lar<;el\ iiicicasecl in ainouiit by previous stiiiiulatiou of the chorda, that is, after the increased supply of ])l(iod produced l)y dilatation of the blood- v^essels. Unless the gland has l)een thus jireviously supplied richly with oxygen, the secretory til)res of the sviupathetic (which are coiiiparati\ely few in number and masked by admixture with vaso-constrictor nerves) are non-effective or nearly so. Langley thus considers that the action of the nerve-tibres on the size of the vessels has more imjjortance than Heidenliain was inclined to give to it ; and that the secretory tibres being in the two nerves mixed with vaso-mot(jr fibres of opposite kinds, explains the difference in the actions of the nerves quite as well as or l)etter than the hypo- tliesis that the secretory fibres are themselves of opposite kinds. Whichever explanation is ultimately shown to be correct - and there is much to be said on both sides — there is little doubt that the parotid and the sublingual are governed by nerAovis influences in the same way as is the submaxillary gland. Stimulation of the sympathetic in the dog produces no secretion of saliva from the parotid gland, or only when the gland has been previously thrown into a state of increased irntability by the previous stimulation of a nerve which corresponds to the chorda tympani in relation to the submaxillary : this nerve is a branch of the glosso-pharyngeal nerve called JacoV)son's nerve, which may be reached within the tympanum, in the tympanic plexus. Paralytic secretion. — This is a thin, watery secretion that occurs about twenty-four hours after section of the secretory nerve. The gland of the opposite side is also affected (antilytic secretion ; Langley). It begins to diminish about the eighth day. It has been explained as a degeneration effect comparable to the fibrillar chyiiiol. de la digestion, 1868. * Ti-eviranus, Biologie, vol. iv. 1814, p. 330; Tieflemanii and Gmelin, Die Verdauung tuicli Versuchen, vol. i. 1826, p. J). ' Hoppe-Seyler, Physiol. Cheni. p. 186. ^ Journ. prakt. Chem. Ixxxvi. 1.">1. See also Sehaer, Zcit. Biol. vi. 467. SALIVA 628 cak'iuin pliospliate and carlxniate, admixed with nuicus and leptotlirix.' The so-called ' tooth-stones ' have the same composition. Qiianfitdtice (Dtuh/sU.—^ The (piantitv of saliva secreted daily by a man varies considerably ; estimates varying between 13 oz. and 3^ lb. have l^een gix^en ; oOO to 800 grammes is another estimate — oxen and horses may secrete 40,000 to 60,000 grammes daily. Its alkalinity averages in man "OS per cent, expressed as sodium CHrbo)iate (Chittenden). Its specific gravity is 1002 to 1006 in man ; 1007 in dogs. It con- tains in man tive parts of solid matter per 1000, of which two are inorganic. - Hun uu iiiixeil saliva Dog's mixed saliva = 12 11=* III.* Water 994-10 995-l(; 994-7 989-63 Solids 5-90 4-84 5-3 10-36 Soluble organic matter 1-42 1-84 3-2 3-57 Epithelium .... 213 l-fi2 — — Sulphocvanirle of potassium 010 006 — — Salts ' 2-19 1-82 103 6-75 Submaxillary saliva. — A cannula is inserted into Wharton's duct, and the saliva obtained by the stimulation either of the chorda tym- pani or sympathetic can be readily collected and examined. The saliva thus obtained is colourless, clear, transparent, and sticky, •^especially if obtained by stimulation of the sympathetic. It is xnarkedly alkaline, and soon becomes cloudy in the air from deposi- tion of calcium cai-boiiate. Its composition is in the main the same as that of mixed saliva ; the mucin is more abundant ; the proteid coagulable by heat is not always present. Ptyalin is present in human submaxillary saliva, except in infants under the age of two months (Zweifel). It is present in most animals, but not in dogs. Potassium sulphocyanide is present in human, but not in dogs' submaxillary saliva.-^ The inorganic salts are calcium carbonate, calcium and magnesium phosphate, potassium and sodium chloride.^ Quantitative analysis (in parts per 1000 dog's submaxillary saliva) : — ' Vergiie, ' Du tartre dentaire et de ses concretions,' These, Paris, 1869. - Frerichs, Wagner's Handworterbuch d. Physiol, ill. 758. ^ C. Schmidt and .Jaeuhowitsch, Ann. Cheni. Pharm. Ixxix. 156. * Herter, Hoppe-Seyler's Physiol. Chem. p. 188. ■^ Zweifel, Untersuchungen ii. d. Verdaiiungsapjxirat . d. Neiigeb. Strasburg, 1874. ^ Longet, Compt. rend. xlii. 480; Oehl, La saliva umana, Pavia, 1864. 624 ALIMENTATION - 1 Bidder an d Schmidt ' Hei-ter- i I 11 Ill IV V VI Water 99604 991-45 994-385 994-969 995-411 991-319 Solids 3-96 8-55 5-615 5-031 4-589 8-681 Organic matters 1-51 2-89 1-755 — Mucin ^ . . — 0-662 — — 2-604 Salts .... 2-45 5-66 3-870 — 7-332 CO., in cbeniical union — — 0-440 0-504 0-654 — Salts" K.,SO, . KCl NaCl . Na.,CO, CaCO, . Ca.3(P0,)., (dog) . 0-209 per 1000 . 0-940 „ . 1546 . 0-902 . 0-150 . 0113 Gases '- (dog) Oxygen . . 0-4 to 0-6 vols, per cent^ CO., (free) . 19-3 ., 22-5 „ O./combined) 29-9 „ 42-2 CNitroo:en . 0-7 ., 0-8 The main facts concerning the difference in the secretion produced by excitation of the two nerves of the gland have been already men- tioned (p. 617). The following are actual analyses (given in per- centages) of the saliva thus obtained : — I. By stimulation of the t-horda tympani.^ Weak stimnlation Strong ,. Organic matters 1-5987 2-5047 0-519 ()-()29 Total s<.Uds 2-1187 31339 A strong stimulus thus produces an increase in the total solids, especially of tlie organic solids, and particularly of the mucin. That the percentage of salts in the saliva also increases with the rate of secretion was also noted by Werther," who obtained as high a percent- age in some cases as 0-77. Langley and Fletcher^ have more recently obtained the same results, both by means of stimulating the chorda, or injecting small doses of pilocarpine, a drug which increases the rate of salivary secretion.^ Such a fact goes to prove that the secretion of even water and salts is au act of the secreting cell, and not simply due to increased transudation from the blood. 1 Bidder and Sclimidt, Ann. Chein. Pharm. vol. Ixxix. - Hoppe-Seyler's Physiol. Chem. p. 191. •5 111 rabbits' submaxillary saliva, mucin is absent. * Herter, Loc. cif. ^ Pfliiger, in Heidenhaiii's Stitdieii des Physiol. Inst. BresluK, Leipzig, Heftiv. p. 25. ^ Heidenliaiii, Ibid. ' Pfliiger s Archiv, xx.xviii. 293. « Phil. Trans. 1889, vol. cLsxx. B, p. 109. 3 A very complete account of the antagonistic action of atropine and pilocarpine on salivary secretion will be found in Journ. Physiol, i. 339 (Langley). Another imjiortant contribution on the influence of nicotine on salivarv secretion, by the same author, will be- found in Journ. Physiol, si. 123. H.UAVA 625 II. By stimulation of tlie sympatlietic (dog). Here we get a small quantity of saliva, which is richer in solids than chorda saliva, especially in mucin and formed elements. Heiden- hain, moreover, found that the percentage of solids falls after prolonged stimulation ; thus : — AiiiDuiit of Porcoiitage sfcretioii of solids flu t lie first 80 mimites .... 0-G774 gramme 3744 \_In 88 minutes, after stimulation had lasted 190 minutes .... 0-8871 „ 1-488 r In the first 40 minutes .... 0-5286 „ 5-864 \ln 30 minutes, after stimulation had lasted 80 minutes .... 0-5330 „ 1-910 Sublingual saliva. — The secretion of the sublingual gland does not TDatei'ially differ from that of the submaxillary. It is, however, the richest of the salivas in solids (2-75 per cent. Heidenhain), formed elements, mucin, and inorganic salts ; it is thus the most viscid and the most alkaline (Heidenhain, Werther, Langley). In certain birds this gland is much enlarged, and secretes the viscid material out of which they build their nests (see Neossin, edible bird's-nest, p. 486). Parotid saliva. — The parotid gland yields a watery secretion, free from mucin, and rich in ptyalin, even in the new-born child. The gland is more highly developed in vegetable feeders than in carnivora. The saliva can be easily collected, especially in dogs, by means of a cannula in Stenson's duct. Its characters, with the exception of slimi- ness, and its constituents, with the exception of mucin, are the same as in submaxillary saliva. It always contains a small quantity of a globulin. Quantitative analysis. — On the next page are some analyses in parts per 1000. The table is compiled by Hoppe-Seyler.^ The Secretimi of the Mucous Meviirane of the Mouth. When the ducts of all the salivary glands have been ligatured a small quantity of very ^ascid secretion is poured into the mouth by the mucous glands of its lining membrane. .Tacubowitsch gives the following analysis of this secretion obtained from a dog : — Solids 9-98 per 1000 Organic solids .... 3-85 „ Inorganic solids .... 6-13 „ It has no dia.static action. The mucus secreted by the tongue of the froo- (an animal with no salivary glands) is, however, diastatic. The pnuon-glands of snakes are modified salivary glands. The secretion is 1 Physiol. Chem. p. 199. S S (526 ALIMENTATION Constituents Human parotid saliva Dog's parotid saliva Horse's I Mitsclierlich II Hoppe- Seyler III Schmidt and Jacubo- witsch IV V VI Herter parotid saliva. vn Lehmami Water .... Solids .... Organic matters KSCN .... KCl NaCl .... CaCO:, .... &83-7 to 985-4 14-6 to 16-3 9-0 0-3 [ 5-0 993-16 6-84 3-44 I 3-40 995-3 4-7 1-4- - 1-2 993-85 6-15 991-527 8-473 1-536 • 6-251 0-688 991-928 8-072 990-0 10-0 2-06 to 6-0 - 4-8 to S-7D Specific gravity 1006 to 1008 1004 to 1007 1005 to 1007 G-ases ' . . . . 1 00 c.c. of saliva yielded 7 CO. of gas (1 c.c. oxygen, 2-5 nitrogen, and 3-5 carbonic an- hydride). By adding phosphoric acid 40 to 60 c.c. of carbonic anhy- dride were obtained. rich in proteids, and the poison is a proteid one (see Proteids as Poisons, p. 137). The specific gravity of snake iDoison is over 1040. Its reaction is in some cases alkaline, in others weakly acid ; it is usually described as yellowish and viscid. 4. THE ACTION OF SALIVA The active principle of saliva is 'ptyalin. This belongs to the class of unorganised ferments, that are called either amylolytic (starch- splitting), or diastatic (resembling diastase, the similar ferment in germinating barley and other grains). Ptyalin may be prepared from a watery infusion of a minced saK- vary gland or from the saliva itself. Dilute phosphoric acid is added, and this is neutralised with lime-water ; the precipitate of calcium phosphate which is formed carries down the ptyalin with it ; this is col- lected on a filter and water added ; the water dissolves out the ptyalin, leaving the phosphate on the filter. The ptyalin is then precipitated from its aqueous solution by adding excess of alcohol. The precipitate may be collected, dried, and preserved for future use. It may be puri- fied by re-dissolving in water, and again precipitating with alcohol. To obtain a glycerine extract, a minced salivary gland is covered with absolute alcohol for twenty-four hours ; the gland substance freed from alcohol is dried, powdered, and allowed to macerate in strong glycerine for several days ; the ptyalin may then be precipitated from the glycerine solution by alcohol as before. R. Kulz, Zeit. Biol, xxiii. 321. SALIVA ()27 The only important clieniical action of saliva is that clue to the presence of ptyalin. It has various physical actions ; it dissolves certain substances, enabling us to taste tliem ; in virtue of its mucin, it lubricates the bolus before it is swallowed ; in virtue of its viscidity and alkalinity, it has a feeble, emulsifying action on fats. The diastatic activity of saliva may be readily demonstrated by the following simple experiment : — A few cubic centimetres of starch solution are placed in a test-tube, and a few drops of saliva added ; the tube is placed in a warm bath at 35° C. and by means of a glass rod a drop is removed every half- minute, and mixed with a drop of dilute solution of iodine on a testing slab. At iirst the drop strikes a deep blue from the presence of starch ; after a few minutes, another drop gives a violet colour ; this is because the starch is gradually disappearing, but some is still left, and the violet colour is produced by admixture of the blue tint, due to starch, and the reddish tint, due to dextrin into which the starch is beiner o converted ; in a few minutes more a fresh drop strikes a reddish brown with iodine, showing that all the starch has disappeared ; and in a few minutes more, a fresh drop, gives no colour at all with iodine, showing that the dextrin which gave the red colour has also gone. If at this stage a little of the fluid be withdrawn and alcohol added in excess, a white precijoitate is jDroduced ; this cannot be starch, as all the starch has long ago disappeared ; it cannot be sugar, as sugar gives no precipitate with alcohol ; it cannot be the dextrin that gave the red colour with iodine, as there is no longer a red colour given with iodine ; if analysed, however, it is found to have the same composition as dextrin, and thus it is called achroo-dextrin ; while the dextrin Avhich gave the red tint is called erythro-dextrin. If we test the liquid at the various stages by means of Trommer's test or Fehling's solution for sugar (p. 95), we shall find sugar present as soon as dextrin appears ; it increases as the dextrin disappears. Achroo-dextrin is, however, only partially, and with great difficulty, converted into sugar. This simple experiment teaches us that starch is transformed into dextrin and sugar, and that ultimately the greater part of the dextrin is also changed into sugar. Nasse ^ was the first to show that this sugar is not dextrose, and called it j^tyalose. v. Mering and Musculus "^ conclusively proved that ptyalose and maltose (the sugar formed by diastase in malting) are identical. "We have already in our consideration of the carbohydrates seen 1 Pfliiger's Archiv, xiv. 473. ^ See Seegen's paper, Pfli'iger's Archiv, xl. 38. SS2 628 ALIMENTATION how this transformation may be represented by a chemical equation ; the equations given by various authors differ according to the view they take of the molecular constitution of starch and the dextrins. The formula given by Brown and Morris is probably more correct than most of the others ; it is : — [starch] [water] [maltose] [aeliroo-ilestriu] [errthro-dextrin] The chief properties of maltose have been already described (p. 103). Small quantities of lactic acid are formed at the same thne.^ Ptyalin acts in a similar way, but more slowly on glycogen ; it has practically no action on cellulose ; hence it is inoperative on uncooked starch grains, in which the cellulose layers are intact. Ptyalin acts best at about the temperature of the body (35° to 40° C.) ; diastase acts most energetically at 60° C.^ Ptyalin acts best in a neutral medium ; a small amount of alkali makes but little difference ; a very small amount of acid stops its activity ; hence the action of the saliva stops when the food reaches the stomach containing acid gastric juice. The gastric juice not only stops its action, but destroys the ferment, so that it does not resume work when the semi-digested food becomes once more alkaline in the duodenum.^ In the human subject and in certain other animals v. d. Velden ^ showed that there is no free acid in the stomach until about three- quarters of an hour after the arrival of the food there. It was there- fore supposed that during this time, the ptyalin was able to exert its activity. This hypothesis was confirmed by the observations of Chittenden and Ely, which showed that saliva neutralised with acid is more active than ordinary alkaline saliva ; the acid first secreted by the stomach is thus presumably used in the neutralisation of saliva, and is consequently an acid to the already powerful ferment, ptyalin, of that secretion. The subsequent experiments of Langley and Eves showed, however, that this is not altogether correct, for in the stomach, when an ordinary mixed diet is being used, proteids are present, and acid proteids or acid peptones have a distinctly retarding action on ptyalin. In all probability, therefore, the conversion of starch into sutfar by ptyalin in the stomach stops after fifteen to thirty minutes, 1 Goldsckmidt, Zeit, physiol. Chem. x. 273. -' See Stutzer and Isbert, Zeit. physiol. Chem. xii. 72. 3 ,J. N. Langlej', Journ. Physiol, iii. 246; Langley and Eves, Ibid. iv. 18; Chittenden and Ely, Ibid. iii. 327 ; Chittenden and Smith, Chem. News, vol. liii. (six contributions). * Zeit. physiol. Chem. iii. 205. SALIVA 629 tliat is before any free acid appears. Free hydrcebloric acid imme- diately destroys the ptyalin. The following is a resvnic of the work of Chittenden and Smith on the subject of the influence of reaction on the activity of ptyalin. For the purpose of testing this action quantitatively a known amount of a 1 or 2 per cent, solution of starch was exposed to the action of a measured quantity of saliva at 40° C. for thirty minutes ; it was then boiled to stop further action, and the sugar in it estimated. The action of a ferment is not proportional to its amount until its solution is much diluted ; when the dilution of saliva is as 1 : 50 or 100, the diastatic action can be taken as a measure of the amount of ferment present. The normal alkalinity of fifteen samples of saliva reckoned in terms of sodium carbonate was 0'097 per cent. When this is neutralised with 0'2 per cent, hydrochloric acid its diastatic action is much increased, especially when the dilution is 1 to 50 or 100, but the difference is still pronounced when the dilution reaches 1 to 2000. There appears to be no proportional relation between natural variations of alkalinity and diastatic action, although the addition of sodium carbonate to neutral saliva retards and finally stops the action of ptyalin in proportion to the amount added. This occurs especially readily in more dilute solutions ; this is not due to simple dilution, but to the thereby diminished percentage of proteid matter, which in the less diluted saliva possibly combines with the carbonate, such proteid compounds having no effect on the ferment. Neutral peptone, on the contrary, has a distinctly stimu- lating effect on the activity of neutral saliva ; and when proportionate amounts of peptone and sodium carbonate are added the distinctive action of the latter is prevented, an alkaline proteid compound being probably formed. These investigators then proceeded to determine quantitatively the amount of acidity necessary to stop diastatic action, the tropfeolin test being used for the detection of free acid.' As a mean of eight determinations, 20 c.c. of neutralised saliva were found to contain proteids capable of combining with 7-74 c.c. of a O'l per cent, solution of hydrochloric acid. When the proteid matter present is saturated with acid the saliva has greater diastatic power than when simply neutralised. Small percentages of acid peptone act similarly, but beyond a certain point (when the amount of combined acid is over 0'006 per cent.) acid proteids retard and finallj- destroy the action of the ferment. A minute trace of free acid in dilute saliva still further increases its diastatic activity ; this trace is, how- ever, so minute as to be for practical purposes inappreciable, for 0"003 per cent. of free hydrochloric acid entirely stops the diastatic activity of saliva. ^ Drops of a saturated solution of tropseolin 00 in 94 per cent, methylated spirit, are allowed to dcy on a porcelain slab at 40° C. A drop of the fluid to be tested is placed on the tropa3olin drop, still at 40° C, and if lij'drochloric acid is present a violet spot is left wlien the fluid has evaporated. A drop of HCl O'OOO per cent, thus leaves a distinct mark. (Danilewsky, CentralhJ. med. Wiss. 1880; Szabo, Mahj's Jahresh. vii. 267; T. d. Velden, Ibid. x. 305.) 630 ALIMENTATION CHAPTER XXX GASTRIC JUICE The juice secreted by the glands in the mucous membrane of the stomach varies somewhat in composition in the different regions, but the mixed gastric juice, as it may be termed, is a solution of a proteolytic ferment called pepsin in a saline solution, which also contains a little free hydrochloi'ic acid. We find, as in the case of saliva, variations in the importance of the gastric juice in different parts of the animal kingdom, the most powerful juice being that obtained from carnivoi'ous animals, whose diet is almost exclusively proteid. The gastric juice of new-born children is quite active (Zweifel). The saliva is a juice which is I'eadily obtainable. The gastric juice, on the other hand, cannot be reached until the animal is either killed, or an operation, that of making a gastric fistula, performed. In man gastric fistuhe are also necessary in cases of disease ; for instance, a tumour occluding the oesophagus would kill its possessor by starvation, unless the stomach were opened, stitched to the wall of the abdomen, and food introduced through the artificial opening. From such cases gastric juice is obtainable, but the result of examining the juice is not absolutely satisfactory. We cannot be sure that it has a normal character if the person from whom it is removed is, as is usually the case, in a depressed state of health. The most celebrated case of gastric fistula, and the first upon which trustworthy observations were made, is that of Alexis St. Martin, a young Canadian who received a musket wound in the abdomen in 1822. He fell into the hands of Dr. Beaumont, who not only saved his patient's life, but took him into his service and then conducted a series of important experiments on him. He was able to collect the juice and to observe the vascular and other conditions of the stomach durincr digres- tion of different foods, during rest, and in minor derangements, of the alimentary canal. The perforation in the stomach, though ordinarily closed by a loose flap of mucous membrane, was 2^ inches broad, thus enabling the painstaking observer ample scope for his investigations GASTRIC JUICE 631 Dr. Beaumont's (liscovei'ies arc described by him in his book on the ' Physiology of Digestion.' ' Since his time somewhat similar cases have been carefully observed by Richet,^ Griinewaldt,^ Schroder,"* and others. The amount of gastric juice secreted daily is differently estimated by various observers. Beaumont by mechanically stimulating the gas- tric mucous membrane obtained on the average H fluid ounce : reckon- ing three meals a day this would give a daily secretion of four to five ounces (135 to 180 grammes). Bidder and Schmidt in dogs obtained about forty times that quantity, and Griinewaldt in his case of human gastric fistula gives a daily mean of 580 grammes. The description of the properties and action of the gasti'ic juice may be now conveniently taken under the four following heads : — (1) The physiology of the secretion of gastric juice. (2) The structure of the cells that secrete the juice. (3) The composition of the juice itself. (4) Its action upon food. 1. THE PHYSIOLOGY OF THE SECRETION OF GASTRIC JUICE Dr. Beaumont writes as follows : — ' The inner coat of the stomach in its natural and healthy state is of a light or pale pink colour, vaiying in its hues according to its full or empty state. It is of a soft or velvet- like appearance, and is constantly covered with a thin, transparent, viscid mucus lining the whole interior of the organ. Immediately beneath the mucous coat, and apparently incorporated with the villous ' membrane, appear small spheroidal or oval-shaped granular bodies from which the mucous fluid appears to be secreted. On the application of alunent the size of the vessels is increased, the colour heightened, and vermicular movements excited. The gastric glands begin to discharge a clear, transparent fluid, which continues rapidly to accumulate as aliment is received for digestion. This fluid is invariably distinctly acid. The mucus of the stomach is less fluid and more viscid, and sometimes a little saltish, but does not possess the slightest character of acidity. On applying the tongue to the mucous coat of the stomach in its empty, unirritated state, no acid taste can be perceived. When food or other irritant has been applied to the membrane, the acid taste is immediately perceptible.' 1 The most important facts made out by Dr. Beaumont are detailed in Dr. Lauder Brunton's book, Disorders of Digestion, 1886. ^ Compt. rend. Ixxxiv. 1514 ; Ixxxv. 156. ^ Ann. Cliem. PJiarm. xcii. 42. ■* Diss. Dorpat, 1853. ^ The use of the word villous here is erroneous; the stomach has no villi. 632 ALIMENTATION This simple statement really contains the essence of all our subse- quent knowledge on the subject. When not excited the stomach is free from gastric juice ; it may be excited, as it normally is, by the presence of food ; but mechanical, thermal, chemical, or electrical stimuli may also be employed. Dilute alkalis, such as the saliva, excite the secretion especially well. Schift' ^ made the statement that active gastric juice is only secreted after absorption of what he termed peptogens : dextrin was one of the most important of these ; soup was another. Schiff's method of experimentation is open to some question, - but these substances do undoubtedly act as excitants of the secretion. Schiff's mistake was to attribute to them the sole power of exciting gastric secretion. There is no doubt that purely nervous (reflex) mechanisms come also into play. Thus the smell, the sight, the thought of food will excite a flow of the juice. In Richet's case of gastric fistula (the oesophagus had been occluded by caustic alkali) the placing of sugar or lemon juice on the tongue caused a secretion of gastric juice ; in this case no saliva could have reached the stomach. CI. Bernard once observed in a dog a flow of the juice on excitation of the vagus nerve. He also once observed that stimulation of the sympathetic nerves passing from the semilunar ganglia to the stomach arrested the secretion. Rutherford has shown that when the vagi are cut during digestion, the lining of the stomach becomes pale ; that stimulation of the perijjheral end. produces no effect, but that stimula- tion of the central end causes the mucous membrane once more to be reddened. In rabbits, however, after division of both vagi below the cesophagus, digestion goes on in a normal way. What is to be learnt from a number of observations of this kind 1 First, that our knowledge is most inexact, and that thorough and consecutive experiments are much to be desired. Secondly, that though inexact they at least teach the fact that the nervous system has some control over gastric secretion. Thirdly, that any direct influence of nerves on gastric secretion, as in the case of the salivary glands, has not been discovered. Fourthly, that what nervous influence has been discovered is exerted rather on the blood-vessels than on the secreting cells of the stomach, the increased flow of gastric juice being secondarily produced by the dilatation of the blood-vessels. We, in fact, possess a considerable amount of knowledge concerning the vaso-motor nerves of the abdominal organs, and also of the nervous mechanism of peristalsis ;, but in a work on chemical physiology we have only to do with these to a very small extent. A theory has been promulgated that local 1 Archives des sciences physiques et ncdurelles, 1877. - Laaigley, Journ. Physiol, iii 291. GASTRIC JUICE 633 centres in the ganglia of the plexuses of the stomach and intestine have an influence on both blood-vessels and secretion. Such a theory would require very forcible backing up before it could be regarded as tenable. All recent research goes to prove the relatively small import- ance of peripheral centres for the carrying out of reflex actions. 2. THE STRUCTURE OF AND CHANGES IN THE CELLS THAT SECRETE GASTRIC JUICE Two kinds of glands are distinguished which diflfer from one another in the character of their enclosed cells, and in the nature of their secretion. The pyloric glands are so called because they are found most numerously in the pyloric region ; they are distinguished by the large size and depth of the gland mouth or duct as compared with the tubules that open into it. The duct is lined by columnar cells con- tinuous with and similar to the columnar epithelium covering the general internal surface of the stomach ; the tubules are lined with shorter and more cubical cells, which are uniformly granular throughout. The cardiac glands (fundus glands of Heidenhain) are so called because they occur most numerously in the cardiac half of the stomach. Their duct is short, their tubules, in proportion, long. The latter are filled with polyhedral cells, only a small lumen being left ; they are more coarsely gi-anular than the corresponding cells of the pyloric glands. These cells were called princijKil cells by Heidenhain,' adelomorphic cellshy Rollett,- and central cells on account of their position. Between them and the basement membrane of the tubule are other cells of a different nature called jxirietal cells (Heidenhain), delomorphic cells (Rollett), or oxyntic cells (Langley).^ They are most numerous in the more superficial portions of the tubules. Their granular appearance is due to a close and uniform intercellular network (Klein). ^ They are readily stained by many colouring agents, especially aniline blue. The changes that occur in these different cells on secretion have been worked at by Heidenhain, Ebstein,'^ and Langley. The following is in brief the substance of Langley's observations : — The use of osmic acid is to be much recommended for studying these conditions, as hardening reagents like alcohol cause the granules to become swollen and indistinct. The central cells exhibit changes similar to those already described 1 Arch.f. viikr. Anai. \\. 368. ^ Centralhl. med. Wiss. 1870, Nos. 21 and 22. ^ Journ. of Physiol, ii. and iii. (o|i)s = acid). They were formerly called peptic cells, a term that must now be discarded. '^^Stricker's Hanclbiich, 1871. 5 Arch.f.7nikr.Anat.\i. 634 -ILOIENTATION r^ as occurring in the salivary glands. Befoi'e secretion they are ' loaded ' with granules ; during secretion they discharge their granules, those that remain being chiefly situated near the lumen, leaving in each cell a clear outer zone {see fig. 84). Hie cells of the pyloric glands undergo similar "changes. In both these cells and the central cells of the cardiac glands some substance readily precipitable by alcohol makes its appeai'ance during discharge, as this re- agent then renders the cells turbid. The oxyntic cells undergo merely a change of size during digestion, being at first somewhat enlarged and then shrink- ing to less than their original volume (Heidenhain). We have in the granules of the central cells another instance of a zymogen or ferment-precursor. It is the precursor of pepsin, and is called pepsinogen. The parietal cells are those which secrete the hydrochloric acid. The evidence upon which this statement rests is the follow- ing : Heidenhain by means of a surgical operation, performed antiseptically, suc- ceeded in making in one dog a cul-de-sac of the fundus, in another of the pyloric region of the stomach ; the former secreted a juice containing both acid and pepsin ; the latter, parietal cells being absent, riG.84.-ACardiacGianriofsimpiefonn secreted a viscid alkaline juice Containing from the Bat's Stomach. Osmic acid i-ipT\ejT-. preparation (Lauglev). c. columnar r r epithelium of ihe surface; n, neck of Briicke showed that the aciditv of the the gland, with central and parietal •' cells;/, base or fundus, ocenpiefi only glands is greatest near their mouth ; here bj" principal or central cells, -which o o exhibit the granules accumuiatefi also the parietal cclls are most abundant ; towards the lumen of the gland. and no doubt the acid is quickly expelled from the glands. CI. Bernard showed this by his well-known experi- ment of injecting potassium ferrocyanide in one vein of an animal, and lactate of iron into another. These substances in presence of free acid strike a blue colour, and he found only the surface of the mucous membrane of the stomach was blue. In the frog there is a well-marked separation of two regions : the oesophageal region, free from pai'ietal cells, secretes an alkaline juice ; the stomach itself, which contains the parietal cells, an acid juice (Langley). GASTKIC JUICE 635 Thus, although there can be but little doubt that the central cells secrete pcpsm, the argument that the parietal cells secrete acid is at present one of exclusion only. The rennet- ferment (rennin or chymosin) appears to be formed by the same cells that manufacture pepsin. Hammarsten • and Langley ^ obtained evidence of the existence of a zymogen of rennin analogous to that of pepsin or ptyalin ; a weak alkaline extract of the mucous membrane contains no rennet ; a weak acid extract contains rennet, and causes clotting in milk, even if the extract be made alkaline. A weak acid is generally found eflfective in converting a zymogen into a zyme or ferment. The seci'eting cells of the stomach, like secreting cells universally, select certain matei'ials from the lymph which bathes them ; these materials are worked up by the pi-otoplasmic actixaty of the cell into the secretion which is then discharged by the cell into the lumen of the gland of which it forms part. The most important substance in a diges- tive secretion is the ferment; in the case of the gastric juice this is pepsin ; we can trace an intermediate step in the process by the visible presence of its precursor, pepsinogen. But another equally important material in the juice is the acid, for pepsin acts only in acid media. We have now, therefore, to consider a little more fully the differences be- tween pepsin and pepsinogen, and, secondly, the important but puzzling problem of the formation of a free acid from the alkaline blood or lymph. Pepsin and pepsinogen. — The following research was earned out by Langley and Edkins.' Their object was to discover a method of determining the relative amounts of pepsin and pepsinogen in any given fluid, and thence to determine whether both exist in the gastric glands. The following two methods were found to give approximate results : — (1) The power of sodium carbonate to destroy pepsin is much greater than its power to destroy pepsinogen. Thus if equal volumes of neutralised acid extract of gastric mucous membrane and 1 per cent, sodium carbonate solution be mixed, ^ to ^ of the pepsin is destroyed in fifteen seconds, and it is unable to digest such a proteid as iibrin. (2) The power of carbonic acid to destroy pepsinogen is greater than its power to destroy pepsin. If an aqueous extract of a frog's oesophagus be taken, and a stream of the gas passed through it for half an hour, ig to |f of the digestive power of the fluid is destroyed ; while if an aqueous extract be warmed with dilute acid in the first instance, to convert the pepsinogen into pepsin, and it is then neutrahsed and the gas passed through it, there is little or no loss of diges- tive power. The passage of carbonic acid through the extracts throws down a precipitate of a globulin ; but pepsinogen, which is thus probably a globulin, is not carried down unaltered, since a solution of the precipitate in dilute hydro- chloi-ic acid has little or no digestive power. Pepsinogen and pepsin are both destroyed at 54° to 57°, the temperature at which the globulin is coagulated. I Maly's Jahresb. 1872, p. 123. ^ Journ. oj I'fiysioL ui. -zbl. 3 Hiid. vii. 371. 636 ALIIVIENTATION r^ The destruction of isepsinogen by carbonic acid is increased by the presence of a small amount of neutral salt, and diminished by small amounts of peptone. The gases oxygen and carbonic oxide have no effect on either pepsinogen or pepsin. On applying the above methods to the oesophageal glands of the frog, it was found that little or no pepsin is present in the cells themselves. The conversion of pepsinogen into pepsin that occurs when the secretion leaves the cells is, no- doubt, the same chemical change as that produced by the action of a dilute acid on the zymogen. The formation of Jiydroch loric acid. — There is at present no thoroughly satis- factory theory to account for the presence of free hydrochloric acid in the gastric juice. Foster ' suggests that it may be formed by the decomposition of some highly complex and unstable chlorine compound formed in the cell by union of organic substances with the chlorine of sodium chloride. Most other observers have considered that sodium chloride is a more direct source of the acid ; but sodium chloride is, as Foster points out, an exceedingly stable substance, and carbonic acid, the only free acid in the blood, is a weak acid. The terms weak and strong as applied to acids are, however, misleading. So-called weak acids- are, by what is termed ' mass influence,' able to unite with bases, displacing acids of greater ' avidity.' Thus the formation of free hydrochloric acid from sodium chloride and carbonic acid is not only a possible, but probably the correct ex- planation of the phenomenon (Bunge, ' Physiol. Chem.' p. 161). Kalfe attributes the production of the acid to the passage of electric currents through the mucous membrane, causing a reaction between sodium bicarbonate and sodium chloride, thus : NaHC03 + NaCl = NajCOj + HCl, but there are no valid grounds for sup- posing that such currents exist. It appears to me more probable that it is lactic acid which is chiefly instrumental in the decomposition of sodiiim chloride. Lactic acid is generally found in the stomach during a meal, especially if the meal contains carbohydrates ; fermentative changes in these produce the lactic acid, which reacting with the sodium chloride produces sodium lactate and hydrochloric acid. This view was first promulgated by Maly.^ Lactic acid certainly will decompose sodium chloride in this way in cold dilute solutions. Drechsel has discovered that the lactates in the blood are increased from 0"01 to 0-02 per cent, during digestion ; a fact that supports Maly's view of the case. The great difiiculty, however, in accepting Maly's theory is that carbo- hydrates are not always present in the food, and that a flow of acid from the gastric glands can be excited by distilled water or mechanical irritation. What, then, is the source of the lactic acid under those circumstances ? This objection is met by Landwehr ^ by the following ingenious theory, in which animal gum (p. 480) plays an important part : the lumen of the gastric glands is always more or less filled with mucus ; when the glands are stimulated a ferment is produced which decomposes the mticin, forming lactic acid from its carbohydrate con- stituent (animal gum) ; this acid reacting on sodium chloride produces free hydrochloric acid and sodium lactate : the former is poured into the stomach ; the latter is absorbed by the blood. If it be admitted that sodium chloride is a direct source of hydrochloric acid, Landwehr's theory of the modim nnprnndi ' 'I'ext-book, 6tti edit. p. 419. 2 Sitzungs. d. Wien. Akad. vol. Ixix. 1874 ; also vol. Ixxvi. In the latter paper a further suggestion is made, viz. the acid originates by the interaction of the sodium chloride and the sodium dihydrogen phosphate of the blood. 5 Che7n. Centralbl. 1886, p. 484 ; Pfliiger's Archiv, xl. 21. GASTRIC JUICE 637 appears to bo a satisfactory one. It Is, however, possible that the sugar of the blood and lymph is the real source of the acid. All attempt to solve the question was made by Kiilz ' : he i administered bromides and iodides, and then sought for free hydrobromic or hydriodic acid respectively in the gastric juice, and found it. As Drechsel -' points out, however, the decomposition might have been effected by the hydrochloric acid of the juice, and not by the metabolic activity of secreting cells. If chlorides are not given in the food, hydrochloric acid disappears from the gastric juice after a time (Cahn^). It is found that as the acidity of the gastric juice increases, that of the urine ■diminishes. This is not because of any diminution of free acid Un urine — as urine contains no free acid — but because the amount of the base liberated by the forma- tion of the gastric acid is increased, and passes into the urine. If sodium lactate is produced it no doubt is changed into sodium carbonate, which passing into the urine tends to render it alkaline. 3. COxMPOSITION OF GASTRIC JUICE Tlie methods of obtaining gastric juice that have been adopted are the following : — Spallanzani ^ fed birds on pieces of sponge to which a piece of string was attached ; after the sponge had remained in the stomach for a sufficient length of time to absorb the juice, it was pulled up by means of the string. Since then gastric juice has been obtained from cases of gastric jdstulffi both in men and animals. The first case carefully observed in a human being was that of Alexis St. Martin ; the first artificial gastric fistula in dogs was made by Blondlot ; ^ Bardeleben,^ Bidder and Schmidt/ Bernard,^ Holmgren,^ Panum/° and many others have since then performed similar experiments. For the investigation of the action of the gastric juice, it has been found that artificial gastric juice acts in the same way as the genuine article, and it is much easier to obtain. Schwann was the first to make an artificial juice, by extracting the mucous membrane of the stomach of a recently killed dog with 0"2 per cent, hydrochloric acid ; v. Wittich was the first to make a glycerin extract of the mucous membrane. The mucous membrane must be allowed to stand twenty-four hours before the extract is made, or treated with a little dilute hydrochloric or acetic acid, or with solution of sodium chloride." By either of these means ^ Zeit. Biol, xxiii. 460. - Ibid. xxv. 396. ^ Zeit. physiol. Chem. x. 522. * Versuch. iiber das Verdauungsgeschdft, iibers. von Michaelis, Leipzig, 1785. 5 Blondlot, Traite analytique de la digestion, Paris, 1843. ^ Arch. f. x>hijsiol. Heilk. 1849, vol. viii. ' Die Verdanungsafte und der Stoffwechsel, Mitau and Leipzig, 1852. s Bernard, Leqons de pliysiol. experiinentale, Paris, 1856. 9 Virchow-Hirsch, Jahresb. 1869, p. 103. i" Ibid. 1879, p. 99. 11 Griitzner, Neue Unters. ii, d. Bildung des Pei)sin, Breslau, 1875. 638 ALIMENTATION pepsinogen is converted into pepsin ; glycerin is then added, and allowed to extract the pepsin for at least eight days. For artificial- digestion experiments, an artificial juice may be made by mixing a little of this extract with 0-2 per cent, hydrochloric acid ; or, better, the pepsin is precipitated from the extract by means of alcohol ; this pre- cipitate is collected and dried at a low tempei'ature, and when required dissolved in hydrochloric acid of the same strength. The gastric juice in those animals in which it has hitherto been examined is either colourless or faintly yellow, clear, not viscid, and acid. It does not coagulate on boiling, but gives abundant precipitates with lead acetate, with mercuric chloride and with alcohol, showing the presence of pepsm ; it gives no precipitate with acetic acid, showing the absence of mucin. The two important constituents of the juice, the acid and the ferment, have already been several times mentioned, and we have endeavoured to trace the manner of their formation by the cells that secrete them. We have now to take up the question of their quantity in the juice itself, the methods of detecting them, and separating them from the other constituents of the juice. Before passing, however, tO' the consideration of these subjects, the following table of analyses may be first given : — ^ Constituents In parts per 1000 Human Dog Sheep Water . 994-404 973-062 986-143 Organic substance; (chieflv pepsin) 1 HCl . ' . 3-195 0-200 17-127 3-050 4-055 1-234 CaCl„ . 0-061 0-62-1 0-114 NaCl . 1-465 2-507 4-369 KCl . 0-550 1-125 1-518 NH^Cl . 0-468 0-478 Ca3(P0,), Mg3(P0,), FePO, 1 f 0-125 1-729 0-226 0-082 1-182 0-577 0-331 The points to be noted in such a table are the following : — 1. The relatively low percentage of solids in the human juice as compared with that of the other animals, particularly the dog. The young woman from whom the juice was removed is spoken of as healthy : this term is a comparative one ; there appears to be little doubt that 1 The above table was constructed by C. Schmidt and his pupils (Bidder and Schmidt, Ann. Chem. PJiarm. xcii. 42). The case of human fistula from which the juice was obtained was that of a healthy young woman named Katherina Kutt under Griine- waldt's care. GASTRIC JUICE 639 in a peifectly liealthy person, that is, a person without a gastric fistula, the percentage of both pepsin and acid would be higher, though of course not so high as in a carnivorous animal like the dog. 2. The great preponderance of chlorides over other salts ; appor- tioning the total chloi'ine found to the various metals present, that which remains over must be combined with hydrogen to form the free acid of the juice. I'lie acids of the (/astric juice. — Beaumont by the tongue, and C. Schmidt by litmus paper, demonstrated the fact that the stomach when not seci'eting was alkaline, but that the juice it poured out on stimulation was acid. In order to guard against error from the occur- rence of acids introduced with or formed from food, Brlicke neutralised the contents of the stomach with magnesia, and on removing this squeezed the stomach and obtained an acid juice. CI. Bernard's ex- periment with potassium ferrocyanide and lactate of iron has been already mentioned (p. 634). Prout ^ was the first to obtain hydrochloric acid by the distillation of the gastric juice. Dungliuson and Emmett obtained the same result from the juice in Dr. Beaumont's case. Lehmann ^ considered that this result was due to the action of lactic acid on the chlorides in the juice, and not to the presence of free hydrochloric acid. Leuret and Lassaigne ^ supported this "view, as they found free lactic acid in the stomach. The analyses by C Schmidt, however, placed the matter on a safe footing, as he showed that the amount of chlorine was greater than would combine with the metals and ammonium present in the juice. There is, however, very little doubt that, though hydrochloric acid is the acid 2)'^'>' excellence of the juice, lactic acid does occur during digestion ; this consists partly of sarcolactic acid derived from meat, and fermentation lactic acid derived from carbohydrates. The amount of lactic acid is much increased in those disordered conditions of the stomach when excessive fermentative processes are occurring. Small quantities of volatile acids, such as acetic and butyric, are also pro- duced in this way. Numerous methods liate ieen devised for the detection of these acids, and the most important of these are the following : — 1 . For free hydrochloric acid. The tropaeolin test has been already described (p. 629) ; other colour reactions extensively used are as follows : Solutions of gentian-violet or methyl-violet are turned blue : this is exceedingly delicate, but is hindered by the presence of peptone. Uffelmann,* who has devoted a large 1 PJiil. Trans. 1824, p. 45. ^ JBer. d. Sachs. Gesell. d. Wiss. Leipzig, i. 100. 3 Becherches j^hi/siol. et chiin. de la digestion, Paris, 1825. 4 Zeit. kli7i. Med. viii. 39'2. 640 ALIMENTATION amount of atteution to this subject, recommends one of the two following reactions : (a) 0'5 c.c. of red Bordeaux wine is mixed with 3 c.c. of alcohol and 3 c.c. of ether ; the mixture is almost colourless, and gives a rose colour with a few drops of a 0'45 to 0'5 per 1000 solution of hydrochloric acid, even in the presence of peptone, albumin, and salts, {h) Bilberries are made into a pulp with water, extracted with amyUc alcohol, and the extract used for colouring paper, which is thereby turned blue' or greyish-blue, fainter than blue litmus. When this is dipped into 0-2 per 1000 hydrochloric acid it is turned red. Lactic, acetic, and butyric acids give the same reaction when present in the proportion of 4 to 6 per 1000 ; a proportion never found in tlie contents of the stomach. Wiesner and Singer have introduced a reagent consisting of 2 parts of phloroglucinol, 1 part of vanillin, and 30 jiarts of rectified spirit. A few drops of liltered gastric juice is evaporated with an equal quantity of the reagent and red crystals, or if much peptone is present, a red paste is formed. The reaction talies place with 1 part hydrochloric acid in 10,000.' The organic acids do not give this reaction. 2. For free lactic acid. A solution is made by mixing 10 c.c. of 4 per cent, carbolic acid with 20 c.c. of water and 1 drop of the liquor f erri perchloride of the British Pharmacopoeia. An amethyst blue clear liquid is formed, which is turned yellow by lactic acid when present in only 1 part per 10,000. The test is best per- formed as follows : Boil the stomach contents and iilter ; extract the filtrate with ether ; evaporate the ethereal extract to dryness, dissolve the residue in a little water, and add a few drops of the reagent (UfEelmann). Hydrochloric acid simply renders the fluid colourless, and must be present in fairly large quantities to do this. The metlioiU devised for estimating the amount of the acids in the stomach are the following : — The oldest method is that of Bidder and Schmidt : this consists in performing an ultimate analysis, apportioning the chlorine to the metals and ammonium present, and calculating the remainder as hydrochloric acid. Rabuteau's method " modified by Cahn and v. Mering,^ consists in driving off the volatile acids by heat and shaking the residue with a large excess of ether, which takes up the lactic acid : this is separated, and cinchonine (Rabuteau used quinine) is added to the remainder until the reaction is neutral. The cinchonine hydrochloride is dissolved out by shaking with chloroform ; the chloroform is distilled off from this extract, and chlorine estimated in the residue. Another method, devised by Cahn and v. Mering, consists in distilling the contents of the stomach with water three times. The volatile acids are estimated in the distillate. The residue is shaken six times with 500 c.c. of ether, and this is evaporated to dryness, and the lactic acid estimated by titration. The residue contains the hydrochloric acid, and this also can be estimated by titration (jsee Acidimetry, p. 16). Another method, used by Seemann * and Hehner,^ consists in neutralising the stomach contents by titrating with sodium hydrate, evaporating to dryness, and carefully incinerating. The ash is extracted with water, and the alkali present in the extract is estimated by titrating with an acid ; the difference between the amount of alkali added and the amount of alkali found gives the amount which must have combined with hydrochloric acid, the lactic and volatile acids being 1 A. Griinsberg, Chem. Gentralbl. 1887, p. 1560. 2 Comptes. rend. Ixxx. 01. ^ Deutsch. Arch. klin. Med. xxxix. 329. 4 Zeit. hlin. Med. v. ^ Zeit. anal. Chem. xvii. 236. oastijk; .iriCE 641 burnt during incineration. This nu'tlKxl gives results a little loo high ; the nthtr methods take a long time and a huge quantity of reagents. A method introduced by Sjciqvist ' gives, according to him. ])erfectly accurati- results, and is sutticiontly simple to use clinically, as in Uw. examination of vomit. The contents of the stomach are evaporated to dryness with barium carbonate, and then incinerated; barium chloride is thus formed and remains unchanged, but the barium salts of the organic acids are burnt to barium car- bonate. The barimu chloride is then extracted with water, and the quantitj' of this salt which goes into solution is a measure of the original amount of free hydrochloric acid. Tht- titration is carried out as follows : The solution of barium chloride is placed in a beaker, and a qxuirter of its volume of alcohol added, then a few c.c. of a 10 per cent, solution of sodium carbonate containing 10 per cent, of acetic acid. A standard solution of potassium dichromate (of which 1 c.c. corresponds to 4'Or) milligrammes HCl) is then added from a burette till the end reaction is obtained ; the burette is read, and the quantity of HCl calculated. The indication of the end of the reaction is the yellow colour which the smallest excess of the dichromate gives to the liquid floating over the white precipitate produced by the interaction of the two salts. A moi-e delicate test for excess of dichromate is, however, Wurster's tetramethylparaphenylene-diamine paper. Potassium dichromate in an acetic acid solution acts in the same way as ozone, to test for which the paper was originally useil ; it turns it blue. The ferments of the yastric juice. Fejisin. — The name pepsin was given to the proteolytic ferment of the gastric juice by Schwann.^ Wasmann ^ was the first who attempted to isolate it, E. Briicke ^ the first who succeeded. Briicke's method consists in extracting the mucous membrane of the stomach with a 5 per cent, solution of phos- phoric acid ; to this lime-water is added, and the precipitate of calcium phosphate so formed carries the pepsin down with it. The precipitate is collected on a filter, washed with water, and dissolved in dilute hy- drochloric acid ; to this solution a saturated solution of cholesterin in a mixture of alcohol and ether (4 : 1) is added in small quantities ; the cholesterin is precipitated, and this, again, carries down the pepsin with it. The precipitate is washed first with a weak solution of acetic acid, then with water, and lastly with ether. The ether disst)lves out the cholesterin and leaves the pepsin undissolved. The pepsin, which by this method is obtained only in small quantities, is then diied at a low temperature. Von Wittich's"' method of precipitating the pepsin l)y alcohol from a glycerin extract of stomach gi\es a lai-ger yield. This is freed from salts and peptones by dialysis. The constitution of pepsin is unknown. The elementary analy.ses of it that have been made [C, 53 ; H, 6-7 ; N, 17-8 ; 0, 2l>-5 (Schmidt) : ' Zeit.jjln/siol. Cheni. xiii. 1. - Arcli.f. Aiiat. ii. Physiol. 1h;J(!, p. iK); Fogg. Ann. xxxviii. o5.S. ^ Diss. Berlin, 1839. * Sitzungsher. Witn. Akad. xliii. 6012. ^ PJJuger's Arcliiv, iii. 1!)3. 642 ALI.MKNTATION C, 51 ; H, 7"2 ; N, \~yi (Chapoteaux)] yield numbers appi-oximately the same as proteids ; the obseivatioiis of Langley and Edkins show also that the temperature at which solutions of pepsin lose theii- activity (57° to 58°) is the same as that at which the proteid in solution is coagulated. Probably pepsin, like other enzymes, is either a proteid or a proteid-like svibstance. Pepsin can be heated to 100° in the dry condition without losing its power.' By standing under dilute alcohol or by precipitation with metallic salts, it does not lose its fermentative activity. Strong alcohol in time destroys its power. It does not dialyse through animal membranes nor through parchment paper. Briicke states that it is precipitated from an aqueous solution by lead acetate and platinum chloride, but not by silver niti-ate, tannic acid, acetic acid, and potassium ferrocyanide, nor does it give the xanthoproteic reaction. Pepsin is active only in an acid medium. It has Ijeen surmised that pepsin and the acid form a loose compound.'^ Other acids can take the place of hydrochloric acid, but are less favourable to the action of the ferment ; nitric acid (O'l to O'i per cent.) and lactic acid are next best ; sulphuric, phosphoric, acetic, foimic, &c. follow at a long interval,^ Pepsin is most energetic at a temperature a little above that of the body (40° C). Pick and Murissier,^ and also Hoppe-8eyler,-^ are inclined to believe that the pepsin of cold-blooded animals is some- what different from that of the warm-blooded animals, as it does not work more energetically at 40° C. than at 0° C. Til e fate, of peptiin. — Like other ferments, pepsin is not exhausted by the work it does, but is always available to perform more. Some of it is doubtless absorbed, as it is found in the tissues {see p. 412) and in the urine. The pepsin which passes (jn into the small intestine is rendered inactive by the alkalinity of the juices there, and according to Langley ^ is destroyed by the trypsin (jf the pancreatic juice. The rennftt ferment. — Hammarsten " states that pepsin and rennin are different ferments fijr the following reasons : — (a) Rennin is destroyed by a lower temperature than pepsin. (6) Though both are precipital)le either by magnesium carbonate or lead acetate, the precipitation of pepsin is complete, that of rennin incomplete. By fractional precipitation they can thus be separated. 1 Al. Sclimidt, Centralbl. med. Wiss. lH7fi, Xo. 29. 2 Chandelon, Bull, de I'acad. roijale, 1887, vol. i. p. 289. ' Davidson and Diebrich, Arch. f. Anat. u. Physiol. 1860, p. G8H; Wolffshiigel, Ffliiger's Arch. vii. 188 ; Ebstein and Griitzner, Ihid. viii. 132. * Verhandl. d. Wurzhiirg. ph;/s. med. Gcs. N.F. iv. p. 120. * Ffliiger's Archiv. xiv. 394. •» Journ. Physiol, iii. 252. ' VJrchow-HirKth, Jahretb. 1873, p. 133. (iAsTinr .iCH'K 643 4. THK ACTION OF GASTHIC JUICE The action of gastric juice can be i-eadily demonstrated Ijy a simple ■expei-iment. Four test-tubes are taken wliich we may label A, B, C, und D, A is half filled with water and a few di-ops of a glycerin extract of stomach iuldcd to it, or a few fragments of pepsin dissolved in it : B is half tilled with 0-2 per cent, hydrochloric acid ; C is half filled with a solution of pepsin in 0*2 per cent, hydrochloric acid, or a few (hops of a glycerin extract of stomach may be added to half a test-tube full of 0-2 per cent, hydi'ochloric acid ; D is lialf filled with the same liquid. A small fragment of a solid proteid, such as a piece of lean meat or a shred of fibrin, is placed in A, B, and C ; D is filled up with a 10 per cent, solution of egg-albumin. All four test-tubes are now put in a warm bath at 40° C, and after about ten to twenty minutes they may be taken out and examined. In A, which contains pepsin alone, the fibrin is unaltered ; in B, which contains hydrochloric acid alone, the fibrin is swollen and transparent (with rather stronger acids, acid-albumin would be formed, or even small quantities of albumoses) ; in C, containing both acid and pepsin, the fibrin will be swollen and pai-tly dissolved ; a little later it will be almost entirely dissolved, the products being acid-albumin, albumoses, and peptone. In D there will be no visible change, but on testing for albumin little or none would be found, it also being transformed into acid-albumin, albumoses, and peptone. On prolonged digestion the acid-albumin is converted into peptone ; a somewhat insoluble product called anti-albumid is, however, first formed ; the albumoses are finally converted into peptones too. The following simple tests will show the presence of the products of gastric digestion : — (a) Colour a small quantity of the liquid with litmus, and neutralise with dilute (0-1 per cent.) alkali ; a precipitate of acid-albumin or syntonin is produced, which dissolves in excess of alkali. (b) Add nitric acid ; a precipitate of the albumoses is produced, which dissolves up on heating, and reappears on cooling. (c) Add a dx'op of very dilute copper sulphate solution ; a precipi- tate is produced ; this dissolves up on adding ammonia, forming a violet solution, or on adding potash or soda forming a rose-red solution. This so-called biuret reaction is given by both peptones and albumoses. Ordinary pioteids (albumins and globulins) give a blue solution with copper sulphate and ammonia, and a violet with copper sulphate and potash or soda. This test should always be performed in the cold. (d) Saturate the liquid with ammonium sulphate (after neutralisa- T T 2 6-14 ALIMENTATKJN tion) ; a precipitate is produced ; filter ; tlie pi-ecipitate consists of the albiimoses ; the tiltrate contains the peptone, which gi^•es no precipi- tate on boiling ; no precipitate with nitric acid ; it, however, gives a yellow colour on being heated with nitric acid, which is turned orange by ammonia ; it also gives the biuret reaction. (When ammonium sulphate is present, a large excess of potash is necessary to get the red tint.) The distinctive and useful feature of a peptone is that, unlike other proteids, it is readily diffusible through an animal membrane, and thus proteids can be absorbed. The peptones are undoubtedly the products of the hydration of proteids ; they and their intermediates, the albumoses, can be formed by other hydrating agents, such as dilute mineral acids or superheated steam. It has also been recently stated that dehydi-ating agents will produce ordinary proteids from j^eptone. ' The earliest experiments of this nature were made l)y Schwann ^ and Lehmann.'' Before their time digestion experiments had l.'een performed in various other w-ays ; the ancients supposed that the breaking up of the food w^as effected by the stomach grinding down the food in the same manner as the gizzaz'd of a bird ; others had an idea that heat and moisture in the stomach produced a kind of jjutre- faction. Reaumur in 1752, and Spallanzani in 1783, fed birds on materials enclosed in perforated metallic balls ; after a time these balls wei'e vomited up, or in Spallanzani's experiments M^ithdrawn by a string ; the examination of the semi-digested food showed them that mechanical grinding could not have produced the effect, and also that there was no odour of putrefaction. Then came the discovery that the juice secreted by the stomach was acid ; the first observers were inclined to attribute the solvent power of the juice to its acid, but, as Dr. Beau- mont showed, an acid of the same strength is a less powerful solvent, and therefore the gastric juice must contain a special solvent principle ; this Eberle supposed to be the gastric mucus, a supposition easily refuted. It was Schwann who discovered this special principle and called it pepsin ; he gave the name alhuminotie to the pi-oduct of its action on albumin ; Lehmann's name peptone, howcAer, has since been generally adopted. Lehmann recognised that peptone is not coagulated by heat as albumin is. Meissner ^ described a number of products which he termed parapeptone, dyspeptone, metapeptone, a, h, and c peptone. Schmidt-Mulheim distinguished between parapeptone, pro- peptone, and peptone. Parapeptone is the acid-albumin ; propeptone is a very good name for what we now call the proteoses. Briicke ^ gave the names liydrophyr to a vai'iety of peptone insoluble in alcohol, and ' Bunge's Phijsiol. Chem. p. 201. - Loc. cit. ^ Lchrhuch, 1850. * Zeit. rat. Med. vii., viii., x., xii., and xiv. * Sitzungsher. Wien. Akad. xxxvii. 17'i ; Ixi. Abt'.u 2. GASTKIC JUICE 645 alJcoph;/!- to ono stated to be soluble in alcohol ; tliis apparent solu- bility ai-o^^e from the fact that the alcohol used was not absolute. MoMenfeld,' Kossel,^ Maly,^ and Herth ' made elementary analyses of the peptones they obtfiined. Gnitzner^ inti'oduced a vcn- valuable method of estimating tlie relative diges- tive arti\-ity of artificial gastric juices. A 0-1 per cent, solution of carmine i.s made with glycerin containing a little ammonia ; this is used for staining finely divided fibrin, which is then well washed with water and preserved for use in ether. Equal quantities of the coloured fibrin are placed in equal quantities of the two liquids to be tested ; as the fibrin is digested, carmine enters into solution, and the liquids are compared with one another, and mth a standard solution of carmine after a given time, with regard to the intensity of their tint. Nearly all of our present knowledge of the chemistry of digestion is. however, due to the work of Kiihne "^ and those associated with him in his researches, particularly Chittenden ^ and Xenmeister.'' A most valuable method of isolating peptone was discovered Vjv "\Venz,9 ! Sheep . •. 26 : 1 — Goat . . 26 : 1 — 654 ALIMENTATION CHAPTER XXXII THE SlJCItETIOX OF THE PAXCKEAS The pancreas is a gland very similar in structure to the parotid gland. Its duct enters the duodenum close to the orifice of the bile-duct. Such knowledge as we possess of the chemical composition of the pancreas as a whole has been given on p. 5.58, We are now more particularly interested in its secretion. Tlie older physiologists were quite ignorant of the vast importance of the pancreatic juice. Claude Bernard (1846) considered that it was instrumental in the emulsifying of fats. Bidder and Schmidt ' were the first to make analyses of the juice, and our knowledge of its action and its ferments is due to the investigations of Heidenhain,^ Bernstein,-* Langley,^ Lea,-^ and especially KUhne.*^ The dog has been the animal from which pancreatic juice has been generally obtained, as the principal duct of the pancreas»in this animal enters the intestine quite two centimetres from the orifice of the bile- duct. A cannula is inserted into this duct, brought through the abdominal wound and carefully stitched to it ; in a few days the wound heals (CI. Bernard). The animal suffers fx'om not being able to carry on intestinal digestion pi-operly, and in consequence the pancreatic juice in a day or two becomes very watery compared to that which is secreted at fii-st. The secretion of pancreatic juice begins in the dog immediately after the introduction of food into the stomach, and attains a maximum three hours later (Bernstein). A large amount of food increases both the quantity and the quality of the juice secreted ; the juice, however, secreted at the commencement of digestion is always richer in solid constituents than that secreted later. The secretion of pancreatic juice is stated to "be continuous in herbivorous animals (Heidenhain "). There is at present nothing known concerning secreting nerves of 1 Die Verdaiiungssafte mid der Sfoffirechscl, Mitau and Leipzig, 1852; Ann. Chnn. Pharm. xcii. 33. - Pfiiiger's Archiv, x. 557. ^ Sitzungsh. d. Akad. d. Wiss. (Leipzig, 1869), p. 96. ■* Journ. Phi/sioL iii. 5 Kiihne and Lea, Vcrhandl. d. Heidelberg, naturhist. med. Vereins, N.F. 1, Heft v. p. 445. '' Arch. f. path. Anat. xxxix. 130; Heidelherg. Vcrhmidlungen.'S.Y . 1. Heft iv.and v. ^ Pfluger's Archiv, xiv. 457. THE SECHETION OV THE l'AN(l{EAS f555 the p.ancrpjis. During digestion the puiicrcas, liowever, like the other abdoiiiin.'il organs, is gorged witli blood from dilatation of its vessels. Induction shocks applied to the organ itself or injections of blood or ■chyle stimulate the secretion (Kiihne and Lea). Pajicreatic juice is secreted under consiflerable pressure ; in the rabbit the pressure in the duct amounts to IG to 17 mm. of mercury (Heidenhain). The quantity secreted by t})e dog is about 2~) grammes per kilo- gramme of body- weight in the twenty-four hours (Bidder and Schmidt). Colin obtained from the horse 175, from the cow 200 to 270, from the pig 1 2 to 15 grammes per hour. It has been calculated that a man secretes about 150 grammes of pancreatic juice per' diem. ^Microscopic examination of the gland-cells in different stages of activity reveals a series of changes comparable to those already ■described in the case of the salivary and gastric cells. Granules indicating the presence of a zymogen,' which is called trypsinogen Fig. 85. — Part of au .Vlveolus of tlie Rabbit's Pancreas : A, before (Uscliargf : B, .iftcr. ( From Foster, after Kuliiie and Lea.) (i.e. the precursor of trypsin, the most important ferment of the pancreatic juice), crowd the cells before secretion ; these are discharged during secretion, so that in an animal whose pancreas has been powerfully stimulated to secrete, as by the administration of pilocarpine, the granules are only seen at the free border of the cells (Kiihne and Lea). For the investigation of the action of pancreatic juice, an artificial juice is now usually employed. A pancreas is allowed to stand at the ordinary temperature of the air for twenty-four hours ; or it may be treated with dilute acetic acid immediately ; either method converts the zymogen into the ferment. It is then minced and placed for some days or weeks under glycerin."^ The glycerin dissolves out the ' Refer to p. 4.51. These gi-anules are not so readily (lestroyed by chromic acid as those in the sahvary glands and stomach. - V. Wittich, Pfluger's Archiv, ii. 193. (;5( ALIMENTATION ferments ; these may be precipitated irom the extract by alcohol, then collected, dried at a low temperature, and preserved for future use. An artificial pancreatic juice may be then made by dissolving this in 1 per cent, sodium carbonate solution ; or a little of the glycerin extract may itself be added to the same alkaline solution ; this, how- ever, acts more slowly because of the presence of glycerin. COMrOSITION OF rANCKEATIC JUICE The normal secretion of the pancreas in the dog is a clear, colourless, viscid, almost syrupy fluid. It has a saltish taste and strong, alkaline reaction. The alkalinity is due to phosphates and carbonates, especially of soda. Tlie pancreatic juice of herbivora is more watery than in the dog ; in one case of human pancreatic juice obtained by Herter the fluid was not viscid but limpid. The following analyses (given in parts ])er 1000) ha\e been made : — Dog Uors^e lluiiiau 1 Collected on first From a periniuieiit 1 opeuing the duct fistula (Krijfrer).' Hoppe-S-^eyler '- Herti'r =" (Schmidt) Mean of 3 analyses Water . . . 1100-76 980-44 982-53 976-0 Siolids . . . 98-92 19-r.(i 1747 24-0 1 Organic matter 90-38 12-7:^ 8-88* 18-0 Salts . . . 8-54 6- S3 8-59t 6-0 KOI ... . — 0-93 NaCl . . . 7-36 2-53 NajPO^ . . . 0-4.5 0-02 * Of this 8-6 cou.xisted of fer- Na„0 . . . 0-32 3-30 ments soluble in water, after Ca;(PO,), . . 0-22(CaO) 0-07 preciiDitation by alcohol Mg.,P.,0. . . — 0-01 t Containing much sodium Mgb '. . . 0-05 0-01 phosphate The organic substances present in the pancreatic ferment are : — a. Ferments : These are the most important, both quantitatively and functionally, of all the constituents ; they are four in number : i. Trypsin — a proteolytic ferment. ii. Amylopsin or pancreatic diastase an amylolytic ferment, iii. Steapsin a fat-splitting fei-nient. iv. A milk-curdling ferment, ' Diss. Dorpat, 1851. Quoted from Hoppe-Seyler, Phi/s/ul. Clicm. p. 25!). - Ibid. p. '2r>".t. ^ Quoted fi'om McKeudriek's Plajsiology, ii. 1'25. TlIK SKCHETION <>1' 'I'lIK 1•A^'CREAS 657 h. A small uinoiiut of proteid wliich is coagulable by lieat. c. A mucin or mucin-like substance. ' (1. Traces of leucine, tyrosine, xanthine, and of soaps have been described. The ft'rmenfs of th' jntiwrrntic Jnio'. — i. .T'/'?/;:**-!/^. Bernard ^ and later Corvisart ^ observed that the pancreatic seci-etion dissolved coagu- lated white of egg. Kiihne studied this action carefully and gave the name trypsin to the ferment that produces the action. Kiihne pre- pared the ferment by means of making an aqueous extract of the pancreas at 0° C, and precipitating the proteids and ferments there- from with alcohol. The precipitate was collected, dissolved in water, and acetic acid added till 1 per cent, of the acid was present in the solution ; the precipitate so produced was again extracted with water and filtered ; the filtrate was again treated in the same way, first with alcohol, then with acetic acid ; the filtrate was made alkaline with soda, digested at 40° C, and filtered. The filtrate was evaporated down, and thus tyrosine crystallised out ; the rest of the tyrosine, leucine, and peptone was dialysed off". Though this method gives a purer ferment than those previously adopted, yet, as Hoppe-Seyler states, the pre- l)aration cannot be regarded as absolutely pure. The substance ob- tained is soluble in water and in glycerin, but not in alcohol ; when a solution in water is acidified faintly and heated, a heat-coagulum is formed. Kiihne ^ has more recently introduced the following method of preparing trypsin. The fresh or dried gland is first digested with 0-1 per cent, solution of salicylic acid for four hours, then with alkaline .solution of thymol for twelve hours ; the acid and alkaline extracts are mixed and the amount of thymol brought up to 0-5 per cent. ; the amount of soda is brought up to the same percentage and the mixture is digested for six days, then cooled, and the tyrosine crystals which have formed are filtered off". It is then neutralised with acetic acid and saturated with ammonium sulphate ; this precipitates the trypsin ; the precipitate is collected, washed with saturated solution of ammo- nium sulphate, and dissolved in 0*2 per cent, soda solution ; this gives a powerful digestive fluid. If one desires to get I'id of ammonium sulphate, this is done by dialysis. A conclusion which appears to be justified from these methods of 1 In two specimens of dogs' iJancreatic juice I have examinecl, acetic acid gave a stringy precipitate. The viscidity of the juice is evidently due to this substance, though whether it is true mucin or a nucleo-albumin I did not investigate. - Lerons, Paris, 1855, p. 334. ^ Snr une fonction 2)ea connne da pancreas, Paris, 1858. •• Ceidralhl. iiicd. Wiss. 1880, No. 3 U 17 658 ALIMENTATJoX preparation is that trypsin is either a proteid ov a substance closely allied, or adherent, to a proteid. Trypsin acts best in an alkaline medium ; it also acts in a neuti'al medium. Stutzer ' obtained equally good results with artificial diges- tion when the alkaline fluid used was 0-'2o, O'li, or 1"0 per cent, of sodium carbonate. Trypsin will not act at all i)i an acid medium, and is destroyed by hydrochloric acid or by the acids it meets farther on in the large intestine (Langley). None passes into the urine, so probably it is entirely destroyed in the intestine. Salicylic acid, howevei', does not hinder the action of the ferment, so that this antiseptic can be added to artificial digestion experiments to prevent the putrefaction so generally associated with tryptic digestion ^ (Kiihne). Trypsin occurs in the pancreatic secretion of new-born children, except in certain cases, and in these cases diarrhoea, generally fatal, is apt to occur. ii. Amylopsin. — The diastatic action of pancreatic juice was first described by Valentin ^ ; the ferment was separated in a more or less pure condition by Kroger,* who found it could be precijjitated but not destroyed by lead acetate, though by more powerful reagents, like mineral acids, acetic acid, and alkalis, it is destroyed. Other attempts to obtain a pure product by means of extracting the gland with lime- water were made by Danilewsky,"' and later by Cohnheim.*^ It diffuses more readily than the other ferments of the juice (v. Wittich). It is a))sent in the pancreatic juice of new-born children (Xorowin,^ Zweifel *). Hence much starchy food is bad for very young children. Amylopsin, like most ferments, acts best at 40° C. Like ptyalin, it converts starch into maltose. It acts better in the presence of bile than by itself.^ Stutzer "^ found that pancreatic fluid acts better on carbohydrates when it is neutral than when alkaline ; yet after the food has been subjected previously to the action of ptyalin and then of pepsin, the best results with pancreatic fluid are obtained when it is feebly alkaline. iii. Steapsin. — The existence of a fat-splitting ferment in the pan- creatic juice is inferred from the action of the pancreatic juice on fats. 1 Zeit. ■phyiiioL Chein. xi. 207. - Other antiseptics often used are ether (Hoppe-Seyler, Pliijuiol. Client, p. 204) and arsenious acid in small quantities (Scluifer and Bcihii, Wiirxbiiry. Vcrliandl. 1872, vol. iii. p. 2538). I have found the latter e.xceedingly useful. s Lehrb. d. Physiol. 2nd edit. 1844. " Loc. cii. s Virchoiu's Archiv, xxv. 279. ^ Ibid, xxviii. 2.51. 7 Centralbl. vied. Wiss. 1873, No. 20. 8 ioc. cii. . '■• Martin and Williams, Proc. Boy. Soc. xlv. 358. More recently (Ibid, xlviii. ICO) these observers have shown that the bile is also fa\ourable to the action of trypsin on proteids. '" Zcit. jiJiysiol. C It c m. xii. T2. TiiK si';(i;i':ii<»N i»i' iiik 1'.\N('i;f..\s 659 Tlu' ferment has never l)ecn separated ; it is destroyed by treating the ghind with alcohol ; it does not dissolve in glycerin as do the other two ferments Ave have described. This ferment probably exists in the secretion of the pancreas of the embryo, for free fatty acids are found in the meconium. iv. Jfilk-ciird/hi;/ fenni'iit. — The addition of pancreatic juice to milk causes clotting, but this action is seldom called into ])lay normally, as the milk upon which t]u> juice has to act has been alicady curdled bv the rennin of the stomach. THE ACTIOK OF PANCREATIC JUICE OX FOODS A few simple experiments can be readily performed with artificial pancreatic juice, which will teach us the chief facts in connection with the action of that juice. Three test-tubes are labelled A, B, and C ; in each is put a few cubic centimetres of the artificial juice ; the fluid in A is heated to 60" and subsequently cooled and a piece of fibrin placed in it ; a piece of fibrin is also placed in B, and a few c.c. of starch solution in C. All are then put in a warm bath at 40° C. The fibrin in A remains unaltered. This experiment illustrates the general truth that ferments are destroyed by high temperatures. The filirin in B undergoes fairly rapid solution ; it is, however, not first swollen and then dissolved, as is the case with gastric juice, but is gradually eroded or eaten away from the edges. The products of (ligestion are mvich the same as in gastric digestion : instead of synto- nin, an albuminate of the nature of alkali-albumin is formed. Albu- moses and peptones (sometimes called try})tones) are present ; the albumoses are more i-apidly converted into peptones than in gastric digestion, and after a time some of the peptones are further decomposed, yielding leucine, tyrosine, and similar substances. The starch in C is rapidly changed into maltose with dextrin as an intermediate product, exactly as in salivary digestion. Another experiment which illustrates some important facts in connection with pancreatic digestion is the following : An ox pancreas, about twenty-four hours after its removal from the animal, is minced finely ; the mincemeat is divided into two parts ; each part is placed in a good-sized flask and a litre of 1 per cent, sodium carbonate added ; the white of an' egg is also added and the mixtures placed in the warm bath at 40° C. for twenty-four hours, another white of an egg being added about the middle of this period. In one flask, however, the process is rendered antiseptic either by the addition of thymol or a little 1 per u u 2 660 ALIMENTATION cent, salicylic acid. The necks of the flasks are plugged with cotton- wool. After twenty-four hours tlie fluid fi-oni each is filtered ott" from the undigested residue. The one to which the antiseptic was added is free or nearly free from odour ; the other has an offensive fiscal odour. This second flask more correctly imitates what occurs in the intestines ; an alkaline medium is the most favourable for the growth of bacteria, and bacteria thus flourish, producing indole, skatole, phenol, &c. in the intestines as well as in our flask. Various bases, amines, and ammonia are produced ; leucine, tyrosine, and other acids — in fact, all the pro- ducts usually derived from proteids by putrefaction. We thus have two processes occurring simultaneously in the intestine, and experi- ments in which putrefaction is prevented have, therefore, to Ije per- formed in order to ascertain whether the production of any particular substance is due to the pancreatic ferment, or to the accompanying putrefactive bacteria. Leucine and tyrosine will be found in the fl.uid in the antiseptic flask, thus showing that these products are produced by the trypsin alone ; they are, however, much more abundant in the flask in which putrefaction was allowed to take place. Indole and skatole are produced by putrefaction only. The presence of leucine and tyrosine may be demonstrated in the following ways : — a. Take some of the fl.uid, add Millon's reagent, and filter off the white precipitate of mercury compounds of proteids that are formed ; the filtrate is turned red on boiling ; this is due to the presence of tyrosine. If the tyrosine is abundant, the filtrate is pink even before boiling. b. Faintly acidify and boil another portion of the fluid, filter ott' any proteid that may be coagulated, and preserve half the filtrate for the next experiment. Evaporate the other half to a small bulk on a water- bath ; mount a drop of this on a glass slide and cover. Crystals of tyrosine will be seen, and, if the evaporation has been carried on long enough, crystals of leucine also (see figs. 32 and 33, p. 83). Tyrosine is less soluble than leucine in water ; hence it crystallises out first. c. Take the other half of the filtrate and add excess of absolute alcohol to precipitate the peptone ; filter and evapoi'ate the filtrate to a small bulk ; it becomes yellowish and sticky from the presence of eucine. Microscopic examination shows abundance of leucine balls. Tyrosine, not being soluble in alcohol, is absent. Leucine gives a well- marked xanthoproteic reaction. d. Other methods of separating out leucine and tyrosine are given on pp. 82, 83. TiiF. sK('i;i;ii(iN oi- I'lii': panckkas OfJl Action OH prot>'lf/s (Old (ilhii iiilitoids. — Trypsin acts like pef)sin, l>ut with i(M-tiiin (litfereiues. The most striking of these differences are — (I. Trypsin acts in an alkaline, pepsin in an acid medium. b. Trypsin acts more ra})idly than pepsin, l)ut the same series of proteoses can be detected as intermediate products in the formation of ])cptone. '•. An all)uminate of the nature of alkali allnimin is formed in tryptic, of the nature of acid-albumin in peptic digestion. d. Trypsin acts more powerfully than pepsin on certain albu- minoids difficult of digestion, such as elastin, and waxy or albuminoid substance. It digests nuclein, which is not attacked at all by gastric juice. Keratin and chitin are, however, indigestible by both ferments. ''. Trypsin acts further than pepsin, decomposing the hemi-jjeptone into simpler products, of which the most important are leucine and tyi'osine, asparaginic acid,^ ammonia,^ and proteinchromogen. I'roicincJtromogoi is a substance, uriginally described by Gmelin, which gives with chlorine or bromine a reddish-violet product, protein chrome. These names are suggested by Stadehnann,^ who has recently examined these substances. Nencki * considered that proteinchromogen is naphthylamine ; a view which Krukenberg^ and Hemala'' showed to be untenable. Neumeister " has suggested tlie name tryptoplian for it. Stadelmann was unable to separate protein- cln-omogen in a state of purity, but its bromine compound (xaroteinchrome) was separated by dissolving it in 90 per cent, alcohol. When dissolved in ether it shows a well-marked absorption-band in the green. Elementary analysis gave results from which the percentage composition of proteinchromogen was calcu- lated (C, 61-02; H, ()-89; N, 13-68; S, -l-es ; O, 13-71). Its reaction and com- position pomt to its being- a prcteid, or a substance closely allied to a proteid. Action on carbohydrates. — The conversion of starch into maltose is the most powerful and the most rapid of all the actions of the pan- creatic juice. Kroger observed that I gramme of dog's pancreatic juice which contained 0-014 gramme of organic material changed 4-C72 grammes of starch into sugar in half an hour at 35° C. The details of the process are exactly the same as already described for ptyalin. Not only boiled but also raw starch is affected. Glycogen is changed in the same way, more slowly than starch, but tiuickly in comparison to the action of saliva. "^ Cane sugar is not affected by eitlier juice. 1 Radziejewski and Salkowski, Bcr. (h-Kf-srli. CJieiii. Ges. IST."., vol. vii. p. 1050; V. Knierem, Zeit. Biol. xi. 19S. - Hirschler, Zeit. phyaiol. Chem. x. 300; Stadehiuuai, Zeit. Biol. xxiv. 201. •' Zeit. Biol. xxvi. 491. -* Ber. deidsch. cliciii. Ges. vii. 1593. •' Vertiandl. 2>ttysik. iiied. Ges. Wiirzburji-, xviii. •^ Chem. Unters. wiss. Med. von C. F. \V. Kralcei'hei-g, Heft ii. •? Zeit. Biol. xxvi. 324. 8 Seegen, Centralhl. iiied. Wiss. 1870, No. IS. 662 ALI3IENTATI0X Action on fats. — The action of the pancreatic juice on fats is a double one : it forms an emulsion, and it decomposes a small quantity of the fats into fatty acids and glycerin. The fatty acids unite with alkaline bases to form soaps (saponification). The chemistry of this process has been already described (p. 492). Ftn- the action of pan- creatic juice on lecithin, sei p. ."331. The chemical action of the pancreatic juice on fats cannot be demonstrated by an artificial juice made from a glycerin extract, as the steapsin is not soluble in glycerin. Either pancreatic juice from a fistula or a watery extract of pancreas must l)e employed. If the watery extract has j)artially undergone putrefaction — which it does very readily — its activity on fats is increased. The fat- splitting action of the pancreatic juice and of the organised ferments (bacteria) of the alimentary tract is identical. If a little butter of neutral reaction be mixed with some pancreatic juice, and the mixture be put in the warm bath at 40° C. for twenty minute.s, and litmus then be added, it will be turned ]-ed by the fatty acids which are liberated. The formation of an emulsion may be studied in this way : Shake up olive oil and water together, and then allow the mixture to stand ; the finely divided oil-globules will soon sepai'ate fi-om and float on tlie surface of the water ; but if a colloid matter, like albumin or gum, be first mixed with the water, the oil separates more slowly. A more permanent emulsion is formed by an alkaline fluid, and especially when a small amount of fatty acid is being continually liberated ; the free acid combines with the alkali to form a soap which forms a thin layer outside each oil-globule. It will be seen that pancreatic fluid possesses all the necessary qualifications for forming an emulsion : — 1. It is alkaline. 2. It is viscous from the presence of proteids. 3. It has the power of liberating free acids, and thus forming a layer of soap on each oil-globule. 0. Minkowski ' has recently found tliat, after extirpation of the pancreas, fats, except those in milk, are not absorbed, although splitting of the fats under the influence of bacterial agency still continues. Acidulation destroys ordinary emulsions made with an alkaline carbonate, the fat running into large drops ; but with the emulsions formed in the body (milk, chyle, pancreatic emulsion) this is not the case. Kiihne has suggested that in pancreatic emulsions, as in milk, each globule has an albuminous envelope which facilitates the adhesion of the fat to the absorbing cells of the intestinal wall. "Whether this be so or not, it is beyond question that some peculiarity of these natural emulsions* exists. 1 Berlin. Min. Wuchoisch. 1890, No. IS. THE SECRETION OK THE TANCRKAS 663 which rendors tlioni easy of absoi'iition. .Minkowski's observations aro also in- teresting as showing that fat is not absorbed to any great extent in the form of soaps. This is also borne out by K. L. IJass' ' experiments with certain ethereal salts which resemble fats in their constitution. Pathological Conditions <>f the Pancreatic Secretion Very little is known on this subject. In the examination of the hiliou.s vomit of a ease of typhoid, Hoppe-Seyler - found the pancreatic ferment to be present. In a case of intestinal obstruction examined by the same observer, the pancreas was almost entirely replaced by a jelly- like substance having the following percentage composition : Water, 97*4 ; solids, 2*6 ; urea, 0"12 ; fat, 0-02 ; extractives, 1"40 ; salts, 0-57 ; substances insoluble in water, 0'49. The alcoholic extract contained much leucine. Concretions are sometimes found in the ducts of the gland ; these consist chiefly of calcium phosphate and carbonate. To judge from experiments on dogs, impaired nutrition leads to a lessening of the solid constituents of the secretion. There can be but little doubt that amemic, febrile, and debilitating conditions generally in man impair the richness and usefulness of the pancreatic juice. Complete extirpation of the pancreas in dogs gives rise to glycosuria, -^ and Hirschfeld has shown that in diabetic patients the absorption of proteid and fat is much impaired. This is, perhaps, an indication that the cause of diabetes is in some cases disease of the pancreas. These observations are of considerable value ; for, although their precise meaning has yet to be worked out, they appear to indicate that the cells of the pancreas do something else than manufacture pancreatic juice ; they appear to play, in addition, an important part in the processes of general metabolism. Concomitant injuries to the nerves of the liver during the operation, as a cause of the diabetes, can be excluded, as partial extirpation of the pancreas, where there is the same risk of injuring nerves, produces no glycosuria. ' Zeit. physiol. Cheiii. xiv. 41(i. - Physiol. Chem. p. '269. •^ Minkowski and v. Mering, Communication to Internat. Fhijsiol. Congress, Basel, 1889. See also Lepine, Lijon medical, Jan. 19 189 664 ALI3IENTAT1()N CHAPTER XXXIII SrCC us ENTER I C US The snccus entericus, or intestinal juice, is the secretion of the tubular glands (crypts of Lieberkiihn) which exist in the mucous membrane of the intestine. It need hardly be pointed out that it is a matter of great difficulty to obtain this secretion unmixed -with other secretions, and hence there are great discrepancies in the observations that have been made on its action. Another source of fallacy is to be found in the fact that most observers have taken little or no care to exclude the presence of putrefactive bactei'ia. The method usually adopted of obtaining the juice is by means of a fistula, but even this is not altogether free from error, as the natural secretion is then apt to be masked by transudation from the capillaries. Thiry's ^ method of making a fistula is to cut the intestine across in two places : the loop so cut out is still supplied with blood and nerves, as its mesentei'v is intact ; this loop of intestine is emptied, one end is sewed up by sutures, ($'\\ tlie other stitched to the abdo- mm. of mercury, which i.s about double that in the portal vein. Altlu)Ugh pressure in the portal capillaries cannot account for the secretion, the rapidity of the flow of blood through these capillaries has an important influence, the activity of the hepatic cells depending on the amount of lilood they receive in a unit of time. The secretion of l)ile is continuous, l)ut it is accelerated under certain conditions ; for instance, after the ingestion of food ; and this is probably by means of a reflex mechanism. Whether it is carried out solely by means of the vaso-motor nervous .system, or by means of special secretory or trophic ner^•es, or whether both factors are called into play, we cannot at present say. All we are acquainted with is the influence of blood-supply on the seci-etion ; no special trophic or secretory nerves ha\e as yet been demonstrated to exist. Pfliiger has long held that nerve-fllaments terminate in hepatic cells, and thouuh Maceallum - states that he also has traced minute nerve- 1 Hermann's Handhiich, 1880. - Quarterly Journ. Micros. Sc. new series, xxvii. 45-2. KILE OTl twigs into llu' interior of the cells, liistolo^ists, ;is ;i rule, do not ;iercentage in the first two cases is about equal : it is only explicaljle partially by the fact that fistula-bile does not stay in the gall-bladder. The low percentage of solids is, as the next table shows, almost entirely due to want of bile-salts ; this can be accounted for in the way first suggested by Schifi" : that there is normaUy a bile circtdation going on in the body, a large quantity of the bile-salts that pass into the intestine being reabsorbed and again secreted. Such a circulation would obviously be impossible in cases where all the bile is discharged to the extenor and so lost. The following table gives analyses of human bile, in the 2nd and 3rd columns of fistula-bile, in the 4th column presumably of normal bile. Fistula bile (healthy Fistula bile (case of woman. Copeman . cancer. Yeo and and Winston) I Herroun) Normal bile (Frerichs) Sodium glycocholate Sodium taurocholate Cholesterin, lecithin, fat Mucus . Pigment Inorganic salts Total solids . AVater (by difference) :■} 0-6280 0099O 0-1725 00725 0-4510 1-4230 98-5570 100-0000 0-165 0055 0-038 [ 0-148 0-878 (including ex- tractives) 1-284 98-716 100-OCMi 9-14 1-18 2-98 0-78 1408 85-92 100-00. ^ Pfliiger's ArcJiiv, ix. 492. - Lehrbuch d.physioh Chem., by Gomp-Besanez, 3 Auf. p. 5-29. BILE f)77 This table illustrute.s the fact that of the two Inle-salts tlie glyco- -cliolate is the more abundant. Many other oljservers who have published analyses of human bile note the same fact. The pioportion of the two bile-salts is thus given in percentages : — Sodium fjlvcocholate Sodium taurocholate S9 ~ AVe see, as before, that the bile from the gall-bladder is more con- 'Centrated tlian that which is freshly secreted, and that this is chiefly shown in the percentage of bile-salts, which in the dog consist almost exclusively of taurocholate of soda. The small percentage of sodium •chloride is due to the fact that the greater part of that salt was dis- solved by the alcohol and not estimated. The amount of taurocholate present may be easily estimated from the amount of sulphur in the dry residue of tlie alcoholic extract, taurocholic acid being the only :substance there that contains sulphur- (see further p. 681). ' J'jilif/cr'.-i Archie, xii. 54. - PhijsJol. Chem. p. 301. 678 AL13IENTATI0X Percentage composition of the bile of various animals : Constituents Ox" Mucin and pigment . 0'30 Bile-salts ~\ Cholesterin, lecithin, ■ I 8-00 and fat . . . , . , J Inorganic salts ... 1"26 Total solids .... 9-56 Water 90-44 Pig^ 1 Kangaroo^! Goose Python ; 1 I' 11^ 0-o9 4-34 2-56 31 0-89 8-38 I 2-23 7-59 14-96 16-4 8-46 109 0-36 0-3 0-03 2-10 2-6 0-20 11-20 14-13 19-9S 22-4 9-58 88-80 85-87 80-02 77-6 90-42 Tlie dry residue of the alcoholic extract contains the following percen- tages of sulphur (Benscli," Strecker) : — Dog . Fox . Wolf . Bear . Ox . Calf . Sheep 6-21 Goat . 5-96 Pig . 5-03 Hen . 5-84 Pike . 3-58 Cod , 4-88 Perch 5-71 Plaice 5-20 0-33 4-96 5-77 5-66 5-99 5-91 The amount of iron in the bile is important. The iron is present as a phosj^hate, and there can be no doubt that it is derived from haemoglobin. The bile-pigment is formed from haemoglobin, but is free- f rom iron. Some of this iron is stored in the liver-cells, some discharged as phosphate in the bile. The percentage of iron in the bile is thus given by various observers : — Observer Human bile Dog's bile Ox-bile Young ^ Hoppe-Seyler" Kinckel'"" . 0004 to 0-010 0-0062 0-016 0 0063 to 0-0078 0-0058 0-003 to 0-006 The amount of iron discharged in the excretions (bile and urine) is small compared with the amount of htemoglobin destroyed to form biliary and urinary pigments. The remaining iron is stored in the liver-cells as a compound with nuclein and proteids. The compound so formed may occur in the form of pigment-granules in the cells, or as a diffuse, colourless, soluble substance. According to Delepine these iron compuunds are once more elaborated into new 1 Berzelius, Lehrbuch, Dresden, 1831. - Gundlaeh and Strecker, Ann. Chem. Pharin. Ixii. '205. 5 Schlossberger, Ibid. ex. 244. * Marsson, Arch. il. Pharin. Iviii. 138.. s Otto, A)in. Chem. Pharm. clix. 18i). ^ Vogtenberger and Schlossberger, Ibid, cviii. GO. ' Ihid. Ixv. 215. ** Journ. Anat. and Phijsioh (2j, vii. 158. ^ Loc. cit. ^"^ Pfli'iger's Archiv, xiv. 353. r.ii.K 679 hsemoglobiii lor \hv yoiiai!: red coi'iJiiscles. This lie (U'scrilu's ms the ferrogcnio function of tiie liver (wo p. 552). The gases of the bile have been examined by Plliiger,' Boguljobow,- and Noel." Oxygen is absent or present in the merest traces; the most important gas is carbonic acid; it, however, diminishes daring the stay of bile in the gall- bladder. The carbonic acid is present in two conditions, one part being free and removable merely by placing the bile in a vaciuim ; the other part is more lirnily combined, and recjuires the addition of some other acid, such as phosphoric acid, to drive it otf. The numbei's given vary very much, the free carbonic acid from 5 to 17 vols, per cent., the combined carbonic acid from 0'(> to 62 per cent. Small quantities of nitrogen are found in addition (^sec also p. ;]92). Bile -mucin Landwelu- ^ was the first to point out that the slimy substance in bile is not a compound of a proteid with a carbohydrate radicle as are the true mucins. He considered it to be a mixture of serum-globulin with the bile-salts. An examination of his analytical results shows that there is some difficulty in accepting this view ; and, although a mixture of sodium glycocholate with serum -globulin has the physical characters of bile-mucin, a mixture of globulin with bile deprived of its so-called mucin does not produce the characteristic viscidity of normal bile. This mucinoid sub.stance can be precipitated from bile by means of acetic acid or by excess of alcohol. It is, unlike true mucin, slightly soluble in excess of acetic acid. PaijkuU ' has under Hammarsten's superintendence prepared the substance by precipitation with alcohol. He found, like Landwehr, that this substance is not true mucin, though it may contain small quantities of true mucin apparently derived from the walls of the gall-bladder. On gastric digestion it yields an in- soluble residue of nuclein. The so-called mucin of bile is therefore chiefly a nucleo-albumin. Whether it is derived wholly from the walls of the ducts and gall-bladder, or is partly formed by the liver-cells, merits a fresh investigation. The Bile-salts The Inle contains the sodium salts of complex amido-acids called the bile-acids. The two acids most frequently found are glycocholic and taurocholic acids. Glycocholic acid {C^r^i-^'^Of^) is especially abundant in the bile of 1 Pfli'tger's Archiv, ii. 173. - Centralbl. vied. Wins. ISCiO, Xo. 42. ^ Etude ghieralc sur les variatiatis des gaz dtt sane/. Thc'se. Paris, 1870. 4 Xrit. plijisioJ. Chciii. viii. 114. 5 Jbid. xii. !!)(!. 4580 ALLMENTATloN herbivora and in man ; its amount is increased by a vegetable diet. By the action of dilute acids and alkalis, and also in the intestine, it takes up water and splits into glycocine or amido-acetic acid and cholalic acid. C^eH.gNO, + H,0=C,H5N0., + C24H,o0.5 [glycocliolie acid] [glycocine] [cliolalic acid] The glycocholate of soda has the formula Co,jH4 3XaN06. Tcnirocholic acid (C2GH45XO-8) is especially abundant in the bile of ■camivora. By the action of hydrolysing agents and in the intestine it splits into taurine and cholalic acid. C26H45NO7S + H,.0=C ^H^NOgN + Cv^H^oOs [taurocliolic acid] [taurine] [cholalic acid] The taurocholate of soda has the formula C26H44NaX07S. Cholalic acid (C25H40O5) is derived from the decomposition of the bile-acids. Its constitution is at present unknown. Choleic acid {C.25H42O4) has been sepai-ated in small quantities from ox-bile ; and fellic acid (C23H40O4) from human bile. It is admixture with fellic acid that renders the cholalic acid of human bile apparently different from that obtained fi'om other sources. Hyo-cholalic acid (C25H40O.5) re- places cholalic acid in liyo-gl ycocliol ic a.\\(\. hyo-tauroclwlic acid, the acids of pig's bile. In the bile of the goose, cholalic acid is replaced by cheno-cholalic acid (C27H44O4). Further particulars concerning the bile- /icids will be found on pp. 86 to 88. The bile-acids may be prepared from bile by the following methods : — Evaporate ox-bile to a thick syrup, stirring it frequently with a glass rod ; digest this with cold absolute alcohol ; this leaves the pigment, mucin, and i^art of the mineral salts undissolved ; boil the extract with animal charcoal to completely decolourise it, and filter. Another method consists in rubbing up bile with animal charcoal into a paste ; this is dried on the water-bath, and extracted with absolute alcohol, and the extract filtered. The extract having been made, the alcohol is distilled off, the residue dissolved in a little absolute alcohol, and ether added till it becomes turbid. In a few hours or days a whitish semi-crj'stalline mass is deposited. This is Plattner's crystal- lised bile, and consists of a mixture of glycocholate and taurocholate of soda. This is dissolved in water, a little ether is added, and then dilute sulphuric acid ; stir well, and glycocholic acid ciwstallises out in shining needles, the taurocholic acid remaining in solution ; the crystals may be collected on a filter, redissolved in dilute spirit, and precipitated ^\-ith excess of ether. Another method is as follows : — Dissolve Plattner's crystals in water ; add neutral lead acetate, and lead glycocholate is precipitated ; collect this on a filter, wash, dissolve in hot alcohol, and remove the lead by a stream of sulphuretted hydrogen ; filter off the lead sulphide ; add water carefully to the filtrate, and •cr^'stals of glycocholic acid will be precipitated. To the previous filtrate from the glycocholate of lead add Ijasic acetate of lead and ammonin : taurocholnte of i;iLE 681 lead is iiroripitattMl, froui which tanrocholic. acid ni:iy lu- prepared, as glycncliolic :icid is from tlio glycucholato of lead. ITiifncr's method, as modified by Marshall,' for obtaining glycoc^holic ;icid is the following : A little hydrochloric acid is added to fresh bile, the mixture shaken, and the mucinoid material so ])recii)itated filtered off. Ethyl ether and hydrochloric acid are added to the filtrate; the i)roportion of liltrate : acid : ether =100 : 5 : 30. The uaxture is shaken and allowed to remain some hours, when crystals form, which are then collected on a, filter, washed with water holding hydrochloric acid and ether in solution, and dried in the air. By recry.stallisation they are obtained perfectly colourless. In the preparation of tanrocholic acid one would preferably use dog's bile. To determine its amount quantitatively take the dried alcoholic extract of a known quantity of bile ; evaporate it to dryness on the water-bath with fuming nitric acid ; the sulphur is thus converted into sulphuric acid ; digest the residue with water, and determine the sulphuric acid by titration with alkali (p. 16) : 98 parts H.^SO, = 32 sulphur; and 1 part sulphur = lfi-8 taurocholic acid. To prepare cholalic acid boil bile with caustic potash for twelve to twenty-four hours; then precipitate with hydrochloric acid; wash the precipitate with water, and dissolve it in caustic soda containingalittle ether; render this acid with hydrochloric acid, and crystals form after a time ; decant, cover the residue with ether; drain ofE the ether in half an hour, and dissolve the deposit in boiling alcohol ; to this solution add a little water till a permanent precipitate appears ; tetrahedric crystals of cholalic acid soon form. To prepare glycocine, glycocholic acid is boiled for a long time with strong hydrochloric acid ; the firm resin (bile-resin) that is formed consists of cholalic acid and dyslysin ; this is filtered off, and the filtrate yields on evaporation hydro- chloride of glycocine (C2H5N0._,.UC1). This is dissolved in water, treated with lead hydrate, filtered, and the soluble lead compound of glycocine in the filtrate decomposed by a stream of sulphuretted hydrogen. The lead sulphide is filtered off, and the filtrate on concentration yields crystals of glycocine. which may be purified by recrystallisation. Taurine is best obtained from dog's bile ; this is concentrated and then boiled several hours with hydrochloric acid ; the bile-resin is filtered off. and with it some sodium chloride which has crystallised out. Evaporate the filtrate to dry- ness, and digest the residue with alcohol, to remove the glycocine hydrochloride if any is present. The residue insoluble in alcohol is extracted with boiling water, and the extract left to crystallise ; more sodium chloride separates, the taurine remaining in solution. Decant off the liquid, and add to it four or five times its volume of boiling alcohol ; this dissolves the taurine, which separates in prismatic crystals as the liquid cools. To pur-ify it, taurine may be redissolved in water, and recrystallised by the addition of alcohol. For the methods of obtaining the rarer forms of bile-aciils the original memoirs {see footnotes, p. 88) must be consulted. Pettenkofer^s test. — The following reaction is given by bile, and by the bile-acids, and is apparently due to the presence of cholalic acid. Spread a drop of bile in a thin film on a porcelain capsule, and mix with it a drop of strong solution of cane sugar and a drop of strong sulphuric acid, and if necessary warm. A deep purplish-red colour appears. ' Zclt. pliysiol. Chein. \\. '1'6'd. 682 ALIMENTATION This should be called t\\e furfur-aldeli i/dt reaction, as it is this substance- formed from the sugar and acid which gives the colour with cholalic acid. It is unfortunately not distinctive of the bile-acids, Vjeing also given by numerous other organic substances. None, however, except o-naphthol give it so readily as the bile-acids ; and the spectroscopic appearances are different in many instances ; the colour produced by bile shows one band between D and E and another at F. A third fainter band near the D line, which fades as the reaction becomes fully marked, is desciibed by MaelNIunn {sff fig. S8, s})eL-trum 6). The Bile-pigments The two principal pigments of the bile are named bilirubin- (formerly known as cholepyrrhin, biliphsein, or bilifulvin) and biliverdin. Bile which contains chiefly bilirubin (such as dog's bile) is of a golden or orange yellow colour, while the bile of many herbivora, which con- tains chiefly biliverdin, is either green or bluish green. Human bile is generally described as containing chiefly bilirubin, but in Copeman and "Winston's case, Vjiliverdin was present in excess. These pigments are undoubtedly formed from haemoglobin : injec- tion of h;eiBOglobin into the portal vein increases the bile-pigments. Bilirubin is identical with the substance called h;ematoidin, crystals, of which form in old extravasations of blood (see p. 293). The above bile-pigments are free from iron, and show no absorption-bands when examined spectroscopically : Imt there is a strong absorption of the violet end of the spectrum in the case of bilirubin, while in that of biliverdin some of the red is absorbed. Bilirnhin has the formula CigHi^N.^Os (Stadeler,' Maly ^j, CgHtjXO., (Thudichum ^) ; the first is the one usually accepted. It has been pre2:)ai"ed by ilissolving it out from the bile by means of chloroform after acidulating,^ and from the gall-stones of men and oxen by Stadeler and others. It sometimes occurs in a crystalline- form in the gall-bladder. It is insoluble in water, slightly soluble in alcohol and ether, readily soluble in chloroform, benzene, acids, and alkalis. Hoppe-Seyler '" recommends the following method of preparing it. Bile is diluted with water and precipitated by milk of lime ; this- carries down the pigment ; a stream of cai-ljonic acid is passed through the mixture till no more precipitate forms ; the precipitate is collected, suspended in water, treated with hydrochloric acid, and extracted with chloroform. From this extract the pigment is precipitated by alcohoL 1 Vierteljahrschr. (1. Ziirich. naturforscli. Ges. viii. 1. - Sitzititgsher. Wieit. Akad. Ivii. and Ixx. '• Joiirn. prakt. Client, civ. 193- 4 Valeutiiier, Ganshurfj. Zcitsch. 185S. . '" Fhijiitvl. Chem. p. 294. lUi.K 68^- "When bilirubin is treated \vitli oxidising agents a series of coloured products ai'e successively formed. This constitutes Gmelin's test for the bih'-pignients. If a drop of bile be spread in a tliin film on a porcelain plate, and a drop of nitric acid containing Jiitrous acid in solution be phiced in tlie centre of it, tlie drop of acid l>ecome.s surrounded by rings of colours, gieen, blue, violet, red, and yellow. The green colour is the first stage, the yellow the last stage in oxidation. The green pigment is l)iliver(lin ; the blue or violet product is called l)ilicyanin ^ ; its composition is unknown ; it shows certain absorption- bands. The red pioduct has also not been further investigated. The end or yellow product was called choletelin by Maly,^ whose formula for it is Ci^HigN.^O,,. It is soluble in water, alcohol, acids, and alkalis, and is amorphous. MacMunn •* describes the spectroscopic- changes that occur as follows : As the blue coloui- appears a broad shading composed of two bands appears at D, then a black band close to F. The two bands first mentioned are separated by a narrow interval in which the D line is seen (fig. 88, .spectrum 1). As the colour changes progress the band after D fades away, then that before D ;. and when the yellow stage is reached one band, that at F, is alone visible (fig. 88, spectrum 2). BUlverdin has the formula CgHgNO.^ (Thudichum). It may occur as such in the bile ; it may be formed by simply exposing red bile to the oxidising action of the atmosphere ; or it may be formed, as in Gmelin's test, by the more vigorous oxidation produced by fuming nitric acid. It gives the remaining colours of Gmelin's test quite well. Maly obtained biliverdin by the action of acetic acid or mono- chloracetic acid on bilirulnn. It differs from bilirubin considerably in its solubilities, being soluble in alcohol, insoluble in chlorofoi-m and in water, almost insoluble in ether. It can be pi-ecipitated fi'oni bile by means of hydrochloric acid. It has never been obtained in a crystalline form. • Haycraft and Scofield^ have recently shown that not only may the bile- pigment undergo changes of an oxidative nature, but that reduction processes may occvir also ; for instance, placing the positive pole of a battery in bile, and then completing the circuit, will cause a series of colour-changes to occur, indi- cating oxidation ; if, now, the negative pole be substituted for this the reverse series of colour-changes occurs, indicating reduction. They also show that under certain other circumstances, especially in the presence of putrefactive organisms,, reduction mav occur in the bile. 1 Herasius and Campbell, Pfliiger's Archiv, v. 497. 2 Sitz. Wien. Akad. lix. Abth. ii. •> Clinical Chemistry of Vrine, London, lSSi>, p. 170. ■* Zeit. pliysivl. Chciii. xiv. 173. -684 ALIMENTATION Ilijdro-hiJiruhin. — If a solution of Lilirulnn or biliverdin in dilute alkali be ti-eated with sodium amalgam, or allowed to putrefy, a rose- red or brown-red pigment is formed which is slightly soluble in water, easily soluble in alcohol, ether, chloroform, salt solutions, or alkaline fluids. Maly ' investigated this substance, and gave it the name of -hydro-bilirubin, and assigned to it the formula C32H44N4O7 ; it thus contains less hydrogen, and rather more oxygen, than bilirubin. Its spectroscopic appearances are as follows : A dark band between h and F, and a fainter band in the region of the D line (fig. 88, spectrum 3). The ammoiiiacal solution oi this pigment gives on the addition of zinc-chloride a well-marked green fluorescence, and then shows three bands instead of two (tig. i^^, spectrum 4). The interest of this substance arises from the fact that many physiologists believe it is identical with the substance called stercobilin by Vaulair and Masius,^ which is the colouring matter of the fa;ces, and according to some also with urobilin, the chief pigment of the urine. We shall see, when discussing those pigments, that hydro-bill- , rubin is not absolutely identical with either. MacMunn^ and Disque"* both regard hydrtj-bilirubin as an impure product. Bilifuscin (CxJl^o^-Pd i^ ^ pigment which has been obtained from brown gall- stones. The gall-stones are powdered and thoroughly extracted with a mixture of ether and alcohol to remove the cholesterin, then with dilute hydrochloric acid to remove calcium salts ; the acid is washed away with hot water and the residue shaken with alcohol. On distilling off the alcohol from the extract, a reddish-brown amorphous pigment is left, which is bilifuscin. It is insoluble in water, chloroform, or ether ; soluble in alcohol. It shows no absorption bands. It does not give Gmelin's test (Stadeler). Mliprasin (C,sH^„NoO,;) is the name given l)y Stiideler to a green pigment which he separated from gall-stones. ]\Ia]y ^ considers it is identical with biliverdin. Bilihvmin is the huraons-like residue left after extracting gall-stones with water, alcohol, ether, chloroform, and dilute acid successively (Stadeler). It is probably an impure substance. CJuilulucmathi This pigment occurs in the l^ile (jf the ox and sheep, and gives a three-banded spectrum (fig. 88, sijectrum ;"3). An ethereal ex-tract of the residue — oVjtained by agitating the acidulated bile with chloroform and evaporating this — is evaporated, and the residue again taken up with chloroform, which is washed in a separating funnel with water. On evaporating the chloroform a dark green jjigment with a musky smell is left. It is considered by MacMunn," who fir.st described it, to be a derivative of hasmatin, jjrobably an intermediate stage in the formation of biliverdin. 1 Centralhl. med. Wiss. 1871, Xo. 51. - Ihhh No. 24. 3 Loc. cit. p. 107. ■* Zi'if. pJtijs/ol. CJiein. ii. '2.^9. ^ An/i. Clie/ii. I'luinii. clxxv. 7(;. '■ Joiini. of Pliijsiol. vi. 22. lilJ.F (')H5 lljL'iiioglobin itself and :i sulistaiK-i' liki' iiu'tliicmoglobin li;i\c hevu y itself. If, however, bile or bile-salts be added to pancreatic juice, that juice will convert starch into dextrin and maltose more quickly than a control specimen con- taining no bile (8. Martin and D. Williams '). How bile favours the action of pancreatic juice it is at present impossiljle to say. Action of fntti. — It is found in cases of jaundice, when no bile enters the intestine, and in cases of biliary fistula also, that the faeces contain a large amount of undigested fat. In the dog, 40 to 50 per cent, of the fat in the food is found in the fsipces. Bile is therefore important in the digestion of fat. Here again, however, it is the combined action of the bile, with the pancreatic juice, that is important. Although the bile is by some said to have a slight emulsifying action, it is if present at all very slight. There is, however, no doubt that pancreatic juice plus bile act on fats Ijetter than pancreatic juice alone.^ Bile is said also to aid in the absorption of fats by lubricating the Tnucous memV)rane of the bowel. If an animal membrane, such as a piece of bladder, or even a filter paper, be moistened with bile, fat will pass through it under less pressure than if they are moistened with vvater.3 It is a little dangei'ous to draw positive conclusions from such an experiment as this. We shall see that aljsorption is not simply a 1 Proc. Boy. Sac xlv. 358. More recently these observers have shown that bile also favours the action of pancreatic juice on proteids {Ihid. xlviii. 160). 2 A recent paper on this subject is' one by A. Dastre, Compt. rend. Soc. hiol. 1887, p. 782. ••j V. "Wistinghausen, Diss. Dorpat, 18.^1; J. Steiner, Arch.'f. Anat. u. Physiol. 1873, p. 137 ; 1874, p. 286. r.ii.K 087 matter of ditfusion or tiltratioii, and is a very different matter from what occurs in dead membranes ; and there is perhaps no substance in which the living activity of tlie cells is so much needed for a])sorption as fat. BUe xs a hi.rative and an antiseptic. — The fieces in animals or human beings who suffer from jaundice or a biliary fistula are extremely hard, and liave an intense putrescent odour. Administra- tion of bile relieves this condition ; it is also known that a larce increase in the flow of bile has a purgative effect as in bilious diarrhcea. The bile itself is readily putrescible, and the power it has of diminish- ing putrescence in the fieces is due chiefly to the fact that by increas- ing peristalsis it hastens the passage of putrescible matters through the bowel.' Copeman and Winston- performed a number t)f cultiAation experiments with bacteria of different kinds, and found that, though bile is able to a small extent to control putrefactive changes, the bacteria grew almost as readily in the tubes to which bile had been added as in those to which no bile had been added. Limbourg ^ made similar experiments, and estimated certain products of putrefaction •(amido-acids and ammonia) in artificial pancreatic digestions with and without the addition of bile-salts. In the specimens whei'e the bile- salt was present, these products were somewhat lessened. The fate of tlie constituents of the bile. — We have seen that fistula- bile is poor in solids as compared with normal bile, and this is explained on the grounds that the normal bile-circulation is not occurring, and hence the liver cannot excrete what it does not receive back from the intestine. Schiff ^ was the first to show that if the bile be led back into the duodenum, or even if the animal be fed on bile, the percentage of solids in the bile secreted is at once raised. It is on these experi- ments that the theory of a bile-circulation is chiefly founded. The bile- circulation relates, however, chiefly, if not entirely, to the bile-salts ; they are found but sparingly in the ffeces; they are only represented to a slight extent in the urine; hence it is calculated that seven-eighths of them are reabsorbed from the intestine, especially the large intestine. This is by no means the least curious of the phenomena of bile-secretion. The bile is a most elaborate secretion ; it is poured into the intestine, and tinds apparently little to do ; it is split into simpler constituents, which then hurry back by the portal vessels to the liver again, when once more they unite to form bile-salts. It is stated that of the two bile-salts, the taurocholate is the more easily decomposed. Small quantities of cholalic acid, taurine, and glycocine are found in the faeces ; some of the taurine 1 McKeiidrick, Fhysiologij, ii. 122. - Jotirn. Physiol, x. 213. ^ Zeit. physiol. Chem. xiii. 196. * F/JUger's Archiv, iii. 598. C88 alimp:xtati()N is absorbed and excreted as tauro-carbaniic acid in the urine (p. 8-!5)_ Some of the glycocine may be absoi'bed and excreted as urea (Salkowski); but the greater part of all these constituents are apparently taken back to the liver to form bile-salts over again. The cholesterin and mucus of the bile are found in the faeces ; the pigment is changed into sterco- bilin. a substance like hydro-bilirubin, but a little different from it. 4. .\BXORMAL AXD PATHOLOGICAL CONDITIONS IN BILE-FOEMATION. Effect of poisons and diseases. — Many poisons, especially metallic ones, are excreted by the liver, particularly antimony, arsenic, copper, lead, and mercury (Oi'fila and others). Sodium indigo-sulphate after injection into the circulation soon appears both in the bile and the urine (Diakonow '). Iodine, grape sugar, oil of turpentine are found in the bile after injection into the circulation (Bernard). Large quantities of water similarly injected leads to the appeax-ance of albumin, both in bile and urine (Mosler -). In ursemia, the quantity of urea (which is present in mere traces in healthy bile) in the bile is increased. In cholera, the bile also contains more urea than normal, and, like the blood, is very concentrated. In febrile conditions generally, the amount of bile, like that of saliva and gastric juice, is diminished. In fatty and in amyloid degeneration of the liver, the total per- centage of solids, and especially of bile-salts, in the bile is greatly lessened (Ritter,'' Hoppe-Seyler ^). In acute yellow atrophy of the liver, the Vjile, like the blood and the ui-ine, contains leucine and tyrosine. In typhoid fever, the bile is after deatli found to Ije sometimes acid. This may anse from decomposition of the lecithin in the bile, or from diffusion of acids from the intestine into the gall-bladder after death ; leucine and tyrosine are also stated to have been found, -^ Ijut these may arise from putrefaction after death. Jaundice and chohfmia. — The small pressure of the bile in the bile- ducts accounts for the fact that a very slight obstruction wiU prevent the bile from entering the intestine ; the fseces are thus almost white (clay-coloured). Bile, however, continues to be secreted, and is absorbed ^ Hoppe-Seyler's Med. chem. Vnters. ii. p. 245. - Mosler, TJeber den Vehergang von Stoffen aiis dem Blute in die GalU, Giessen, 1857. See also Heideiihain, Studien des Physiol. Inst. Breslau, 18C3. ^ Journ. de Vanat. et de x>hijsioh 1872, p. 181. * Physiol. Chem. p. 318. 5 Fiei-iclis, Wien. ined. Wochensch. 1851, p. 30. liiLK 689 Ijy tlie lymphatics, ;viul entering into tlie circulation stains the skin •and mucous membranes yellow, and passes into the urine ; in the urine bile-pisj^nient may be easily recognised by Gmelin's test ; bile-acids are more dilhcult to discover, and seem to be often absent in the urine of such cases. Non-obstructive jaundice and ehubonia are described in' Chapter XVI (p. 311). C/toh'nti'ra'iniu.— FlinV considers that the separation of chole.sterin by the bile IS essential for the maintenance of the healthy activity of the nervous system, and that derangements of this secretion lead to nervous symptoms, which he designates by the name cholestercemia . These observations require fuUer investigation before they can be accepted. Gall-stones. — These are concretions that may occur in the biliary passages, or more frequently in the gall-bladder. They consist chiefly of cholesterin, with a smaller amount of calcium cai-bonate. They may, or may not, be infiltrated with bile-pigment, v. Planta and Kekule ^ analysed some gall-stones which contained 90 per cent, of dry cholesterin. In some cases, however, the most important constituent is bile-pigment. Thudichum'' found that the bile-pigment is chiefly bilirubin, not, however, free bilirubin, l)ut a calcium compound of the pigment called bilirubin-calcium. In some gall-.stones of this nature Maly ^ found 28 to 4-5 per cent., and Phipson^ 61 per cent, of bilirubin. Stadeler found in addition biliverdin, bilifuscin, biliprasin, and bili- humin. The nucleus or central portion of a gall-stone appears to be chiefly mucus."' Other constituents occasionally found in gall-stones are zinc (Thudichum and Maly), iron, copper, and manganese (Bley,^ Wurzer'), fats (v, Planta and Kekule), silica (Pleischl,^ Bley), uric acid ■(Stockhardt,^ Marchand'°), and in cases of typhoid fever and tuber- culosis fat-globules (Gorup-Besanez"). The Secretion of the Gall-hladdcr B. Birch and H. Spong '* obtained this secretion in two cases of biliary fistula in human beings, in which the bile charmels were completely shut off from the gall-bladder. The amount secreted daily was 20 to 30 c.c. The fluid had the same characters in both cases ; it was clear or faintly opalescent, viscid, and had a specific gravity of 1011 or 1012. It was always distinctly alkaline. It contained 2 per cent, of solids; 1-2 per cent, organic (mucin and a trace ' Austin Flint, jun. Becherches exp. sur line nouvelle fonctiou du foie, Paris, 1868. - Ann. Chem. Pharm. Ixxxvii. 367. " Quart. Journ. of the Chem. Soc. 186-t. * Ann. Chem. Pharm. clxxv. 70. '• Lelimann-Gmehn's Lehrbuch, viii. 4.5. * Journ. prakt. Chem. i. 115. ' Scliweigg, Journal, viii. Go. ■* Kastner's Archie, viii. 300. '•* Diss. Lipsise, 183'2. '0 Zeit. rat. Med. iv. 114. ii Lehrbuch, p. .53.5. '- Journ. of Physiol, viii. 378. Y Y <)*J0 ALIMENTATION of proteid) ; 0-S per cent, inorganic, of which tlie most abundant salt was .sodium chloride. They do not regard this fluid as playing any important part in the digestive jirocess. The Invertehrate Liver The so-caUed liver of invertebrate animals appears, in those cases in which an examination of its properties has been made, to fulfil the functions of a pancreas. A. B. Griffiths ' found that the secretion of the ' liver ' of the limpet, like that of cephalopods, converts starch into sugar, forms an emulsion with fats, and a soluble femient extracted from the glands converts fibrin into peptone, leucine, and tyrosine. The secretion itself contains proteids, leucine, and tyrosine, but no biliarj- acids. Glycogen also could not be detected in either the organ or its secretion. The glycogenic function of the vertebrate liver is performed in molluscs by the connective-tissue-cells (Blundstone '-). Whether the gland tliat secretes the ink in sepia corresponds to a liver is a matter of doubt. The secretion is not digestive ; it is used to colour the sea-water and cover the flight of the animal. It has been investigated by Schwartzenbach ■' and Hosacus,' who find the black pigment is its chief constituent (80 per cent, of the dry solids) ; there are also small quantities of a mucinoid substance, carbonate of calcium and magnesia, sulphate and chloride of sodium. Nencki and Sieber^ have recently separated from the pigment an acid sepiaic acid, containing carbon, hydrogen, oxygen, nitrogen, and sulphur. 1 Froc. Hoy. Hoc. xhi. 392 ; Proc. lioy. Sac. Edin. xiii. 120. - Proc. lioij. Soc. xxxviii. 442. " Liebifj's Jahresh. 18(>2. p. 539. * Arch. d. Pharm. (2), cxx. 27. 5 Xeiicki and Sieber, Chem. Cenfrcilbl. 1888, p. .587. GDI CHAPTER XXXV PrTllKFACTIVE PROCESSES T\ Tlfi: IXTESTTXE TiiK ancients regarded the whole digesti\e process as one of the natuie of putrefaction ; they used the term no douht in a loose sense, but the earliest experiments of Reaumur, Spallanzani, and Beaumont showed that in the stomach at least there is no formation of malodorous gases, the presence of which is the most palpable evidence of putrefaction. This has since then been a matter of ccmimon observation ; in certain disordered conditions of the stomach, gas-forming fungi may flourish and cause flatulence and eructations (p. 650), but during the normal diges- tive process in the stomach these are absent. .Since we have known more a1)out putrefaction and its causes, it has Ijeen found that bacteria do not flourish I'eadily in acid media ; a priori then we should not expect them to be acti\e in the stomach. The actual inAestigation of the question has been made by Harris and Tooth,' who, using the latest bacteriological methods, have been able to demonstrate satisfactorily that the general belief in the absence of the activity of micro-organisms during gastric digestion is well founded. 8traus and ^^'urtz- ha\e found that gastric juice is an actual germicide, and destroys the bacillus anthracis, the cholera bacillus, and many others. In the intestine, however, especially in the large intestine, putre- factive processes always occur. The bacteria are introduced with the food, but escape the direct action of the gastric juice. They may be diminished by purging, which produces rapid removal i if the products of putrefaction, or by the administration of antisejjtics ; the use of these, however, in man is limited ; large doses of iodoform or calomel, such as Baumann^ and Moran^ administered to dogs with success, would be exceedingly dangerous to use in human beings. We have already seen that these processes are kept within normal limits by the natural antiseptic, the bile. Within such limits putre- faction is probably a useful process, acting on food very much in the same way as does the pancreatic juice. In many cases the organisms exert a peptonising action, and only seldom a diastatic action (W. Miller '"'). ' Joiirn. Physiol, ix. 220. - Archives dc mid. experimentale, 1890; see Brit. Med. Jourii. vol. i. 1890, p. 25'2. See also Falk, Virchow's Arcliiv, xciii. 117; Frank, Deufsch.vied. Wochensch. 1884, No. 24. 3 Zeif.physiol. Chem. x. 123. ■» Hid. p. 318. 5 Chem. Centralbl. I88fi, p. 580. Y Y 2 692 ALLMENTATIOX Vignal ' separated an enorinours number of microbes from tlie faeces, six of which are found in tlie mouth also, and many of them have consider- able digestive action. Many are fat-splitting. Other organisms Ijring about the formation of leucine and tyrosine, indole and skatole, lactic and butyric acids, itc. A useful function fulfilled by the organisms appears to be the destruction of poisonous substances, such as choline, the alkaloid derived from lecithin. It is possible that if other alkaloids (leucomaines) are formed by the processes occurring in the intestines, these also are destroyed, for they are absent in the normal excretions. It need hardly be said that an excessive amount of putrefactive change in the intestines is injurious, producing distension of the abdomen by the gases which accumulate, and other forms of discomfort. The amount of putrefactive change occurring in the alimentary canal or elsewhere in the body as in putrid abscesses is best measured Ijy the amount of certain products in the urine. These are termed ethereal sulphates ; the indole, skatole, cresol, phenol, and a few other sub.stances formed by putrefaction are absorbed in very large measure, and excreted in the urine as combined sulphates. The methods of estimating these will be described under Urine. The gases of the intestinal canal have been analy.sed by Planer,^ Huge,'' and Hofmann.' They vary a good deal with the diet. The following are Planer's numbers (from dogs) in 100 volumes of the mixture of gases: — Gases 1 1 Small intestine Large intestine | Meat die Bread diet Vegetable diet Meat diet j Vegetable diet C0„ . 401 38S 47-2 74 2 65-1 II, . 13-9 6-:^, 48-7 1-4 2-9 H,S . — — — . 0-8 — 0, . 0-5 0-7 — — — N, . 45-0 54-2 4 0 23-6 5-9 The following are Ruge's figures ; the gases were obtained from human beings : — Gases Milk diet Meat diet ' Vegetable diet 1 - CO^ .... H3 . . ■ • CH, . . . • N, . . . . 9 to 16 8 to 13 ' 21 to 34 43 to 54 0-7 to 3 I 1-5 to 4 0-9 26 to 37 44 to 55 36 to 38 45 to 64 10 to 19 ! i 1 Compt. rend. cv. 311. 5 Ihid. xliv. * Sitzungsher. Wien. Akad. xlii. * Wien. 7ned. Wochensch. 1872, No. 24. PrilJKI'ACTlVK IMIiH'KSSKS IN IIIF. I NTK.STl Ni: 693 Oxygen .-ind sulpliuretti'd liydiogcii were fimiid in traces only ; Hofniann found no marsh gas in raljbits. Th<' c(irho}iic acid, as is seen in the above tables, is always present ill large (juantities, especially in the large intestine, and especially wlien till' diet is \egetable. Its sources are the decomposition of carbonates, acetates, and lactates in the food, the alcoholic fermentation of dex- trose in the intestine, the putrefaction of carl xihyd rates (especially celluhise) and proteids, the butyric fermentation of lac-tic acid, and the putrefaction of choline. The enormous (]uantity of gas discharged in i-ases t»f hysterical flatulence consists largely of carbonic acid ; it is ])ossible it may have simply diffused from the blood-vessels. T/ic hydro;/ fu is most abundant on a milk diet : its source is the i)utyric acid fermentation of lactic acid (p. 103). Tlie marsh gas is derived from the decomposition of acetates and lactates. Hoppe-8eyler ' represents the decomposition of calcium acetate by the equation (C2H302).,Ca + H20 = CaC03 + C02 + 2CH4. It is also derived from the decomposition of cellulose (Hoppe-Heyler,- Tappeiner,^ Henneberg and Stohmann "*). Hoppe-8eyler's .formula for the reaction is C,jH,o05 + H20=3COo + 3CH4. Henneberg and 8tohmann con- sider that hydrogen, acetic acid, and butyric acid ai^e also formed, their equation for the reaction being 21C6H,oO,5+ llH2O = 26CO2 + 10CH4 -1-6H2 4- I9C2H4O2 + 13C4Hg02 ; whichever equation is correct, the fact remains unaltered that a vegetable diet is that which yields most marsh gas. A third and small source of marsh gas is from the choline of lecithin (Hasebroek ^). The nitrogen is derived chiefly from the swallowed air ; the oxygen is lai'gely absorbed ; nitrogen is also contained in the ammonia, which is the result both of pancreatic digestion and putrefaction of proteids. Tlie hydrogen sidyliid'' is derived wholly from the putrefaction of proteids. We may in conclusion briefly glance at the matter of putrefaction from another point of view, namely, its action on each class of the proximate principles of food. Action on fats. — This is a fat-splitting action, exactly similar to that pi'oduced by the steapsin of the pancreatic juice. Putrefaction in addition produces lower acids (valerianic, butyric, Arc.) of the fatty series. Lecithin is similarly decomposed into its acid (glycero-phos- phoric) and choline which then breaks up into carbonic acid, marsh gas, and ammonia. Action on carholiydrates. — The chief fermentation here is the lactic acid followed by the butyric acid fermentation {»ee p. 103). 1 Zeit.phijsiol. Cheiu. ii. 561. - Ibid. x. 201, 401. ^ Zeif. Biol. xx. 52; xxiv. 105. * Ibid. xxi. Giy. -^ Zcit. j'hysiol. Clicm. xii. 148. 694 ALi:\IENTATI()N With regard to cellulose it may l)e here stated that putrefaction is the odIv known change that tliis constituent of food undergoes in the alimentary canal. ^ Henneberg and Stohraann nevertheless consider it a source of enei-gy. An important practical point in cattle feeding, Avhether cellulose economises the decomposition of proteid, has not yet passed beyond the regions of dispute (v. Knierem,"'^ "NVeiske, and others •'). Action on proteids. — The antipeptone is decomposed with more difficulty than the hemipeptone. The products of putrefaction of proteids are ammonia, sulphuretted hydrogen, ammonium sulphide, volatile and fatty acids ; amiiies and amido- acids, especially leucine and tyrosine : indole, skatole, phenol, and cresol, phenyl-propionic, and phenyl-acetic acids, and the aromatic oxy-acids, hydroparacumaric and parahydroxyphenylacetic ;icids. The presence of these numerous acid compounds, especially of lactic acid, gives the contents of the large intestine, as a rule, an acid i-eaction. The presence of indole and skatole gives the fa?ces their characteristic odour: they are, however, very largely absorbed and excreted as ethereal sulphates in the urine. With regard to the production of indole, Harris and Tooth * found that its appearance, and that of its allies, is capricioiis, and can be easily prevented in artificial pancreatic digestion. The smallest amount of mercuric chloride or phenol, even if not sufficient to render the fluid aseptic, prevents the formation of these substances. Whenever indole is present, however, large numbers of all sorts of bacteria are present also ; stiU it may be absent even if swarms of micro- organisms are present. It thus appears that there are special indole-forming organisms. As a result of inoculation experiments, it was found that indole was formed from peptone, not from leucine and tjTOsine. It is interesting to note that certain products of putrefaction, especially phenol or carbolic acid, and cresol are antiseptics ; the microbes thus produce comiJouuds which, if allowed to accumulate, would ultimately destroy their life. It is considered by certain observers that the production of poisonous alkaloids is a normal process in the alimentary canal, that these are absorbed, and if ex- cessive in amount may produce self-poisoning or ' auto-intoxication.' As a rule, they are excreted, however, by the kidney, and thus the body generally escapes their poisonous action (Bouchard). Such a doctrine must be considered unproven for the present. The most careful of the numerous researches in this direction entirely negative the idea. Ptomaines are absent, not onlj- in normal urine and faeces, but also in these excretions in various diseases. There is, however, some evidence of their formation in typhoid fever, cholera, and cystinuria. We have seen that under normal circumstances choline, a typical instance of a poisonous animal alkaloid, is broken up into simple non-poisonous products by the intestinal bacteria ; and it is exceedingly probable that if other alkaloids are produced by bacteria in the intestine, they also are promptly destroyed by other species of the same micro-organisms (see also Chapter XIII). * Bunge surmises {Physiol. Chem. 192) that the epithehum cells of the intestine may have a similar action on cellulose. He also dwells (p. 81) on the important action of cellulose as a mechanical stimulus to peristalsis. - Zeit. Biol. xxiv. 293. ^ Ibid, x.xii. 373. < Jouni. of Physiol, is. 220. 095 c'hai^'J't-:j{ XXX VI THJ-: fj:cj:s The fa'c-es consist of the indigestible and undigested portions of the food, products formed from food-stuffs by the digestive ferments (indole, skatole, soaps, itc), and certain constituents of tlie digestive secretions (mucin, altered bile-pigment, itc). The amount of the faeces varies with the amount and character <»f the food. Over-eating entails voluminous excrements, since, though nmch of the food taken may be digestil)le, it escapes digestion and absorption simply because its amount is too great for the digestive ferments to act upon, or foi- the absorbing surface to come in coutact Avith. On a mixed diet of moderate amount in man, Liebig calculated that the weight of the fjeces is one- seventh to one-eighth of the food taken. Calculating both food and" fa3ces in the diy state, Bischoff and A'oit found in dogs that Avith a nitrogenous diet the fteces weighed one- tenth to one-fourteenth, with a bread diet one-sixth to one-eighth of the food. The amount of Avater in the faeces varies considerably in health from 68 to 82 per cent. In diarrhoea it is more abundant still. The constituents of the faeces may be classified as follows : 1 . Undigested foods : fats, carbohydrates, and proteids, if any of these are present in excess in the food. On a moderate diet, unaltered proteid is never found. '1. Indigestible constituents of the food: cellulose, keratin, mucin,' chlorophyll, gums, resins, cholesterin. 3. Constituents digestible with difficulty : uncooked starch, tendons, ■elastin, nuclein, various phosphates, and othei- salts of the alkaline earths. •i. Products of decomposition of the food : indole, skatole, phenol, kc. ; fatty acids, formic, acetic, butyric, isobutyric,- caproic, valerianic: other acids, lactic, malic, succinic, kc. Some of these acids are free; some in •combination with ammonia and other bases ; hsematin from lu^moglo- bin ; ^ soaps, especially calcium and magnesium soajDS of oleic, palmitic, And stearic acids (the soluble soaps are, of course, to a large extent 1 Mucin which has been separated out hy means of lime water and acetic acid is readily digestible by artificial pancreatic juice [see p. 4Sl). Mucin as contained in nmcus, however, appears to be quite unaltered by the natural juices. - Brieger, Ber. deutsch. cheiu. Ges. x. 1027. s Hoppe-Seyler, Physiol. CJiem. p. 330. 696 ALDIEMATioN absorbed) ; stercorin, a product of decomposition of cholesterin : this substance was described by Fbnt,' but its existence is very doubtful ; excretin (CooHggO), another doubtful substance described by Marcet.- 5. Bacteria of all sorts and debris from the intestinal wall : cells, nuclei, mucus, etc. L. Hermann ^ found in a loop of intestine separated ill the manner <)f Thirv and Yella that at the end of some weeks it was filled with Ijacteria, cellular debris, and often fat, the whole mass having a ffecal appearance. 6. Bile residues : these are mucin, traces of bile-acids and their products of decomposition: cholesterin and lecithin, the latter in traces only are also fi)und : these two substances also partly owe their origin to the ingested foods. The bile-pigments as such are not present, but are changed into a substance like hydro-biliruVjin, which is called sterco- bilin.* Stercobilin may originate also from the ha-matin in the food (MacMunn-^). Hoppe-8eyler,^ however, who made experiments on dogs, found that hsematin is easily discoverable in the fjeces, and regards it as improbable that stercobilin originates from the haemoglobin of the food. This subject merits renewed study, and the experiments should be made on animals in which no bile is allowed to enter the intestine. The meat of the food cannot, however, be a large contributor to the pigments of the fseces, as the stools of jaundiced persons are clay- coloured even if they are on a meat diet. Hydro-bilirubin and sterco- bilin are usually considered to be produced by reduction processes : MacMunn, however, regards the formation of stercobilin as one of inter- mediate oxidation : by further oxidation it may be transformed into a substance like choletelin, the most highly oxygenised product of the bile- pigment with ^\hich we are acquainted. T. J. Walker' has recorded two cases in which the liver was apparently healthy, but the pancreatic duct was occludefl ; the f;eces in these cases were free from sterco- bilin, being clay-coloured as in jaundiced persons. He therefore con- cludes that the pancreatic ferment is in some way necessary for tlie formatioii ot tlie f;ecal pigment. Stercobilin may l)e most readily prepared by extracting the fseces with acidulated alcohol (17 parts of rectified spirit to 3 of sulphuric acid) ; the extract is diluted wdth water, and shaken with chloroform : the chlorofonn dissolves out the pigment and may be driven ofi" by evaporation. 1 Eecherche.i c.rp. siir inic nonvelle fonction du foie, Paris, 18GH. 2 Ann. de chem. ef de phys. lix. 91. ^ Du Bois Bei/iiiond's Archiv, 1889. * Vanlair and Masius, Centr. med. Wiss. 1871, No. 24. 5 Journ. of PJn/siol. x. lir>. ^ PlujsioL Chem. p. 339. ' Medico-Chiriirgical Trans, vol. Ixxii. 1889, p. 257. TIIK I'.ICCKS 697 Before jiroccotrmg to dcscriln.' tlif spectroscopic api)fiiniuces of this substance it must be acknowledged that as yet spectroscoijic analysis is the only method yet applied to this and related [)igraents (iiydro-bilirubin, urobilin, Sec); it is possible in the future that other methods of investigation maj^ confirm or correct the knowledge obtained by the sjiectroscope. Another possible source of error is the admixture of unchanged hiematin witli such ])igments, and a third difficulty arises from the fact that there arc probably intermediate products between bilirubin and stei'cobilin which occur in different proportions in different prepara- tions. This last assumption is confirmed by the differences obtained in measure- ments of the bands of stercobilin in different preparations. One of these inter- mediate products appears to be absorbed, carried to the liver, and there excreted into the bile as biliary ui-obilin (p. 085) ; by further oxidation biliary urobilin can be artificially changed into a pigment closely resembling stercobilin. The absorption spectrum of stercobilin is practically identical with that of hydro-bilirubin (fig. 88, spectnim B, p. 685). We have seen, however, that hydro- bilirubin after treatment with zinc chloride and ammonia shows a green fluor- escence and a three-banded spectrum ; stei'cobilin, on the contrary, though it shows the same fluorescence, gives a four-banded sjjectrura. There are also certain differences in the spectra of the two substances after treatment with other reagents, such as soda, or zinc chloi-ide by itself, or ammonia by itself. The spectroscope thus teaches us that the two substances cannot be identical. Still more does the spectroscope teach us the non-identit}' of either of these pigments with vxrobilin. Jaffe' and Maly- first described urobilin, and considered that it originated from bilirubin, that bilirubin was changed into hydro-bilirubin in the intestine, and then partly absorbed and excreted in the urine. Subsequent investigations have, how- ever, shown that there are two pigments or their chromogens in the urine which have each received the name urobilin ; one is normal urobilin, which shows the same spectrum as choletelin, that is one band only (at F) ; the other pathological urobilin which occurs in certain diseased conditions is possibly identical with stercobilin, and no doubt originates in the intestine as Maly considered. Normal urobilin does not necessarily arise in the intestine from stercobilin ; in Copeman and Winston's case of biliary fistula,^ no bile entered the intestine, but the urine was not colourless ; it contained ordinary urobilin. In cases of ex- travasation of blood, the destruction of blood-pigment may give rise to pathological urobilin in the urine,^ and moreover normal urobilin was obtained artificially by MacMunn by acting on acid heematin with hydrogen peroxide. Pathological virobilin is regarded by MacMunn as a less highly oxidised product than normal urobilin. It thus appears that if the urine pigment be formed in the liver, it is unnecessary for it to go through the stage of bile-pigment, though this stage probably occurs under normal circumstances. This subject will be more fully flealt with under Urine (Chapter XL I). Meconium The meconium, or the contents of the intestine of new-born children, is a greenish-brown, ahnost black, viscid material. Its reaction is generally acid. On microscopic examination it shows leucocytes, often stained green, coluiiinar epithelium cells from the ' CentraJhh vied. Wiss. 180.3, p. 241. - Aim. CJieiii. PJuiriu. clxi. 308 ; cl.xiii. 77. "' Journ. Physiol, x. 213. ' 4 Cases recorded by MacMunn, Ihid. p. 83. <)5>S ALIMKNTATIOX intestinal wall, fat-globules, and crystals of cholesterin. Zweifel found it contained 20 to 27 per cent, of solids, of which 1 per cent, was inorganic, the remainder organic : the percentage of fat and fatty ■acids was 0-7o : that of cholesterin was also 0'75. Tlie chief organic constituents are the bile-salts, more or less changed : the bile-pigments bilirubin and Inliverdin, not changed at all, and mucin. The inorganic constituents are phosphatas and sulphates of magne- sium and calcium, oxide of iron and sodium chloride. The most remarkable difi'ei'ence between meconium and fteces is in the pigment. In meconium, .stercobilin is absent ; in addition to liiliverdin and bilimbin, it contains a small quantity of a pui-plish ]>igment, which gives a narrow aVjsoi-ption Vjand before D, and another darker and wider between I) and E, which is probably an oxidation product of bilirubin (Hoppe-Seyler). ' In fact, meconium is, as Mott - puts it, little else but concentrated Inle. Patlwlogical Alterations in the Fences Section of the nerves going to a loop of intestine paralyses the blood-vessels, and causes an abundant watery exudation. If these nerves contain fibres which are secretory in function, this incre^ised flow of fluid may be in part a paralytic secretion of the intestinal glands (Moreau''). Purgatives act in various ways, .some exciting a flow of fluid into the intestine, some increasing i:)eristalsis, others acting in 1x)th ways. The excito-secretory action of saline purges, like magnesium sulphate, is probably due to their irritant properties, and not simply t'> osmosis : the low diffusibility of the salt, however, impedes the ab.sorption of the secrete^l fluid (Hay^). The diarrhcea of certain diseases (typhoid, cholera, dysentery, kc.) is probably due to specific poisons produced Vjy bacteria. Ordinary diarrhcea is due to the irritant action of bad or indigestible food, or to accumulation of hard fa-ces, or it may l>e produced in certain forms of emotion. The rice-water stools of cholei-a contain a low percentage of solids, very little proteid, little or no Ijlood, a vast amount of intestinal epithelium, leucine, and tyrosine, and perhaps certain ptomaines. Blood and pus appear in dysentery, and occasionally in typhoid fever. If the blood is small in amount, tlie hsematin which is formed gives the stools a dark, almost black colour. If the amount of blood ' Physiol. Cliem. p. .340. - Practitioner, Aug. 1890. ^ Comjjt. rend. 18-58, p. 554. * Brunton's Materia Medica, p. 342. is large, ;is in ulceration into ;i Mood -vessel, the corpuscles and hajnio- i,dobin are for the mosc i)art unchanged. Typhoid stools contain abundance of annnoniuni carl)onate, and amnionio-niagnesiuni phospliate often in crystals. Skatole is absent (Brieger'). In intestinal catarrh the faeces are watery, and contain albumin, an increased percentage of salts, urea, and alloxan. The urea is also increased in urannia ; it may, ho\ve\or, be converted into annnoniuni cai-bonate. The chief salts in the stools in all cases of diarrhoea, cholera included, are chlorides of sodium and potassium ; the amount of chlorides in the urine is correspondingly diminished (Schmidt '^). Tn jaundice, bile is absent, and the stools are hard and clay-coloured. T. J. Walker recorded two cases in which clay-coloured stools occurred, though the liver was apparently healthy ; the pancreatic duct was, however, occluded. In some forms of diarrha^a there is an excessiAc amount of bile present. In cases where either the bile oi- pancreatic secretion is diminished or absent, the fat of the food is in great measure not digested, and passes away in the f?eces. In some of these cases the fats or their soaps are found in a crystalline condition in the f;eces (Oesterlein,^ .Stadelmann ^). The administration of the salts of mercury or iron causes the tVeces to be black from the formation of the sulphides of tlutse metals. Gall-stones may be present, having passed from the gall passages into the intestines. Scybahe are hard masses of faeces containing a good deal of dried mucus. Intestinal concretions consist generally of earthy phosphates ; but they may contain chiefly organic matters, fat, hair, vegetable fibres, and in some animals (antelopes) two special components, named lithofellic (CooH3604) a.nd ellagic (C,4H,;0>^-f2H20) acids, have been described (Ettling and Will, Gorup-Besanez '). Such very briefly is an enumeration of the pathological conditions met with in the fteces. To the practical physician, the subject of properly recognising these is a matter of paramount importance, and for further imformation on the subject, the reader is referred to works on materia medica, medicine, and pathology. 1 Ber. d. chcni. Gcs. 1877, p. 1031. -' Clmractcrisiil; der Cholera, Leipzig, 1850. 5 Mitth. a. d. Died. Klinik in Wiir;:bur!j, i. 1. * Archivf. klin. Med. xl. 372. •'' Lehrhitcli. 1S74, p. 5.57. 700 ALIMENTATION CHAPTER XXX^'II ABSORPTION Food is digested in order that it may be absorbed. Certain changes are produced by the action of the digestive secretions on the food, by means of which it is reduced to such a condition that it may pass more easily into the blood-vessels and lacteals of the intestinal walls. In the mouth and oesophagus, the thickness of the epithelium and the quick passage of the food through these parts reduce absorption to a minimum. Absorption takes place rapidly from the stomach ; it is. stated that most of the peptone formed in the stomach is absorbed before the chyme passes into the duodenum. The small intestine with its folds, and villi to increase its surface, is, however, the great place for absorption ; and although the villi are absent from the large intestine^ absorption occurs there also, but to a less extent. Some foods, such as water and certain salts (sodium chloride, *fec.), are not acted on by digestive juices, but ai-e absorbed unchanged. The organic foods, however, undergo a change from a colloid to a diffusible condition : thus proteids are changed into peptone, and starch into sugar : these soluble substances diffuse into the neighbouring vessels. The fats undergo a double change : the smaller amount is saponified, and soluble soaps are absorbed like other soluljle materials ; the greater part of the fat is, however, emulsified, that is, reduced to a fine state of subdivision ; the minute fat-glolndes pass into the vessels by a mechanism which will require special description. The question as to whether the lymphatics are the only absorbents was settled by Magendie, who showed that if the thoracic duct of an animal be ligatured, and a soluble poison introduced into the intestine, the animal dies quickly because the poison has Ijeen taken into the blood-vessels. Absorption is partly a physical process, namely, that of diffusion. "Water, salts, and sugar pass out of the intestinal canal into blood or lymph, when the fluid in the intestine is richer in those substances than the blood or lymph ; and the greater the difference between the- contents of the intestine and that of the vessels, the more rapidly does diffusion occur. The process is thus not simply one of filtration under- pressure caused by the movements of the intestine. The rate of diffusioix ABSORPTION 701 is increased by the fact that all the Huids cmicernecl are in motion, and so new layers of fluid are successixely being brought into juxtaposition. Absorption is by no means a mere physical process ; we must also take into account the fact that the cells through whitli the fluids pass are living, and have a power of, not oidy selecting materials for aVisorption, but also of changing th<^se substiinces while in contact with them. It is in the alisorption of proteids and fats particularly that the vital properties of the cells come into play.^ The cells are of two kinds : (1) the columnar epithelium that chjjsiol. Ghem. 1881, p. 27. ABsi)i;i'ii(»\ 70;-^ Later Hofmeister ' found in tlic Ijlood of aniniuls during digestion small quantities of peptone ; hut these were sometimes al>sent. Sflunidt-Mulheini, Plosz and (ivorgyai - and Drosdoff^ had before this found traces of peptone in i)ortal blood. This was, however, in the days before the use of ammonium sulphate as a i-eagent for the separation of peptones. Neumeister,' who has emi)loyed this reagent, proves most conclusively that both [leptones and allnimoses are always absent from both blood and lymph, even during the most active peiiods of digestion. It is as well for us that they are, as they are most violent poisons, causing a rise of temperature, a fall of blood - pressure, and a change in the blood, rendering it uncoagulable. Ncuiueister also found that, although it is possible after injection to recognises the presence of albumoses and peptones b.v their effects in rendering the blood uncoagulable, they cannot be demonstrated there by chemical means; their secretion by the kidneys begins ten minutes after injection. In the dog he found that the albumoses underwent hydration before appearing in the urine, the primary albumoses appearing as deutero-alburaose, the deutero-albumose as pi'ljtono. Probably this digestion occurs by means of the pepsin secreted by the kiilnoys in the urinnry tubules, where there is momentarily a formation of free acid. In the rabbit no such change occurs ; the urine contains no pepsin in this animal, and the albumoses injected into the circulation are secreted as such. Where, then, during normal digestion does the change from peptone into the blood proteids occur? It must occur dui-ing the actual pi'o- cess of absorption ; though whether the epithelium-cells, or the lymph- cells, or both are the active agents in producing the dehydration there is at present no evidence to show. There is, however, evidence to show that the mucous membrane as a whole has this power, Hofmeister^ found that the mucous membrane of the stomach and intestine are the onl}' parts of the body in which a supply of peptone is always found during digestion. A stomach recently removed fi-om an animal has also the power of reconverting peptone into native proteid. V. Ott " and others, who have carried out researches in Kronecker's laboratory, use tlie word seruvi- albumin synonymously with the proteids of the blood-plasma. The actual proof of the obtaining of serum-albumin is in their case by no means satisfactory ; it is not a chemical, but a physiological one. A solution of ' serum-albumin' artificially circulated through a frog's heart has the power of keeping it beating ; this power is not possessed by a simdar solution of peptone. If, however, the solution of peptone be placed into the stomach of a living dog, and withdrawn in a few minutes, it is again capable of keeping the heart beating. This is regarded as sufficient proof that the stomach had in this time reconverted or regenerated the; ' peptone ' into ' serum-albumin.' Miss Popotf ' showed that the same result followed if, instead of putting the solution of peptone into the stomach, it were I Zeit.physiol. Chem. 1882, p. 51. ^ Pflilger's ArcMv, x. .536. ■' Zeit.physiol. Chem. 1877-8, p. 210. * Zeit. Biol. xxiv. 272. ^ Zeit. Physiol. Chem. iv. v. vi. ; Arch. f. cxp. Patli. it. Pliarm. xix. Sec also Salvioli, Du Bois BeymomVs Archie, Suppl. 1880, p. 11-i. 8 Archiuf. Physiol. 1883, p. 89. ' Zeit. Biol. XXV. 427 ; see also Miss Brinuk, Ibiil. i')'d. 704 ALIMENTATION allowed to remain in a loop of intestine separated from the rest of the alimentary tract by a Vella's tistula ; or even if it were placed in contact with pieces of mncous membrane removed fi-om a recently killed animal. Peptone produced by the pancreatic ferment, however, was not regenerated in this way. These experi- ments are not altogether satisfactory, as they entirely leave out of account Ringer's important results, showing tlie great effect produced by minute doses of salts on the frog's heart {see p. 256). All these fcacts taken together constitute a very strong chain of evidence that under nor- mal circumstances pep- tone is 'regenerated,' not while it .still remains in the cavity of the stomach or the intestine, and not after it reaches the blood or lymph, and still less the liver, but during its passage through the cells of the mucous membrane. The absovjitioii of/at.' The way in which minute fat-globules pass from the intestine into the lacteals has been the subject of much contro^"ersy. In- stead of entering into this controversy I propose Fig. 89.— Section of tlie Villus of a Kilt kilkilduriusrfat-aVisorii- Uara fo trive a reSUmC tion (E. A. Schafer) :>";>, epithelium; .itate of chloric acid, and add sola- Imriumsuliiliiitp, insoluble tion of barium chloride in nitrii- lui'l The chief sulphate is potassium sulphate. Hy- drochloric acid is added to prevent precijiit^ition of plios])hates anil car- bonates Make urine alkaline with , White flakesof earthy (Ca In addition to tlie earthy potash or ammonia, and and Mg) phosphates are I phosphates, phospliates of warm precipitated; soluble in " acetic acid Acidulate with nitric acid, add nitro-molybilate of ammonia, and warm Calcium Magnesium , Sodium and potassium Acidulate with acetic acid, and add solution of am- monium oxalate Treat the above filtrate with ammonia and a few drops of sodium jihos- phate A yellow crystalline pre- cipitate A white precipitate of cal- cium oxalate is formed ; filter this oflE Evaporate the lu-ine to dryness : incinerate the residue ; dissolve the asli in water ; evaporate this down, and test by the flame reaction Anmionio-magnesiimi phosphate separates out Sodium gives a yellow, potassium a violet flame : potassium gives a yellow precipitate in neutral solu- tions -ivith platinum chlo- ride : sodium does not Carbonic acid Heat the urine gently in a Tlie paper is tm-ned blue, test-tube, holding a piece j and regains its colotir on of red litmus paper over i gently wanning it the mouth of it Place the urine in a tightly closed flask connected wit h a second flask, in which lime or barj-ta water is , placed ; the second flask is I exhausted by an air-pump. Warm the urine gently sodium and potassium also occur, wliicli vary in composition witli tlie re- action of the urine. A depositof phospliates may often occur in alkaline or neutral urine, or may come do\ra on heating. This is soluble iu acetic acid, and is thus distin- guished from albumin This is soluble in acetic acid Ammonia is only present in appreciable quantities in stale urine A white precipitate of caJ- 1 cium or barium carbonate, \ respectively is formed in the second flask Hold a piece of moist blue I The paper is turned red, litmus paper over the andregainsits colour when unne and warm Jn- Trea Evaporate urine to a third of its hulk, and aiid nitric acid Crystals of urea nitrate separate out Proceed as above with Crvstals of urea oxalate oxalic acid 1 separate out Add alkaline solution of An evolution of bubbles of sodium hypobromitc j nitrogen takes place If the urine is albuminous render aci-,OJ. On adfling potash after the ammonia the spot becomes j purplish blue. If potash j or scla alone is used, in- stead of ammonia, a violet colour appears, which dis- I appears on heating A black spot of reduced j silver appears | A solution of uric acid or Produces a reddish preci- j Hence urates may, when lu^te warmed with copper pitatc of cuprous oxide | in excess in urine, be mis- sulphate and caustic : taken for sugar potash I These dissolve on warming tlie tirine Th^ are coloured pink by urinary pigment, forming the so-called lateritions deposit. The urates oc- ciuring in sediments are acid urate of soda, of potash, and ammonia The separation of uric acid from this is effected by This urine is whitepowder finely powdering and dissolving in warm soda ; filter : when dry, creamy when render the filtrate acid with hy.lrochloric aci'l. A fresh ; it consists chiefly white crystalline deposit of uric acid separates out j of ammonium urate riiK riMNH 719 SuVistftiioe Hippurio aciil Creatinine Test 250 e.c. of fresh wine are evniMirateil down to 25 o.c, and i)o«ilereil pyi>suin aililed until it forms a thick iiaste : this is aciili- fied witli iwetic aciil and extracted with pure ether ; distil the ether off from ethereal extract : dissolve residue in hot water, and filter Evaporate the urine with nitric acid, and heat the residue in a dry test-tube Urine is evaporated to a quarter of its bulk, and after cooling poured off from residue, if any ; this is precipitated by acetate of lead : excess of lead re- moved by H .S ; filter oft' lead sulpiiide ; nearly neu- tralise filtrate with soda, and add concentrated mer- curic chloriclc solution Take 250 c.c. of urine ; add milk of lime and calcium chloride in excess to pre- cipitate pliosphates ; filter, and eva]X)rate to small bulk : to this add 50 c.c. absolute alcohol, and let mixtiuv stand six hours. Add 10 to 15 drops of alcoholic solution of zinc chloride lieactioii On cooling, hippurio acicl crystallises out from the filtrate. The crystals may be purified from benzoic acid by petroleum ether, which dissolves H. acid, leaving 1!. iU'id insoluble. Distil oft" petroleum ether from extract, and dissolve residue in water as before A smell of oil of bitter almonds is given off .1 precipitate of creatinine with mercuiic chloride is produce!} ; this is sus- pended in water ; a stream of US is passed through it; and the lead sulphide filtered off. The filtrate is decolourised with animal charcoal, and eva|iorated to a small bulk. The re- maining mass of creatinine hydrocldoride is crystal- lised out twice from strong alcohol, and the HC re- moved by boiling with lead oxyhvdrate Itcinarks There is very little hip- puric acid in human urine. It is abundant in the urine of horses and other herbivora Crystals (rosettes) of zinc chloride-creatinine form in the com-se of a y the action of ammonia on carbony] chloride, by the hydration of cyanamide, from ammonium carbonate, and by several other methods. Preparation from urine. — Urea was first pi-epared in an impure condition from urine by Rouelle, then by Fourcroy and Vauquelin.' The following methods are those now generally adopted : — (1) Evaporate the uriin' to a small bulk. Add strong, pure nitric uc.id in excess, keeping the mixture cool during the addition of the acid. Pour off the excess of fluid from the crystals of urea nitrate wliich are formed ; strain through muslin, and press between filter paper. Add to the dry product barium carbonate in large excess, and mix tiioroughly with sufficient methylated spirit to form a paste. Dry on a water-bnth, and extract with alcohol ; filter ; evaporate the filtrate on the water-bath, and set aside to crystallise. The product may be decolourised by animal charcoal and purified by reerystallisation. (2) Tlie following method is well adapted for the preparation of microscoisic specimens of urea and urea nitrate : Take 20 c.c. of urine ; add ' baryta mixture ' (two volumes of barium hydrate solution and one volume of barium nitrate solu- tion, both saturated in the cold) until no further precipitate is produced ; filter ; evaporate the filtrate to a thick syrup on the water-bath, and extract with alcohol; pour off and filter the alcoholic extract ; evaporate it to dryness on the water-bath, and take up the residue with w^ater. Place a drop of the aqueous solution on a slide, and allow it to crystallise ; crystals of urea separate out. Place another drop on another slide, and add a drop of nitric acid ; crystals of urea nitrate separate out. Properties of urea. — It is readily soluble in alcohol and in water, but not in ether. Its taste is saltish ; it is odourless, and neutral to litmus paper. It crystallises in silky four- sided prisms with oblique ends, or in delicate white needles, when rapidly crystallised (fig. 91). When treated with nitric acid, nitrate of urea (CON.^H^.HNO;^) is formed; this crystallises in octahedra, lozenge- shaped tablets, or hexagons (fig. 92«). AVhen treated with oxalic acid, flat or prismatic crystals of oxalate ofurea(CONoH^.H2C204-fH.,0)are formed (fig. 92 h). These crystals may be readily obtained in an impure form by adding the respective acids Fig. 91.— Crystals of Urea ; «, four-sided prisms : h, indefinite crystals, such as are usually formed from alcohol solutions. ' Anil dc cli'nn. xxxii. S(i. 3 A 722 EXCRETH >N to urine wliicli lias been concentrated to a third or a cjuarter of its bulk. Other compounds of urea w'nh acids have been also described ; thus phospliate of urea (CONoH^.H^POj) was said by Lehmann' to occur in small quantities in urine ; a compound of urea with uronitrotoluolic acid, with the formala C,4H,c,N30,o, was found by Jaffe" in dog's urine after the administration of orthonitrotoluol ; the greater part of the urea in urine is, however, free. Urea also forms compounds with salts ; the most important of these is with mercuric nitrate ; with this substance it forms a white precipitate, with the 0„^ Fig. 92.— f(, nitrate ; h, oxalate of urea, formula CON.,H4.Hg(N03)., + 3HgO. This compound is important, as Liebigs volu- metric process for the estimation of urea depends on its formation (see p. 810). Drechsel* has described a compound of urea with palladium chloride (PdCl., + 2COK„H4). There is also a crystalline compound of urea with sodium cliloride (COXoH .NaCl + H.,0), which may be obtained by evaporating to dryne,ss a solution of these two substances, such as occurs, for instance, in ordinary urine. Urea may be decomposed in various ways : — (1) When heated to l.">0° to 170° it melts, and gives off ammonia : the substance which remains is termed biuret * (2C0N„Hj -NH3 = C^OJS'jHj). Biuret [urea] [biuret] with caustic potash and copper sulphate gives a characteristic rose-red solution. AVhen biuret is heated it gives off ammonia, and cyanuric acid is left (■.SC.,0.,N H- — 3NH3 = 2C3H3N,03). Cyanuric acid gives a violet solution with [biuret] " [cyauuric acid] caustic potash and copper sulphate. (2) Bv means of an organised ferment, the torula or micrococcus ureae, which o-rows readily in stale urine, urea takes up water, and is converted into ammonium carbonate (CON,H, + 2H,0 = (NHJ.COJ. (3) Bv means of nitrous acid, urea is broken up into carbonic acid, water, and nitrogen, CON^H^ + 'S.,Oj = CO. + 2H.p + 2X.,. This may be used as a test for urea ; add fumino- nitric acid to a solution of urea; an abundant evolution of gas bubbles takes place. 1 Client. Centram. 1K(>(>, p. 1119. 3 Journ.in-nld. Chcm. X.F. xx. 409. - Zeit. jjJii/aiol. Chem. ii. 50. ■* Poggeiulorfis Annalen, Ixxiv. G7. TKEA 723 (4) Chlorine water anises a >omewhut similar decomjiosition (CON.H, + H,U -r 3CL, = CO, + N, + 6HC1 ). (5) Hypochlorite or hypobromite of soda decomposes urea in the followinj^ way: (COX,H, + 3N:iHrO = CO,+ X, + 2H,0 + 3NaBr). This reaction is impoitant, as upon it depends one of tlie best metiiods of estimating the quantity of urea in urine {stee p. 811). Quantity of urea in nrinr. — The (juautity of urea iu uiiiie varies a good deal, the chief cause of variation being tlie amount of pioteid food ingested. In a man who is in a state of eijuililjrium, and on an ordinary mixed diet, the quantity of urea secreted daily is between 25 and 40 grammes, the average being 33 grammes (.300 grains). On a diet poor in proteids it may sink to 1 5 to 20 grauuues, and in a diet rich in proteids it may rise to 100 grammes per diem. We have seen that the concentration of the urine varies considerably in health, and thus the percentage amount of urea varies also. It may be roughly said that the quantity of urea in normal human urine is 2 per cent. ; in dogs it may be 10 per cent. Women secrete rather less than men ; children absolutely less than adults, but in proportion to their weight more. Uhle gives the following table, which repre- sents the amount of urea secreted in twenty- four hours jjer kilo, of body- weight at ages— From 3-6 years . . . about 1 gramme „ 8-11 „ ... „ 0-8 „ 13-16 „ ... „ 0-4-0-6 „ Adults 0-37-0-6 „ The excretion of urea is usually at its maximum three hours after a meal, especially after a meal lich in proteids. The quantity of urea does not, however, necessarily depend on increased production of urea ; a long-continued increase in urea indicates increased tissue- metabolism, but a temporary increase may be merely produced by an increase of the urinary secretiim, by which the urea collected in the body is quickly passed off. In the same way diminished excretion of urea may be due either to diminished metabolism, or to retention of urea in the body, as in ura?mia. These considerations are especially useful in determining the influence of food on urea excretion. In the first place the urea does not come direct from the food ; the food must be first assimilated and become part of the body Ijefore it can break down to form urea.^ The urea is increased by food, first because food stimu- lates the tissues to acti%-ity, and so metabolism is increased ; and, secondly, stimulates the kidneys to acti^•ity, and so waste accumulated products are got rid of. 1 An exception to this rule is probably to be found iu the case of the amido-acids, especially leucine. (.See further under Metabohsm, p. 845.) :j A 2 724 EXCRETION Camerer ' carried out experiments on four person.-, each of whom partook of only one meat meal in the twenty-four hours, and whose urine was collected at intervals of three hours ; these samples were analysed separately, the urea-nitrogen and the extractive nitrogen (Le. total nitrogen minm urea-nitrogen) being estimated. The increase in both kinds of nitrogen commenced almost immediately after the meal, the urea-nitrogen reaching its maximum in from seven to ten hours, while that derived from the extractives was greatest in the first four hours after the meal. The quantity of urine was smallest during the firet four hours, and greatest seven to ten hours after the meal ; the least concentrated urine was that accom- panied by the secretion of the greatest total amount of urea. A large nunil:>er of ob.servations on the influence of other varj-ing conditions on the amount oi urea excreted, have been recorded, and may be tabulated as follows : — Circamstances prodncing An iiicrea.=e of urea A decrease of urea Administration of — Dilute sulphuric acid (Kurtz -). potas- siimi chloride (Dehn^), ammonium I salts, especially with food.^ small doses of phosphorus, arsenic, antimony, mor- phia, codeia (Giithgens ^), large doses \ of quinine (Oppenheim *). Poisoning by — Phosphorus (Storch,' Bauer *•), arsenic (KosseP). Application of cold to the skln(Voit '•). Hot baths (Schleich '•). Increase of oxj-gen inspired (Fran- kel '2). Excessive muscxdar work (xfe ]>. 43fi). Diseases :— At the commencement of acute febrile diseases, up to the acme of the fever. Dnrintr the parox]i-5ms of intermittent fever (ague). Administration of — Small doses of quinine (Oppenheim), In diabetes. Diseases : — During the sinking of the fever. In most chronic and debilitating diseases (anaemia, svphilis, phthisis, dropsical affections, &c.) Towards the fatal termination of most fliseases (.5 to 6 grms. daily). In ursemia ; thesecretion may entirely cease. In diabetic coma. j In an degenerative changes of the liver, especially in acute yellowatrophy. The formation of urea. — The formation of urea occurs through the ' Zeit. Biol. xxiv. 306. - Kurtz, Dhi. Dorpat, 1874. ^ Diiss. Rostock, 1876. * Hallervorden, Arch. ex. Path. u. Pharm. x. 124; Feder and Voit, Zeit. Biol. xvi. 177. ■• Quoted by MacMunn, Clin. Cheni. of Urine, p. 36. *■ Pfliiger's Archiv, xxiii. 446. '' Den acute Phosphorforgiftning, Copenhagen, 1875; Arch.f. hlin. Med. 1867, vol. ii. * Zeit. Biol. vii. 71. » Arch.f. exper. Pathol, v. 128. "^ Zeit. Biol. xiv. 57. " Dj«s. Leipzig, 1875. '- Arch.f.j>athol. Anat. Ixvii. 1 : Ixxi. 117. IRK A 725 whole of healthy extia-utcriiif, life. It is also formed in the fd'tus, but there its place is, ttt a large extent, taken by another substance called nllanfoiii. The important questions in lelation to the formation of urea are, first, where is it formed 'I and secondly, from what is it formed 1 Where is urea foi-nied i The older authors considered that it was formed in the kidneys, just as they also einmeously considered that carbonic acid was formed in the lungs. Prevost and Dumas' were the first to show that afte)- complete extirpation of the kidneys, the formation of urea goes on, and it accumulates in the blood and tissues. Similarly in those cases of disease, in which the kidneys cease work, urea still continues to )>e formed and accumulates in the body. If, then, the kidneys are not specially the seat of formation of urea, where is this special seat, or is there any special seat 1 If we look to the most abundant tissue of the body — the muscles — we find urea absent, or nearly so ; there can, however, be no doubt that some inter- mediate steps in the process takes place in the muscles.- In the muscles we find the place of urea taken by creatine ; some of this is undoubtedly excreted as creatinine. Whether some is further changed into urea is a matter of doubt, and has already been discussed in connection with muscle (pp. 419, 439). The liver is now generally supposed to be the chief place where urea is formed ; this view was originally put forward by Meissner ; ^ but, although contradicted by Gschleidlen,^ Munk,'* and Pekelharing,^ it is supported by the more recent experiments of Brouardel,^ Roster,** Schroeder,^ and Minkowski.^ It is, however, very probable that other cellular organs like the spleen, lymphatic and secreting glands par- ticipate in the formation of urea. The urea passes into the blood, is carried to the kidneys, and is there excreted. The facts of pathology point very strongly in support of the theory that urea is formed in the liver. Diabetes is sometimes a disease of the liver in which the metabolism of its cells is much increased, leading to an abundant formation of sugar which passes into the blood and urine ; and in these cases the urea is also increased. 1 Ann. lie chim. et de 'phys. xxiii. 90. Tliis observation has been since confirmed by many observers, e.g. Tieclemann and Mitscherlich, Poggendorfs Annaleii, xxxi. 303; Marchand, Journ. jjraM. Ghent, xxi. 260. ■ Selachian fishes form an exception to this rule. The kidneys appear to be sluggish and urea accumulates in the blood to an enormous extent ('2-1) per cent.) ; the muscles contain 1-9 and the liver 1-3 per cent, of urea (Zeii. phijsiol. Chnii. xiv. ."JTO). ■"• Zeit. rat. Med. N.F. xxxi. 234. •* Studien il. d. Urspriing den Harnstoffs, Leipzig, 1871. ■'' Pfliiger's Archiv, ii. 100. ® I^''^- V- ^'^'^■ "^ Arch, de physiol. norm, et pathol. (2), iii. 373, 551. ■^ Italian paper quoted by Hoppe-Seyler, Physiol. Chem. p. 807. 'J See pp. 727, 735. 72G ExcKpyriox In the opposite condition when degenerative clianges occur in the liver, we have a lessened formation of urea : this has Ijeen recently pointed out by Noel Pa ton,' who sliows that two functions of the liver, bile-formation and urea-formation, bear a direct relationship to one another. In excessive degeneration, such as occui's in acute yellow atrophy of the liver, the urea in the urine is very small, or may be absent, its place being taken by leucine and tyrosine. From what is urea formed ? Urea is formed from the proteid con- stituents of the body. The intermediate steps in the process are, how- ever, practically unknown ; the laboratory of the human body is very opaque, and it is difficult to find out much more than the beginning and the ending of many metaljolic plienomena. Chemists ha^■e not succeeded in oljtaining urea from proteids outside the body.- Creatine has been considerefl by some as an important intermediate product in the formation of urea : urea can be obtained artificially from creatine from the cyanarnide radicle which it contains {)iC(^ ]). 419). Uric acid also has been regarded as another of these intermediate products ; this is supported by the fact that urea can be artificially obtained from uric acifl, as will be fully described in connection with uric acid. It is, however, not regarded by physiologists as an import- ant precursor of urea in the body. When cyanui'ic acid (the i-elation of which to urea has been already described, p. 722) is administered internally, the urea in the urine is increased.'^ There is, however, no evidence that this occurs normally. The amido-acids, glycocine, leucine, and tyrosine, have also been, placed in the same category : there is no evidence that tyrosine acts thus ; injection of tyrosine into the circulation, or feeding with tyro- sine, produces no increase in the urea eliminated.' The introduction of glycocine and leucine, however, int(» the bowel, or into the circula- tion ()Salkowski), increases the amount of urea. No doubt these sub- stances are carried to the liver, and there the final transformation takes place. In acute yellow atrophy the appearance of amido-acids in the urine, in place of urea, lends some support to this theory. If ui-ea is not derivf-fl clirectly from jiinido-acids it may orioiiiatf from certain simpler substances, wliicli cither sprino- from tlic amirto-aciils or liave a common 1 Brit. Med. Journ. vol. ii. ISSC, p. 207. 2 The statement of Bechamp iAnii. de chiui. ct dc phifH. (:)), xlviii. :-i4Si, tluit he has siK'eeeded in obtaining urea hy oxidising albumin with potassium permanganate, has been disproved by Stiideler {Jouim. prakt. Chem.\-x.xV\. 2.51), Loew (Il/id. N.F. ii. 28!)), Tappeiuer {Siichs. Alifid. Bcr. 1871), and others. Bechamp, however, still maintains the c-orrectnesf- of his original statements {Conij^f. rend. Ixx. 8Gfi). 3 Coppola, Chem. Cciiir. vf)l. ii. 1889, p. 375. < Jaffe, Zeit-jihij-iiul. Chnn. vol. vii.; Baas. Ihid. ii. 48.j ; C'ohn, ILid. kW. 18!). vine ACID 727 origin with tlicni. I[(i]i|ii-Se_\lrr.' wlm.^c niiiiiiijii is of great wei.iilit in llicsc matters, states tluit tlicre are live possibilities regarding,' the origin ol' urea from simple decomiiosition iiroducts of proteids ; tliey are as follows : — (1) From ammonium carbonate : that urea may originate from ammonia and carbonic acid with loss of water [(NH,)oCO^ — 2H,X)--=C0N._.I-I,] was first advanced as a possibility by Schmiedeberg.'-' Tiiis, however, never occurs outside the bt)dy at so low a tomi)eratnre as tlio body-ti'niperature.and probably it also never occurs within the bod v. (2) From anununiuui carbamate ; this view has been advanced by Drechsel.* He has found traces of earbamic acid (amido-formic acid, CHaNO.,) in the blood, and has also obtained it by the artificial oxidation of glycocinc and leucine, and lastl}' by electrolysis he has produced small quantities of urea from anmionium carbamate. (3) From cyanic acid. AVe have already seen that J'lliiger's view of the con- stitution of a living proteid is that it contains cyanogen radicles (p. 115) ; wu have also seen that by heating urea, biuret and cyanuric acid are formed, so that it also contains the elements of cyanogen. This view, though theoretical like the others, has thus a certain amount of probability about it. We must suppose that either two molecules of cyanic acid and one of water luiite to form urea and car- bonic acid (2C0.NH + H.,0 = C0N.J-I, + C0.j), or that two molecules of cyanic acid and two of ainnumia unite to form two of urea (2C0.NH + 2NrI., = 2CON._,H.,). (4) From cyananiide (CN.NH.^). This is regarded by Hoppe-Sejder as highly improbable, and tluis lie gives no coiuitenance to the theory that urea originates from creatine. (5) From ethereal carbonates and ammonia. 'J'his view is also regarded by him as so improbable as not to merit discussion. From the foregoing it will be seen that Hoppe-Seylcr regards cj^anic acid as the substance which is most probably the antecedent of urea. Recent experi- ments by Schroder,^ however, point very strongly to the fact that ammonium carbonate is at least one of the urea-precursors. These observations may be briefly summarised as follows : (1) After excision of a dog's kidneys, the urea in the blood increases fourfold in twenty-four hours. (2) If blood mixed with ammonium carbonate is passed through the excised kidneys, the urea in this blood is not increased. (;i) If the mixture of blood and ammonium carbonate is passed through the muscles of the lower limbs, again there is a, negative result. (4) But if the mixture is passed through the liver, it will then be found to contain an increased quantity of urea. (5) If the blood from a fasting animal is passed through the liver, no urea is formed ; if the blood is taken from an animal during digestion, the urea is slightly increased, though not so much as when it is mixed with ammonium carbonate. (6) In cirrhosis of the liver, where the liver cells are injured by the pi-essure of new connective tissue, the urea in the urine is greatly diminished, while the ammonia is greatly increased. (7) The administra- tion of ammonium-salts with the food increases the quantity of urea in the urine. URIC ACID Uric acid (C3H4N4O3) is, iu uiammals, next to urea, the medium by which the hirgest quantity of nitrogen is e.Kcreted from the body. It is, however, in birds and reptiles the principal nitrogenous constituent 1 Physiol. CJiriii. p. SOS. - Afch.f. cxpcr. Path. viii. 1. 3 Joiiru. prakt. Choii. X.F. xii. 417 ; xxii. 47(). •* Arch. E.vpcr. Phariii. innl I'dth. x\. ;!tU ; .\ix i'.T!!. 728 EXCKETION of their uinne ; it has also Ijeen found in tlie organs of many inverte- brates that correspond to the vertebrate kidney ; e.g. the green glands of Crustacea,' the Malpighian tubes of insects, and the nephridiaof cer- tain molluscs.- It is more abundant in carnivorous animals than in man.^ In hei'bivora, though replaced to some extent by hippuric acid, it is, nevei'theless, fairly abundant.^ Prejiaration from urine. — If 5 c.c. of hydrochloric acid be added to 100 c.c. of urine, and the mixture be allowed to stand for twelve to twenty-four hours, crystals of uric acid separate out, and either fall to the bottom of the containing vessel, or adhei'e to its sides. These crystals are coloured dark-red by the urinary pigment, and may be obtained fairly free from it by repeated solution in caustic soda or potash, and re-precipitation by hydrochloric acid. If, however, one wishes to prepare pure uric acid, the solid lu-ine of a reptile or bird, which consists principally of the acid ammonium salt, should be selected ; one has not then to separate any pigment. It is boiled with 10 per cent, caustic soda or ammonia; diluted, and then allowed to stand. The clear fluid is decanted, and poured into a large excess of water to which 10 per cent, of hj-dro- chloric acid has been added ; after twenty-four hours, crystals of lu-ic acid are deposited. These may be purified by washing, re-solution in soda, and re-pre- cipitation by acid. Properties of uric acid. — Pure uric acid crystallises in colourless rhombic rectangular plates, or in rectangular prisms. In striking con- trast to urea, it is a most insoluble substance, requiring for its solution ^.^y^ 1,900 parts of hot and 15,000 parts of cold ^,^^^^^^=5ss^ y — . *^~^--^ water. It is very slightly soluble in alcohol and ether. The urates are also very in- soluble substances. The precipitate of uric acid obtained in cases of gravel, and also that produced by the decomposition of urates which occurs when acid is added to the urine, is always deeply tinged red c^r brown by urinary pig- ment, the deposit having a cayenne pepper- _^^ m. JWU. like appearance. The forms which ui-ic acid ■PP ^^ "^P <^^^ assumes under these circumstances are very various, the most frequent being the whet- FlG. 93. rrie Aoi.l Crystal.. ^^^^^ ^i^.^^^ . ^j^^^.^ ,^^^ ^^^^ sheaf-like Or baiTel-shaped collections of needles ; some of the bundles take the form of dumb-bells (see tig. 9-3). 1 Griffiths, Chcm. Ncns, li. 121. - MacMunn, Journ. of Physiol, vii. 128. ■"' But not universally. Sec Sanarelli, Cheni. CentraJhh 1887, p. 804. ■* Mittelbach, Zcit. physioJ. Chem. xii. 403. luic ACID 729 Uric :ici(l has been prepared synthetically by Hnrbaczewski ' by heatinor fflvcocine and urea together (C,H.-,N02 + 3CON2H4 = C.5HjN^O:, + 3NH3 + 2H20) ; and by Behrend and Roosen"^ by [uric acifl] other methods. Its constitution is, however, but little known ; and many various views are held, the latest being that it is a derivative of acrylic acid. It, however, appears, undoubtedly, to contain cyanogen radicles. The murexide test and SchiflTs test liave been already described (p. 718). Uric acid reduces alkaline solutions of cupric .salts on boiling ; before .--eduction occurs, howe^-er, a white precipitate (a compound of uric acid with the copper) is first formed. Uric acid is dibasic. Decompimtions of urir or and urea and oxalic acid are formed : 2C4H„N A + •iH.,0 + O, = iCON.,H , + 2C.,H.,0 ,. [allantoin] [urea] [oxalic acid] (5) The following decompositions are interesting, as the mnrexide test is the chief characteristic test for uric acid. By oxidation with nitric acid, alloxan and urea are formed : 2C5H,NP3 + 2H„0 + 0., = 2CjH.,N.,0j + 2C0N.,H,. By heating or by electrolysis, alloxan splits into alloxantin, parabanic acid, and carbonic acid : 3C,H„N„0^ = C,H ,N ,0; + C:,H,N.,0, + CO., ; [alloxai,] [alloxautin] [parabanic acid] and on treating alloxantin with annnonia the purjjle colour due to murexide or purpm-ate of ammonia appears : C,H ,N^0, + 2NH, = CsH,N,0, + H.O. [alloxantin] [mnrexide] Compounds of uric acid. — Uric acid is dibasic, and thus there are two classes of urates, the normal urates and the acid urates. A normal urate is one in which two atoms of the hydrogen are replaced by two atoms of a monad metal like sodium (C5HoNa2N403) ; an acid urate is one in which only one atom of hydrogen is thus replaced ; the acid urate of sodium has, therefore, the formula C,5H3NaN4()3. The urates of the alkalis are those which are obtainable from the- urine. The mo.st abundant urate obtained from human urine is the normal sodium urate ; small quantitie.s of those of potassium and cal- cium also occur. The acid ammonium urate is the chief constituent separable from the excrement of birds and reptiles (tig. 95). The urates, like uric acid, are insoluble substances, and hence if excess occurs in the urine, they will be precipitated when the urine cools, after it is passed. This will especially occur if what is called the acid fermentation takes place, the acid urate of sodium being much more insoluble than the normal salt. The reaction that occurs may be thus represented : ri;ic \(ii» TBI ■2C5H2Na,N403 + J I ,U + C( K=2C^l l.,N,iN .Oa + Na,CO;,. [iioniiiil soilimn l:u'h\ sdilinm unite] [sf)(linui ;irntf] carboimtf] Tlie acid sodium urate (Mg. 94) is, indeed, the chief component of the pinkisli deposit of urates (often called lithates) that occurs in con- centrated, cold, acid ui-ine. This deposit, sometimes called the Jateri- tioifs deposit, from its resendjlance to brick-dust, is generally amorphous, or only partly crystalline. The pink colour is derived from the urinary pigment, and is called uroerythriu. This deposit can be readily distin- guished from other urinary sediments V)y the fact that it dissolves upon warming the urine to the temperature of the body. A certain amount of calcium oxalate crystals (octahedra) will often l)e found mixed with the urates. The close relationship of uric and oxalic acids is apparent from the forniube on pp. 729, 730. Fn;. 04.— Aeiil Sculiiiiii I'ratc. Fii;. 1)5. -Acid Ainmoiiium Uratf. The following table ' gives concisely certain facts relating to the urates : — iluliilitj' in watur ])ciHi>itiMla.s Acid ammoninm urate Normal sodiiiiii ,. Acid sodium ,, Normal potassium ,. Acid Normal calciuiu ,. Acid Acid lithium ,, C,R,N,,0,.(NHO C,H..N,0,.Na., C,H,N,0.,.Na C,H,N,0,.K, C-,H,N,0,.K 0,H.,N,0.,.Ca (C,H3N,0,),Ca C,H.,N,0.,Li 1 in IfiOO Amorphous or spiked globular masses Nodular masses Amorphous ; rarely erystalline Amorphous : or in fine needles 1 „ 800 1 ,, 1.500 Fine granules 1 ,. COO Amorphous ; or in fine needles 1 „ tiO Ditto 77 1200 4-1 The greater soluljility of potassium and lithium urates has led to the adminis- tration of potash and lithia in cases where uric acid or ui'ates are in excess in the urine, and it is desired to dissolve them up. The calcium urates occur in mere traces in lu'ine, birt have been found in gouty deposits, in addition to sodiumj urates {are p. .510). 1 Balfr's msraftrs of thr Kidiioj!^, ISS,', p. SI. 732 EXCRETION Sir W. Roberts ' lias recently investigated the urates of the urine, and the following is a ri^sume of his paper : ' The presence of uric acid in human urine is somewhat anomalous. As a vehicle for the elimination of nitrogen, it is not needed. Its place is taken by urea, which, by its easy solubility, is better adapted to the liquid urine of mammals. Perhaps uric acid is a vestigial remnant in mammalian descent. But, although physiologically insignificant, ui'ic acid is pathologically the most prominent component of the urine, this being chiefly due to its tendency to form concretions. ' All acid urines tend inevitably to depo.sit their uric acid sooner or later. The time of onset of precipitation varies from a few hours to five or six days, or even longer. The inference from this is that patho- logical gravel is due to an exaggeration of conditions which exi.st in a less pronounced degree in health. To get at an explanation of this spon- taneous precipitation it is necessary to examine the states of combina- tion of uric acid in uiine. ' Uric acid (C5H4N403= H._,U) is a bibasic acid, and forms two regular orders of salts, namely, ne,utral or normal urates (M2U) and acid urates or hiurates (MHlJ).^ But in addition to these it forms a series of hyperacid combinations, first discovered by Bence Jones, and tenned by him qiuidrurates (WHM .^.^). The neutral urates are never found in the animal body, and are only known as laboratory products. The biurates are only encountered pathologically as gouty concretions. The quadrurates, on the other hand, are especially the physiological salts of uric acid. They constitute the exclusive combination in which uric acid exists in solution in normal urine, and they become visible some- times as the amorphous urate sediment. The urinary excretion of birds and serpents is composed exclusively of quadrurates. The quad- rurates can, moreover, be formed artificially under conditions which prevail in the animal body. The special and characteristic reaction of the quadrurates is that they are immediately decomposed by water into free uric acid and biurates. ' They exist in acid urine in the presence of water and of super- phosphates. These conditions necessarily involve the ultimate libera- tion and precipitation of uric acid. The first step is the breaking up of the quadrurate by the water of the urine into free uric acid and biurate according to the following equation : — (MHU.H.,IJ) -f- H,0=(H2U) + (MHU). [(luailripn'^.-] [free uric a<-iil] [liinrate] ' This explains the liVjeration of half the uric acid. But the biurate 1 Proc. Med. Chir. Soc. 1«'.)0, p. K.".. - In these formula;, the sjTiibol M represents a monad metal, and the symbol U, the radicle C-^B.^'S^O^. line ACID 7;{;{ thus formed is fortliwith chuugt'tl in Liu; presence of si4K!X'plio.sph;ites into quadrurate. Thus : - 2(MHU) + (MH,,P04)=(MHU.H2U) + (M,HP(),,). [biiirato] [siiiicriiliosiiliiitc] [(luailniriitf] [diiMctallic iiliii>iil]iitc] By these alternating reactions all the uric acid is at length set free. * Seeing that uric acid exists in acid urine (that is, for some sixteen hours out of the twenty-four) amid conditions which, if the quadi'urates stood alone and uncontrolled, would lead to its immediate precipitation, and yet that in the normal course no such early piecipitation occurs, it is obvious that the urine must contain certain ingredi(!nts which in- hibit or gi-eatly retard its water from breaking up the quadrurates. These inhibitory ingredients consist chiefly of (1) the mineral salts, (2) the pigments of the urine. 'The conditions of the urine which tend to accelerate the precipi- tation of uric acid, as in the formation of concretions and deposits, are (1) high acidity, (2) poverty in mineral salts, (3) low pigmentation, (4) high percentage of uric acid. The converse conditions tend to retard precipitation. On the interaction of these factors the occur- rence or non-occurrence of uric acid gravel appears to depend, and pi'obably the most important of these factors is the grade of acidity.' Q,U(intit]] of uric acid in the divine. — The quantity excreted by an adult man varies from seven to ten grains (O'o to O'TT) gramme) ; during hunger it sinks to four grains. ^ Parkes gives the average per- centage of uric acid in human urine as 0"03 to 0"05, and the pro2:)ortion of urea to uric acid as 45 : 1. Haig- states that the excretion of uric acid is much affected by food, being' greatest during the ' alkaline tide ' that follows a meal. He regards this as chiefly a washing out of the uric acid accumulated in the liver and spleen in the period between meals (acid tide) ; but there can be little doubt that, as the activity of these organs is increased after food, increased metabolism in general and increased production of uric acid in particular also take place. Certain morbid conditions increase, certain others diminish, the excretion of uric acid ; the appearance of a sediment of irrates, however, does not necessarily mean an increased formation of urates ; it may be due to increased concentration of the urine. A deposit of urates occurs when the urine is concentrated, as after violent exercise, and consequent profuse sweating ; it also occurs in the concen- trated urine of fever ; after indigestion, inflammation of joints, in certain heart and lung affections, in cirrhosis of the liver, and occasionally in catarrh of the bladder where the acid fermentation is taking place. Free uric acid in crystals occurs in the urine of person.s with a uric acid diathesis ; a condition allied to 1 Estimations of uric acid secreted in health have been made by Becquerel {Gmelin's Handh. viii. 327), H. Eanke {Ansscheidung der Harnsaure hcim Menschen, Miinclien, 1S58), Neubauer [Text-hool-, p. 3H1), J. Ranke {Grundzi'ige der PJiijsioI. Leipzig), Beneke {Pathol, des Stoffwecliseh, Berlin, 1874), and many others. - Juurii. of PhijsioL viii. 211. 734 EXCKETION but fee p. 307). It is also important to remember that the formation of urea, uric acid, and similar substances is, as Pfliiger points out, very l;irgely synthetical, as the number of nitrogen atoms they contain in proportion to carV>on atoms is greater than in proteids- (see pp. 115, 544). XANTHINE This substance has the formula C^H^XjO, ; that is, it contains one atom of oxygen less than uric acid. Its chemical characters have been already described (p. 89), and the method of prei^aring it from urine is the same as that already described in connection with extracts of muscle (p. 421). A large quantity of* urine must, liowever, be taken and evaporated down, for Neubauer found onlj- 1 gramme of xanthine in 300 litres of normal human in-ine. It was first described in urine by Bence Jones,' and occasionally occurs there as a crystalline sediment, and in certain rare forms of urinary calculus. Diirr and Strohmeyer state that its amount is increased in the urine by sulphur baths. Its nitrate and chlorate have characteristic crystalline forms. HYPOXANTHINE This substance (C^H^N^O) is a third member of the same group, and contains one atom of oxygen less than xanthine. It is sometimes called sarcine. This substance, which has been already fully described (pp. 90 and 421), is absent in normal urine, but appears in the urine of cases of leucocythiiimia. GUANINE This substance has the formula CjHjN^O, and is thus closely related to the preceding (^see also p. 90). It is absent from human urine, but occurs in guano and in the excrements of spiders, in the organ of Bojanus of the mussel, and the green glands of crustacea (Will and Gorup-Besanez '-). ALLANTOIN This substance, which has the formula CjIIgN^Og. can be obtained artificially from uric acid (p. 729), and has been prepared synthetically hj Grimaux ■' by heating together a mixture of glyosylic acid and urea(C.H.,03 + 2C0X.^H^ = C^H^N^Oj + H.p). It crystallises in colourless prisms which are soluble in hot water, slightly soluble in cold water, and insoluble in alcohol or ether (fig. 40, p. 90.) It is precipitated from its solutions by mercuric salts. It may be separated from urine by precipitating with lead acetate, filtering, passing sulphuretted hydi-ogen through the filtrate, filtering again, evaporating the final filtrate to a syrup, and letting it stand for several days. Allantoin then crystallises out. It occurs in mere traces in normal human urine, except directly after birth, but is increased by a flesh diet, and increased after the administration of tannic acid.^ It was first described in the amniotic fluid of the cow by Vauquelin,^ then by ' Quarterly Journal Chejn. Spc. xv. 78. - For references to literature see Weinlancl, Zeit. Biol. xxix. 300. •" Comjit. rend. Ixxxiii. 62. ■* Kiihler and Schottin in Lchinann's Handh. 1«59, p. 93. •'■' Ann. cle chim. xxxiii. 269. ri;ic ACIJ), AMI Ai.i.ii'.i) sii'.s'iANcKs 737 Las.siigm-' in the alliiiitoio lliiid of the saniu aiiiiual ; there is little doubt that it owes its origin here to the fu'tal urine. It was found by ^^'6hler■- in the urine of new-born calves, and since then in the urine of new-born children and other animals by many observers. Salkowski" found that it, together with urea and oxalic acid, is increased in the urine of dogs liy the a(biiinis(ratii)n of uric acid. OXALUiaC ACID This substance, which has the formula CaH|N/)|, is an oxidation product of uric acid, and was stated to occur in traces in combination with ammonia in luMuan urine by Schunck.* Hoppe-Seyler,'* however, regards it as possible that it nuvy be formed during the analytical processes required to separate it. CREATININE The chemical characters of creatinine (C^H.NgO^ creatine ~H„0) have been . 01. 5 Ber. d. deutsch. chem. Ges. ix. 71!) ; xi. 500. * Proc. Bay. Soc. vi. 140. * Fhi/siol. Chcm. p. 810. 8 Ann. Chem. Pharni. cviii. 354. For recent researches on this question, see G. S. Johnson, Proc. liuij. Soc. xlii. 365, xliii. 493. ' Arch. path. Aiiaf. xlviii. 358. 8 Deutsche Klinik, 1802, p. 209. » Zctt. rat. Med. (3), xxiv. 100; xxxvi. 225. '" Physiol. Chem. trans, by Wooldridge, p. 3'J7. " Arch. 2>ut]i. Anat. xviii. 422. '- Wien. mcd. Wochensch. 1803, No. 22. '^ Ber. dcufsvh. chem. Ges. v 578. " Arch. path. Anat. Ixix. 354. '-^ PjiiUjcr's .irchic, xv. 350. 3 E 738 EXCRETION CHAPTER XL THE AIWMA'JTC SUBSTAXCJJS IX UBIXE The aromatic .substances which occur in urine belong to four classes : — (1) Hippuric acid, and similar aromatic compounds of glycocine. (2) Combinations of glycvironic acid with aromatic substances. (3) Aromatic oxy-acids. (4) Ethereal sulphates. HIPPUKIC ACID This substance is chiefly of interest, as it is one of the best instances of synthesis occurring in animals. The method of prepaiing it from urine has already been given (p. 719), and its relation to aromatic bodies has been also briefly described (p. 77). It appears in human urine in abundance after ingestion of benzoic acid, of other aromatic substances related to benzoic acid, or of vegetable tissues containing such substances. Its large amount in the urine of herljivora is thus due to the benzoic acid in their food ; the benzoic acid taken into the body unites with glycocine to form hippuric acid and water. C^HgOg + C2H,N0.2=C9HyN03 + H,0. [benzoic aeiil] [glycocine] [liiii[iuric aclil] Hippuric acid occurs in the urine to a small extent, however, even in starving animals, and is tlius a product of the metabolism of the animal tissues. A third .source of hippuric acid is as a result of putrefactive processes in the alimentary canal. The pro- ducts of putrefaction are partly alis(jrbed, and pass thence to the urine ; an intermediate .stage in tlie formation of hippuric acid in this way is phenyi propionic acid (Salkowski, ' Tappeiner ^). Properties of hippuric acid. — It is a monoba.sic acid, which cry.stallises in transparent, colourless, four-sided prisms (fig. 96). It has a bitter taste, but no smell. It is readily soluble in hot alcohol or ether, but only slightly soluble in water. Under the action of '■ Ber. dent sell, chcni. Ges. xi. 500. - Zeit. Biol. .vxii. 236. Till-: AROMATIC yrj{.STAN(KS IX IJUNK 739 mineral acids it takes up water and splits into its components, jsiol. Chem. vii.) by feeding animals with tyrosine found no increase of aromatic or any other substances in the urine. Intravenous injection of tyrosine was followed by a similar negative result (Cohn, Ibid. xiv. 189). THE AROMATIC SUBSTA^X'ES IN VU^E 74S After the medicinal or surgical use of carbolic acid the amount of the phenol- sulphate in the urine is increased ; two substances are also formed by the breaking up of carbolic acid, called pyrocatechin and iij'droquinon. 'J'hese become in alka- line urine dark brown on exposure to the atmosplieric oxygen, and it is this that liroduces the well-known colour of the urine in so-called ' carboluria.' C'rfsol sulj/Juitc of potassium. — This is not so abundant as the phenol-salt, but may be obtained in the following way from the urine of an lierbivorous animal like the horse: 3 or 4 litres are evaporated to a syrup; this is extracted with absolute alcohol, filtered, and the filtrate precipitated with an alcoholic solution of oxalic acid ; tlic precipitate is removed by filtration, and the filtrate made slight!}' alkaline with caustic jiotash. This produces a precipitate, which is liltered off; the filtrate is evaporated to a thin syrup, which is then kept at a temperature of 0° C. Leafy crystals of the potassium cresol sul])hate separate out, and may be purified by recrystallisation out of absolute alcohol (Krukenberg'). The formula for cresol is CjH.(HO) ; that for the potassium sulphate of cresol is OjHjO.SOjK. Cresol, like phenol, gives a red colour with Millon's reagent, but no violet colour with ferric chloride. Of the three isomerides called cresol, that known as paracresol is the most abundant ; ortho- and metacresol also combined as sulphates are present in mere traces. (The meaning of the prefixes ortho-, meta-, and para- in connection with aromatic substances is explained on p. 76.) Some of the paracresol formed in the intestine is further changed into phenol and excreted as the phenol-sulphate of potassium (Baumann). CatecJiol-siilphate of jJotassiuin. — Catechol or pyrocatechin, CgHgO.^, has two isomerides, named hydroquinon and resorcin. These were first found as ethereal sulphates in the urine of dogs after they had been fed on dihydroxyl <_;ombinations of benzene.^ This salt of pyrocatechin has since been found normally in traces in human urine, especially in children,^ but more abundantly in that of the horse. It becomes darker when the urine putrefies. This, how- <'ver, is only apparent when catechol appears in abnormally large quantities (see Carboluria, Alkajatonuria). Protocatechuic acid both free and as an ethereal sulphate is also present (Baumann,' Preusse"'). Tndoxyl- sulphate of potasslnw. — -The pai'ent of this substance, named indole (CgH-N), is formed in the intestine ; indoxyl, a radicle Klerived from this, has the formula CgH^NO ; this united with SO.jK forms the indoxyl-sulphate which is found in the urine (CgHglSrO.SOgK). This substance forms white glancing tablets and plates, easily soluble in water, less so in alcohol. By oxidation, indigo-blue is formed from it (2CaH6KNS04 + 02=2C,H5NO + 2HKS04). [iiidox. sulpli. of potassium] [iiicUgo-blue] [jiot. liyd. sulpliate] The indoxyl-sulphate of potassium has received the unfortunate name of indican, under the mistaken idea that it is identical with plant indican. This latter substance is a glucoside, and only resembles the indican of urine, in that (me of its decomposition products is ' Grundriss der med. chem. Anat. p. 87. - Baumann and Herter, Zeit. physiol. Chem. i. 244 ; ii. 335. ■"' Ebstein and Miiller, Arch. path. Anat. Ixii. 554; Furbringer Berlin. Min. Woch. 1875, Nos. 24 and 28; Fleischer, Ibid. Nos. 39 and 40. * Pfiiiger's Archiv, xii. 63; xiii. 16. ' Zcit. ph^ysiol. Chem. ii. 329. 744 EXCRETION indigo blue {see pp. 78, 79). The following methods have been devised for obtaining indigo fi'om urine : — (1) Jatfe's method' : — Equal parts of urine aiul hydrochloric acid are mixed; to this mixture a few drops of a saturated sohition of 'bleachinu- powder' are added cautiously till the maximum of blue colour appears. Tlu; mixture is then agitated with chloroform, which takes up the blue pigment ; on evaporating off the chloroform the indigo is left. If this is weighed an approximate quantitative estimation of the amount of indigo in the volume of urine originally taken can be made. Albumin, if present, must be separated before performing this test, as it develops a blue colour with hydrochloric acid. (2) MacMunn's method- : — Equal parts of urine and hydrochloric acid with a few drops of nitric acid are boiled together, cooled, and agitated with chloroform. The chloroform is generally \iolet, and shows an absorption band before D, due to indigo blue, and another after D, due to indigo red {.see fig. 97, spectrum 4, J). 748). Tliis method is preferable to Jaffe's, as 'bleaching powder' destroj's small quantities of indigo. The quantity of indigo in the urine of starving dogs was found by Halkowski '^ to he 4 to 5 milligr. in three days. After a])undant meat meals it rose to 16 to 17 milligr. per client. In loOO c.c. of normal human urine, Jafie found from 4 to 19 milligr. of indigo. Horse'-s urine contains twenty-three times as much. The greater abundance of indigo, like that of other aromatic substances in the urine of herbivora, depends on the diet. It is absent in the urine of new-born children.^ When indol is injected under the skin, or given by the stomach, it apj^ears in the urine as indoxyl sulphate of potassium (Jatfe,-^ Nencki and Masson,'^ Christiani") This salt is also increased in the urine in intestinal obstruction, peritonitis, typhus, cholera, cancer of the liver, long-standing suppuration, and Addison's disease. No doubt in many of these cases this is due to increased absorption of putrefactive products. Many aromatic drugs, like turpentine, oil of bitter almonds, and creosote, also increase its amount in the urine. Sometimes urine shows when decomposing a bluish red pellicle of microscopic- crystals of indigo blue and red, owing to the decomposition of the indoxyl- sulphate (Hill-Hassal, Stirling). A calculus composed of such crystals has been rnce described (Ord). Skatoxyl - sulpliate of pota>i.sium. — 8katole is methyl-indole CjjHg(CH3)X. Like indole, it is foimed 1 )y the putrefaction of proteids in the intestine ; some of it is absorbed, and passes into the urine as the skatoxyl-sulphate of pota.ssium (CgH^NO.SOaK). It is rather- more aljundant in human urine than the indoxyl-salt.'' 1 F/liUjir'a Arcliir, iii. 44S. - Clinical Client, of Urine, p. 97. ■' JBe?\ d. dctitsch. chem. Ges. ix. 138. ■• Senator, Zeif. jjJii/siol. Chetn. iv. 1. 5 Centr. med. Wiss. 1872, No. 1. « Malij's .Jahresb. 1874, p. 221. ^ Zeit. iiliysiol. Chem. ii. 273. * G. Hoppe-Sej-ler, Zeit. phtjHiol. C/teiit. xii. 1. F.Hoppe-Seyler, however, states that he lias found gi-eat variations in the relative and actual amounts of both salts without assignable cause {Physiol. Chem. p. 84(5). TIIK AROMATIC SlllSTANCKS IN I1{1NK 745- Ottu' obtained a ivcl pifinient wliicli he considered to be formed from the skatoxyl-sulpliate as indigo is from the indoxyl sulphate. Mester,'- however, who fed a dog on skatole, found only ti'accs of the skatoxyl salt in the urine,' but abundance of the skatoxyl-pigment. He gives certain reactions of this pigment, and considers it identical with those previously described under the names of urorubin, urorosein, uroerythrin,&c. In the urine it exists as a chromogen of un- known nature, perhaps a compound of skatoxyl witli glycuronic acid, analogous to indoxylglycuronic acid. On adding mineral acids to the urine containing it, it became red or reddish violet, especially on waruiinii-. The pigment is probably an oxidation product of the chromogen. The following table (adapted from Hoppe-Seyler) will be found convenient for the separation of some of these substances : — Urine is evaporated to one-third of its volume, and shaken with takes up pyrocatechin and hydroquinon jther ; this A. The ethereal extract Evaporate off the ether, and dissolve residue in water. Add lead acetate. This produces a precipitate. Filter this off Precipitate Dissolve in water ; pass a stream of sulphuretted hj'- drogen throutrh it to preci- pitate lead. Filter off the lead sulphide ; concent rate the filtrate, shake it with ' ether ; evaporate the ether from the ethereal extract. ' Residue = Pyrocatechin Filtrate S'epai'ate lead as before. Shake final filtrate with ether ; evaporate ether from ethereal extract ; the resi- due is Hydrochixon B. The residue after extraction with ether. Render acid with sul- phuric acid ; put it into retort and distil. In the distillate Phenol will be found Thc' following table gives in a concise way some important reactions of these substances : — • Suljstancc sought Test Reaction Remarks Pyrocatecliiii . This may be obtain al from urine directly by evapo- rating to a sjTui\ extract- ing with.aicoliol, evapo- rating the alcohol from the extract, and taking up the residue with ether. Evaporate off the ether from the extract, and take up residue with water It is this substance and hydrochinon which give | to alkaline urine a dark colour at the surface (due to oxidation ). It is present in abundance in ' carboluria ' 1 Vflilger's Airhiv, xxiii. G14. - Zeit. plnjisivl. C'hciii. xii. 130. •^ Using G. Hoijpe-Seyler's method of isolating these substances {Zrif. physiol. Chem* vii. 423). 746 EXCEETION Substance sought Pyrocatechiii Hydrochinoii Oxy-acids All J weak ferric chloride Render the solution alka- line Add lead acetate Will be found in the ethereal extract, made as in preceding table It is coloured green, which passes into violet on add- ing acetic acid and am- monia On exposure to the air it becomes yellow, then brown, or even black It is precipitated A watery solution, with ammonia added Is coloured Ijrown Sulilimcd Obtained by shaking urine made acid with strong mineral acid witli ether, evaporating off the ether from the extract, and taking up the residue with water. AddMiUon's reageut 500 c.c. of urine are treated with excess of bromine water Distil urine acidulated with sulpluiric acid Ilemarks Yields an indigo-blue sub- limate An intense red colour A turbidity appears^ at first, passing on standing for several hours into a distinct yellow precipitate of tribromophenol Warm the distillate with Millon's reageut Add ferric chloride to the distillate Decolourise urine ^vith animal charcoal ; dip a pine chi]) in hydrochloric acid containing a little potassium chlorate, and moisten it with the urine Phenol will be found in the distillate ; add bro- mine water ; a precipitate of tribromophenol ap- pears A cherry red colour Cresol gives the same reactions except that with ferric cliloride. The method of separating it from urine is given on p. 743 A deep violet colour It turns blue in sunliglit 747 CHAPTER XLI THE PIGMEXTS OF THE UlilNE The pigments of the urine have Ijeen described under different names 1)y different observers, and in the followino; account of them I shall follow MacMunn ' very closely. It will also be convenient here to describe, not only the normal pigments, but also those occurring in disease. XOrniAL UROBILIN This is the principal colouring matter of normal urine. It may be obtained from the urine by adding neutral and then basic lead acetate until there is no further precipitate. The precipitate consists of the chloride, sulphate, and urate of lead, and it carries down with it most of the pigment ; it is filtered off ; the filtrate is clear and almost colourless. The pigment is extracted from the precipitate by alcohol ncidulated with sulphuric acid ; the extract is filtered off from the remainder of the precipitate which is insoluble in this reagent. The ■extract has a deep yellowish colour ; it is agitated with chloroform. This reagent dissolves out the pigment, which is obtained in an approxi- mately pure condition by evaporating the chloroform from the chloro- formic extract. Normal urobilin thus obtained is amorphous, yellowish brown in colour, freely soluble in alcohol, chloroform, acids, acidulated water, and partly soluble in ether and benzene. An acid solution of it shows spectroscopically one absorption -band close to and enclosing the F line. If the solution be made neuti'al by alkalis, the band disappears. If the absoi'ptioii spectrum of normal urobilin (fig. 97, spectrum 1, p. 748) be compared with that of choletelin (fig. 88, spectrum 2, p. 685), it will \>e found that the two are practically identical. It would, however, be premature to say that the two substances are identical, until spectro- scopic analysis is supported by other analytical methods. When normal urobilin is separated out and dissolved in alcohol, and treated with zinc chloride and ammonia, the solution shows a green fluor- escence, which, however, is not nearly so well marked as that obtained ' Clinical Chemistry of Urine, pp. 104-112. 748 EXCRETIOX by treating a solution of hydrolnlirubin in the same way. The spectroscopic appearances are also different. Spectrum 4 (fig. SS) gives the absorption -bands of hydrobilirubin after this treatment ; the band at F becomes narrower, and shifts nearer to the b line, and there are two other bands near the C and D lines respectively. The spectrum of urobilin, treated with zinc cliloride and ammonia, is very similar. The band at F shifts nearly to the b line, becoming somewhat narrower at the same time, and two new bands at the red end of the spectrum make their appearance. They are fainter and narrower than the similar bands just described in connection with hydrobilirubin, and they have a slightly different position. Origin of normal urobilin. — The theory formerly advanced as to the oridn of normal urobilin Avas that bilirubin entering the intestine with BC D' E5 F Gr p: ^J I ' ^ \ j YiCr. 07.— I, Ali~i'r|iti.iii--|'.rti-iiiii Ml iHiniml Unil'iliii ; intili- 'i -[iirlt and snlpluiric acid extract from the precipitate- obtaiiU'il by treatiuar normal urine witlineutral ami basiclead acetate ; 2. Absorp- tion-spectrum of pathological urobilin : solution prepared in the same way : 3. Absorption-spec- trum of urohsematoporphjTin : solution prepared in the same way ; 4. Indigo blue and indigo red from normal urine. The urine was boilel with an equal bulk of hydrochloric acid, and when cold asitated with ch'oroform. The same spectnim may be obtained by treating the urine by .laffe's metliod and agitating ^v-ith chloroform. The bluer the chloroform, tlie ilarker is the band before D ; the nearer it approaches ral the d.arker that after D. A third banil at F may be also- seen, due to lu-obilin (after ilacilunu). the bile was acted upon by nascent hydrogen generated by putrefac- tion processes, and that a reduction product was formed, which Maly ' considered was identical with one which he had prepared artificially from Ijilirubin by the action of sodium-amalgam, and which he called hydrobilirubin. The name stercobilin was given to the pigment of the fjeces by Vaulair and Masius.^ It was further supposed that the pig- ment of the faeces was in part absorbed from the alimentary canal, carried to the kidneys and there excreted. Hydrobilirubin, sterco- l)iliu, and urobilin wei'e thus considered to be different names for the 1 Aim. Chcm. Pliariii. clxi. 308; clxiii. 77. 2 Centralhl. rneih Wiss. 1871, No. 24. Till-: I'lCMKNTS dl" IIIK IKMNK 74U same pimiUMit ; but recent speetioseopic n'scaivli lias sliown dilTerenees Ijetween them (>•»'' also p. ()U7). MacMunn is iiiflincd to regard the foniiatiun uf iiminal urobilin rather as the result of oxidation processes by means of the nascent oxygen in the intestine or elsewhere in the body than as due to reduc- tion processes. This view is based chiefly nn the fact that by the acti(»n of hydrogen peroxide on acid htvniatin, he is able to prej)are an artificial product which shows tlie same spectroscopic ajjpearances as normal urobilin.' Hoj^pe-Seyler - had pre^•iou sly prepared an artificial urobilin from hiemoglobin, and also from hjematin, by the action of tin and hydrochloric acid. AVhether stercobilin and urobilin are to he looked upon as products of reduction or oxidation must, therefore, still be regarded as unsettled. The most important point to notice, how- ever, is that urobilin may originate either from bile-pigment or from blood-pigment. We have seen that stercobilin and urol)ilin are different spectro- scopically. The question next arises, are they different also in origin, or is urobilin simply stercobilin which has l:)een some nv hat changed in the processes of absorption and excretion ? This question cannot be answered positively ; there are, however, certain facts which seem to point to the conclusion that the processes that form the two pigments are, to a certain extent at any rate, independent of one anothei-. These facts are as follows : — (1) In animals with a l)iliarv fistula, no bile enters the intestine; still the urine contains urobilin. (2) In Copeman and Winston's ^ case of biliary fistula in a woman, in whom no bile entered the intestine, and whose f;eces were uncoloured by stercobilin, the urine still contained urobOin. (3) Some cases recorded by Mott (see p. 552) seem to locate the formation of normal urobilin in the liver. For when the destruction of red corpuscles is excessiAe in the portal circulation, the li\ er con- tains more iron than usual, and the iron-free residue of ha-moglobin appears in the urine as urobilin in abnormally large quantities. The quantity of uroljilin in the urine seems tt.) be increased by oxidation, for instance, by a little dilute potassium permanganate, by hydrochloric or nitric acid, or by the occu)rence of the acid fermentation. It is supposed that the greater part of the urobilin present is in the form of a colourless chromogen, which, on oxidation, is converted into the pigment (Hoppe-Seyler, MacMunn). 1 Journ. of Fhijsiol. x. 11-2. - Pfliigcr's Archie, x. 208. ^ Journ. uf Phi/siol. x. 21. 750 EXCRETION' PATHOLOGICAL UKOBILIX This substance is sometimes termed febrile urobilin, as it appears in certain febrile conditions. It is prepared from urine by the same method as that already described for normal urobilin, and is soluble in the same reagents. Its spectrum is shown in tig. 97, spectrum 2. The band at F i& darker and wider than the corresponding band of normal urobilin ; there are in addition two other bands in acid sokitions, one between D and E and the other just before D. On treatment with zinc chloride and ammonia a deep green fluorescence is developed, as with stercobilin and hydrobiliiubin, and the spectrum then seen is a three-banded one practically identical with that obtained by treating normal urobilin in the same way, and also like that obtained by the similar treatment of hydrol)ilirubin. Stercobilin, on the other hand, after this treatment gives a f(.)ur-banded sjiectrum. Pathological urobilin can be artificially prepared from artificial normal urobilin by the action of reducing agents. We may therefore infer that pathological urobilin is a less oxidised stage of the same material which under normal circumstances passes into normal urobilin. Pathological urobilin, like normal urobilin does not necessarily arise from bile-pigment. It may arise from the blood-pigment. After extensive extravasations of blood into the tissues, or into the peritoneum, the urine becomes dark, like jaundiced urine, but the pigment is found to be pathological urobilin.' rEOH.EMATOPOllPHYEIX This pigment has been found by MacMunn and subsequently by le Kobel ^ in certain diseased conditions, aIz. Addison's disease, acute rheumatism, cirrhosis of the liver, pneumonia, jiericarditis, peritonitis, measles, meningitis, typhoid fever, and Hodgkin's disease. 3Iac^Iunn considers it probably closely related to Baumstarks •'' urorubrohsematin and urofuscohsematin. Like urobilin, it may exist in the urine partly in the form of a chromogen (named urobilinoidin by le Nobel), which on oxidation is transformed into the pigment. It can be prepared from the urines that contain it by the same method as that already described for urobilin. It can be prepared artificially from hjtmatin (not from bile-pigments) by the action of zinc and sulphuric acid, sodium-amalgam, and other reducing agents. ' Cases of tliis condition which occurred in Univ. Coll. Hosp. under Dr. Ringer's care are described by MacMunn, Joiirn. Physiol, x. 83. - Pfluger's Archiv, vol. xl. 18«7. ^ Ibid. ix. 568. TIIK riCMENTS OK THK TRINE 751 Til acid solutions tliesj)ectrum is char;icteristic (fig. 97, spectrum .'}); the bands are four in number : a narrow one before and touching D, another darker between D and E, a feeble shading between these twcj, and, lastly, a band at F, jtractically identical with that of normal urobilin. If the pigment is dissuhed in alcoh(jl, and ammonia added, a five-banded spectrum like that of neutral hjematoporphyrin i.s obtaineil. Treated with zinc chloride and ammonia, a faint green fluorescence appears ; the band at F becomes a little narrower and shifts a little towards the red end of the spectrum, and there are two other bands between D and E. This pigment thus i-e.sembles the pigments we have pi-eviously discussed, but shows certain differences from all of them. More recently MacMunn (Proo. Physiol. Soc. 1890, p. xlii.) found in throe specimens of morbid urine a pigment ])robab]y intermediate between nroha;ni;i- toporphyrin and hicmatoporphyrin.' These urines were of a deej) Burgundy red colour, contained no proteid, and, on the addition of a drop of sulphuric acid, showed the spectrum of acid hajmatopoqahyrin (fig. 59, spectrum 10, p. 277). The table on the next page collects together the chief distinguishing characters of these various but still closely allied pigments. OTHER UEIXAIiy PIGMENTS Indigo. - See p. 744. Shatole jnijment. — See p. 74.5. Urorvhin, urorosein, imrpurin. — Prol);ibly identicjil with the skatole-pigment (Mester). Uroerxjthrin. — This is the pigment wliich colours deposits of urates a brick- red tint. Mester considers that this also is identical with skatole-pigment. MacMunn, however, states that it gives certain characteristic reactions. It may be extracted from the urates by boiling alcohol. This solution gives two ill- defined bauds before F. In the solid state it becomes green with caustic soda or potash. Its origin and its relation to urobilin are unknown. Uroclirome. — This was the name given by Thudichum- to what he considered to be the chief urinary pigment. It is possibly impure urobilin altered by the method of preparation. It may be prepared as follows : — Precipitate about 50 c.c. of urine with lead acetate and a drop of ammonia. Filter. The filtrate is colourless. Scrape the precipitate into a capsule, mix it with a few drops of sulphuric acid, and add to the pasty mass a little alcohol. Filter. A yellow alcoholic solution of urochrome comes through. Boil this with excess of sulphuric acid, and dilute the acid liquid with water. Black flocculi are formed : these do not consist of carbon produced by chan-ing, as they are readily soluble in ammonia, from which it can be again precipitated by sulphuric acid. The name uromelanin was given to this black pigment. Thudichum described another derivative of urochrome, which he named uropittin (S^^^i^.,0.^). These experiments have now a merely historical interest. ' See also Ranking and Pardington, Lancet, ii. 1890, p. 607. - Brit. Med. Journ. Xovember 18(34, p. 509. V 5ii KXCRETION I'lgment HiidrobUiruhin Stercohilin Xormul urobilin The pigment of normal urine Pathological 1 Uroluetnalv- urobilin porphiinn Definition A jjigment arti- ficially pro- duced from bili- rubin by reduc- tion witli sodium-amal- gam The pigment of the faeces Pigments occurring in the urine in certain diseases, cliiefly of a febrile cliaracter Origin in the body A reiluction- proiluct from bilirubin. (An oxiilation-pro- duct from bili- rubin, and partly from the hajmoglobin of the food MacMunn) A reduction- product from bile-pigment or blood-pigment. (Oxidation-i)ro- duct — MacMuun) A less highly oxidised pro- duct from bile- or blood-pig- ment (MacMunn) A reduction- pioduct from blood-pigment (MacMunn) These pigments all exist in the urine mostly as chromogens, which by oxiilation are conyerted into the pigments Spectro- scopic ap- pearances Two bands, one at D, the other .between b and F (fig. 88, spec- trum 3) Two bands, one at D, the other Ijetween&and F One band at F (likecholetelin) (fig. 97, si)ec- truui 1 ) Three bands, one just before D. one between D and E, the third (lark and wide at F ( ihiil. spectrum 2 ) Four bands, one just before D, two between D and E.tlie fourth at F (/Mrf. spectrum 3> On treat- ment with zinc chloride and ammonia Well-marked green fluores- cence. Sped rum: three bands, one after C, one at D, the third between h and F (fig. 88, spectrum 4) Well-markeil green fluores- cence. Spectrum : four banils, one after C, one at D, one between D and E, the fourth between 6 and F Fairly well- marked green fluorescence. Spectrum : three bauds, like tl lose of hydro- bilirubin Well-marked green fluores- cence. Spectrum : three bands, like those of hydro- bilinibin Faint green fluorescence. Spectrum : three liands, two be- tween D and E, the tliird be- tween 6 and P Melanin. — This substance must not be confused with the artificial product named uromelanin just described. Melanin, or n chromogen called melanogen, converted into melanin by oxidation, occurs in the urine in some cases of melanotic sarcoma {see p. 499), and the term mclamtria may be employed to le world, the formation of a humous substance. These humous substances are allied to carliohydrates, and also to .aromatic bodies, as on fusing with potash they yield pyrocatechin, protocatechuic acid, as well as volatile fatty acids. Some apjicar to be nitrogenous, and such a one is formed, according to Udranszky,' from urea .and the normal carbohydrates of the urine on heating with ;i mineral acid. The dark colour of herbivorous urine, and also that in carboluria, is stated by Udranszky to be due to similar Immous pigments, and according to him the pyrocatechin obtainable from such urines is due to the decomposition of the humous substances they contain. The experiments, how- ever, do not appear to me to fully boar out these .statements, and until fuller light is thrown on the subject, Udranszky 's conclusions must be accepted with caution. Zeif. phijsivl. Chew. ii. 5:37; xii. 33. 3c 754 EXCEETIO^' CHAPTER XLII OTHER OIIGAXIC CO.YSTITUEX'TS OF THE UBJyE A NUMBER of organic constituents, in addition to those already described, may occur in small quantities in the urine. We may divide these into the following groups :— (1) Non- nitrogenous acids : oxalic, succinic, and lactic acids. (2) Fatty acids. (3) Glycero-phosphoric acid. (4) Carbohydrates : dextrose, animal gum. (5) Ferments : especially pepsin. (6) Mucin. (7) Cynurenic and urocanic acids. Oxalic acid (C2H2O4). — The free acid never occui's in the urine, but it is united with calcium to form an oxalate, which, under ordinary circumstances, is held in solution in the urine by the acid phosphate of sodium. Schultzen ' found that 0-1 gramme (TS grain) was excreted daily by men. Neubauer in some cases found it wholly absent. It is much more abundant in the urine of horses and pigs. It occurs in excess in the urine after the ingestion of rhubarb and cabbage, the former of which contains an especially large amount of the acid. It is increased in a condition called ' oxaluria,' in which the most prominent subjective symptom is nervous depression. Oxaluria occurs in a variety of ailments ; an increased secretion of uric acid is generally accompanied with an increase of oxalic acid. It also occurs in excess in certain cases of catarrh of the urinary passages. When present in excess it is in the form of a precipitate of crystals of calcium oxalate. Such crystals are frequently found in the ' lateritious deposit ' of febrile urine. Such crystals form, as a rule, after the urine has stood a few hours, especially if it contains excess of mucus, or spermatozoa, as in spermatorrhoea. This deposition of crystals is probably the result of an acid fermentation. Crystals of calcium oxalate (C2Ca04 + 211,0) are distinguished by their form, quadratic octahedra with a short principal axis ; these are 1 Arch.f. Anaf. u. Phijsiol. 1868, p. 719. OTliKi; ORGANIC CONSTITUENTS OF THE TRINE 755 often termed ' envelope crystals ' (tig. 98). Occasionally dumb-bell foi-ms are seen. The crystals are further characterised by their extreme insolubility ; they are insoluble in ammonia, in acetic acid, and soluble with ditliculty in dilute hydrochloric acid. On account of the insolubility of calcium oxalate, oxalic acid is generally estimated as the calcium salt. The origin of oxalic acid in the body is uncertain. Frerichs and Wohler found ^ that dogs fed on uric acid had an increase of calcium oxalate in their urine. The close relation- ship between uric acid and oxalic acid appears to be undoubted, as may be seen by consulting the -f,r„ 98. -Crystals of ,, T . n • • 1 / f7on\ ('aloium Oxalate. account already given or uric acid (p. 1 2\)). Calculi consisting of calcium oxalate (mulberry calculi) are exceed- ingly hard and insoluble. Stones consisting of a mixture of uric acid and calcium oxalate are fairly common. Succinic acid (C^Hj^O^), the third term of the series of acids of which oxalic acid is the first, has been occasionally found in the urine (Meissner '), especially after the ingestion of asparagus (Hilger -). Salkowski,^ v. Longo * (after ingestion both of asparagus and asparagine), and Baunaann^ (after ingestion of sodium succinate) failed to find it in the urine. Lactic acid (CaH^Oj).— This probably does not occur in normal urine. It has been found in the urine combined with bases after great muscular activity;" according to Cola.santi and Moscatelli ' the form of lactic acid wliich then occurs is sarcolactic acid. It has also been found in cases of trichinosis," acute yellow atrophy of the liver,'' liver cirrhosis,"* diabetes," phosphorus poisoning,'- rickets,'^ leucocYthajmia,'^ osteomalacia,'^ and in animals after extirpation of the liver '« (see p. 735). Fatty acids.— These occur in normal urine in mere traces (0008 gramme daily). They consist of formic, acetic, butyric, and propionic acids, and the amount in the day's urine can be increased by treating the urine with oxidising agents to 0-9 to TS gramme (v. Jaksch "). • Meissner and Sliepard, Untersuchungen ii. d. EntstehenderHippursdure,'H.a.nover, 1866. - Liebig's Ann. clxxi. 208. ^ Pfiuger's Archiv, iv. 95. * Zeit. physiol. Chem. i. 213. ^ jj j^. p. 215. « Spiro, Ibid. p. 117. 7 Gazz. Ital. xvii. 548. The occuiTence of lactic acid in the urine (except in frogs) after muscular work is denied by Marcuse, Bied. Centralbl. 1887, p. 92. 8 Simon and Wibel, Ber. d. deutsch. chem. Ges. 1871, p. 139. 9 Schultzen and Riess, An)i. des Charite Krank. xv. 1. '0 Bunge, Phijsiol. Chem. trans, by Wooldridge, p. 345. " Bouchardat, Malij's Jahresb. 1870, p. 155. '- Schultzen and Riess, loc. cit. 15 Gorup-Besanez, Lehrbuch, 1878, p. 606. 1^ Kijrner and Jacubasch, Arch. f. path. Anat. xliii. 196. 15 Mcers and Myk, Zeit. anal. Chem. 1869, p. 520 ; Arch. f. klin. Med. v. 480. "^ Minkowski (in geese). Arch. exp. Path, und Pharmak. xxi. 41; Marcuse (in frogs\ Pfliiger's Archiv, xxxix. 425 ; Nebelthau (in frogs), Zeit. Biol. xxv. 123. The acid appears to be sarcolactic (Nebelthau). ^^ Zeit. physiol. Chem. x. 536. 3 c 2 7r)f*) EXCRETIOX The amount of fattj' acids also increases during the occurrence of the ammo niacal fermentation of urea (Salkowski).' The amount of fattj- acids in the urine is increased in certain febrile condi- tions to 0-06 gramme, and in certain liver diseases to 0 6 to 1 gramme per dkni. This condition is called lipnciduna by v. Jaksch. The acids are apparently free in tlie urine. Glycero-phosphoric acid (CjHsPOg) occurs in normal urine^ to the extent of 15 milligrammes yjer litre. It is increased in nervous diseases (Lepine) and after chloroform narcosis (Zuelzer). Carbohydrates. — Dextrose. — The occurrence of aVjundant quantities of grape sugar in the urine is one of the most prominent symptoms of the disease called diabetes. The question, however, we have now to consider is, Does sugar occur in normal urine 1 The answer has been sought by many observers, a large number of whom state that sugar is, and a nearly equally large number of whom state that it is not present.^ The conclusion one would draw from a hst of references such as is given below is, that if sugar is present at all, it occurs in very small quantities. The difficulty of the investigation is increased by the fact that urine contains several substances that reduce alkaline solutions of cupric hydrate ; these are uric acid, hippuiic acid, pyrocatechin, glycuronic acid, and creatinine. Xone of these, however, undergo the alcoholic fer- mentation on the addition of yeast, and this does take place with the reducing substance of normal urine (Abeles). .Some of the older opponents of the view that urine contains sugar said that, even if sugar is formed, it is the result of the decomposition of indican ; this, of course, was when physiologists held the idea that the indican of urine, like that of plants, was a glucoside. AVe have already seen (pp. 79 and 743) that the so-called indican of urine is not a glucoside. The balance of evidence appears to me to be clearly in favour of the existence of a small quantity of dextrose in normal urine. The most recent work at the subject is that of Wedenski. He shook up a large quantity of normal urine with benzoic chloride ; by this treatment insoluble benzoyl compounds of carbohydrates, if present, separate out. ' Zclt. Physiol. Chcin. xiii. 264. Salkowski considers that during the fermentation they origiuate from the carbohydrates of the urine. See also Tanigati, ibid. xiv. 471. ^ Kliipfel and Fehhng, So'tnichewski, Zeit. jihysiol. Cliem. iv. 214. 3 For most of the following references I am indebted to Hoppe-Seyler's Physiol. Chem. p. 828. Tliose who state sugar is present are : Briieke {Wien. Akad. Sitzujigsher. xxix. 34G), Bence Jones {Chem. Soc. Quart. Journ. xiv. 22), Tuchen {Virchow's Archiv, xx^-ii. 2G), Ivanoff (Diss. Dorpat, 1861J, Meissner and Babo {Zeit. rat. Med. (3), ii.), Pavy, {Chiy's Hasp. Pep. xxi. 413), Abeles [Centralbl. med. Wiss. 1879, Nos. 3, 12, and 22), Udranszky {Zeit. physiol. Chem. ii. 537; xii. 33), Wedenski {Ibid. xiii. 122), Salkowski {Ibid. xiii. 264), Hagemann {PJJiiger's Archiv, xliii. 501). Those who state that sugar is absent are: Friedlander {Arch. d. Heilk. vi. 97), Maly {Wien. Akad. Sitzintgsb. Ixiii. 2), Seegen (Tbid. Ixiv. 2), Kulz {Pflvger's Archiv, xiii. 269). (tTllKK oKCANir CoNSTn TENTS OF TlIK IIJINH 7.>7 ►Such ;i precipit.itiun ilues <»ccur. Elementary analysis showed the probable presence of two carbohy(h"ates ; these were separated by treatment with soda ; part remains undissrilved, and gives the reactions of dextrose ; the part that dissolves gives the characters of animal gum. Aninuil gum. — Tliis carbohydrate radicle of mucin (*ft' j). 480) was originally found in tiie urine b}' Landwehr.' Milk sugar. — This often occurs, but in small and variable quantities, in the urine of nursing mothers (Blot,- de Sinetz,* Hofmeister,' Kaltenbach *). Hofmeistor {jrecipitated urine with lead acetate and ammonia, filtered, decom- posed the filtrate with sulphuretted hydrogen to get rid of lead, filtered, shook the filtrate with silver oxide, filtered, decomposed the filtrate with sulphuretted hydrogen to get rid of silver, filtered ; to the final filtrate barium carbonate was added, and the mixture evaporated to dryness. Alcohol removed milk sugarfrom the residue, and characteristic crystals of it were obtained by evaporating off the alcohol. Kaltenbach further showed that this substance was milk sugar, as he obtained galactose and mucic acid from it. Inosite.—'^Taa.W quantities of inosite have been found in normal urine by Cloetta,* Gallois,' Strauss,** Kiilz " ; in the urine of cases of Bright's disease by C'loetta, and in diabetic urine by Mosler and Schwanert.'" Dahnhardt" obtained 01 gramme of inosite from 8 kilos, of oxen's urine. Feeding with inosite does not increase the amount in the urine (Kiilz). It may be detected in the urine as follows '- : — Several litres of urine feebly acidified are completely precipitated with lead acetate and filtered. The filtrate is warmed and completely precipitated with basic lead acetate. After standing forty-eight hours the precipitate is collected, washed, suspended in water, and treated with a stream of sulphuretted hydrogen ; the lead sulphide is filtered off. Uric acid separates from the filtrate after some hours ; this also is filtered off. The solution is then evaporated to a syrup on the water-bath, and absolute alcohol added. The precipitate is dissolved in hot water, and three or four times the volume of 90 per cent, alcohol added. Ether is cautiously added till a permanent cloud appears ; the inosite crystallises out, and may be collected ; it will then give its characteristic tests (p. 101). Ferments. — Pepsin. — Several observers (Briicke, Sahli,'^ kc.) have found pepsin in the urine. The following is an abstract of Leo's ''' work on the subject. Small pieces of fibrin soaked in the urine absorb the pepsin therefrom ; on rerQO\'ing them to 0*1 per cent, hydrochloric acid thev are rapidly digested. Control experiments with fibrin not pre- viously soaked in urine gave negative results. Morning urine is richest in pepsin. ' Centralbl. med. Wiss. 1885, p. 369. - Compt. rend, xliii. 67tt. 5 Gaz. med. Paris, 1873, p. 573. * Zeit. pkysiol. Chem. i. 101. 5 Ibid ii. 360. « Ann. Chem. Pharm. xcix. 289. ^ These, Paris, 1864. 8 Diss. Tubingen, 1870. 9 Centralbl. wed. Wi.^s. 1875, p. 933. 10 Arch, pathol. Anat. xliii. 229. 11 Arbeit aus d. Kieler jihysioL Inst. 1868, p. 157. 1- Salkowski and Lenbe, Lehre vom Ham. 15 Pfliiger's Archiv, xxxvi. 209. i* Ibid, xxxvii. 223; xxxix. 246. 758 EXCRETION Neumeister • found pepsin in the urine of the dog, but not in that of the rabbit. Neumeister and Stadelinann ^ both showed that the ferment in the urine is true pepsin ; it forms peptone and all the intermediate proteoses from fibrin just as pepsin does. Trypsin. — Except Sahli, all observers agree that trypsin is absent from the urine. Sahli's results were probably due to the non- prevention of putrefaction in his experiments. Pieces of fibrin soaked in urine, according to Leo's method, are not digested in 1 per cent, sodium carbonate solution, thymol being added to prevent putre- faction. As trypsin is not found in the blood or tissues, Leo concludes that it is entirely destroyed in the alimentary canal ; while the pepsin is not wholly destroyed there, but is partly absorbed, and passes into the blood, tissues, and urine. By making extracts of the different parts of the intestine, Leo draws the conclusion that pepsin disap- pears in the second third and trypsin in the lower third of the small intestine. Biastatic ferment. — Holovotschiner^ states he has obtained small quantities of ptyalin or a similar diastatic ferment from urine. i?e«7;^^^. — Holovotschiuer and Helvves*both obtained from urine traces of a ferment wliich curdles milk. Mucin. — This is the chief constituent of the mucus derived from the urinary passages. It occurs in normal urine in small quantities ; in catarrhal diseases of the urinary passages it is increased. It is slightly soluble in neutral and alkaline urines, and may be precipitated there- from by acetic acid (insoluble in excess) or by alcohol ; it is not pre- cipitated l>y boiling, and so may be distinguished from albumin. It is probably the source of the animal gum found by Landwehr in the urine. Cynurenic and urocanic acids. — Tliese are two peculiar acids, the characters of wfiich are dsscribed on p. 91. and which hitherto have been found only in the urine of dogs. The former may be precipitated in crystals from urine by nitric acid. They are found in the urine of starving dogs, and so must be products of metabolism, and not the result of putrefaction in tlie intestines.' Kryptophanic acid (C^H^NO^) was described by Thudichum as a normal con- stituent of urine, but has not been found by anyone else. Nephrozymase is a substance iDrecipitated by alcohol from urine by Bechamp. According to him, it is proteid in nature. Normal urine, however, is absolutely free from proteids. Uretliaii (ethyl carbamate) is found in small quantities in the alcoholic extract of normal urine. It is, however, an artificial product of the action of alcohol on urea (Jaffe and Cohn)." 1 Zeit. Biol. xxiv. 272. - Ihid. .\xv. 208. 5 Chem. Centralhl. 1886, p. 327. * Pflilger's Archiv, xliii. 38i. * Baximann, Zeit. physiol. Chem. x. 123. '' Ihid. xiv. 395. '59 CHAPTER XLIII Tin: ISOllGAXIC CUASTITUENTS of VlilNE The inorganic constituents of the urine are chiefly chlorides, carbonates, sulphates, and phosphates ; the metals with which these are in com- bination are sodium, potassium, ammonium, calcium, and magnesium. Small quantities of fluorine, silicic acid, and iron also occur ; and the free gases present are carbonic acid and nitrogen, with traces of oxygen. The total amount of salts varies from 9 to 25 grammes daily. The inorganic salts of the urine are derived from two sources : first, from the food ; the salts pass into the blood, and then are excreted by the kidneys ; secondly, as a result of metabolic processes ; this is especially the case with the phosphates, and more particularly still with the sulphates. The salts of the blood and those of the urine are much the same, with the important exception that, whereas the blood contains only traces of sulphates, the urine contains abundance of these salts ; the sulphates are derived fi'om the changes that occur in the proteids of the body ; the nitrogen of the proteids is excreted as urea and uric acid ; the sulphur is oxidised to form sulphuric acid, which passes into the ui'ine chiefly combined with metallic bases, but to a small extent also in ethereal combinations with organic radicles to form the ethereal sulphates we have already considered (p. 740). The excretion of sulphates, moreover, runs parallel to that of urea. The tests for the chief salts are given on p. 717. Their estimation is described in Chapter XLY. THE CHLORIDES The principal chloride in the urine is that of sodium. Small quantities of potassium chloride and traces of calcium and magnesium chloride are also present. Sodium chloride is, in fact, the most abun- dant salt in the urine, as it is in the blood and in most other fluids of the body. Yogel gives the daily amount of chlorine excreted as 6 to 8 grammes, which would correspond to 10 to 13 grammes of sodium chloride. The ingestion of sodium chloride in the food is followed by its appearance in the urine, some on the same day, some on the next day 760 EXCKETloN (Dehn ^) ; some, however, is decomposed to form the hydrochloric acid of the gastric juice. The sodium chloride, however, does not merely pass through the body without making its effect felt ; it stimulates metabolism and secretion, as has already been pointed out (p. 61). The urine is richest in sodium chloride after a meal ; poorest at night time.'' Drinking large quantities of water or beer increases it; a rich secretion of gastric juice causes a temporary decrease in the chlorides of the urine.^ Certain chlorine compounds other than chlorides causes an increase of the urinary chlorides : chloroform narcosis,^ and the administration of ethyl trichloracetate act in this way ; whereas certain other chlorine compounds (chloral.^ carbon tetrachloride, methyl chloride, &:c.) do not have this effect (Blast"). The quantity of chlorides excreted varies greatly in disease : — It is diminished in most febrile diseases ; the cause of this is unknown, but is, perhaps, partially due to diminished intake of the salt, or in some cases to diaiThoea, by means of which a certain quantity of salt passes out per rectum (see p. 699). The decrease is especially marked in pneumonia, pleurisy, and typhoid fever, and runs x>arallel to the height of the fever. In pneumonia the chlorides may entirely disappear from the urine, their reappearance being one of the signs of improvement. It is diminished in cholera, chorea, and pemphigus. It is increased in diabetes, poly- uria, and some forms of Bright's disease, where a large amount of urine is excreted. The relation of sodium and potassium salts in some of these conditions has been investigated by E. Salkowski.' In health the ratio is variable, depending to some extent on diet. In febrile conditions in which the total chlorides are diminished the excretion of the potassium salt rises above the average. Zuelzer » states that the same occurs in conditions of excitement. THE SULPHATES The sulphates in the urine are of two kinds, ordinary sulphates of potassium and sodium (pre-formed sulphuric acid), and ethereal sulphates (combined sulphuric acid). They are derived in small measure from the food (administration of sodium or magnesium sulphate increasing the quantity of sulphates in the urine), ^ but chiefly from the meta- bolism of proteids in the tissues. The ethereal sulphates are the result of putrefaction of proteids in the intestines or elsewhere, as in a putrid abscess. The sulphates of the urine may vary in amount from 1 -5 to 3 grammes daily (Furbringer,'" Neubauer''). The administration of free sulphuric acid to dogs increases the urinary sulphates '2; in rabbits this is not the case.'^ 1 Pfluger's Archiv, xiii. 353. - A. Hegar, Ueber d. Ausscheidung d. Chlor diirch den Ham, Giessen, 18.52. 3 Chem. Centralbl. 1887, p. 1561. ■* Zeller, Zeit. physiol. Chem. viii. 70 ; Kast, Ibid. ii. 277. * Chloral passes into the urine as urochloralic acid (v. Mering). * Loc. cit. ^ Pfluger's Archiv, iii. 351. ^ Centralbl. ined. Wiss. 1877, Nos. 42 and 43. » Sick, D/.SS. Tubingen, 1859. "^ Arch. path. Anat. Ixxiii. 39. 1^ Neubauer and Vogel's Text-book. 1- Frey and Gahtgens, Centr. med. Wiss. 1872, No. 53; Kurtz, Diss. Dorpat, 1874. ^^ Salkowski, Arch, jjath. Anat. Iviii. 1. TJIK ]N()I((iANJC CONSTITIKNTS oF IHINK 701 The variations uf tl>e aiuount of uriiiaiy sulphates in disease can be almost guessed after the statement has been made that their amount runs parallel to that of the urea excreted. In conditions where metabolism is increased (fever, diabetes) the suli)hates are increased ; in conditions where metabolism is diminished (convalescence from fever, most chronic affections) the sulphates are diminished. Bence Jones states that an increase occurs in various forms of delirium ; also in acute inflammatory diseases of the brain and .spinal cord. THE CAEBOXATES Carlxmate and bicarbonate of .sodium, calcium, magnesium, and ammonium are generally present in fre.sh, alkaline urine. They arise in the organism from carbonates of the food, «jr from lactic, malic, tartaric, succinic, and other vegetable acids in the food. They are thus most abundant in the urine of herbivora and vegetarians, whose urine, we have ah'eady seen, is thus rendered alkaline. Urine containing carbonates is either cloudy when passed or, like saliva (see p. 622), soon becomes .so on standing ; the deposit, if allowed to settle, will, on examination, be found to consist of calcium carbonate and also phosphates. THE PHOSPHATES Phosphoric acid in normal urine occurs in the form of two classes of phosphates : — (1) Alkaline phosphates. Phosphates of sodium are the most abundant ; those of potassium scanty. (2) Earthy phosphates. Phosphates of calcium are the most abun- dant ; those of magnesium scanty. The composition of the phosphates in urine is liable to variation. In acid urine, the acid salts are generally present, and give the urine an acid reaction. These are chiefly sodium dihydrogen phosphate (NaH2P04) and calcium dihydrogen plio.sphate [Ca(H.,P04).2]. In neutral urine, in addition to these, phosphates wuth formula? NajHPOj (disodium hydrogen phosphate), CaHP04 (calcium hydrogen phos- phate), and MgHPOj (magnesium hydrogen phosphate) are also found. In alkaline urine there may be in addition to, or instead of some of, the above the normal phosphates of sodium, calcium magnesium [NagPO^, Ca3(P04)2, Mg3(P04).2]. In addition to these, phosphoric acid may be united to the bases ammonia, urea, and creatinine. - The earthy phosphates are precipitated by rendering the urine alkaline by ammonia, potash or soda, or in the ammoniacal fermenta- tion that occurs in decomposing urine. The alkaline pho.sphates remain in solution after the earthy phosphates have l>een precipitated in this way. The phosphates found most frequently in the white creamy 762 EXCRETIOX precipitate that occurs in decomposing urine are (1) the triple phos- phate (ammonio-magnesium phosphate, NH4MgP04 -(- 6 H^O), which crystallises in triangular prisms, or so-called ' coffin-lid crystals ' (fig. 99), and occasionally in feathery stellate crystals ; (2) calcic phosphate, often called ' stellar phosphate,' which crystallises in star-like clusters of prisms. In acid urine a crystalline calcium phosj^hate occasionally separates out ; it may also be obtained by adding calcium chloride to urine, or, after the internal administration of lime-water, or potassium carbonate. It has the composition CaHP04-|-2HnO (Hill Hassal,! Stein 2). ■%/ Normal urine gives no precipitate when it is ^Magnesium^orT?rpie boiled. N'eutral, alkaline, and, occasionally, faintly Phosphate. add urine give a precipitate of calcium phosphate when boiled ; this precipitate is amorphous, and is liable to be mis- taken for albumin ; it may be distinguished readily from albumin, as it is soluble in a few drops of acetic acid, whereas coagulated proteid does not dissolve. Salkowski^ showed that the jDrecipitated phosphates often redissolve when the urine cools. There have been various explanations advanced to explain the pre- cipitation of phosjjhates by heat. They all, however, may be summed up by saying that the phenomenon is the result of unstable equilibrium among certain phosphates, the balance of solubility being easily dis- turbed by changes of temperature and reaction, and possibly modified by the kind and amount of other salts in solution (W. G. Smith,"* Stokvis-5). The precipitation was believed bj' some to be due to evolution of carbonic acid. Salkowski attributes it to the decomposition of a compound of calcium an sodium phosphate. Keynolds suggests that the change produced by heat may be represented as follows : — 2Ca,H,(rO,),-f CaH,(P0J, = Ca3^P0,), (insoluble) + 2CaHj(P0 J, (soluble). A. Ott" speaks as follows on the subject. Erlenmeyer' has shown that acid calcium phosphate is soluble in 700 parts of water. But the urine is able to hold more than this in solution, in virtue of the presence of other salts. Similarly the normal phosphate is more soluble in urine than in water, such salts as potassium phosphate and sodium chloride aiding its solution. By heating an aqueous solu- tion of the two phosphates the acid phosphate is changed into the normal phos- 1 Proc. Boy. Soc. x. 281. ^ Liebig's Annaleii, clxxxvii. 90. ' Zeit.physioL Cheni. vii. 119. * Dublin Journ. Med. Sci. July 1883. ■'• Chem. Centralbl. 1884, p. 42. « Zeit.physioL Cliem. x. 167. ' Ber. d. deutsch. clievi. Ges. ix. p. 1839. TlIK lX(>K(iANJC CONSTITUENTS ol' 'I'llE I'lUNE 763 phatc, and is precipitatt^d, pliosphoric acid passing into solution. But in normal urine no such ])recipitation, or only a slight one, occurs, because of the jjresence of the other salts just alluded to. W, however, the normal relation between these .salts be upset, IIhmi we get precipitation of the normal calcium phosphate. Ori(jhi (if, caul variations in, the tiritinri/ pJio.sjJiafes. — The pho.s- phoric acid in the urine is partly derived from the food, and is partly a decomposition j^roduct of lecithin and nuclein. The amount of the acid in the twenty-four hours' urine varies from 2-r) to 3"5 grammes, of which tlic earthy phospliates constitute about half (1 to l"") gramme). The excretion of phosphoric acid varies in amount with the food taken : after the midday meal, especially if it consists of meat, it rises, reaching its maximum in the evening ; it falls during the night, reaching its minimum at midday. The average of Ott's analyses shows that the ratio of F.fi^ combined as normal phos- phate to that combined as acid phosphate was as follows :— Evening urine (2 to 10 P.M.), 91 : 100; night urine (10 p.m. to 8 a.m.), 58 : 100 ; morning urine (8 A.M. to 2 P.M.), 69 : 100. An interesting point in connection with this subject is whether activity of the nervous system produces an increased output of phosphoric acid from increased metabolism of lecithin. Mendel ' found it diminished in chronic brain diseases. Vanni and Pous,- with certain reservations, came to the same conclusion. Mairet ' concludes that brain work increases the excretion of alkaline phosphates. The question, however, appears to me to be an especially difficult one to investi- gate ; the quantity that arises from decomposition of brain substance must under any circumstances be small, and such small differences are particularly hard to recognise when one remembers, as Mairet himself points out, that effective brain work is difficult on an insufficient diet. The increase noted may just as probably be due to the food taken to sustain mental activity as to the mental activity itself. Several observers have found that muscular work increases the output of phcsphoric acid (Mosler, ''Lehmann ^), while others have found that it does not (Pettenkofer and Voit,"^ Byassan," North ^). In various pathological conditions the output of phosphoric acid varies : it is diminished in gout," in most acute diseases, probably because only a small amount of food is taken (Vogel), in kidney disease,'" in the intervals of intermittent fever," after large doses of chalk, ether, and alcohol, and during pregnancy (owing to the formation of foetal bones). It is increased after copious draughts of water, after sleep produced by potassium bromide or chloral hydrate (Mendel '-), in inflamma- tion of the brain, in chorea, acute atrophy of the liver, phthisis, and leuco- cythaemia. An increase of phosphates in the urine is termed 2)hosj)hati(ria. A deposit of earthy phosphates may be due to disturbance of the unstable equilibrium of the 1 Arch./. Psi/cJiiatrie, vol. iii. 1872, p. 636. ^ chem. Coitralhl. 1887, p. 1526. ' Compt. rend. xcix. 282. * Beitrnge zur Kenntniss der TJrinabsonderang, Giessen, 18,53. 5 Arch.f. Anat. u. Physiol. 1871, p. 14. " Zeit. Biol. ii. 4,59. ^ Essai, Paris, 1868. ^ Proc. Roij. Soc. xxxi.x. 443 (see also p. 437 of this book). 9 Stokris, Centr. med. Wiss. 187.5, No. 47. 10 Brattler, Ein Beitrag zur Urologie, Miinchen, 18,58. 11 Haxthausen, Diss. Halle, 1860. i-' Loc. cif. 764 EXCRETION urinary phosphates, and not to actually increased excretion ; a careful analysis of the twenty-four hours' urine should always be made. In true phosphaturia, of which the chief symptoms are nervous irritability and digestive troubles, the amount of PoO^ in the twenty-four hours may ri.se to 7 to 9 grammes.' Calculi consisting wholly of calcium phosphate are exceedingly rare ; imc acid calculi, however, are often covered with a coating of phosphates ; the presence of the stone in the bladder sets up inflammation, the urine is thus rendered alkaline, and calcium phosphate is precipitated. OTHER IXOKGAXIC SUBSTANCES Iron occurs in small quantities : the compound in which it is present is un- known (Hamburger-). Traces of silicic and nitric acids,^ derived from drinking water, have been found. Traces of fluoiine are sometimes present. Fi'ee ammonia occurs in mere traces also, but is increased when putrefaction sets in. Hydrogen peroxide was found in traces in fresh urine by Schoubein." Sulphuretted hydi'ogen develops in putrid urine, probably not from the sul- phates, but from other combinations of sulphur, such as sulpho- (thio-) cyanic acid,^ and cystin (^see Cystinuria, Chap. XLIT) ; hyposulphites may occur in t^'phoid fever urine.^ The gases. — The following table represents the chief analyses that have been made. The numbers are volumes per cent. : — Gases Plauer ' Pfluger' 1 Ewald ■■' Strassburger "* 1 Human iirine 1 Carbonic acid free . „ ,, com- bined . Oxygen . Nitrogen . 4 to 'J 13 to 14 ^ 2 to 5 OltoO-7^ 0-2 to 0-6 0-O7to008 0-7 to 0-8. 0-8to0 9 1 TT. , . c The pressure of Higherinfever ^.^ n i - „p * i ., "^ . , ,, CO., = 9lo of an at- than in health - , i,- i, • mosphere. which is 004 higher than in the 0-9 blood 1 Tessier, Du diabete j)l'osphatique, Lyons, 1877. - Zeit. phijsioJ. Chem. ii. 191; iv. 248. ^ Rohmann, Zeit. physiol. Chem. v. 94. * Sitzungsher. d. Bayer. Akad. d. Wiss. vol. i. (2), 1864, p. 115. * Munk, Arch. f. path. Anat. Ixis. 354; Gschleidlen, Ffliigefs Archil, xiv. 401 ; xv. 350. 6 Miiller, Che?n. Centralbl. 1887, p. 807; Berlin, klin. Wuch. xsiv. 405. " Zeif. d. Gesellsch. d. Aerzte in Wien, 1859, p. 465. » Pfliigefs Archie, ii. 165. '•• Arch.f. Anat. u. Phgsiol. 1873, p. 1. i*' Pfluger' s Archiv, vi. 93. 7()5 CHAPTER XLIA' AJiXOllMAL A\D PATHOLOGICAL URINE Morbid conditions of the urine are exceedingly numerous. The urine naay contain excess or diminution of one or other of its normal con- stituents. These conditions have been already described in the pre- ceding chapters ; we may thus have urea, urates, phosphates and other salts in gx-eater or less abundance than usual. The alterations in the pigments of the urine have already ]>een described. We have, however, now to consider alterations in the urine, in which substances normally absent from, occur to a greater or less extent in, that secretion. We shall also have to take up in a rather more connected way than we have done hitherto the deposits that occur in the uinne. The substances which occur in the urine under abnormal conditions are those introduced into the body with food or in the form of drugs ; and those which are due to the presence of disease of the urinary tract or other pai-ts ; among these blood, pus, bile, albumin, and sugar are the most important. It will be convenient to describe the heterogeneous group of cases we have to consider in the following order : — 1. Substances that appear in the urine as the result of the admi- nisti'ation of drugs. 2. Deposits of various kinds that may occur in the urine. 3. Urinary stones, or calculi. 4. Blood and blood-pigment in the urine. 5. Bile in the urine. 6. Proteids in the urine. 7. The urine in diabetes. 8. Glycuronic acid in the urine. 9. Fats in the urine (chyluria) 10. Alkaptonuria. 11. Alkaloids in the urine. DRUGS IN THE URINE Inorganic salts. — Iodide, bromide and chloride of potassium or sodium, appear in great measure unchanged in the urine. Salts of 766 EXCRETION cfesium, rubidium, lithium, and thallium behave similarly, as also do nitric, boric, and chloric acids. Compounds of arsenic and antimony and lead pass only in slight amount into the urine. The excretion of lead is increased by the use of potassium iodide. Mercury and silver, and other heavy metals, pass into the urine in mere traces, or after prolonged administration. The alkalis and their carbonates pass into the urine, diminishing its acidity or making it alkaline. Acids combine in the body with Ijases, and pass into the urine as salts. Iodine appears as an iodate, sulphur as a sulphate. Organic substances. — Alcohol when taken in excess appears in the urine only in traces. Chloral appears as urochloralic acid (Jaffe ') ; chloroform, partly as uVochloralic acid, and partly is decomposed increasing the amount of chlorides. Vegetable acirh are, as a rule, changed into carbonates. Gallic and pyrogallic acids are partly excreted as such, partly as pyrogallol, pyrocatechin, and other suVj- stances, which turn brown on exposure to the atmospheric oxygen in alkaline urine. Tannin appears chiefly as gallic acid ; benzoic acid and allied benzoyl compounds combine with glycocine to form hippuric acid. The fate of other aromatic substances has been already described (Chapter XL.). Quinine, strychnine, and morphine are excreted for the most part unchanged, though sometimes morphine may be totally destroyed in the organism. URINARY DEPOSITS The different deposits that may occur in urine are chemical substances and formed elements. The chemical sicbstances are uric acid, urates, calcium oxalate, cystin, leucine, tyrosine, xanthine, phosphates, and indigo crystals. The formed or anatomical elements consist of blood-corpuscles, pus, mucus, epithelium cells, spermatozoa, casts, fungi, and entozoa. The methods of examining urinary deposits are partly chemical, partly microscopical. We can recognise the chemical substances by their characteristic reactions ; the microscope even here comes to our aid, for it enables us to see whether the deposit is crystalline or amorphous, and if the former, the shape of the crystals is often diagnostic. In the recognition of the anatomical elements, the microscope is the principal method of examination, chemical tests being of secondary importance. In the examination of urinary deposits, it is important to note whether the urine contains the deposit immediately after being passed, ' See Glycuronic Acid in Urine (p. 793). ABN'OK.MAL AND PATHOLOGICAL URINE 7hijsiol. Cheml xiv. 109. 3 There appear to be different isomerides of cystin which differ in the readiness with which they give up their sulphur (Goldmann and Baumann, Zeit. plujsioh Chem. -an. 254). ■* Ahst. Chem. Soc. Journal, 1885, p. 830. ^ Lqc. cit. AI?N()H."\IA1, AND I'ATIK )!.( )( i ICAl. IHINH 709 On coiii|iiiiin*. - These are of various kinds, according to the situation of the disease ; cells from the renal tubules, from the pelves and ureters, from the bladder and urethra. They are larger than pus- cells, their nucleus is apparent without treatment with acetic acid, and their form is characteristic of the situation fi"om which they come {see Chapter XX T, Epithelium). Cells from cancerous and other growths of the urinary tract may occasionally be found in the urine. (5) Spermatozoa may be found if semen has passed into the urethra, or bladder, or into the vagina. (6) Ca.^ts. — These moulds of the renal tubules only occur in urine in kidney disease, and the urine containing them is albuminous. They may be classed as follows : — a. Hyaline casts, produced by the escape of blood-plasma into the tubules; this coagulates, and is carried into the main urinary stream by the jn-essure of fluid behind. If the epithelium of the tubule has been previously removed, larger hyaline casts are obtained. b. Epithelial casts. — These are hyaline casts to which the detached epithelium of the tubule adhei-es. c. Blood-casts. — These are casts formed in tubes into which hiemorrhage has occurred. They contain entangled blood-corpuscles, or may sometimes appear to consist wholly of blood-corpuscles. d. Granular casts. — These are casts containing the debris of degenerated epithelium cells, or of blood-corpuscles ; in the latter case they are yellowisli. 3 D 2 772 EXCRETION e. Fatty casts. — These are casts dotted with fat-granules or globules, either within epithelial cells or free, i.e. after the disintegration of epithelial cells. f. Albuminoid casts. — These are casts formed of waxy or albuminoid material which form in waxy, lardaceous, or albuminoid degeneration of the kidney. They are stained brown by iodine. g. Mucus casts. — Casts of mucus, often of great length, sometimes occur, and do not necessarily indicate disease. h. Seminal casts. — Casts of the seminal tubes of the testes. These contain spermatozoa. , i. Leucocyte casts. — These are formed of adherent leucocytes, and occur in suppuration, which involves the renal tubules. j. Urate casts. — Acid sodium urate occasionally takes the form of a cast of the tubule in which it was excreted. This does not necessarily indicate disease. It dissolves on the application of heat, (7) Parasitf.-<. — Healthy urine i.s free from microbes. They may be introduced from without, as by the use of dirty catheters ; they may arise from disease, such as tuberculosis of the urinary passages ; ov they may even come from more distant jiarts, passing into the ui-ine from the blood, as in anthrax, or carried l)y the blood to the kidneys in diphtheria, typhoid fever, ulcerative endocarditis, and other diseases. Sarcinaj and saccharomyce.s have Ijeen found in diabetic urine. Hook- lets of Tceuia echitiOcoceus, ova of the fluke BiUtarzia hremtitnhia, embryos of filaria sanguinis Jiominis, and thread-worms from the vagina in women are in.stances of entozoa occurring,' in the ui-ine. Many of the preceding deposits occur but rarely in the urine. The following scheme for the preliminary examination of a urinary deposit takes into accoimt only the commoner forms of sediment. It is also necessary to recognise in such a scheme that proteids, especially serum-albtniiin in solution in the urine, raaj' occur in conjunction with a deposit. I. Ascertain the reaction of the urine by means of litmus jDaper. II. Note the naked eye appearance of the deposit, and perform the following tests : — Remove some of the deposit from Confirmatiiry tests to the bottom of the urine glass witli a Nakeil eye appear- be applied to the sedi- Reaction of pipette, and shake it up witli some of ance of dejiosit ment removed from the urine the supernatant urine urine Ijy filtration a. Fill a test-tube lialf full of the turbid urine and heat i. The turbidity disappears — C'UATES Brick-red deposit Murexide test As a rale acid 11. The deposit remains almost in- soluble— Vmr Acid Cayenne pepper Murexide test Acid ill. The turbidity increases — Calcium Carbonate White deposit Dissolves in acetic acii;.mal .\ni> i'.\)"ii"ti.i)(;ic,\i, riiiNH m two Inst u imcfl arc also rare. Tak ^v,•lnn iHlute hydrochloric acid. a. It dissolres romjilefcl;/. Cystin or a fivsli imrtinii of the powder, and digest with b. It dlgmlrex inconipletcli/. Filter ; wash the residue with water. Tr}' the niurexide reaction. Both uric acid and ammonium urate stones give this reaction. To test for am- monia proceed as follows : Warm a fresh portion of the powder with sodium carbonate solution. Ammonia is given off, and may be recognised by colour is produced, its smell, blueing of red litmus paper, which becomes red- and fumes with a glass rod wetted with hydrochloric' acid. If ammonia is present, the stone consisted of Aji- MOXiUM Urate ; if absent, of Uric Acid. B. The powder becomes black, owing to the organic substances present, but leaves a considerable residue. The stone may then consist of urates of sodium and potassium, or earthy phosphates, or calcium oxalate, or carbonate. (1) Take a fresh portion and place on platinum foil. It melts under the blowpipe flame. Fusible Calculus. Confirm by dissolving in dilute hydro- chloric acid and testing for phosphates. (2) Take a fresh portion of the powder ; add dilute hydrochloric acid, a. It dissolves comph'telij. | b. It dissolves incompletely. Uric acid is absent. I Uric acid is present, and may be If it dissolves with effervescence, j tested for in the residue in the man- Dissolve a fresh portion in ammonia and filter : evaporate off the ammonia from the filtrate ; hexagonal crystals of cystin form. The crystals may be also obtained by adding 4icetic acid to the ammoniacal solu- tion. Xaxthine Dissolve a fresh portion in nitric acid ; evapoi'ate to dryness on a por- celain dish ; add caustic potash when cool ; a red dish ■\iolet on heat ing. •C'ALCluil CARBO^'ATE is present. If there is no effervescence, it is absent. If it dissolves without effervescence, and a fresh portion of the powder be incinerated, and then effervesces with hydrochloric acid, the stone contains or •consists of Calcium Oxalate, which, by the process of incineration, had Taeen converted into the carbonate. If it dissolves without effervescence either before or after incineration then the portion of stone taken consists wholly of Phosphates. If, however, there is effervescence, phosphates are •not necessarily absent ; they may be mixed with the carbonate, or more rarely with the oxalate of calcium, which gave the effervescence before and after incineration resjoectively. Phosphates may be •tested for by the nitro-molybdate and other well-known tests. The raetals ■calcium and magnesium may be tested for in the usual way (p. 717). Stones often contain a .small percentage of Iron. This also passes into the hydrochloric-acid solution ; render it feebly alkaline with ammonia : cool ; acidu- late with acetic acid ; yellowish white flocculi of ferric phosphate separate out. Collect these on a filter, dissolve them in hydrochloric acid, add ferrocyanide of potassium and Prussian blue is developed. ner described above (A b). The stone then consisted of Urate of Soda. Traces of that of potash may be also present. Phosphates, calcium carbonate, or calcium oxalate may have been mixed with the urate, but these pass into solution in the dilute hydrochloric acid used ; these may be tested for as described in the accompanying column (a). Sodium and potassium, calcium and magnesium also pass into solution if present as chlorides. These metals maj' be detected as already described (p. 717). 77C EXCRETION BLOOD AND BLOOD-PIGMENT IN UEINE When ha-iiiorrliage occui/s in any part of the urinaiy tract, blood appears in the urine. Blood which comes from the kidneys, as in acute Bright's disease, cancer of the kidney, scurvy, purpura h.emorrha- gica, endemic luematuria (due to the parasite Billiarzia), and after the use of turpentine, caiitharides, and other drugs, is generally mixed uniformly with the urine. Blood which comes from the Ijladder is often clotted, and usually comes in greatest abundance towards the termina- tion of the act of micturition, while that from the urethra stains most deeply the urine that is passed at the commencement of micturition.- Blood from the prostate is generally uniformly distiiliuted in the urine, so resembling blood from the kidneys. If a large quantity of blood is present, the urine is deep red, espe- cially if it is alkaline ; the microscopic examination of the sediment reveals the presence of blood-corpuscles ; on spectroscopic examinatitm, the bands of oxyhsemoglobin are well seen. If only a small quantity of blood is present, and is uniformly mixed with the urine, that secretion has a characteristic reddish-brown colour, which clinical observer's have termed ' smoky.' This is especially seen when the urine is acid. The precise cause of the smoky tint has received but scant attention ; it may be in some cases merely due to admixture of the oxyhsemoglobin and urinary pigments ; more often it is due- to the formation of metha^mogloljin. Methtemoglobin is genei*ally formed in small quantities in acid urine which contains blood after its- removal from the body. In other cases still, the pigment occurs in a condition more akin to ha?matin • than to haemoglobin. In some cases very little of the blood passed, dissolves in the urine ; the most charac- teristic spectrum wdiich is then obtainable from the deposit is that of haemochromogen.- The deposit collected on a filter is dissolved in rectified spirit containing ammonia, and a drop of ammonium sulphide added. The two characteristic bands of hl.-., (>ls, (>54). * Physiol. Chein. p. 8(j'2. ■' Clin. CJicmistri/ of Urine, p. 164. 778 KXCKKTIoN faint band between C and D, hut ncaier C, due to metli.'emoglubiii (tig. 59, spectrum 6), and the t\\\> ty])ical bands of oxyh.'eraoglobin, i)i addition, between 1) and E. On ;iddinmogl()bin band fades, tlie oxyhfemoglobin bands get a little darker, and then are rapidly re- placed by the single Imnd of i-educed luemoglobin (fig. 59, spectrum 3). In one case, Neale- found oxyhpemoglobin cj'ystals in the urine. In some cases bile-pigments have been found, in addition to the blood- pigment^ ; in other cases they are absent.^ On })oiling the urine of paroxysmal luvmoglobinuria, a brown coagulum is formed. This is due to the coagulation of globin, the pro- teid constituent of htenioglobin. In three of the cases I have examined, serum-albumin was present in addition, and in one of these serum- albumin occurred in the urine at the connnencement of the attack, previous to the appearance of blood-])igment in that secretion.-^ I5ILE IX UlilXE ]^»ile appears in the urine in the condition known as icterus, or jaundice. The varieties of jaundice and their causes have been already described in connection with the blood (p. 311) and the bile (p. 688). Bile-pigments in urine give it a gi-eenish-yellow, or greenish-brown, colour, which in severe cases may app)-oach black. In two cases bili- rubin crystals have been found in tlie mine.'' X^rine containing excess of urobilin may simulate icteric ui'ine, and so the following tests should always be perfoi'ined : — ' The urine must always be examiiied iinniecliately after ammonium sulphide has boeii lidded, for in a few mhiutes it becomes cloudy and opaque. - Lancet, voh ii. 1870, p. 725. ^ Hoppe-Seyler, Loc. cit.; Kiihne, Virrlmu-'a Arcliir, xiv. :)li) ; Herrmann, DinH. Berlin, 1859; TarchanofE, Pfliigei-'s Archie, i.x. 58. 4 Naunyn, Arch.f. Anat. unci PJtij.iiol. 18(i8, p. 4(»2: Steiner, Ibid. 1873, p. lOO. 5 This case was under Dr. Ringer's care in Univ. Coll. Hosp. and is recorded \>y Barton, Clin. Soc. Tram. 1890, p. 80. Foulerton (Lanrrt, ii. 1890, p. 709) is inclined to consider that the not infrequent ass(X'iation of ha?moj;lobinuria with oxaluria is not merely accidental. •^ Hoppe-Seyler and v. Recklinghausen noted the appearance of bilirubin crystals in the urine of a boy into whose vessels lamb's blood had been transfused ; and Ebstein (Dctiiscli. Arch. klin. Med. 1878, ]>. 115) found similar crystals in urine which contained Wood in a case of pyonephrosis. .\l!N(>i;.MAl, AND I'A ril (1) llineHna nuction. This tost consists in pouring the urine on to tlie sur- face of some nitric acid in a test-tube ; the play of colours, green, blue, red, yellow, is produced at the junction of the two liquids. The test may be also performed on a white plate ; a droj) of nitric acid placed in the centre of a film of the urine on the plate becomes surrounded with coloured rings if bile-pigments are present. The spectroscopic appearances of these colour- changes are characteristic (p. G83), a,nd may thus serve to distinguish bile in doubtful cases from other pigments which give certain colours with nitric acid. Ordinary urine always becomes rather -(.larker on the addition of nitric acid from o.xidation of its chromogens ; urine con- taining excess of skatoxyl ])igmeiit (p. 74.3) becomes red; urine containing excess of indoxyl sulphate of potassium becomes blue or violet from the formation of indigo. Gmelin's test is so delicate and so perfectly characteristic that it is seldom necessary to separate out the pigments. This, however, may be done by Hoppe- .Seyler's method as follows ' : Add milk of lime to the urine, and pass a stream of •carbonic acid through the mixture till no further precipitation takes place. The jirecipitate carries down with it the bile-pigment, if present, while urobilin and indiain are left in solution. The precipitate is collected ; a little water and a little chloroform acidulated with acetic acid are added, and the mixture shaken ; biliverdin colours the water green, and bilirubin colours the chloroform yellow. Both solutions give Gmelin's reaction. (2) MarcchaVs test.'- — A few drops of tincture of iodine (B.P.) are carefully poured on to the surface of the urine in a test-tube. If bile-pigments are present ■a green colom- appears below the red layer of iodine tincture. If the urine is very dark it should be diluted with water before applying the test. The Bile-salts. — Yogel and Diagendorff"' found 08 gramme of these .salts in 100 litres of normal urine. In other words, they are for prac- tical purposes absent from normal urine. It is also very difficult to ur<; ' t(i\es the percentage as even higher; Oertels,* i>n the othei- hand, places it at '^ pei- cent. The tests applied in these cases (especially in those of Chateauhourg) were not wholly satis- factory, and tlie subject demands reinvestigation. It is, however, probable that \igorous nnisculai- action, especially in those at all prone to kidney disease, jnay ])roduce a tenipoiary congestion of that organ and lead to temporary nlbuininuiia. A somewhat siniila)- condition sometimes occui-s aftei- tlie application of cold to the body,^ as after a cold l)ath. Tlie blood is diiven into the interioi- of the body from the >kin. and the renal vessels are thus over-filled. In some cases, again derangements of the nerAOUs system (which interfere with the Aaso- motor nerve regulation of the kidney vessels), and derangements of digestion, and anaemia (which altei" temporarily the composition of the l)lood), mav lead to a similar temporary or functional albuminuria. Experimental aJbuminuria. — The following experimental inter- ferences with the kidney cii-culation are followed by tlie appearance of the serum-proteids in the urine : — (1) Pressure upon (not closure of) the renal Aeins ' : the pressure in the glomeruli is thus increased. (2) Clo.suie of the renal ai'tery, and subsequent re-establishment of the circulation : this is due to interference with the nutrition of the renal cells. (3) Ligature of the aorta below the one kidney, and extirpation of the other. This raises the pressure as in (1). (4) Ligature of the aorta above the giving off of the renal arteries. (5) Compression of the trachea : this leads to asphyxia and the consequent lise in blood-pressure. We thus see that conditions of increased blood-pressure in the kidneys lead to albuminuria. Runeberg,'^ from a curious misunder- standing of some of his experiments, stated that lessened blood -pressure produced this result. His mistake was demonstrated l)y Gottwalt.'' Under the heading of experimental albuminuria must also be in- cluded the appearance of egg-albumin in the urine after its injection ' Becherches siir Valbuviinurie 2}hysiologiqiie, 1883; see also Senator. AJbinninuria in Health and Biscase,^ev> Syd. See. translation, 1884 ; Saundby, Glasgow Med.Journ. June 1884 ; Ralfe. Diseases of the Kidneij, p. 533. - Ziemssen's Handhitch der aUgemein. Thera2>ie, iv. •' Lassar, Arch, patlt. Anat. vol. Ixxix. 1880. •• Perls, Arch.f. exper. Pathol, vi. 113. •' Arch. d. Heilk. xviii. 1; Denfsch. Arch.f. klin. Med. xxiii. 41, 225. ^ Zcif.jihgsiol. Chem. iv. 423. 782 EXCKETloX into the vessels of animals, or after too great ingestion of eggs both in animals and men. Albtcminuria in disease. — The pressure of tuinimrs oi- of the pregnant uterus on the renal veins will cause albuminuria, as in the experiments just mentioned. Venous congestion in heart disease will act in the .same way. In certain conditions of the 1)lood, allnimin appears to- pass more readily than normally through the renal cells, and appears, in the urine ; e.g. in antemia (this is perhaps partially explicable by the lessened nutrition of the renal cells) ; in the first stage of con- valescence from cholera, just when the blood after being viscous and almost at a standstill in the kidneys, is l)ecoming more fluid again. Phosphorus-poisoning, and the excessi^•e use of morphia, and the poisons of certain diseases cause albuminuria ; this is especially seen in scarlet fever, which may be followed in bad cases by Bright's disease ;. to a less extent in typhoid, diphtheria, and pneumonia. It also may occur in diabetes.' The most frequent cause of alljuminuria is, however, Bright's- disease, in which the kidneys are actually diseased, and may even cease work altogether, producing ursemia (p. 315i. The forms of Bright's disease are — (a) Acute Brights disease. Much albumin, and often blood. (h) Large white kidney ; a fatty degeneration of the kidney-cells. Much albumin as a rule. {(■) Granular contracted kidney ; an overgrowth of connective tissue pressing on the renal tubules. Much urine secreted ; quantity of albumin smaller than in (a) and (h). (d) Albuminoid kidney. Albuminoid or waxy degeneration of the kidney substance. Quantity of urine increased ; quantity of albumin comparatively small. In Bright's disease renal casts can generally be discovered in the urine. The amount of albumin in urine rarely exceeds 1 per cent. ; it may, however, rise as high as 4 per cent. (Hoppe-Seyler).^ The word albumin has been used, as is usual with clinical observers, as the name of the proteid in urine. It should, however, be remembered that the proteid serum-globulin is nearly always present as well. The relation of albumin to gloljulin in the blood and lymph is termed the proteid- quotient (p. 341). If this is high or low, the pi'oteid-quotient in the urine is high or low also respectively. ^ The relation of albumin to globulin in uriae has not been much investigated, for, so far as we 1 Maguire, Brit. MnJ. Junrn. vol. ii. iSHCi, p. oio. - P/njsiol. Ghent, p. 858. ^ Pigeaud, Hoffmann, Maly's Jahreah. xvi. 474; Noel Paton, Brit. Med. Juiirn. n. l.s'JO, p. 196. Al?Ni>i;.M.\l. ANI» l'.\TII(i|,ii(;ic.\i, I KiNK 783 know ;iL present, the iiiiitlcr is one of nicic theoretical interest. The practical point a physician wislu's to ascertain is whether or not there is in the urine a proteid Avhieh is coaf^ulated by heat. Both allnmiin and globulin are precipitated on heating the ui'ine, the size of the L'oagulum giving a rough indicatioji of the amount of proteid present. In order to ascertain wiietlier or not serum-globulin is present, the- urine must be neutralised, and then saturated with magnesium sul- phate. A precipitate indicates the presence of serum-globulin.' Thi.s precipitate may be collected on a filter, dissolved by the addition of water. The solution so formed coagulates at 75° C. The albumin coagulates about the same tempei-ature, but is found, not in the preci- pitate produced by magnesium sulphate, but in the filtrate from which that precipitate has been removed.^ When a large quantity of serum-globulin is jsresent, the following- test of Sir W. Roberts may be applied: A glass vessel is filled with water, and a few drops of the urine allowed to fall into it. Each drfip leaves a milky trace behind it, and when a number of drops have been added, the water becomes opalescent. On adding acetic acid it again becomes clear. Occasionally serum -globulin is found without albumin in the urine ; sometimes it exceeds the serum-albumin in quantity. Usually the serum-albumin is in excess. Senator finds that the quantity of glolDulin is greater in waxy degeneration of the kidneys than in other forms of Bright's disease.^ In severe organic disease of the kidney, and in the albuminui'ia that occurs in diabetes, Maguire ^ finds the proportion albumin : globulin = 2 "5 : 1 a common one. In testing for these substances, the account already given of the proteids in Chapters X and XV should be consulted. The following li.st of tests may, however, be found useful, as it embodies all the chief reactions by which albuminuria is discovered. Towards these tests, albumin and globulin behave alike. If the urine is cloudy, it should be filtered first. The best and most trustworthy test is undoubtedl}=" the first in the following list ; it is, moreover, the simplest. Proteoses in urine.-^A.\\ albumose was first described in urine in a case of osteomalacia by Bence Jones.'' It has been since found in the urine in this disease by Kiihne'' and others. Hoppe-Seyler ~ ' Some albumoses iire also preeipitatecl by this salt, but give certain ether special tests to be mentioued later. - Mr. F. Smith (Array Veterinary Department) recently pointed out to me that normal urine gives a precipitate on saturation with magnesium sulphate. Wliat this, precipitate consists of is at present unknown ; it is not proteid in nature. ^ Noel Paton {loc. cit.) was not able to confirm this observation. * Brit. Med. Journ. vol. ii. ISSO, p. 54:J. '" Phil. Trans, vol. i. 18iS, p. 55. « Zcit. Biol. xix. 209. • Physiol. Chem. p. 858. 784 EXCRETION (1) Heat to 73° to 75° C., or boil; the top of a loug column of Tu4ne in a test-tube should be heated ; the difference between this and the clear urine below is then well seen (2) Add nitric acid The proteid is If the urine is alkaline it must i coagulated > be acidulated with a few drops of weak acetic acid, either before or after Vjoiling. The precipi- tate which is formed is insoluble in acetic acid, and can thus be : distinguished from phosphates. Nitric acid should not be used in this test, as coagulated albu- min is sUghtly soluble in nitric acid The proteid is precipitated. (Oc- casionally in very concentrated urine a crystalline preci- pitate of urea ni- trate is produced, but this is most exceptional) On subsequently boiling, this precipitate either does not dis- solve at all or onlv slitrhtlv (3) Heller's nitric acid test. The urine is poured j gently on to the surface j of some nitric acid in a ! test-tube • (4) Johnson's picric j acid test. A concen- [ trated solution of picric i acid is poured on to the '■ surface of the urine in a test-tube A ring of white This test is especially appli- precipitate at the cable to ivrines containing only junction of the two a small quantity of albumin liniiids is seen A ring of white precipitate occurs at the junction of the two liquids; this increases on heating (5) Potassio-mercuric iodide (Taurefs re- added to tlie Causes a white precipitate agent ') urine This method of applying the test is applicable to urines con- taining a small amount of albu- min. The test may be also per- formed by mixing the two re- agents together, or by adding a little solid picric acid to the urine. A precipitate forms in both cases. Peptones and albu- moses are precipitated by this reagent, but the precipitate re- dissolves on heating This reaction may also be per- formed in Heller's method. This reaction is the most delicate of the various reagents investigated by a committee of the Clinical Society.^ It is, howe\er, inferior to Heller's nitric-acid test, as it precipitates peptone and albu- ' Mercuric chloride, 1-35 giamine; ^wtassium iodide. 3-32 grammes; acetic acid 20 c.c. water Hi c.c. -' Clin. Soc. Trans, six. 339. AKNiiKMAl, AND I'ATlli >Li »i i K'Al. I IMNK /bo Test Reaction Ilemarks mose (this precipitate, however, redissol ves on heating), alkaloids, and also bile-salts (these may be extracted from the precipitate by ether) ' (fi) Sir W. Roberts" test. Acidulated brine (one ounce of hydro- chloric acid to a pint of saturated socliuni chlo- ride solution) added to the urine Causes a white precipitate of the proteid (7) Other precipitants of proteids, . e.fi-. sodium tungstate with or with- out citric acid, ferro- cyanide of potassium, metaphosphoric acid, &c. have been from time to time recommended For convenient bed-side test- ing these reagents are sometimes used in the solid form. Pavy's pellets consist of citric acid and Ijotassium ferrocyanide. Papers in-eviously soaked in potassio- mercuric iodide, potassium f en-o- cyanide, sodium tungstate, and, lastly, picric acid have been pre- pared by Dr. Oliver. The Clinical Society's committee report that of these,, potassio-mercuric iodide papers are the best fV)und it in several cases of atrophy of the kidneys, Lassar ^ in the urine of people rubbed with petroleum, Oertel ■* in a few cases after severe exertion. Both it and peptone are found in the urine of animals into whose circulation they have been introduced.^ The origin of this substance in the body is unknown. Virchow ^ found a similar substance in the red marrow in cases of osteo- malacia. Kiihne and Chittenden ^ found that in elementary composition the proteose of the urine resembles hetero-globulose more closelv than any other proteose, and suggest it may arise from serum - globulin. Another proteose sometimes found in the urine is deutero-pro- teose, which closely resembles peptone in its reactions, and is often mistaken for or mixed with peptone. It will be more appropriately considered in connection with peptonuria. 1 Brasse, Coiiijjt. rend. soc. biol. (8), iv. 369. ^ Ziemssen's Handhnvh d. Therapie, 188i. s Arch. path. Anat. iv. 309. - Arch. path. Anat. Ixxvii. 164. ^ Neumeister, Zeit. Biol. xxiv. 272. ^ Zeit. Biol. xxii. 409. 3e 786 EXCRETION Hetero-proteose (whether it be hetero-albumose or hetero-globulose), if it occurs alone in the urine, may be detected in the following way : — 1. Heat the urine to 65^. A precipitate forms iu neutral or faintly alkaline urine, which, unlike coagulated albumin or globulin, dissolves in a few drops of dilute hydrochloric acid. This precipitation does not occur in acid urine. 2. Add nitric acid to the urine ; a precipitate forms which dissolves on heating and reajipears on coohng. 3. Add a drop of dilute copper-sulphate solution and excess of potash ; a rose- red colour forms (biuret reaction). Albumin and globulin give a Ariolet colour. i. Saturation with magnesium sulphate causes ]Drecipitation. 5. Picric acid, potassio-mercuric iodide, mercuric chloride (in acid solutions), sodium tungstate, all give white precipitates. If a proteose is present mixed with peptone, saturation with ammonium sul- j)hate precipitates the former and leaves the latter in solution. If proteoses or peptones, or both, are present mixed with albumin and globulin, heating the urine (after acidulation if the urine is alkaline) jirecipitates the latter and leaves the two former in solution. As, however, there is risk of the formation of a small amount of primary proteoses (proto- and hetero-) by the hydrating action of the acidified hot liquid, a better method of separation consists in adding to the urine ten or twelve times its volume of absolute alcohol. A pre- cipitate of all the proteids is produced : by the end of two or thi'ee weeks the alcohol renders albumin and globulin insoluble ; the proteoses and peptones are soluble, and may be dissolved out with distilled water. The properties of proteoses, and tables for their sejjaratiou from one another, and from other proteids, will be found fuUy given in Chapter X. A table for the separation of the various proteids that occur in the urine will be found at the end of this section. Peptonuria. — There appear to be many conditions in which peptone has been described in the urine. ^ In healthy urine no peptone is present. In decomposing albuminous urines it may be formed from the albumin after the urine is passed. Many of the observations that have been made on this subject were published previous to the appearance of Kiihne and Chittenden's recent work on proteoses and peptones, and many of the older experiments therefore now require careful revision. It is only within the last few years that we have been furnished with accurate data for the identification and separation of these substances. The proteid most liable to be mistaken for peptone is deutero-proteose. They may be readily distinguished and separated from one another by saturating the urine (slightly acidified with acetic acid) with ammonium sulphate : this salt precipitates the deutero- proteose, and leaves peptone in solution. Any proteid that remains in solution after filtering ofi" the precipitate produced by thorough saturation with ammonium sulphate must be peptone. Tliis is, in fact, the only method by which peptone can be identified - with 1 It was first described m luiae by Gerhardt, Deutsch. klin. Arch. Med. v. 215. - Martin Brit. Med. Journ. vol. i. 1888, p. 842. Ai;Nt)l{M.\L AND rATIIOLOdlCAL 1 KINK 7'S7 certainty. A solution of peptone so obtained gives no precipitate with nitric acid, nor with copper sulphate ; it is not precipitated by heating ; it is ])recipitated by alcohol, but not coagulated by that feagent. Tt is also precipitated by tannin, potassio-uiercuric iodide, phospho-molybdic acid, phospho-tungstic acid, and picric acid. It gives a well-marked xantho-proteic reaction (yellow colour on boiling with nitric acid, turned orange or brown by ammonia), and biuret reaction (rose-red colour with copper sulphate and caustic potash). Martin has found that most of the so-called cases of peptonuria {especially in purulent diseases) are really cases of deutero-proteose in the urine. The following table contrasts the behaviour of these two substances in various reactions : — jDeutero-alhmnose | Pejrtone (1) Gives no precipitate with uitric acid under aav circumstances. (1) Gives no precipitate witli nitric acid unless a considerable amount of salt be also added. This precipitate < insi2ndut< is a disease in which there is a very abundant secretion of very watery urine, and is j^robably dependent on a derangement of the vaso-motor nerves of the kidney or their centre. The disease which, however, we have now to deal with is called, in contradistinction to the above, diabetes menitu.% and is characterised by a very almndant secretion of urine ' witli a high specific gravity 1 The quantity in the twenty-four hours rarely falls below two litres ; it may rise to eight and even ten litres. Ar.NORMAL AND I'A rilol.oi ; K'Al, linXK 7H!) <(1030 to 1050), and containinn' dextrose. Su!' carlninic acid is dl'lcn (il)taiiic(l rcdiii iiii|iiii-i(ics in the yo.ist . Another method of i)crrormiiiNOK^r.\l, AND I\\TTI0L(W1CAL lin.NK 795 FATS IX THE I'ltlNE Fats may be present in the urine umlcr three conditions : — (1) When no disease of the kidneys is present. a. From excess of fat in the food ( Bernard, ' Wiener, - 8eriba •'). h. After administration of cod-liver oil (Sir W. Roberts ^). c. In fat-embolism occurring aftei- fractures. d. Tn the fatty degeneration of the livei- that occurs in phospliorus- poisoning. I'. Tn cases of long-standing suppuration, phthisis, and pya-mia ; here doubtless fatty degeneration of the pus-cells occurs. /. In diabetes mellitus, when there is often a lipjeniic condition ' (see p. 315). In all these ca.ses the excess of fat passes into the blood, and thence to the kidneys. (2) In disease of the urinary organs. ff. In Bright's disease ; fatty casts are often seen. /i. In pyonephrosis (Ebstein^). This comes under the same cate- gory as the cases included under e. (3) In a peculiar disease, which occurs in the tropics, known as diyluria. This is sometimes accompanied with the formation of tumours containing lymph, in the regions of the scrotum and thighs {see p. 349). Its most marked symptom, however, is the passing of milky urine. It is produced by a jDarasitic worm called the filaria sanguinis hominis ; this inhabits the lymphatics, especially of the scrotum and lower limbs. It is also found in large numljers in the kidneys. Not only chyle, but also blood may be found in the urine. In chylous urine the following abnormal constituents are found : Fibrinogen, serum-globulin, serum-albvimin, finely divided fats, traces of soaps, lecithin, and cholesterin ^ ; in fact, all the constituents of chyle are present.^ Chylous urine usually coagulates when j^assed or deposits strands of fibrin. It is difficult to explain the occurrence of chyluria without supposing that a fistulous communication exists l)etween the lacteals and the urinary jiassages ; and though sometime.s- ' herons, Paris, vol. ii. 1859, p. 8G. - Arch. 2)afh. Anai. ii. ^ Dentsch. Zeit.f. Chir. xii. 118. * Quoted by MacMunn, Clin. Chemistrij of T'rinr, p. 203. 5 Robert, Diss. Halle, 1880. « Detitsch. Arch./, klin. Med. xxiii. IIS. ' Cholesterin lias also been found in the urine in fatty degeneration of the kidnej"s (Beale, Archives of Medicine, 18,57), in diabetes and jaundice (Salisbury, Amer. Joiirn. Med. Sciences, 1863), and in the urine of an epileptic treated with potassium bromide (Pohl, Petershurger rued. WocJi. 1877, p. 171). ^ For analyses of chylous urine see Hoppe- Sevier, P7?//.'?io/. Clteiu. ji. 870: Eggel, Dentsch. Arch. klin. Med. vi. 421; Brieger, Zeit. j)hijsiol. Chem. iv. 407. 796 ' EXCKKTIoX a post-mortem examination lias failed to reveal the presence of such, in other cases it has been found (Odennis and Lang, ' Hensen ^). For the recognition and estimation of the fat, lecithin, and cholesterin, the same methods may be employed as have already been described in connection with nervous tissue (p. 533). They are all soluble in ether; the ether is evaporated off, and they are found in the residue. The characteristic reactions of fats are described on p. -487, of lecithin p. 520, and of cholesterin p. 531. ALCAPTOXUUIA We have already seen that, after the administration of carbolic acid, gallic acid, and other aromatic compounds, the urine if alkaline becomes dark-brown on exposure to the air. This condition is produced by the oxidation of pyrocatechiu, hydroquinon, pyrogallol, and similar substances. The same compounds probably cause a similar darkening that occurs in the urine of herbivora, the diet of these animals contain- ing a large amount of aromatic substances. In certain cases, the pathology of which is nut well understood, an excess of these same aromatic compounds appears in human urine Avithout the administration of drugs : the urine consequently, as in so- called carboluria, darkens on exposui-e to the air. Cases of this kind were first described by Bodeker,-* and he called the substance alcapton, and the condition alcaptonuria. Gorup-Besanez ^ was the first to sug- gest that alcapton and pyrocatechin are the same thing. Salkowski and Leube, Epstein and Miiller," take the same view. W. G. Smith" and Preusse " believe that, in addition to pyrocatechin, protocatechuic acid is largely present in these cases. Udranszky® believes that botli of these are derived from an organic compound, which he calls a humous substance. In some cases examined h\ Kirk,^ he separated an acid from the urine, which he calls uroleucic acid (CgHmOg), which, accord- ing to Huppert, is probal)ly pyrogallolpropionic acid. It is possible that in different cases we may have different aromatic compounds present ; they all darken on oxidation : and they all reduce Fehling's solution, and must not lie confounded with dextrose. ALKALOIDS IN THE URINE The absorption of alkaloids from the alimentary canal sometimes occurs ; these are carried to the kidneys and excreted there unchanged; 1 Yircliow-Hirsch, Jahresh. vol. ii. Iti74, p. (574. - Pfiiiffer's Archir, x. 9-t. ^ Zeit. rat. Med. ^-ii. 128. ■* Lehrbuch, ii. o"24. ° Virchow's Archiv, Ixii. 554. ^ Dublin -Tourn. Med. Set. Jan. 1882. ^ Zeit. jjJiysiol. Che/ii. ii. 324. * Ibid. xi. 537 ; xii. 33. ^ Brit. Med. Journ. vol. ii. 1888, p. 232 ; vol. ii. 1889, p. 114S. Ar.N('l;M.\[, AND r\lll(>I,(»(ll('AL riMNK 1U7 iitropiiif, (iiiiniiu', and sti yrliniiic, and sninctinies niorphiiie, may thus, after their adn)inistratii)ii to a patient, be found in his urine. Whether ptomaines and leuconiaines, formed by putrefaction in the intestine, pass normally in the same way into the urine is very doubt- ful. If such alkaloids as neuiine and choline are thus formed, they are probably wholly destroyed by further putrefaction before there is time for absorption to take place (see Chapter XXXY). Tn cases of disease, symptoms may be sometimes explained by supposing that absorption of jioisonous animal alkaloids is occuiring. That such is the case remains to be proved in most cases. Normal urine is free from all alkaloids, except creatinine, and the same is true for most cases of morbid urine. Diamines have, however, been found in cases of cystinuria, cholera, and pernicious antemia. The toxicity of normal urine has Ijeen explained by Ponchet and Bouchard as due to the presence of ptomaines. Btadthagen has, however, shown that it is more pi'obablydue to the potassium salts the urine contains. A fuller discussion of the subject with reference to literature will be found in Chapter XIII. DIAZO-EEACTION IX URINE The diazo-reaction, sometimes called Ehrlich's reaction, is as follows : Two solutions are necessary ; (1) a concentrated solution of sulphanilic acid ; (2) a solution of sodium nitrite (1 in 200). 200 c.c. of (1) are mixed with 10 c.c. of pure hydrochloric acid and 6 c.c. of (2). Equal quantities of this mixture and the urine are mixed and rendered •strongly alkaline with ammonia. A bright carmine-red colouration constitutes the reaction. After standing twelve to thirty- six hours, a deposit occurs, the upper part of which is gi'een or black. The reaction is never given by healthy urine. Rutimeyer (see ' Lancet,' ii. 1890, p. 413), who has examined 260 urines, states it is of special value in the diagnosis di typhoid fever, as it is given by typhoid urine, but not in the urine from cases of intestinal cataxTh. It is also given by the urine from cases of acute tuberculosis (due to the absorption of caseous matter ?) and in certain other diseases. The exact diagnostic value of the test is a matter for future clinical research, and a good deal of correspondence on the subject Avill be found in the medical journals. 71)8 EXC']{ETION CHAPTER XLV QUANTITATIVE ANALYSIS OF URINE ESTIMATION OF THE TOTAL SOLIDS a. The amount of total solids may be obtained approximately by calculation from the specific gravity. The last two figures of the specific gravity- are multi- plied by 2-33 (for adults),' or by 1-66 (for children). Thus, if a man passes in the day 1500 c.c. of urine with a specific gravity of 1021, then 21 x 2-33 = 48-93 g;rammes in 1000 c.c , or 73-39 grammes of solids in the day's urine (1500 c.c.) b. A more accurate process is the following : Take 5 c.c. of urine in a weiglied F I KINE 7<)9 b. The ariility ul' urine is due lUJi-uiall}- lu acid pliosphatc of soda. The acidity of urine nuiy tiierefore he reckoned more correctly in terms of phosphoric acid. The ])roportion of phos])horic acid combined as acid phosphate to the total amount of pliosplioric acid present may be taken us a measure of the acidity of urine, and may be estimated in the following waj' (Hnppert): — The following reagents are necessary : — i. The reagents necessary to estimate the amount of total phosphoric acid (see p. 802). ii. Standard caustic soda solution containing 10 grammes of caustic soda in a litre of distilled water : 1 c.c. = 0-00591 gramme of phosphoric acid (PoO^). iii. Standard sulphuric acid solution. This contains 12-25 grammes of sulphuric acid (HoSO,) in solution. It maj' be obtained by taking 7-5 c.c. of ordinary sulphuric acid and diluting it to a litre. It must be still a little further diluted until a known volume of it exactly neutralises the same volume of the soda solution. The amount of extra dilution which is necessary is ascertained by titration. iv. Saturated solution of barium chloride. V. Neutral litmus solution. Method. — The total phosphoric acid is first determined with uranium nitrate («ee p. 802) ; 200 c.c. of urine are then taken, and rendered strongly alkaline with the soda solution from a burette, the quantity used being noted Call it a. Chloride of barium is then added till no more precipitate occurs. The liquid is filtered. The filtrate is coloured with neutral litmus solution. It is then rendered neutral with the sulphuric-acid solution cbropped into it from a burette, the quantity used being noted. Call it ^. Calculation. — When the urine has been rendered alkaline in the way described the unsaturated phosphoric acid (i.e. that combined as acid phosphate) com- bines with soda, so that phosphate of soda is formed. The barium chloride gives a precipitate of barium phosphate. After the removal of this by filtration the alkalinity of the fluid is determined by the sulphuric-acid solution. The difference in the amounts of acid and alkaline fluids used will correspond to the amount of unsaturated phosphoric acid ; from this the quantity of acid combined as acid phosphate can be calculated by the follo\\-ing formula : — a; = quantity of phosphoric acid combined as acid phosphate ; a = quantity in grammes of phosphoric acid which is unsaturated in the urine. = (a-)8)x-0059 & = total quantity of phosphoric acid in urine (found by the uranium nitrate method) ; that is, one has to subtract a third of the total phosphoric acid from the amount of unsaturated phosphoric acid and multiply the difference by 2. The acid phosphate contains in its molecule two parts of phosphoric acid, while in the total phosphates there are three per molecule. One, therefore, finds the relation of the acid phosphate to the total phosphates in molecules by divid- ing the quantity of phosphoric acid combined as acid phosphate by 2, and that contained in the total phosphates by 3. Example. — 200 c.c. of urine taken. Total phosphoric acid (Zi) = 0-2 gramme. Another 200 c.c. taken. 30 c.c. (a) of soda solution added. 800 EXCKETION After filtering- off the barium phosi^hate it was found tliut 9-7 c.c. (3) of the sulphuric acid solution were necessary to neutralise the filtrate. a- y8 = 30 -9-7 = 20-3 c.c. Each c.c. corresponds to 000591 gramme of phospiioric acid. «-(a-)3)xO'00591 = 20-3xO'00591 = 0'12 gr. ; .r = 2(« - J) = 2(0-12 - ^"^ = 0-108 gramme. The proportion of total phosphate to acid pho.-^phatcs in molecules 0-2 0-108 „„ r, 3 • 2 ESTIMATION OF CHLOKIDES The chlorides in the urine consist of those of sodium and potassium, the latter only in small quantities. The method adopted for the determination of the total chlorides consists in their precipitation by a standard solution of silver nitrate or mercuric nitrate. a. Mohr's method, — Precipitation by silver nitrate. The following solutions must be prepared :-- i. Standard silver nitrate solution. Dissolve 29-075 grammes of fused silver- nitrate in a litre (1000 c.c.) of distilled water ; 1 c.c. = 001 gramme of sodium chloride. ii. Saturated solution of neutral potassium chromate. Analysis. — Take 10 c.c. of urine ; dilute with 100 c.c. of distilled water. Add to this a few drops of the potassium chromate solution. Drop into this mixture from a burette the standard silver nitrate solution ; tlie chlorine combines with the silver to form silver chloride, a white pi-ecipitate. When all the chlorides are so precipitated, silver chromate (red in colour) goes down, but not while any chloride remains in solution. The silver nitrate must therefore be added until the precipitate has a pink tinge. Read off the quantity of standard solution used, and calculate therefrom the quantity of sodium chloride in the 10 c.c. of urine taken, and thence the- percentage. Sources of error and corrections. — A higli-coloured urine may give rise to diffi- cult}'- in seeing the pink tinge of the chromate of silver : this is overcome by diluting the urine more than stated in the preceding- paragra])h. 1 c.c. should always be subtracted from the total number of c.c. of the silver nitrate solution used, as the urine contains small quantities of certain compounds more easily precipitable than the chromate. b. To obviate such sources of error the following modifications of the test, as described by Sutton,' is used : 10 c.c. of urine are measured into a thin porcelain capsule and 1 gramme of pure ammonium nitrate added ; the whole is then evaporated to dryness, and graduallj' heated over a small spirit lamp to low red- ness till all vapours are dissipated and the residue becomes white. It is then dissolved in a small quantity of water, and the carbonates produced bythe com- bustion of the organic matter neutralised by dilute acetic acid ; a few grains of 1 Volumetric Analysis, p. o09. QrANrnATlVF, analysis of IKINK 801 pure calcium carbonafL- to remove all free acid are then added, and one or two drops of potassium ehromate. The mixture i> then titrated with decinormal silver solution (If) ItfiG gv. of silver nitrate per litre) until the end reaction, a pink colour, appears. P^ach c.c. of silver solution represents 0-U05837 gr. of salt ; consequently . if 12-5 c.c. have been used, the weight of .salt in the 10 c.c. of urine is 0'072l)G gr., or 0-7296 per cent. If 5fl c.c. of urine are taken for titration, the number of c.c. of silver solution used will represent the number of parts of salt per 1000 parts of urine. Pribram' uses potassium permanganate at a boiling temperature to destroy organic matter instead of ammonium nitrate. Other methods for estimating chlorides are those of Volhard,-' Habel and Fernholz,' Arnold,' and Zuelzer,^ but none are so good when applied to urine as Mohr"s. c. Liebigs method. — Precipitation b}- mercuric nitrate. The following solutions must be tirst prepared: — i. Standard mercuric nitrate solution : — Dissolve 20 grammes of pure mercury in boiling nitric acid ; then dilute to nearly a litre. To dilute this to the right strength, preliminary experiments must be performed with a standard solution of pure sodium chloride, 20 grammes to the litre. Take 10 c.c. of the standard sodium chloride solution, add to this 2 c.c. of a 4 per cent, solution of urea, and 5 c.c. of a saturated solution of sodium sulphate. Into this mixtm-e allow the mercuric nitrate solution to flow from a burette, stirring the mixture the while. A precipitate forms, which redissolves on stirring ; add the mercuric nitrate solution till a permanent precipitate (not an opalescence) forms ; the reaction is then complete. The strength of the mercurial solution is thus determined, and it is then diluted so that 20 c.c. = 0"2 gramme of sodium chloride = 10 c.c. of the standard sodium chloride .solution : 1 c.c. therefore corresponds to 001 gramme of sodium chloride, or 0-00ti059 gramme of chlorine. ii. Baryta mixture. — This is made by adding two volume*^ of barium hydrate solution to one of barium nitrate solution, both saturated in the cold. iii. Dilute nitric acid (1 in 20). Analysis. — Take 40 c.c. of urine. Add 20 c.c. of baryta mixture. Filter off the precipitate which forms, which consists of sulphate and phosphate of baiium. Take 15 c.c. of the filtrate; this corresponds to 10 c.c. of the original urine. Render this slightly acid with dilute nitric acid. Run in the standard mercuric nitrate solution from a burette, stirring the mixture well until a pennanent precipitate appears. Read off the number of c.c. used ; multiply by 0()1. This gives the amount of chlorine as sodium chloride contained in 10 c.c. urine. Explanation and corrections. — This test depends on the fact that when mercuric nitrate and sodium chloride in solution are mixed, sodium nitrate and mercuric chloride, which are both soluble in water, are formed. It is not till all the chloride in the urine is so decomposed that mercuric nitrate begins to com- bine with the urea present to form a permanent white precipitate. Hence the necessity of estimating the chlorides when using Liebig's method for the deter- mination of urea. In order to obtain the exact point at wiiicli the precipitate becomes a per- 1 Zeit. anal. Chem. ix. 428. - See Sutton's Vol. AimI. p. 310. ^ Pfliiger's Archiv, xxiii. 85; xxiv. 2. ■* Ibid. xxxv. 541. ^ Her. deutsch. chem. Ges. x^-iii. S". 3f 802 EXCKETIOX manent one, the process must be repeated in another specimen. The advantage of tliis process is its simplicity ; its disadvantage is that the end point is rather obscure. If tlie urine used is albuminous the albumin must be first removed by boiling, after the addition of a few drops of acetic acid, and filtering off the precipitated albumin. ESTIMATION OF THE PHOSPHATES The phosphoric acid in the urine is combined with soda, potash, lime, and magnesia. a. Estimation of the total phosphates. For this purpose the following reagents are necessary : — i. A standard solution of uranic nitrate. The uranic nitrate solution contains 35-5 grammes in a litre of water ; 1 c.c. corresponds to 0 005 gramme of phos- phoric acid (P.jOj). ii. Acid solution of sodium acetate. Dissolve 100 grammes of sodic acetate in 900 c.c. of water ; add to this 100 c.c. of glacial acetic acid. iii. Solution of potassium ferrocyanide. Method. — Take 50 c.c. of urine. Add 5 c.c. of the acid solution of sodium acetate. Heat the mixture to 80° C. Run into it while hot the standard uranium nitrate solution from a burette until a drop of the mixture gives a distinct brown colour with a drop of potassium ferrocyanide placed on a porcelain slab. Read off the quantity of solution used and calculate therefrom the percentage amount of phosphoric acid in tlie urine. b. Estimation of the phosphoric acid combined with lime and magnesia (alka- line earths). Take 200 c.c. urine. Render it alkaline with ammonia. Lay the mixture aside for twelve hours. Collect the precipita'ed earthy phosphates on a filter ; wash with dilute ammonia (1 in 3). Wash the precipitate off the filter with water acidified by a few drops of acetic acid. Dissolve with the aid of heat, adding a little more acetic acid if necessary. Add 5 c.c. of the acid solution of sodium acetate. Bring the volume up to 50 c.c, and estimate the phosphates in this volumetrically by the standard uranium nitrate, as before. Subtract the phosphoric acid combined with the alkaline earths thus obtained from the total quantity of phosphoric acid, and the difference is the amount of acid combined with the alkalis soda and potash. c. Instead of uranium nitrate a standard solution of uranium acetate may be used. The directions for the making of these standard solutions will be found in ' Sutton's Volumetric Analj'sis.' As a rule, it is less troublesome, and not much more expensive, t> purchase standard solutions ready made. ESTIMATION OF THE SULPHATES The sulphates in the urine are of two kinds : the pre-formed sulphates, viz. those of soda and potash, and the combined or ethereal sulphates. a. For the determination of the total amount of sulphuric acid (SO^) (i.e. pre- formed and combined sulphuric acid together) in the urine one of two methods is adopted : — 1. Volumetric method. i'. Gravimetric method. QUANTITATIVK .\N.\I,VS1S ()K IIIIXI'; 803 1. Volumetric determination. — Tliis process coDsists in adding? to a given volume of tho mine a standard solution of cldoride of barium so long as a preci- pitate of barium sulphate is formed. The following solutions are necessary : — i. Standard barium chloride solution : 30-.5 grammes of crystallised chloride of barium in a litre of distilled water; 1 <:.r.. of this solution corresponds to 0*01 gramme of sulphuric acid (SO3). ii. Solution of sulphate of potash : 20 per cent. iii. Pure liydrochloric acid. Met/wfl. — 100 c.c. of urine are taken in a flask. This is rendered acid by 5 c.c. of hydrochloric acid, and boiled. The combined sulphates are thus converted into ordioary sulphates, and give a precipitate like them with barium chloride. The chloride of barium solution is allowed to drop into this mixture as loiig as any precipitate occurs, the mixture being heated before every addition of barium chloride to it. After adding 5 to 8 c.c. of the standard solution, allow the preci- pitate to settle ; pipette off a few drops of the clear, supernatant fluid into a watch- glass ; add to it a few drops of the standard barium nitrate solution. If any pre- ci])itate occurs, return the whole to the flask and add more barium chloride ; again allow the precipitate to settle, and test as before ; go on in this way until no more barium sulphate is formed on the addition of barium chloride. Excess of barium chloride must also be avoided ; when only a trace of excess is present a drop of the clear fluid removed from the flask gives a cloudiness with a drop of the potassium sulphate solution placed on a glass plate over a black ground. If more than a cloudiness appears, too large a quantity of barium' chloride has been added, and the operation must be repeated. From the quantity of barium chloride solution used, the percentage of sulphuric acid in the urine is calculated. 2. Gravimetric determination (i.e. by weight). — This method consists in weighing the precipitate of barium sulphate obtained by adding barium chloride to a known volume of urine; 100 parts of sulphate of barium correspond with ;i4"33 parts of sulphuric acid (SO3). Metliod (Salkowski). — 100 c.c. of urine are taken in a beaker. This is acidified with 5 c.c. of hydrochloric acid as before. Chloride of barium is added till no more precipitate occurs. The precipitate is collected on a small filter of known ash, and washed with hot distilled water tiU no more barium chloride occurs in the filtrate, i.e.until the liltrate remains clear after the addition of a few drops of hydric sulphate. Then wash with hot alcohol, and afterwards with ether. Remove the filter, and place it with its contents in a platinum crucible. Heat to redness. Cool over sulphviric acid in an exsiccator; weigh, and deduct the weight of the crucible and filter ash ; I he remainder is the wei^fht of barium sidphate formed. Error. — When the experiment is carried out as above there is a slight error from the formation of a small quantity of sulphide of barium. This may be cor- K-cted as follows: After the platinum crucible has become cool add a few drops I if pure sulphuric acid (H.,SO J. The sulphide is converted into sulphate. Heat :iL;ain to redness to drive off excess of sulphuric acid. b. The following is Salkowski's ' method of estimating the combined sulphuric acid ; that is, the amount of SO3 in ethereal sulphates : — 100 c.c. of urine is mixed ' Zeit. vhysiol. Cliem. x. 346. This method is a modification or Baumann's original lethod, Ihid. i. 71. •3 P 2 804 EXCRETION with 100 cc. of alkaline barium chloride solution, which is a mixture of two volumes of .solution of barium hydrate with one of barium chloride, both saturated in the cold. The mixture is stirred, and after a few minutes filtered ; 100 cc. of the filtrate ( = 50 cc. of urine) are acidified with 10 cc of h.ydrochloric acid, boiled, kept at 100° C. on the water-bath for an hour, and then allowed to stand till the precipitate has completely settled ; if possible, it should be left in this way for twenty-four hours. The further treatment of this precipitate ( = combined sul- phates) is then carried out as in the last case. Calculation. — 23.3 parts of barium sulphate correspond to 98 parts of H2SO4, or 80 parts of SO3 of 32 parts of S. To calculate the H^SO^, multiply the weight of barium sulphate by-!-^ =0-4206 ; to calculate the SO3 multiply by — = 0-34835; 32 to calculate the S multiply by _^ = 0-13734. This method of calculation ap- 233 plies to the gravimetric estimation both of total sulphates and of combined sulphates. c To obtain the amount of pre-formed sulphuric acid subtract the amount of combined SO^ from the total amount of SO3. The difference is the pre-formed SO3. Example : 100 cc of urine gave OS gramme of total barium sulphate. This 80 multiplied by - =0-171 gr. = total SO3. Another 100 cc of the same urine gave 0-05 gr. of barium sulphate from ethereal sulphates ; this multiplied by -?2. = 0017 gr. of combined SO,. Total SO,-combined 803 = 0-171-0-017 = 0-154 233 ^ gr. of pre-formed SO3. ESTIMATION OF THE CARBONIC ACID Carbonic acid occurs in the urine both in the free state and also combined with alkaline metals to form carbonates. a. Estimation of the free carhonie acid (Marchand). — 100 cc of urine are put into a glass flask closely fitted with a doubly perforated cork. Through one opening a tube is passed which dips into the urine, and at the other end is con- nected with a tube containing pieces of quicklime. Through the other opening in the cork one arm of a doubly bent tube is passed ; this does not dip into the urine ; the other arm is introduced into an empty flask through a well-fitting cork. This flask is connected by a similar tube with a second flask filled with clear barjia-water, and this with a thinl and fourth filled with baryta-water. The urine is then heated to 100° C. over a water-ljath ; any portions of it that boil over go into the empty flask. The carbonic acid comes off and forms a white precipitate of barium carbonate in the flasks filled with baryta-water. Air is then drawji through the apparatus ; any carbonic acid in the atmosphere being removed by the quicklime. The carbonate of bai-yta formed is collected on a filter, washed with distilled water, dissolved in hydrochloric acid, precipitated again by sul- phuric acid, and weighed as barium sulphate. From the quantity so obtained the amount of carbonic acid in the urine can be calculated ; 196-65 parts of barium caj-bonate correspond to 232-62 parts of barium sulphate, and 44 parts of carbonic acid. b. Till' total carbonic acid is similarly estimated after strongly acidifying the urine with hydrochloric or phosphoric acid. The comlnned carbonic acid is the difference between the total and the free carbonic acid. QIANTITATIVK ANALYSIS OF IKINE 805 ESTIMATION OF THE POTASH AND SODA a. Of the 2>otash aiul soda together. — 30 c.c. of urine are mixed with 30 c.c. of baryta mixture (two volumes of barium hydrate solution to one of barium nitrate solution, both saturated in the cold). The precipitate which forms is filtered ofif, 40 c.c. of the filtrate (corresponding to 20 c.c. of urine) are evaporated to dryness in a platinum capsule in a water-bath. The residue is incinerated, heating gently at first till nearly all the carbon is burnt. To the residue boiling water is added and then carbonate of ammonia as Ion"- as a precipitate is thrown down. The precipitate is filtered off, washed, and the filtrate and washings, acidified with hydrochloric acid, evaporated to dryness in a platinum crucible of known weight. The dried residue is gently heated to drive oflE salts of ammonia, cooled over sulphuric acid in an exsiccator, and weighed. The weight, mimts that of the crucible, is that of the total .sodium and potassium combined with chlorine. b. Cf the potash alone. — Dissolve the two chlorides obtained as above in a little water. Add excess of platinic chloride, and evaporate almost to dryness in a water-bath. Treat the residue with 80 per cent, alcohol, and allow it to stand some hours. The sodio-chloride of platinum alone dissolves. Collect the undis- solved potassio-chloride of platinum on a filter of known weight, wash with 80 per cent, alcohol, dry at 100° C, and weigh ; 100 parts of potassio-chloride of platinum coiTespond to 30-51 parts of chloride of potassium. From this the per- centage of chloride of potassium can be calculated. The combined weight of the two chlorides, minus that of the potassium chloride, gives the weight of the sodium chloride. From this the amount of potash (K.,0) and soda (>^a.^0) can be calculated, one part of chloride of potassium corresponding to 0-6317 of potassic oxide (KjO), and one part of sodic chloride corresponding to 0-5302 of sodic oxide (Xa^O). ESTIMATION OF LIME Lime may be estimated either by a volumetric method or by a gravimetric method. a. Voltunetric method. — The Ume is precipitated by oxalate of ammonia as oxalate of lime : by heat this is converted into caustic lime and carbonate of lime, the amount of which is ascertained by a standard acid solution. The following reagents are necessary : — i. Standard hydrochloric acid solution. — 60 c.c. of hydrochloric acid are diluted nearly to a litre ; it is then placed in a biu-ette, and diluted until it is found that 1 c.c. of it just neutralises a solution of caustic soda containing 20 grammes to the litre ; ] c.c. of this acid solution corresponds to 0 014 gramme of Ume (CaO). b. Standard caustic soda solution ; 20 grammes to the litre, ii. Ammonia solution. iii. Oxalate of ammonia solution. iv. Acetic acid. V. Neutral litmus solution. Analysis. — Take 200 c.c. of urine. Add ammonia till a large precipitate occurs. Collect and redissolve the pre- cipitate carefully by acetic acid, adding only a few drops of acid in excess. To 806 EXCRETION this add oxalate of ammonia, and allow it to stand six or eight hours till a preci- pitate of oxalate of lime settles. Syphon off the clear, supernatant fluid, and collect the precipitate on a small filter, and wash with hot water. N.B. — Preserve the supernatant fluid, filtrate, and washings for the estimation of the magnesia, Incinerate the filter with the precipitate ; lime and carbonate of lime are thus formed. To this residue add 10 c.c. of the standard acid solution, and heat carefully to expel all the carbonic acid. By this means all the calcium present is combined as chloride. Colour the liquid with neutral litmus solution, and estimate the acidity by the standard soda solution. Subtract the number of cubic centimetres of soda solution used from the 10 c.c. of the acid solution employed. The remainder is the number of c.c. of acid solution employed to saturate the lime present. Then calculate from this the percentage of lime. b. Determination by weight. — One proceeds as above till a precipitate of oxalate of lime is obtained from 200 c.c. of urine. This is collected on a filter of known ash, well washed, and incinerated till the weight becomes constant in a platinum crucible of known weight. Cool over sulphuric acid in an ex.siccator, and weigh. Subtract the weights of crucible and filter ash, and the remainder gives the amount of lime (CaO) present in 200 c.c. of urine. ESTIMATION OF MAGNESIA This is best determined by weight. The fluid separated from the oxalate of lime in the preceding experiment is treated with ammonia till alkaline. In this way all the magnesia is thrown down as ammonio-phosphate of magnesia. Allow some hours for this to settle ; collect on a filter of known ash, and wash with dilute ammonia (1 in 4). Incinerate in a platinum crucible till white ; cool over sulphuric acid, and weigh. The incineration is hindered by the presence of uric acid, but can be hastened by adding a small piece of nitrate of ammonia moistened with distilled water to the precipitate. Heat converts the ammonio-phosphate into pyrophosphate of magnesia, 100 parts of which correspond to 3fr03 of magnesia (MgO). ESTIMATION OF AMMONIA (Schloesing) The following solutions are necessary : — i. Standard sulphuric acid. — This contains 49 grammes of sulphuinc acid (H.^SO^) in the litre. It may be made by adding about 30 c.c. of concentrated sulphuric acid to a litre of water, and then by titration this is fuilher diluted till one volume of it exactly Deutralises one volume of a standard solution of caustic soda which contains 40 grammes to the litre ; 1 c.c. of the acid solution corresponds to 0-017 of ammonia (NH3). ii. Standard soda solution containing 10 grammes to the litre. iii. Milk of lime. iv. Neutral litmus solution. Method. — 20 c.c. of urine fi'eed from mucus by filtration are placed in a beaker. A triangle made of glass rod is laid upon it, and upon the triangle is (il ANrri'.\ll\K ANALYSIS ol' IIMNK 807 placed a sliallow xcu^sel contaiainy; 10 c.c. of the staiK.lard sulpluiiiu acid solution. The two are i)laced on a glass plate, and covered with a bell-jar, of which the edges are well greased. Kaise the bell-jar, add quickly to the urine 10 c.c. of milk of lime, and ininiediatcly replace the bell-jar. In forty-eight hours the whole of the ammonia is driven off from tlic urine and absorbed by the sulphuric acid. The sulphuric acid is then coloured with neutral litmus solution. Its acidity is then measured with the soda solution, four volumes of which corresponds to one of the acid solution. Divide the number of c.c. of soda solution used by 4 ; subtract this from the 10 c.c. of acid solution used. The remainder is the number of c.c. of acid employed to saturate the ammonia present, each c.c. of acid so used correspond- ing to 0"017 gramme of ammonia (NH^). Control cvperiment. — Perform a similar experiment with urine to which no milk of lime has been added, and thus estimate the amount of ammonia which has formed in forty-eight hours from the decomposition of urea. Subtract this from the quantit^y found in the tirst experiment. As a rule, however, fresh, healthy urine, if freed from mucus, does not decom- pose in forty-eight hours. ESTIMATION OF TOTAL NITROGEN This is best accomplished by Kjeldahl's method {see p. 28). 5 c.c. of urine and 20 c.c. of the mixed acids are measured into a flask of about 300 c.c. capacity, and heated to boiling. The heat is continued till all vapours cease to come off, and the fluid possesses a clear yellow tint. Twentj'-flve to thirty minutes generally suffice. The flask is then allowed to cool, diluted, and the liquid distilled with caustic soda and zinc into a known volume of standard acid, as already described. The loss of acidity, ascertained by titration with standard alkali, is a measure of the amount of ammonia given off by distillation, and from this the amount of nitrogen is calculated. ESTIMATION OF URIC ACID a. An approximate, and, for most clinical purposes, sufficiently accurate pro- cess is the following (Heintz' method) : — Take 100 c.c. of urine. Add to this 5 c.c. of hydrochloric acid. Lay the mixture aside for twenty-four hours. Collect the crystals on a weighed filter paper, wash with dilute hydrochloric acid, dry at 100° C, and weigh. The increase in weight will give the percentage of uric acid. b. In some cases, however, urine containing uric acid gives no precipitate in this way, and many attempts have been made to find a thoroiighly trustworthy method. Haycraft ' invented a method based on the fact that uric acid combines wdth silver as silver urate : the silver urate is collectetl, dissolved in nitric acid, and the silver estimated volnmetrically by Volhard's method.- From the amount of silver found the amount of uric acid is calculated. Herrmann ^ obtained good results by this method, and Czapek * slightly modifying the process found a large error. Salkowski •'■ also regards the process as of little value, as the composition ' Bvif. Med. Joiirii. December 1885. - Ltebig's Annalen, cxe. 1. ^ Zeif.pJn/sioJ. CJieiii. xii. 496. 4 JHfj, p. 502. 5 Ihid. xiv. 31. 808 EXCRETION of the silver urate is not constant; in this opinion he is supported by Gossage." As, therefore, there is a doubt as to the applicability of Haj'craft's method to urine, I do not propose to give an account of it here. c. Folilier's method'^ (modified by Salkovvski •*) is as follows : -200 c.c. of urine ai-e made strongly alkaline with sodium carbonate, and after an hour 20 c.c. of a concentrated solution of ammonium chloride are added. The mixture is allowed to stand at a low temperature for forty-eight hours, the precipitate which forms collected on a weighed filter, and washed. The tilter is filled with dilute hydro- chloric acid (1 in 10), and the tiltrate collected ; this operation is repeated till all the acid urate on the tilter is dissolved. The filtrates are mixed, allowed to stand for six hours, and the uric acid which then separates is collected on the same filter, washed twice with water, then with alcohol, till all acid reaction disappears, dried at 110° C, and weighed. To the weight obtained add 0-03 gramme, and subtract the Wt;ight of the tilter ; the remainder is the weight of uric acid in 200 c.c. of urine. d. Camerer's method.* — Camerer has subjected to a most thorough examination all the various hitherto proposed methods for the estimation of uric acid, finds none thoroughly satisfactory, and proposes the following new one, which appears to be the best up to the 2?resent : — The twenty-four hours' urine is mixed with a measured amount of dilute solution of caustic soda (500 c.c. of water containing from 0'4 to 1 gramme of soda was found to be the best proportion). This preci- pitates the earthy phosphates, which are then filtered off. The mixture is then diluted with water till its specific gravity is 1010 to 1011 ; if, however, the urine is rich in uric acid, the dilution must be greater ; if poor, less. To 300 c.c, of this diluted urine are added 50 c.c. of Salkowski's^ magnesia mixture (1 part of crystallised magnesium sulphate, 2 parts of ammonium chloride, 4 parts of ammonia solution of si^ecitic gravity 0-924, and 8 parts of water) to precipitate the rest of the phosphates ; filter this off. The first 30 c.c. of the filtrate are used to wash out the measuring glass, the next 175 c.c. (= 150 of diluted urine) are used for analysis ; place this in a beaker containing 0'5 gramme of finely divided calcium carbonate ; then about 5 c.c. of a 3 per cent, solution of silver nitrate ; the preciiDitate which forms is allowed to settle ; the supernatant liquid is tested for silver ; if it contains none, more of the silver nitrate solution must be added. When the supernatant solution gives evidence of excess of silver, proceed with the analysis. The precipitate is collected, the calcium carbonate preventing it going through the filter ; it is well washed with water till quite free from silver and from chlorides, and then dried over sulphuric acid in an exsiccator. An esti- mation of the nitrogen in this precipitate is then made by KjeldahTs method (see p. 23) ; each part of nitrogen found corresponds to 3 parts of uric acid. This gives the amount of uric acid in 150 c.c. of the diluted urine. The amount of dilution being known, the i)ercentage of uric acid in the urine is easily ascer- tained, and from this the quantity in the day's urine is then found. C!amerer ^ points out that there are two possible objections to this method : (1) A loss due to imperfect filtration ; this can be easily obviated by the use ot Schleicher and Schull's papers. (2) Xanthine compounds are reckoned as uric acid. This latter objection is a serious one. Camerer therefore compared his method with that of Ludwig, in which pure uric acid is separated out, silver ' Proc. Boy. Soc. xliv. 284. - PJJufjer's Archiv, x. 153. ^ Virchow's Archiv, Ixviii. 401. See also Pott, PflVigefs Arch. xlv. 389. * Zcit. Biol. xxvi. 84. ^ pftHger's Archiv, v. 319. « Zrit. BioJ. x.wii. 113. tn'ANTlTATIVK AN.\1,VS]S (>F IKINK 809 being got rid of by the u«e of hydrogen suljjhide. Ludwig's method is a tedious and laborious one. The mean difference between Camerer's and Ludwig's method was found to be 11 per cent. Canierer suggests that, knowing this, the real percentage of uric acid can be found bj' calculation ; and the results thus obtained are remarkably accurate. To give an illustration : the silver pre- cipitate of 150 CO. of urine yielded 1439 milligr. of nitrogen or 9-6 milligr. per llX) c.c. 9-6 X 3 = 288 = percentage of uric acid by Camerer's method. 28-8 — (28 8 X Oil) = 25-6 -percentage of true uric acid by calcukition. This is verj- close to 26tK>, which was the percentage found by Ludwig's method. ESTIMATION OF HIPPUEIC ACID The method of estimation con.-^ists in the preparation of pure hippuric acid from a known quantity of urine, and weighing it. Jfetho(/ {Bunge and Schmiedeberg). — 200 c c. of urine are taken. This is rendered alkaline with sodic carbonate and evaporated to dryness. The residue is extracted with cold alcohol, and the extract distilled until all the alcohol has passed off. The remaining water}' fluid is rendered acid with hydrochloric acid, and shaken at least tive times with fresh portions of acetic ether. The acetic ether is washed by shaking with water, and evaporated at a moderate temperature. The residue con- sists of hippuric acid, benzoic acid, and fat. It is extracted with petroleum ether (light petroleum) ; the hippuric acid alone remains undissolved. The residue of hippuric acid is dissolved in a little warm water, the solution passed through animal charcoal. It is then evaporated to dryness on a weighed capsule at a tempera- ture of 50° to 60° C. The crystals consist of hippm-ic acid. Weigh ; the weight, minus that of the capsule, is the amount of hippuric acid in 200 c.c. of urine. ESTIMATION OF OXALIC ACID This is a gravimetric process, the oxalic acid being weighed as oxalate of lime. Method (Xeubauer). — 400 to 600 c.c. of urine are taken. Solution of chloride of calcium is added. Excess of ammonia is added, and the precipitate which forms, dissolved in acetic acid, excess being avoided. Oxalate of lime, however, remains undissolved. Let this settle for twenty-four hours. Some small amount of uric acid is generally deposited also. Collect the precipitate on a small filter ; wash with water; then place the tilter together with the precipitate in hydro- chloric acid, and warm ; the uric acid is not dis-solved, the oxalate of lime is. Filter off the undissolved uric acid, wa.sh with dilute hydrochloric acid, and add the washings to the filtrate. Neutralise this with dilute ammonia ; crystals of oxalate of lime separate out, and are collected on a filter of known weight, weighed, and the quantity of oxalic acid calculated therefrom, 100 parts of calcic oxalate corresponding to 70'31 parts of oxalic acid (CoHoOJ. Or after the crystals of oxalate of lime are obtained, the process may be modified as follows (Czapek) :— Collect ihe cr^"stals on a filter of known ash. Wash with dilute acetic acid, and then with distilled water. Incinerate precipitate and filter paper in a platinum crucible until no more weight is lost. The oxalate is first changed into carbonate of lime (carbonic oxide being given off), and then into lime or oxide of calcium, carbonic acid being given off. CaC204 = CaO + C02-l-CO. About twent}- minutes is generally sufficient for the decomposition. From the final weight of the contents of the crucible the amount of filter ash is deducted ; the remainder is that of the lime formed from the oxalate of calcium. This, mul- tiplied by 1-6071, gives the amount of oxalic acid in the quantity of urine used. 810 EXCRETION ESTIMATION OF UREA If albumin is present it must be first separateiT b}' boiling after acidulation with acetic acid if necessarj^, and filtering off the flakes of coagulated proteid. The two chief methods of estimating urea are: — a. The mercuric nitrate, or Liebig's metliod. b. The hypobromite, or Hiifner's method. a. Liebig's method. — The combination between urea and mercuric oxide has been alluded to in the account just given of Liebig's method of estimating chlorides ; this combination [(CON„HJ„Hg(N03)2+ 3HgO] forms a white precipitate, insoluble in water and weak alkaline solutions. It is, therefore, necessary to prepare a standard solution of mercury', and to have an indicator by which to detect the point when all the urea has entered into combination with the mercury, and the latter slightly predominates. This indicator is sodium car- bonate, which gives a j'ellow colour with the excess of mercury, owing to the formation of hydrated mercuric oxide. Theoretically, 100 parts of urea should require 720 parts of mercuric oxide ; but, practically, 772 of the latter are necessary to remove all the urea, and at the same time show the yellow colour with alkali ; consequently the solution of mercuric nitrate must be of empirical strength in order to give accurate results. The following solutions must be prepared : — i. Standard mercuric nitrate solution. Dissolve 77'2 grammes of red oxide of mercury (weighed after it has been dried over a water-bath) or TIB gr. of the metal itself, in dilute nitric acid. Expel excess of acid by evaporating tlie liquid to a syrupy consistence. Make up to 1000 c.c. with distilled water, adding the water gradually. This solution is of such a strength that 19 c.c. will precipitate 10 c.c. of a 2 per cent, urea solution. Add 52-6 c.c. of water to the litre of the mercuric nitrate solution, and shake well ; then 20 c.c. (instead of 19) = 10 c.c. 2 per cent, urea solution, i.e. 1 c.c. = -01 urea. ii. Baryta mixture. — This is a mixture of two volumes of solution of barium hydrate with one of solution of barium nitrate, both saturated in the cold. jUialysis. — Take 40 c.c. urine. Add to this 20 c.c. baryta mixture and filter off the precipitate of baryta salts (phosphates and sulphates). Take 15 c.c. of the filtrate (this corresponds to 10 c.c. of urine) in a beaker. Run into it the mercuric nitrate solution from a burette, until on mixing a drop of the mixture with a drop of a saturated solution of sodium carbonate on a white tile a pale lemon colour appears. Then read the amount used from the burette, and calculate thence the percentage of urea. Corrections. — This method only approaches accuracy when the quantity of urea present is about 2 per cent., which is about the normal percentage of urea in i;rine. The chlorine in the urine must also be estimated, and the quantity of urea indicated reduced by the subtraction of 1 gramme of urea for every 1"3 gramme of sodium chloride found. If the urine contains less than 2 per cent, of urea, O'l c.c. of mercuric nitrate solution must be deducted for every 4 c.c. used ; if more than 2 per cent, of urea, a second titration must be performed with the urine diluted with half as much water as has been needed of the mercurial solution above 20 c.c. Suppose, then, 28 c.c. have been used in the first titration, the excess is 8 c.c. ; therefore 4 c.c. of water must be .added to the urine before the second titration is made. When ammonium carbonate is present, first estimate the ui-ea in one portion of urine, and the ammonia by titration with normal sulphuric acid in another; 0-017 gramme of ammonia = O'OSO of lU'ea. QUAXTITATIVK ANALYSIS OF I'KINH Hll Tlie cquivalcnl nf ;iiimi()niii in tcniis of iircii must be adtli'd to tlic urea round in tiie first portion of urine. Mod ijj cations. — Kautenberg ' and Pfliiger - have devised modifications of Lie- big's original method. Rautenberg's method consists in maintaining the urea solu- tion neutral througliout by successive additions of calcium carbonate. Pfluger's method is as follows : A 2 per cent, solution of urea is prepared ; 10 c.c. of this are placed in a beaker, and 20 c.c. of the mercuric nitrate solution are run into it in a continuous stream ; the mixture is then brought under a burette containing normal sodium carbonate, and this is added with constant agitation until a per- manent j-ellow colour appears. The volume so used is noted as that necessary to neutralise the acidity produced by 20 c.c. of the mercurial solution in the presence of urea. A plate of glass is then laid on black cloth, and some drops of a thick mixture of sodium bicarbonate (free from carbonate) and water placed upon it at convenient distances. The mercurial solution is added to the urine in such volume as is judged appropriate, and from time to time a drop of the white mixture is placed beside the bicarbonate, so as to touch but not mix completely. A point is at last reached when the white gives place to yellow ; both drops are then rubbed quickly together with a glass rod, and the colour disapjDcars ; further addition of mercury is then made to the urine till a drop rubbed with the bicarbonate remains permanently yellow. Now is the time to neutralise bj- the addition of the normal sodium carbonate to near the volume found necessary in the preliminary experi- ment. If this is quickly done a few tenths of a c.c. of mercuric nitrate will be found sufficient to complete the reaction. If, however, much time has been lost, it may happen that, notwithstanding the mixture is distinctly acid, it gives, even after the addition of sodium carbonate, a permanent yellow, although no more mercuric nitrate be added. The analysis must be under those circumstances re- peated, taking the first titration as a guide to tlie quantities which are necessary. Pfiiiger's correction for concentration of urea is different from Liebig's, and is as follows : — V' = volume of urea solution + volume of sodium carbonate solution + volume of any other fluid free from uvea whicli may be added. V2 = volume of mercuric nitrate solution used. C = correction = -(V'-V-) x 0-08. This formula holds good for cases where the total mixture is less than three times the volume of mercuric niti'ate solution used; with more concentrated solu- tions the formula gives results too high. Pfliiger and P)leibtreu ('Pfliiger's Archiv,' xliv. p. 1) have recently in a series of papers introduced fresh methods of urea analj-sis of so complex a nature that they are quite unsuitable for ordinary clinical work. b. The hypobromite method This is a far more accurate and easier method. The method consists in decomposing urea into water, carbonic acid, and nitrogen by means of an alkaline solution of hypobromite of soda ; the carbonic acid combines with the soda, and the nitrogen which is evolved is measured, and the quantity of urea therefrom calculated. There are many kinds of apparatus for performing this operation, but the best yet devised are those of Dupre * and Gerrard.* The apparatus and reagents one requires for the determination are as follows : — 1 Ann. Chem. Phariii. cxxxiii. 55. ^ Zeit. anal. Chem. xix. 375. Pfeiffer {Zeit. Biol. xx. 540) lias made a careful comparison of the different methods proposed. ^ Dupre, Journ. of the Chem. Soc. May 1877. * Lancet, ii. 1884, p. 952. 812 EXCRETION i. A Dupre's apparatus ' or a Geriaid'.< appavatus.-' ii. A 5 cubic centimetre pipette. iii. A strong glass cylinder with a well-fitting glass stopper, iv. A -10 per cent, solution of caustic .soda. V. Tubes containing 2 and 4 c.c. of bromine. The two last-named reagents are required for the making of the In-pobromite solution, which spoils by keeping (bromate of soda being formed). It should, therefore, be prepared fresh before eveiy dt termination. The hvpobromite solution is made by introducing 23 c.c. of the soda solution into the elass cvlinder, then gently dropping in a tube containing 2 c.c. of bromine. The tube is then broken by shaking the cylinder, which is stoppered ; the bromine escapes, and combines with the soda. This method prevents any inconvenience ainsing from fumes of bromine. The quantity of solution so prepared is .sufficient for one estimation. This procedure is. as Dupre points out, one of the most valuable points about his method ; the solution can be made with perfect safety by the bedside. Method 1 (Dupre). — Measure 5 c.c. of urine, and introduce it info the test-tube attached to the caoutchouc stopper seen on the upper left- hand side of fig. 100 : this will be found simpler to use than the pipette {e.f) figured below. Pleasure 25 c.c. of hypiobromite of soda solu- tion, and introduce it into the bottle, c. Close the bottle carefully with the stopper just mentioned, taking care to upset none of the urine in the test-tube attached to it. This stopper is perforated by a glass tube, which is connected by indiambber tubing to the tube, a, by a T-piece. Open the pinch-cock, d, and ^ I I' 'V\ lower the tube, a, until the surface of the water r^ ;■ .r^:—!-^^^ with which the outer cvlinder is filled is at I'll I ' p iA I '* * " — '^^^ — the zero point of the graduation. Close the pinch-cock, d, and raise a to j^certaiu if the apparatus is air-tight; then lower it again. Tilt c so as to upset the urine, and shake well for a minute or so. Immerse c in a large beaker containing water of the same temperature as that in the cylinder. After two or tliree minutes raise the measuring- tube, n, until the surfaces of the liquid inside and Fig. 100.— Dnijre'i Trea Apparatus. outside coincide. Read off the quantity of nitrogen by means of the gi-aduations on a that results from the decomposition of the 5 c.c. of urine. Some of the tubes of Dupre's apparatus are graduated in divisions corresponding to percentages of urea. The total quantity of urine passed in the twenty-four hours being measured, the total amount of urea excreted in the day can be calculated. If the nitrogen is measured in c.c, 35-4 c.c. of nitrogen corresponds to 01 gramme of urea. 1 How and Co., Farringdon Street. - Gibbs, Cuxson, and Co., "Wednesbory. QrANTlTA'ri\K ANALYSIS ol- I 1;1NK 81H lleacthms and corrrctU>ns.—'\'\w. n-actioii by wliicli urea is decoiuposed in this proceeding may be denoted by the followiu'r formula : CONoH, + :$XaBrO = CO., + N, + 2H..0 + :5N!iBr. From I gramme of urea ()-4(! gramme of nitrogen = 372-7 c.c. are obtained. In practice, however, it is found that only 354'3 c.c. are obtained,' except in diabetic urine, in which the urea yields nearly the normal amount of nitrogen. Moreover, urine contains small quantities of creatinine and urates, which yield some of their nitrogen when acted on by sodic hypobromite. When great exactitude is required these must be removed — creatinine by an alcoholic solution of zinc chloride, and tlie m-ates by acetate of lead followed by .sodic pliosphate (Yvon). 5 c.c. of a 2 per cent, solution of urea in urine yield 35-4 c.c. of nitrogen. This quantity is taken as representing 2 per cent, of urea, and serves as a basis for the graduations of the tubes which an- marked in percentages. When very great exactitude is re- quired the quantity of nitrogen must be measured in cubic centimetres, and the volume obtained corrected for tempera- ture, pressure, and tension of aqueous- vapour by the formula given on p. 35. Method 2 (Gerrard). — In the method the hypobromite solution is prepared a> before, and introduced into the bottle, u (fig. 101). A stout test-tube containing 5 c.c. of urine is carefully lowered by forceps into this. By means of the short tube, c, the long graduated one is filled with water up to the zero mark, a is now connected to this latter tube by indiarubber tubing, as in Dupre's ap^iaratus. The urine and hypobroinite are mixed by tilting the bottle, a ; the nitrogen comes off, and is measured in percentages of urea by the graduations on the tube, U. After waiting ten minutes to allow the tempera- ture of the apparatus and the contained gas to reach that of the atmosphere, the water in the two tubes is brought to the same level by lowering the tube, c ; the reading is then made, and corrected, if necessary, for temperature, pressure, and tension of aqueous vapour as before. Fig. 101. — Gerrard's Urea, Apiiarutus. ESTIMATION OF CREATININE The crystalline compound which creatinine forms with zinc chloride is employed in estimating the quantity of creatinine in urine, 100 parts of the compound corresponding to t)2-42 of creatinine. 1 The cause of this loss of nitrogen has been investigated by Luther, Zeit. physiol. Chem. xiii. 500. He finds part is combined as a nitrate, and part in an unknown organic compound which gives off ammonia when distilled with alkali. 814 EXCRETION Miithod. — Take 250 c.c. of urine. Add milk of lime and calcic chloride in excess to precipitate the phosphates. Filter, and evaporate the filtrate to a small bulk ; to this add 50 c.c. absolute alcohol, and let the mixtare stand for six hour.s. Then add 10 or 15 drops of an alcoholic solution of zinc chloride ; the crystals form, and after two or three days' standing in a dark place may be col- lected on a weighed filter. Wash with 90 per cent, alcohol, dry and weigh, and thence calculate the per- centage of creatinine. ESTIMATION OF SUGAR The quantitative determination of sugar in urine may be made by the different processes already described under the heading Dextrose in Chapter IX. Estima- tions by the saccharimeter can only be made when the urine is perfectly clear and free from other substances that rotate the plane of polarised light. The fermen- tation method is so inaccurate that it should now be altogether discarded. Fehling's method is practically the only one novv in use ; if the urine is albu- minous the albumin must be first separated by acidulating with dilute acetic acid, boiling, and filtering. Most diabetic urines are so rich in sugar that it is necessary to dilute them to ten or twenty times their original volume before placing them in the burette ; this must, of course, be allowed for in the subsequent calculation. The method of analysis itself will be found described on p. 98, and the composition of Fehling's solution on ]j. 95. The following formula will be found useful in estimating the amount of sugar in urine when the English weights and measures are employed : — a; = number of grains of sugar in twenty-four hours. 7> = number of c.c. of urine used from burette to decompose 10 c.c. of Fehling's solution (equivalent to 0 05 gramme = 077 grain of sugar). a = number of ounces of urine in twenty-four hours. 28%39G = number of c.c. in 1 oz. ie = l* X 28-396 v 0-77 ="' x 21-8G5. h h Pmnfs modi fi cation of the above test consists in the addition of ammonia to the copper solution. The composition of Pavy's solution is 34-65 grammes of copper sulphate, 170 gr. of Kochelle salt, 170 gr. of caustic potash dissolved to 1 litre with distilled water ; to every 120 c.c. of this mixture lOO c.c. of ammonia (specific gravity 088) are added, and diluted to 1 litre with water. 1 c.c. of this = 10 c.c. of Fehling's solution. The resulting- solution is a deep blue one, like Fehling's solution. The test is performed as in Fehling's method ; the diabetic urine (diluted to a known extent if necessary) is run into the hot Pavy's solution from a burette until all the blue colour disappears ; there is, however, no formation of a red or yellow precipitate, as the ammonia holds the reduced oxide in solution ; the blue colour simply gradually fades from the solution until when enough sugar is present all blue has disappeared. The disadvantage of this test consists in the fact that the ammonia fumes coming off from the liquid, which must be kept boiling, are so unpleasant that it must be performed in a flask closed with a cork through which two holes are bored ; through one of these holes a short piece of glass tube is passed ; this is connected to the burette by a piece of india- rubber tubing; through the other hole a long piece of glass tubing is passed through which the ammonia fumes pass out ; but these are in great measure con- densed in the tube, and return to the flask. (ilAN'ril'A'l'lXK ANAI-VSIS ol' riJlNK 815 Another iiio G 2 820 EXCKETK )N different parts of the body, and is due to volatile fatiy acids ; its taste is saltish, its specific gravity about 1005. Analyses have been made l)y numerous observers (Anselmino,' Schottin,^ Favre,'^ L. Wolff/ O. Funke,'^ and Leube ''), and there appear to be great variations in the composition of the sweat. In round numbers the percentage of solids is 1*2, of which 0'9 is organic matter. The following taljle from Charles' ' Physiological Chemistry ' ^ is a compilation from several analyses : — Water 98-88 per cent. Solids M2 Salts 0-57 NaCl 0-22 to 0-33 „ Other salts 0-lb (alkaline suli:)hates, phosphates, lactates, and potassium chlo- ride) Fats 0-41 (including fatty acids and cho- lesterin) Epithelium 0*17 ,, Urea 0-08 The salts are in kind and relati\ e quantity very like those of the urine. Funke was unable to find any urea, but most other observers agree on the presence of a minute quantity. It appears to become quickly transformed into ammonium carbonate. The volatile fatty acids present are formic, acetic, propionic, and butyric.^ The proteid which, according to Leube, is present, is probably derived from epithe- lial cells of the epidermis, sweat-glands, and sebaceous glands, which are suspended in the excretion. F. Smith'-* and Leclerc,'^ however, state that in profuse perspiration in the horse there is albumin actually in solution in the sweat. Abnormal, unusual, or pathological conditions of the sweats Drugs. — Certain drugs (sudurifics) favour sweating, e.g. pilocarpine, Calabar bean, strychnine, picrotoxine, muscarine, nicotine, morphine in small doses, camphor, ammonia. Others diminish the secretion, e.g. atropine, and morphine in large doses. Large quantities of water, by raising the Ijlood pressure, increase the perspiration. ' Wagner's HanduiJrterbuch. d. Plajsivl. Art. Haut. 2 Arch. f. Physiol. Heilk, xi. 73. ^ Comj)t. rend. xxxv. 721. * Diss. Greifswald, 1856. ^ Moleschott's Untersuch. zur NaiurleJtre, iv. 36. 6 Arch. f. pathol. Anat. xlviii. 181; 1. 301; Arch. Min. Med. vii. 1. 'P. 349. * Favre mentions a special acid in addition with formula C'loHigNoOij, which he terms sweat-acid, but which requires reinvestigating. » Veterin. Jotirii. Oct. 1888. i" Compt. rend. cvii. 123. 8ECRP:T10NS dF THK skin and AI.I,IEI» STIU'CTLRES 821 Some suhstauces introduced into the body rea])j)ear in the sweat, e.g. benzoii-, tartaric, and succinic acids readily, quinine and iodine with more difficulty (Schottin). Compounds of arsenic and mercury behave similarly (Bergeron and Lemattre '). Dis>'(i)if's. — Cystin has been found in some cases (Gamgee and Dewar'^); dexti-ose in diabetic patients (Semmola, Griessinger, Koch, Kiilz,^ and many others) ; bile-pigment in those with jaundice (as evidenced by the staining of the clothes) ; indigo in a peculiar condi- tion known as chromidrosis (Bizio,^ Hoflniann •^) ; blood or luematin derivatives in red sweat ; albumin in the sweat of acute rheumatism, which is often very acid ; urates and calcium oxalate in gout ; lactic acid in puerperal fever, and occasionally in rickets and scroful^i. Kidneij di) Zeit.physiol. Chem. iv. 345. <> E. Schulze and Marker, Joiirn. prali. Chem. cviii. 200; E. Schulze, Ihid. N.F. vii. 162; ix. 321; Schulze and Barbieri, .Jviirn. f. Landa-irthsrh. 1870, p. 125. Kossel and Obermiiller, Zeit. physiol. Chem. xiv. OOO. SECKKTJONS Ol' TIIK SKIN AND ALLIKI) STIilUyrUHKS S'lii diffi'ivnt kinds of slieop from 7 lo 34 per cent. Among tlie solids .aic cholestcriu and an isomeride of cholest(M-in, called iso-cholesterin. Tliere is a third alcohol of high molecular weight, which lias not been obtained jjiire. Cariiis ' described a compound, which he called hya;nic acid (C.^HjoO.J, whicli may be a derivative of this alcohol. The secretion of the coccygeal glands of birds has been analysed by de Jongc- ; he (inds it contains 40 per cent, of solids, of which about 15 consist of proteids and nuclcin, 19 to 24 of substances soluble in ether, and small (piantities of other organic substances and inorganic salts. The proteids are casein and albumin. The substances soluble in ether are chiefly fatty acid (es])ecially oleic acid) com- pounds, not of glycerin, but of cetyl alcohol ; there is also a trace of lecithin. This secretion is used for the lubrication of the feathers. The glands are absent in the running birds, and are most highly developed in water- fowl. The fatty secretion of the skin of the salamander contains, in addition to fat, pro- teid, lecithin, and cholesterin, an alkaloid with formula C.^Hc^N^Os (Zaleslcy •''). The secretion of the skin of most amphibia is a watery one, and appears to be more akin to sweat than sebum. The glands are in structure, however, very different from sweat-glands. The secretion itself has never been thoroughly examined. Cerumen. — The wax formed in the external auditory meatus appears to be a mixture of .sweat and a secretion from certain glands similar in structure to sebaceous glands. Petrequin and Chevalier ^ find it contains 10 to 11 per cent, of water, 26 to 30 per cent, of fat, 40 to 50 per cent, of potassium soaps, and traces of inorganic salts. Its reddish pigment has not been examined. Tlie secretion of the Meibomian glands of the eyelid, which are similar in struc- ture to the sebaceous glands, and also that of the sebaceous glands round the eyelashes, become mixed. That of the Meibomian glands appears to be the less watery secretion of the two. Tears, the secretion of the lacrymal glands, have been but little investigated. The secretion is more akin to saliva than to that of any otlier glands ; and the lacrymal gland is in structure like a salivary gland. It is, however, convenient to mention them in this place. Stimulation of the fifth cranial nerve produces a clear, that of the .sympathetic a cloudy, more alkaline secretion (compare Salivary Glands, p. G17). Lerch found in 1000 parts by weight of tears 980 parts of water, 13 of sodium chloride, and 5 of proteids. The chief proteid appears to be a globulin, as when dropped into water the tears give a cloudy precipitate. 1 Anyi. Chem. Pharni. cxxix. 168. -' Zeit. physiol. Chem. iii. 225. ^ Hoppe-Seyler's Med. chem. Unters. Heft i. j). 100. 4 CojiqJt. rend. Ixviii. No. 10 ; Ixix. No. 19. PART VI GENEE.\1 METABOLISM 82- CHAPTER XLVll i:xcnA\(n: of ^rATEiiiAL The word metahoHsm has been often employed in the preceding chapters, and, as there explained, it is used to express the sura total of the chemical exchanges that occur in living tissues. The chemical changes have been considered separately under tlie headings Alimenta- tion, Excretion, Respiration, Szc. We have now to put our knowledge together, and consider these subjects in their relation to one another. The living body is always • giving oft' by the lungs, kidneys, and skin the pi'oducts of its combustion, and is thus always tending to lose weight. This loss is compensated for by the intake of food, and of oxygen. For the material it loses, it receives in exchange fresh substances. If, as in a normal adult, the income is exactly equal to the expenditure, the body-weight remains constant. If, as in a grow- ing child, tlie income exceeds the expenditure, the body gains weight ; and if, as' in febrile conditions, or during starvation, the expenditure exceeds the income, the body wastes. The lirst act in the many steps which constitute metabolism is tlie taking of food, the next digestion of that food, the third absorption, and the fourth assimilation. Food, diet, digestion, and absorption have already been dealt ^vith, and it is only necessary to refer the reader to the chapters where these are described. In con- nection with these subjects, it is important to note the necessity for a mixed diet, and the relative and absolute quantities of the various proximate principles which are most advantageous. Assimilation is a subject which is exceedingly ditiicult to describe ; it is the act of the living tissues in selecting, appropriating, and making part of themselves the substances brought to them Ijy the nutrient blood-stream from the lungs on the one hand, and the alimentary canal on the other. The chemical processes invohed in some of these transactions have been already dwelt on in connection with the functions of the liver and other secreting organs, but even there our information on the subject is limited ; much more is this the case in connection with other tissues. The interesting theory of Pfliiger, in connection with the behaviour of the nitrogen in a food-proteid when it becomes part of a living proteid, should be read also in this connectiim (p. 115). 828 GENERAL METABOLISM The functions which we call digesticjii, absorption, and assimilation are the three ste^xs in nutrition, or the building up of the li\ing tissues ; these may, to use Gaskell's exj^ression, be spoken of as anabolic. Supposing the body to remain in the condition produced by these anabolic processes, what is its comj^osition ? A glance through the chapters on the cell, the blood, the tissues, and the organs will con- vince the inquirer that difterent parts of the body have very different compositions : still, speaking of the body as a whole, Volckmann and Bischoff state that it contains 64 per cent, of water, 16 of proteids (including gelatin), 14 of fat, 5 of salts, and 1 of carbohydrates. The carbohydrates are thus the smallest constituent of the body ; they are the glycogen of the liver and muscles, and small quantities of inosite, and dextrose in various parts. The most important, because the most abundant, of the tissues of the body is the muscular tissue. Muscle forms about 42 per cent, of the body-weight,' and contains, in round numbers, 7o per cent, of water and 21 per cent, of proteids ; thus about half the proteid material and of the water of the body exist in its muscles. The body, however, does not remain in this stable condition ; even while nutrition is occurring, destructive changes ai'e taking place simultaneously ; each cell may be considered to be in a state of unstable equilibrium, undergoing anabolic, or constructive processes, on the one hand, and destructive, or katabolic, processes on the other. The katabolic series of phenomena commences with combustion ; the union of oxygen with carbon to form carbonic acid, with hydrogen to form water, with nitrogen, carbon, and hydrogen to form urea, uric acid,, creatinine, and other less imj^ortant substances of the same nature. The formation of these last -mentioned substances, the nitrogenous metabolites, is, however, as previously pointed out, partly synthetical. The discharge of these products of destructive metabolism by the ex- pired air, the urine, the sweat, and fceces is what constitutes excretion ; excretion is the final act in the metabolic round, and the composition of the various excretions have been considered in some of the later chapters of this book. An examination of the intake (food and oxygen) and of the output (excretions) of the body can be readily made ; much more readily, it need hardly be said, than an examination of the intei-mediate steps in the process. A contrast Ijetween the two can he made by means of a balance-sheet. A familiar comparison may be drawn between the ^ The following is in round nuniljer^ the percentage proportion of the different structural elements of the body : skeleton, 10 ; muscles, 4'i ; fat, Is ; viscera, 9 ; skin, S ; brain, 2 ; blood, 5. KXCHANCE OF MATERIAL 829 affairs of the aniuial body .intl those of a eommei-cial company. At the end of the year the company presents a report in which its income and its expenditure are contrasted on two sides of a balance-sheet. This sheet is a summary of tl.e monetary affairs of the undertaking ; it gives few details, it gives none of the intermediate steps of the manner in which the property has been employed. This is given in the preliminary parts of the report, or may be entered into by still further examining the books of the company. In the parts of this book that precede this chapter I have en- deavoured to give an account of the various transactions that occur in the body. I now propose to wind up by presenting a balance-sheet. Those who wish still further to investigate the affairs of the body may do so by the careful study of works on physiology ; still, text-books and monographs, however good, will teach one only a small amount ; the rest is to be learnt by practical study and research ; and we may compare physiologists to the accountants of a commercial enterprise, who examine into the details of its working. Sometimes, in lousiness undertakings, a deficit or some other error is discovered, and it may be that the source of the mistake is only found after careful search. Under these conditions, the accountants should be compared to physicians, who discover that something is wrong in the working of the animal body ; and their object should be to discover where, in the metabolic cycle, the mistake has occurred, and subse- quently endeavour to rectify it. The construction of balance-sheets for the human and animal body may be summed up in the German word Stoffwechsel , or, as Dr. Burdon- Sanderson ' translates it, ' exchange of material.' A large number of investigators have applied themselves to this task, and from the large mass of material published, I shall only be able to select a few typical examples. The subject has been worked out specially by the Munich .school, under the lead of Pettenkofer and Voit. The necessary data for the construction of such tables are : — (1) The weight of the animal before, during, and after the experi- ment. (2) The quantity and composition of its food. (3) The amount of oxygen absorbed during respiration, (4) The quantity and composition of urine, fseces, sweat, and ex- pired air. (5) The amount of work done, and the amount of heat developed. (The subject of animal heat will be considered in a separate chapter.) Water is determined by subtracting the amount of water in- ' SijUalus of Lectures, 1879. 880 GENERAL METABOLISM gested as food from the quantity lost by bowels, urine, lungs, and skin. The difference is a measure of the combustion of hydrogen. Nitro(jeii. — The nitrogen is derived from proteids and albuminoids, and appears chiefly in the urine as urea and uric acid. Minute quan- tities are eliminated as similar compounds in sweat and ffeces. From the amount of nitrogen so found, the amount of proteids which have undergone combustion is calculated. Proteids contain, roughly, 16 per cent, of nitrogen; so 1 part of nitrogen is equivalent to 6*3 parts of proteid ; or 1 gramme of nitrogen to 30 grammes of flesh (Voit). Fat. — Subtract the carbon in the metaliolised proteid (proteid con- tains 54 per cent, of carbon) from the total carbon eliminated by lungs, skin, bowels, and kidney, and the ditFerence represents fat that has uudergone metabolism. Fat contains 76*5 per cent, of carbon ; hence the carbon, which represents fat, multiplied by 1"3, gives the amount of fat which has undergone combustion. The Discharge of Carbon The influence of food on the rate of discharge of carbonic acid is immediate. The increase after each meal, which may amount to 20 per cent., reaches its maximum in about one or two hours. This effect is most marked when the diet consists largely of carbohydrates. About 95 per cent, of the carbon discharged leaves the organism as carbonic acid. The total insensible loss ( = carbonic acid -|- water given ott— oxygen absorbed) amounts in man to about 25 grammes per hour. Of this total hourly discharge of carbonic acid, less than 0*5 per cent, is cutaneous. The hourly discharge of carbonic acid in a man at rest is about 32 grammes, the weight of oxygen absorbed being 25 to 28 grammes in the same time. The houi'ly discharge of watery vapour is about 20 grammes. As a volume of carl^onic acid (CO.,) contains the same weight of oxygen as an equal volume of oxygen (O,), it is obvious that, if all the absorbed oxygen were discharged as carbonic acid, the 'respiratory quotient' (by volume) = ^ "," ^, \ would be equal to 1. This, how- O., absorbed ever, is not the case, the volume of oxygen al)Sorbed being in excess of the carbonic acid discharged. In animals which feed exclusively on carbohydrates (this would only be possible for a short time) equality is approached. The excess of oxygen is greatest when the diet consists largely of fats. On a mixed diet, comprising 100 grauuues of proteid, 100 of fat, and 250 of carbohydrates, with a carbonic acid discharge of 770 grammes daily, and a daily assumption of 066 grammes of oxygen, EXCHANGK OV MATERIAL 831 5G0 grammes of the oxygen aiv discharged in the carbonic acid, about i) in urea, and 97 grannne.s in the form of water (of which 7S grammes are formed from the hydrogen of the fat) ; the respiratory quotient i^ then 0'84. In liibernation the respiratory quotient sinks lower than in any other known condition (often less than 0"5), for the animal then lives almost entirely on its own fat. The discharge of carbonic acid is increased by muscular work, and the respiratory quotient also rises. Diminution of the surrounding temperature causes inci'eased discharge of carbonic acid. (These points ai-e all discussed more fully in Chai:)ter XTX.) The Discharge of Nitrogen In man the minimum daily allowance of nitrogen is 15 grannnes, or 0'02 per cent, of the body- weight : in the carniAora about 0*1 per cent. ; in the ox, as an instance of a herbiA< >rous animal, 0"00n per cent. In certain races of mankind (e.g. coolies) the nitrogen require- ment is less than in Europeans. The reason why this is so is not understood. The bearing of this fact on vegetarianism is pointed out in the chapter on food (p. 599). Some recent experiments by Hirschfeld ' have shown that for a short time nitrogenous equilibrium can be maintained on a smaller daily supply of nitrogen than 15 grammes. Experiments extended over a, longer time have previous to this shown that sooner or later the l)ody begins to waste if the 15 grammes daily are not supplied in the food. In an animal fed exclusively on flesh the discharge of nitrogen at first incresises pari passu with the absorption of proteid, the absorption of oxygen being proportionately increased at the same time. The animal, however, gains weight from increase of fat, the proteid Ijeing split into what is called a nitrogenous moiety, which is burnt off. and a non-nitrogenous moiety which is converted into fat. The discharge of nitrogen is but little influenced by muscular work (see p. 436) ; the increased combustion that occurs in working as com- ' pared with resting muscles falls on their non-nitrogenous constituents. The questions of the nutritive value of gelatin, the origin of fat fi'om proteids and carbohydrates, and the conditions of nitrogenous discharge in starvation, fevei-, and other disordered conditions will be dealt with later in special sections. ' Pfliiger's Archiv, xli. 533. 832 GENERAL METABOLISM Balance of Income and Discharge in Health In Chapter XXVII (p. 604) tables are given of adequate diets ; these will in our balance-sheets represent the source of income ; the other side of the balance-sheet the expenditure consists of the excretions. Exchange of mater Ird on an adequate diet (Ranke's table).' Income « Expenditure Foods Kitrogen Carbon 1 Excretions ' Nitrogen Carbon Proteid, lOOgi-. Fat, 100 „ C'rb'hydat's,250 „ 15-5 gr. 00 ., 0-0 „ 15-5 „ 53-0 gr. 79-0 „ 93-0 „ 225-0 ,. Urea, 31-5 gr. "J , , , Uric acid, 0-5 .. / Faeces ... 1-1 Respiration (CO.J . 0-0 15-5 616 10-84 208-00 225-00 In man the discharge of nitrogen per kilo. <>f body- weight is 0-21 C gramme, and of carbon 3-03 granmies, the quotient -.x = 14-5. In carnivorous animals, which, according to Bidder and Schmidt, use 1*4 C N and 6-2 C per kilo, per diem, -^j = 4-4. In the human being on a flesh diet vr = •'5"2, the exchange thus approaching the condition of the carnivora. This is illustrated by the following balance-sheet (Ranke): — Income Expenditure 1 - Nitrogen Carbon — 1 Nitrogen Carbon Food Disintegration tissues of 62-3 gr. 62-3 279-6 45-9 325-5 Discharged by e.x- cretion . Retained in store . 44-0 18-3 62-3 263-0 62-5 325-0 The details of the above experiment may be given as illustrating the method of working out a problem in exchange of material : 1832 gi-ammes of meat used as food yielded 3-4 per cent, of nitrogen, i.e. 62-3 gr., and 12-5 per cent, of carbon, i.e. 229-3 gr. ; 70 gr. of fat added to the food yielded 72 per cent, of carbon, i.e. 1 The above table was constructed from data derived from the observations of Prof. Ranke on himself. For it I am indebted to Prof. Sanderson's Sijllahus of Lectures, which is also the source of most of the statements in the rtHumt of the chief facts relating to the discharge of carbon and nitrogen just given. EXCHANGE OE .MATKliJAL 88:} 50-3 gr. : 2293 + oO-3 = 279 6 = total carbon in fond. Dining the same period 8fi-:)gi-. of urea were discharged, containing 46-(i per cent., i.e. 40-4 gr. of nitrogen, and 20 per cent., i.e. 173 gr. of carbon, to which must be added 2 gr. of uric acid, containing 33 per cent., i.e. 0-6fi gr. of nitrogen, and 35 per cent., i.e. 07 gr. of carbon. Further, 2-9 gr. of nitrogen and 14 gr. of carbon were discharged in the fajces, and 231 gr. of carbon as carbonic acid in the expired air. Hence the total discharge of nitrogen = 40-4 + O-fiG + 2-9 = 44 gr., and the total discharge of carbon = 17-3 + 0-7 + 14 + 231 = 263 gr. Deducting the quantity of nitrogen dis- charged from that taken in, 18-3 gr. must have been retained in the bodj-, as 108 gr. of proteid, and consequently 53 per cent, of that weight = 62-5 gr. of carbon. were also retained. Comparing the quantity of carbon disposed of in the twenty- four hours with the quantity introduced as food, we find the latter is in excess b,v 45-9 gr.. which must have been derived from the disintegration of the fat of tlie body. Another table of exchange of material on adequate diet may be quoted from tlie work of Pettenkofer and Voit. This take.s into account the elimination of water as well as of carbon and nitrogen. In the first experiment the man did no work. Income Expenditure Food Xitrogen Carbon Excretions Kitrogeu Carbon Water i Proteid, 137 gr 1 Fat, 117 „ I 19-5 Carljohydrate, 352 ., J Water, 201(i „ — • . i 315-0 ' Urine Fseces Lungs 17-4 2-1 19-5 12-7 14-5 248-6 275-8 1279 83 828 2190 Here the body was in nitrogenous equilibrium, and it eliminated more water than it took in by 174 gi-ammes, this being derived from oxidation of hydrogen. It stored 397 grammes of carbon, which is equivalent to 52 gi-ammes of fat. The next table gives the results of an experiment on the same man on the same diet, but who did active muscular work during the day : — Espemlituie Urine Fjeces Lungs Xitrogou Carbon Water 17-4 12-6 1194 2-1 U-5 94 309-2 1412 19-0 336-3 2700 It is important to notice that the discharge of nitrogen was unaltered, while that of both carbon and livdrogen was increased. 3 H 834 GENERAL METABOLISM Inanition The conditions of metabolism, both as regards exchange of material and the production of heat during starvation, have been investigated in animals by CollarddeMartigny,' Chossat,- C. Schmidt,'' Schuchardt,^ Frerichs,^ Bischoff and Yoit ^ ; in human beings by Pettenkofer and Toit,' J. Ranke,^ Schultzen,9 Seegen,io Falck,i' arfd Schimanski.'^ The income from without is, under these circumstances, nil ; expenditure still goes on, as a result of the disintegration of the tissues ; the amount of disintegration is measured by the discharges in the manner already described. The following table from Ranke's experiment on himself represents the exchange for a period of twenty- four hours, twenty-four hours having elapsed since the last meal. lucome 1 Expend] ture Disintegration of tissue Nitrogen Carbon Excretions Xitrogeu i Carbon Proteid, 50 gr. . . Fat, 199() gr. . . 7-8 00 7-8 26-5 157'5 1840 Urea, 17 gr. . . . Uric acid. 0*2 sr. Respiration (CO.,) . } - 00 1 7-8 3-4 180-6 1840 Tlie discharge of nitrogen per kilo, of body-weight was reduced to O'l, s^ being 23-o. In carnivorous animals : in prolonged inanition, the ^' C dischai'ge of nitrogen per kilo, is OD, and ^ = 6*6. During starvation the man or animal gradually loses weight, the temperature, after a preliminary rise, sinks ; the functions get weaker bv degrees, and ultimately death ensues, the total weight lost being from 0-3 to Oo of the ox-iginal body-weight. The age of the animal influences the time at which death occurs, old animals withstanding the effects of hunger better than young ones. 1 Journ. de jjhysiol. exjierim. et path. voL viii. 1828, p. 1.52. 2 ' Eecherclies sur rinanition,' Mem. de VAcad.Moy. des Sciences, vol. i-iii. Paris, 1843. 5 Bidder and Sclimidt, Die Terdauungssi'ifte und der Sfoffwechsel, Mitau and Leipzig, 18.52, p. 202. 4 Diss. Miuburg, 1847. ^ Arch.fiir Anat. u. Physiol. 1848, p. 469. 6 Bischoff and Voit, Die Gesetze der Erniirung des Fleischfressers, Leipzig and Heidelberg, 1860, p. 42 ; Zeit. Biol. ii. 307 ; v. 369. 7 Hid, 8 Arch.f. Anat. u. Physiol. 1862, p. 311. 9 Ibid. 1863, p. 31. '° Wien. Aiad. Sitzungsb. March 16, 1871. 11 Beitriige zur Physiol. Hygiene, dc. vol. i. Stuttgart, 1875. 12 Zeit.physiol. Chem. iii. 396. KX('IIAN'(;K '>|' MATKKlAl 835 This statement was originally made by Hippocrates, and was borne ^lut by the experiments of Martigny and Chossat. Young animals lose w eight niore quickly, and die after a smaller loss of weight, tlian old ones. The exci'etion of nitrogen falls quickly at the commencement of an ♦experiment ; it reaches a minimum which remains constant for several days ; it then rises when the fat of the animal has been used up, and then quickly falls with the onset of symptoms of approaching deatli (Yoit, Falck, Schmidt, Schimanski). The sulphates and phosphates in the urine show approximately the same series of changes (Bidder and Hchmidt). The discharge of carbonic acid and the intake of oxygen fall, but not so quickly as the body loses weight ; it is not until quite the last .stages that these are small in proportion to one another. The f;eces become smaller and smallei- in quantity until no dis- ■charge fi'om the rectum occurs at all. The amount of bile secreted also falls ; but bile is found in the gall- bladder and intestine after death. Chossat, Schuchardt, Schmidt, and Yoit liave constructed tables which show the loss of weight that occurs in different organs. Taking the total loss of weight as 100, the loss due to that of individual organs may be stated as follows (Yoit) : — Boiu' . . . 5-4 Pancreas . . O'l -Muscle . . . 42-2 , Liing-s . . . O'S Liver ... 48 Heart . . . 0-0 Kidneys . . 0-6 Testes . . . Q-l Spleen . . . 0-6 Intestine . 2-0 Some organs thus lose but little weight ; the loss of weight is greatest in the muscles, fat, skin, liver, and blood. Of the muscles, the great pectorals waste most (Chossat). Demant ' found an increase of creatine and a diminution of lactic acid in the muscles of starving birds. The following are Chossat's ol)servatii>ns on the body-temperature in pigeons : Brain and cord 0-1 Skin and liair 8-8 Fat 26-2 Blood . 3-7 Other ]i;irts . .5-0 ConiUtiim of uniinals Temperature Midday ! iliduiglit Healthy pig-eons 1st tliird of starvation period . 2nd ., „ ,. . . . 3rd „ „ „ . . . 1 4222° C. 42-1 1° 11 -ST^ 41-37° 41-48° C. 3D-8= 38-7° 37-3° ' Zeit. physioh Chem. lii. 381. 830 GENERAL METABOLISM There is thus the greatest fall of temperature iu the night — that is, when even under normal circumstances the temperature and the vitality of the body are least. The frequency of respii'ation and the discharge of carbonic acid run parallel to the tempei'ature. Faick obtained a similar result in dogs. Death may be delayed somewhat by artificial warmth, but ultimately occurs from asthenia, sometimes accompanied by convulsions. Exchang-e of Material with various Diets The reasons why a mixed diet is necessary have been already explained (p. 602). Numerous experiments have, liowever, been made- in the study of metabolism on abnormal diets. Feeding nitli meat. — The chief facts concerning this form of nutrition in regard to man have been stated on p. 603. The same is in the main true for animals. The principle that underlies Banting's method of treating obesity is to give meat almost exclusively : the individual then derives the additional supply of carbon necessary for combustion from his own adipose tissue. We have alread}' seen that this may be and often is counteracted by the laying on of fat which comes from the non-nitrogenous moiety of the proteid. Feediwj mth fat. — If an animal receives fat only, the nitrogenous excreta are derived from the disintegration of tissue without any corresponding quantity of nitrogen being supplied in exchange in the food. When fat only is given, or a large excess of fat exists in the food, the respiratory quotient falls. By feeding- dogs with a mixture of fat and flesh, Pettenkofer and Volt's ' experiments gave such inconsistent results that no conclusion can be drawn from them F. Hofmann,- however, was more successful. After a period of inanition a dog was fed on a mixture of a large amount of fat and a small amount of proteid. After death the quantity of fat found in the body was such that only a small part could have been derived from the proteid, the greater amoimt being directly derived from the fat of the food. The animal, moreover, lays on fat in which palmitin, stearin, and olein are mixed in a definite proportion ; this proportion is often different in the fat of the food. In addition to this an animal wiU fatten (laying on fat with its usual composition) on fatty food, such as spermaceti, which contains no glycerides. Feedinrj with carhohydrate^. — The respiratory quotient approaches unity when carbohj'drates alone are taken. So far as regards nitrogen the animal is in a state of inanition, as when fat alone is taken. If given in combination with other foods, both carbohydrates and fat act as proteid-sparing foods {see- p. 603). The table on the next page is from I'ettenkofer and Voit,''and illustrates what happens in a dog on a mixed diet of flesh and carbohydrates. Even when the diet consists wholly of carbohydrates, fat is laid on ; the fat laid on when meat and starch are both present in the food comes partly from the 1 Ze.it. Biol. ix. 30. - Ibid. viii. 153. ^ Ibid. ix. 435. EXCHANGE oF MATEKIAL 837 KdoiI CImiiges iu the bO'ly Flesh Starch 0 37!) 0 608 400 210 400 400 344 500 167 500 soo 379 1500 172 IsOO 379 25IH1 — Sugar 227 Amount ' of proteid decom- posed cal- culates! from urea excreted 1 17 211 22 193 10 436 393 6 413 6 530 1 — 537 14 608 4 1475 1 10 1469 1 - 2512 Proteid ;raine Compare Bunge's view, p. 706. See also Hofmaim's experiments, p. 836. 838 GENERAL METABOLISM 3. The fat stored up appears as granules or drops deposited in the cell-substance, and the increase of fat in the celLs is accompanied fiist by a growth, and subsequently by a consumption of the cell-su Instances. Feedlnr/ mitJi r/elati/i.—A diet containing gelatin alone will not support life This fact is somewhat remarkable when one considers ttie closely allied chemiail nature of gelatin and proteids. WTien gelatin alone is given the body wastes, and the urea excreted is diminished, as in inanition. If an enormous amount of gelatin is given the urea increases. Gelatin, however, like carbohyrlrates and fats, appears to be a • proteid-sparing ' food, and if given mixed with proteids seems to protect the proteids from oxirlation. Gelatin can thus be substituted for a part of the proteid in the food. The following table is much abbreviated from the fidler ones given by Voit ' j it illustrates the facts mentioned above : — Fowl Daily loss or gain ui ^rrammes of flesh Meat Fat Oelatui " 0 0 -338 0 200 20. Meat 7. Gelatin . 8. Meat 6 2 3 2 1 4 3 4 2-4 gr. 2-4 „ 2-4 „ 2-4 „ 2-5 „ 21 „ 2 3 „ 21 „ 1-9 gr. 1-8 „ 1-9 „ 1 8 „ 1" „ 1-7 ,. 2-8 „ 1-7 „ -t- 0-5 gr. + 0-»J „ + 0-5 „ + 0-6 „ + 0-8 „ + 0-4 „ -O-.j „ + 0-4 „ Eflfect of Varying External Conditions on Exchange of Material Efect of atmospheric temperahire. — In warm-blooded animals the ettect of a low surrounding temperature is to increase katabolism, or combustion in the body ; the body loses more heat, and therefore more must be produced to keep the animal's temperature within normal limits. The effect of a rise of atmospheric temperature is the reverse. The effect of cooling the skin and the con-esponding increase in metabolism are well shown by an observation of Weiske's ' on the shearing of sheep. After shearing they excreted daily 1 gramme of urea more than before shearing. In cold-blooded animals, i.e. animals whose temperature varies with that of the surrounding atmosphere, a rise or fall of the latter is accompanied respectively with a rise or fall of combustion in the body. Injinencc of lUjht (see p. 211). AUcrations of bodi/-tem2}erature.—li the changes of the external temperature are so great as to cause a rise (as in steam-baths— Bartels,'- Xaunyn,^ Scheich *) or a fall (as in hibernation) of body-temperature, the metabolic changes are in- creased and decreased respectively as in cold-blooded animals. Effect of compressed and rare fed air.— 'Yhe influence of these factors on the respiratory exchanges has been described on p. 37S. Experiments relating to their effect on the discharge of urea are contradictory, Bert ^ and Hadra"^ finding that compressed air caused an increase in the excretion of urea, Friinkel ' finding no such increase. In rarefied air Frankel found in some of his experiments an increase, in others no increase in the urea excreted. In dyspnoeic conditions, where the supply of oxygen is deficient, the urea increases, and the respiratory CO quotient - becomes greater than unity (Frankel,* Herter '). Effect of removal of blood from tlie body. -The chief effect of a removal of blood from the body is the speedy formation of new blood-corpuscles. The intake of oxygen and discharge of carbonic acid are lessened, and the output of urea is increased (Bauer,'" Jolyet and Ftegnard"). The menstrual flow and epistaxis in strong, healthy people cause no alteration in exchange of material. ' Hoppe-Seyler's Med. chem. Unters. Heft iii. p. 418. - Pathol Untersuch. 1864. ^ Arch. f. exper. Path. iv. 82. ^ Diss. Strasburg, Berlin, 1870. 8 Centr. ined. Wiss. 1875, No. 44. 10 Zeit. Biol. \\u. 567. Arch.f. Anaf. n. Physiol. 1870. ^ La pression harometrique. Paris, 1878, p. 823, " Zeit. Mill. Med. ii. 1. 9 Hoppe-Seyler's Phi/siol. Chem. p. 054. " Gaz. ined'.de Paris, 1877, pp. 179, 190. 840 GENERAL 3IETABOLIS3I Effect of increasing the volume of Hood. — The injection of the blood from one animal into that of another causes a greater or less destruction of the first animal's blood-corpuscles. The eifect, however, varies much with the species of animal used," and apjiears to be due to the solvent effect of the second animal's serum ; it is especially marked when the two animals belon* to different species. The effect on the urine is a slight increase in the discharge of urea : the effect is rather greater when serum instead of defibrinated blood is injected (Forster -). Excliange of Material under the Influence of Organic and Inorganic Substances used as Foods. Drugs, or Poisons Lactic, acetic, tartaric, and succinic acids, asj)aragine, and glycerin are oxidised in the body to form carbonic acLd and water, and, like carbohydrates and fat, are ' proteid-sparing ' materials (Hoppe-Seyler,^ Weiske,^ Lewin '). Ghxerin passes partly as such into the urine (Tschirwinsky *). Glycerin increases the liver glycogen (Weiss, Luchsinger, Salomon ; sec p. 543). Pure glycerin increases the excretion of mic acid (Horbaczewski '). Pheiiijlacetic acid produces increased proteid metabolism (Salkowski*). Alcohol in small quantities diminishes (Beck and Bauer ^), in large doses increases the output of carbonic acid (Parkes '"). The effect on urea is small or none at all (Parkes, Munk "). Coffee produces the same effects as alcohol (Hoppe-Seyler,'- Yoit '^). The effect of various drugs on the output of urea is gi\en on p. 724. Water stimulates metabolic activity, and also assists in washing out the pro- ducts of metabolism fiom the tissues to the place where they are excreted. Sodium chlvride is also essential for the due discharge of metabolic function (^sce also p. 61). Phosphates appear to be equally necessary {see pp. 62, 256). Calcium andjwtassium salts, especially the former, are normal constituents of the body, and without a due supply of these the body wastes. Phosphor us-j}oiso?ii?ig is accompanied with a rapid fatty degeneration of the liver, and the appearance of leucine and tyrosine in the urine. The body wastes quickly. There is an increased output of nitrogen, both as m-ea and uric acid (Storch,'^ Bauer,'5 Friinkel '^). Arsenic and antimony poisoning produce the same effects, but in a much less marked manner (v. Boeck,'' Weiske,'* Gahtgens," Kossel™). In very small doses antimonious oxide produces little or no increase in the discharge of urea ^ See E. A. Schlifer, Beport on Traiisf union. Trans. Obstet. Sac. xxi. - Sitzungsb. d. Bayer. Ahad. d. Wiss. July 3, 1875. 5 Physiol. Chem. p. 957. * Zeit. Biol. xv. 261 (on asparagine). 5 Ibid. p. 243. See also Munk. Arch, pathol. Anat. Ixxvi. 119 (on glycerin). ® Zeit. Biol. xv. 252. "• Monatsh. Chem. vii. 105. -' Zeit. physiol. Chem. xii. 267. 9 Zeit. Biol. x. 361. 10 Proc. Boy. Sac. 1870, Nos. 120 and 123. 11 Verhandl. d. physiol. Ges. Berlin, 1879, No. 6. 1- Physiol. Chem. p. 958. 1^ Unters. u. d. Einjluss d. Kochsah, Kaffee, itc. Miinchen. i860, p. 67. " 1* Der acuten Phosphorgiftutig, Copenhagen, 1865. 15 Zeit. Biol. \ii. 63; xiv. 527. i* Zeit. i)hysiol. Chem. iv. 430. 1' Zeit. Biol. xii. 512. i8 Journ.f. Landwirthsch. xxiii. 317. 19 Centr. med. Wiss. 1876, pp. 321, 833. -"o Arch./, exper. Path. v. 128. KXCIIANOK OF MATEKIAL 841 (Chittenden and Blake '). In >mall doses arsenic diminishes the excretion of carbonic acid (Chittenden and Cuniniins -). Among other substances that simihirly diminish tlie output of carbonic acid, Chittenden and Cummins place uranium salts, copper sulphate, and tartar emetic; morphine, quinine, and ■cinchonidine having little or no effect. Ferric chloride, according to llabuteau,' increa.^cs the excretion of urea, according to Munk ' it does not. Exchange of Material in Diseases Fever. — Fever \i a condition in which the temperature of the body is raised above the normal, and the degree to which it is raised is a measure of the intensity of the febrile condition. A rise of temperature may be produced either by increased production of heat, due to the increase of kataboHc processes in the body, or to a diminished loss of heat from the body. A mere increase in the production of heat does not necessarily produce fever. By adminis- tering an excess of food, combustion is increased in the body; but in the healthy individual this does not produce a rise of temperature, because pari passu with the increased production, there is increased loss of heat. Similarly, diminution in the loss of heat, such as occurs on a hot as compared with a cold day, does not produce fever, because •the production of heat within the body is correspondingly diminished. In fever there is increased production of heat, as is seen by the study •of exchange of material ; the intake of food is, as a rule, very small ; the discharge of nitrogen and carbon results from the disintegration of tissues, which, as compared with that in simple inanition, is large ; "the tissues are said to be in a labile condition, that is, they are easily broken down. In most febrile states, the skin is dry, the sweat-glands, like most of the secreting organs of the body, being comparatively in- •active, and so the discharge of heat is lessened. The skin may, how- ever, sometimes be bathed in perspiration, and yet high fever be present. The essential cause of the high temperature is neither increased for- mation nor diminished discharge of heat, but an interference with the reflex mechanism, which is health, operates so as to equalise the two. Increased nitrogenous metabolism in fever has been observed by Huppert and Eiesell ■' in pneumonia, by Schimanski ^ in pya?mic con- ditions, by Xaunyn " and Sydney Ringer '^ in other febrile conditions. Ringer showed the correspondence in temperature and output of nitrogen very clearly in intermittent fever (ague). ' Studies from Lab. Physiol Chem. Yaie Unii: ii. 87. - Ihid. 200. * Compt. rend. Ixxxvi. 1169. •* Verhandl. d. physiol. Gesellscli. zu Berlin, June 3, 1879. 3 Arch. f. Heilk. vi. 236 ; %-iii. 343 ; x. 329. <= Zeit. physiol. Chem. iii. ilO. ■? Arch.f. Anat. u. Physiol. 1870, p. 1.59. ^ Med. Chir. Trans, xlii. 361. 842 GENERAL METAP,OLIS:\r What is known as the epicritical increase of urea ' is the greatly increased secretion of urea that occurs at the commencement of the- defervescence of a fever. It is probably not due to an increased for- mation of urea, but to the removal of urea which has accumulated, owing to the fact that the kidneys have been acting sluggishly during the height of the fever. Increased output of carbonic acid in fever was shown to exist in guinea-pigs by Plliiger and Colasanti {see pp. 373, 374), in dogs by Frankel,^ in men by Liebermeister and others.'^ Other changes noted in fever are a rapid loss of the liver glyco- gen, a lessening of chlorides in the urine {see p. 760), and the appearance of pathological instead of normal urobilin in the urine {see p. 750). The following table illustrates exchange of material in fever, no food being taken (Burdon-Sanderson ^) : — Income 1 ExpeniUture "*''^Se'°"°* ^^trogen' Carbon Excretions Xitrogen Carbon 8-3 212-7 221-0 1 Proteid, 120 jrr. 18-6 63-6 Fat, 205-7 gr. . 0-0 loT"! 18-G 221-0 Urea and uric acid. 40gr. . Respiration (CO.^) TbO gr. . t 1 18-6 00 18-6 This table should be compared with that on p. 834. Diahetf'S iiieJIitus. — In addition to the presence of sugar in the urine in this disease, the most marked symptoms are intense thirst and ravenous hunger. As a rule, diabetic patients digest their food well. The thirst is an indication of the necessity of replacing the large quantities of water lost by the kidneys ; the hunger, that of replacing- the great waste of tissues that occurs. For not only does the urine contain sugar, but, in addition, a great excess of urea and uric acid. The carbonic acid output is somewhat smaller than in health. In health the carbohydrates, after assimilation, give rise, by oxidation, to car- bonic acid ; in diabetes, all the carbohydrates do not undergo this change, but pass as sugar into the urine. Xot that all the sugar of the urine is derived from carbohydrates, for many diabetics continue to pass large quantities when all carbohydrate food is withheld ; under 1 Colmheim's PatJiologij, '2nd German edit. vol. ii. ^i. 532. - Arch. j}at]ioL Anat. hixvi. Vd6. 5 For references, see Senator, Uuters. ii. d. ficherhaften Process uud seine Behand- lung, Berlin, 1873. ■* Practitioner, April, May, June, 1876. KXCIIANdK ol' .M.Vl'KinAL 818: tlie.so circumstances it must be derived from the destiuction of proteid matter. in spite of abundant nourislimcnt, the body of a diabetic [)atient wastes ; there is not equilibrium ; the output is in excess of the intake. This is especially marked in the case of the excretion of nitrogen. ' Lfin-o<-!jtli((iit'i((. — This appears to be the only other disease in which systematic obsei'vations have Ijeen made on metabolic exchanges.. Altliough, as in all chronic and debilitating diseases, there is a general loss of vitality, and a corresponding lessening of metabolic change, observers have failed to find anything very special or charac- teristic in this disease. This is somewhat remarkable, as so metny important parts of the body may be effected, especially the blood and the blood-forming organs. There is, however, always an increase of uric acid in the urine.^ With regard to urea the statements of various investigators are contradictory "^ Luxus Consumption In former portions of this book we have insisted on the fact tliat the food does not undergo combustion, or katabolic changes, until after it is assimilated, that is, until after it has become an integral part of the tissues. Formerly the blood was supposed to be the seat of oxidation ; but the reasons why this view is not held now have been already gi\'en. AVhen a student is first confronted with balance- sheets, representing metabolic exchanges, it is at first a little difficult for him to grasp the fact, that although the amount of nitrogen and carbon ingested is equal to the amount of the same elements which are eliminated, yet the eliminated carbon and hydrogen are not derived ' The following references to the chief papers on metabolism in diabetes are takers from Hoppe-Seyler's Physiol. Cliem. p. i)71 : M. Traube, Virchmc'a Arcliiv, vol. iv. ; Seegen, Wiener med. Wochensch. 18()3, No. 14; also Der Diabetes meUitus, Berlin, 1875; F. Nasse, Arch. f. iihysiol. Heilk. 1851, p. 52; Reich, Diss. Greifswald, 1850; Rosenstein, Virchow's Archiv, xii. 414; C. Giihtgens, U. i!. Stoffwechsel eiiies Diabetikers, Diss. Doi-pat, 1866; E. Kiilz, Beitriige zur Pathol, ii. Tiwrajne d. Diab. mell., Marburg, 1874-5; Arch. f. experim. Pathol, vi. 140; Pettenkofer and Voit, Sitsuugsb. Bayer. Akad. November 1865 ; Zeit. Biol. iii. 380 ; C. Schmidt, C'liarakteristik der epid. Cholera, Leipzig and Mitau, 1850, p. 160; v. Mering, Deiitsch. Zeit. f. prakt. Med. 1877, No. 18. - The following references are again derived from Hoppe-Seyler's Text-book: Virchow's Arch. f. path. Anat. v. 108; H. Ranke, Beobachtungen iind Versuche it. d.. Aiisscheidung d. Harnsailre, Mtinchen, 1858; Pettenkofer and Voit, Zeit. Biol. v. 326; ' E. Salkowski, Virchow's Ai'chiv, 1. 174, Iii. 58; K. B. Hofmann, Wien. med. Woch. 1870, Nos. 42, 43, 44; Schmutziger, Mosler, Fleischer, and Penzoldt, Deutsch. Arch. f. klin. Med. vol. xxvi. 1880, p. 1. All the foregoing agree in the statement that the output of uric acid is greater than normal. •" Salkowski states there is a lessening, Mosler an increase, Pettenkofer and Voit, Fleischer and Penzoldt, say there is neither increase nor decrease. 844 GENEEAL :^IETA1'.0LIS3I from the food direct, but from the tissues ; the food becomes as- similated, and takes the place of the tissues thus disintegrated. Again, let us suppose the food to be inci'cased in quantity ; the excretions are also increased. Here the increased intake of food stimulates the tissues to increased combustion, pushing, as it were, the old out of the way to make room for the new. Let us suppose we have a tube open at both ends and filled with a row of marbles : if an extra marble is pushed in at one end, a marble falls out at the other ; if two marl)les are introduced instead of one, there is an output of two at the other end ; if a dozen, or any larger number be substituted, there is always a cori-esponding exit of the same number at the other •end of the tube ; the marbles that fall out are equal in number to those put in at the other end, but the marbles that fall out are different ones from those put in. This veiy rough illustration may perhaps assist in the comprehension of the metabolic exchanges. The dithculty just alluded to, which a student feels, was also felt by the physiologists who first studied metabolism : and Yoit ' formu- lated a theory, of which the following is the gist : All proteid taken into the alimentary canal appears to aftect pioteid metabolism in two ways ; on the one hand, it excites rapid disintegration of proteids, giving rise to an immediate increase of urea ; on the other hand, it serves to maintain the more regular proteid metabolism continually taking place in the body, and so contributes to the normal regular dischai'ge of urea. He, therefore, supposed that the proteid which plays the first of these two parts is not really built up into the tissues, does not become living tissue, but undei'goes the changes that give rise to urea, somewhere outside the actual living substance. Consequently, he divided the proteids into ' tissue-proteids,' which are actually built up into living substance, and ' floating or circulating proteids,' which are not thus built up, but by their metabolism outside the living substance set free energy in the form of heat only. It was at this time errone- ously supposed that the exclusive use of proteid food was to supply proteid tissue elements, and that vital manifestations other than heat had their origin in proteid metabolism, the metabolism of fats and cai'bohydrates giving rise to heat only. Hence, when it was first sur- mised that a certain proportion of proteids underwent metabolism, which gave rise to heat only, this appeared to be a wasteful expendi- ture of precious material, and the metabolism of this portion of food was spoken of as a ' luxus consumption," a wasteful consumption. There were many deductions from this general theory to ex2:)lain particular points ; of these two may be mentioned : (1) In inanition, the urea 1 Zeit. Biol. X. -l-li. EXCIlANdK (U- .MATKIJIAL 845 discharged fur the lirst few day.^ is inucli greater than it is subsequently: this v,as supposed to be due to the fact that in the first few days all the floating capital was consumed ; (2) the effect of feeding with a mixture of gelatine and proteid (see p. (S38) was supposed to be due to the fact that gelatin w^'^s able to I'eplace ' floating proteid,' but not 'tissue proteid." This theory of N'oit's, ingenious and plausible at first sight, has met with but little general acceptance, because so many observed facts are incompatible with it, and I cannot do better than conclude this chapter by giving the opinions of three eminent physiologists on the (luestion. , Professor Michael Foster ' writes as follows : ' The evidence Ave have tends to show that in muscle (taking it as an instance of a tissue) there exists a framework of what we may call more distinctly living substance, whose metabolism, though high in quality, does not give rise to massive discharges of energy, and that the interstices, so to speak, of this framework are occupied by various kinds of material re- lated in diflferent degrees to this framework, and therefore deserving to be spoken of as more or less living, the chief part of the energy set free ..loming directly from the metabolism of some or other of this material. Both framework and intercalated material undergo metabolism, and have in different degrees their anabolic and katabolic changes ; both are concerned in the life of the organism, but one more directly than the other. We can, moreover, recognise no sharp break between the intercalated material and the lymph which bathes it ; hence such phrases as "tissue proteid " and " floating proteid " are undesirable if they are understood to imply a sharp line of demarcation between the " tissue " and the blood or lymph, though useful as indicating two different lines or degrees of metabolism.' Professor Burdon-Sanderson - writes as follows : ' The production of urea and other nitrogenous metabolites is exclusively a function of " living material " ; and this process is carried on in the organism with an activity which is dependent on the activity of the living substance itself, and on the quantity of material supplied to it. No evidence at present exists in favour of a " luxvis consumption '' of proteid.' Professor Hoppe-Seyler,^ after stating that he can make out no clear distinction between the two varieties of proteid from Yoit's own writings, proceeds as follows: ' Voit states that the circulating proteid is no other than that which is dissolved in the tissue juice, which is derived i Tea-^iooA-, 5th edit. pp. S-i"), 820. - SijUabus of Lectures, -p. 37. '■ Flujsiol. Chcm. p. 974. 846 GENERAL METAEOLIsM from the Ijmph-stream, and ultimately from the circulating blood. He (Voit) furth(T says : "As soon as the proteid of the blood-plasma leaves the blood-vessels, and circulates among the tissue elements themselves, it is then the proteid of the nutrient fluid or circulating proteid. It is no longer proteid of the blood-plasma, nor yet is it the pro- teid of the lymph -stream."' The place where Yoit situates his circulating proteid is beyond the ken of the anatomist : it is in a mysterious sjmce between tissue-elements, blood -^•esseLs, and lymph-vessels ; the chemist meets with equal difiiculties, as there is apparently no chemical diflfer- ence between tissue proteid and circulating proteid. I can, therefore, ai-rive at no other conclusion than that these terms are not only useless, but unscientific, and are the outcome of speculations in a region where there is as yet no positive knowledge. These criticisms on Yoit's theories do not, however, by any means, lessen the importance and high value of the immense amount of practical research carried on by Yoit and his pupils." I have placed Professor Foster's view first because it takes intn account certain facts which tend to show that there are degrees in metabolism. The most important of these seems to be the formation of amido-acids in the intestine. The fate of tyrosine is uncertain : but it is an undoubted fact that by feeding an animal on leucine, the urea is increased. The transformatiim of leucine into urea occurs in the liver. It can hardly be supposed that leucine becomes to any great extent an integral part of the living framework of the liver cells, but like other extractives, and like aromatic compounds absorbed from the alimentary canal, it becomes a part of what Foster terms the inter- calated material. Here it undei'goes the final change, and is ulti- mately and aj^parently very rapidly discharged in the urine. Dr. Sheridan Lea ' has recently discussed in a very able manner the probable role of the amido-acids in the animal economy, and he com- pares it to the part played by the salts of the food. Neither salts nor extractives simply pass into the urine without fulfilling a useful pur- pose on their way : but the exact and specific use of each, whether on the synthetic or analytic side of metabolic phenomena, must be the subject of renewed research. ' Journ. of Physiol, vol. xi. '262. 847 CHAPTER XLVUl -lyiJ/AL HEAT Among the most important results of the chemical processes we sum up under the term metabolism, is that of the production of heat. Heat, like mechanical work, is the result of the katabolic side of metabolic processes ; the result, or accompaniment, that is to say, of the forma- tion of carbonic acid, water, urea, and other excreted products. As regards temperature, animals may be divided into two threat classes : — (1) Warm-blooded or homoiothermal animals, or those which have an almost constant temperature. This class includes mammals and birds. (2) Cold-blooded or poikilothei'mal animals, or those whose tempera- ture varies with that of the surrounding medium, being always how- ever, a degree, or a fraction of a degree, above that of the medium. This class includes reptiles, amphibians, fish, and probablv all inver- tebrates. The temperature of a man in health Aaries but slightly, beino- be- tween 36-5° and 37-5^ C. (98° to 99= F.) Most mammals have approxi- mately the same temperature : horse, donkey, ox, 37-5° to 38° • do*' cat, 38-5° to 39° ; sheep, rabbit, 38° to 39-5° ; mouse, 40° C. Birds have a higher temperature, about 42° C. The temperatuje Aaries a little in different parts of the body, that of the interior being creator than that of the surface ; the blood coming from the liver Avhen oxida- tion is very active is Avarmer than that of the general circulation the blood becomes rather cooler in its passage through the lungs. The temperature also shows slight diurnal A'ariation.s, reachino- a maximum about 3 p.m. (37-5° C.) and a minimum about 3 a.m. (36-8° C.)- that is, at a time Avhen the functions of the body are least active . If howeA-er, the habits of a man be altered, and he sleeps in the day. working during the night, the times of the maximum and minimum temperatures are also inA'erted. Inanition causes the temperature to fall, and just at the onset of death it may be beloAv 30° C. Active muscular exercise raises the temperature temporarily by about 0-o° to PC. Diseases may cause the temperature to vary considerably especially those which we term febrile (s^e p. 841). 848 GEISERAL METABOLISM Although certain mechanical actions, such as friction, due to move- ments of various kinds, may contribute a minute share in the pi'oduc- tion of heat in tlie body, yet we have no knowledge as to the actual amount thus generated. The great source of heat is, as already stated, chemical action, especially oxidation. Any given oxidation will always- produce the same amount of heat. Thus, if we oxidise a gramme of carbon, a known amount of heat is produced, whether the element be- free or in a chemical compound. The following figures show the approximate number of heat-units produced by the combustion of one- gramme of the following substances. A heat-unit, or calorie, is the amount of heat necessary to raise the temperature of one gramme of water 1° C. {see also p. 605 et seq.) : — Hydrogen . . . 3450 j Fat .... 9069' Carbon . . . .8100 Urea .... 2205 Albumin . . . 4998 Cane sugar . . . 3348- Starch .... 3898- The question of the heat-value of various foods has been already discussed (p. 606). It is, however, most important to remember that the 'physiological heat-value' of a fodd may be different from the 'physical heat-value,' i.e. the amount of heat produced by combustion in the body may be different from that produced when the same amount of the same food is burnt in a calorimeter. This is especially the case with the proteids, for they do not undergo complete combus- tion in the body, for each gramme of proteid yields a third of a gramme of urea, which has a considerable heat- value of its own. Thus albumin,, which, by complete combustion, yields 4998 heat-units, has a physio- logical heat-value=4998 minus one-third of the heat-value of urea (2205)=4998- 735 = 4263. Of the heat produced in the body, it is estimated by Helmholtz that about 7 per cent, is represented by external mechanical work^^ and that of the remainder about four-fifths are dischai'ged by radiation and evaporation from tlie skin, and the remaining fifth by the lungs. and excreta. The following table ^ exhibits the relation between the production and discharge of heat in twenty -four hours in the human organism at rest, estimated in calories.^ The table conveniently takes the form of a balance-sheet in which production and discharge of heat are con- 1 See Dr. Sanderson's Syllabus of Lectures, p. 4'2. - The calorie we are taking is sometimes called the small calorie ; by some the word calorie is used to denote the amount of heat necessary to raise one kilogramme of water 1<^ C. ANIMAL JlEA'l' 849 trasteil ; to keep the iKxly-teiiipemtui'o normal these must be equal. The basis of the table in the left-hand (income) side is the same; as llanke's adequate diet {s''f' p. 604 .and p. S;3l.') : — J'ri'fhictioii of iK'iif j Dhchtirge of hrat Consumiition n:' Calnries I (^'iilorir< I'roteid (100 ar.) . ]00 x 4263 = 426,300 Fat ( lOU gr. ) . . 100 x i»069 = 906,900 Carbohydrates (250 gr.) 250 x8s98 = 974,500 2,307,700 Warniini;- water in food, 2'6 kilos. X 25° C- 6.">,0()0 Wanning air in respiration, 16 kilos. X 25° X 0-24 = 9i;,000 Evaporation in lungs, 630 gr. X 582 = 366,660 Radiation and evaporation at surface, = 1,780,040 2,307,700 The figures under the heading Production are obtained by multiplying the weight of food by its physiological heat-value. The figures on the other side of the balance-sheet are obtained as follows : The water in the food is reckoned as weighing 26 kilos. This is supposed to be at the temperature of the air taken as 12° C. ; it has to be raised to the temperature of the bodj-, 37° C, that is through 25° C. Hence the weight of water multiplied by 25 gives the number of calories expended in heating it. The weight of air is taken as weighing 16 kilos. ; this also has to be raised 25° C, and so to be multiplied by 25 ; it has further to be multiplied by the relative heat of air (0'24). The 630 grammes of water evaporated in the lungs has to be multiplied by the potential or latent heat of steam at 37° C. (582). The portion of heat lost by radiation and evaporation from the skin constitutes about four-fifths of the whole, and is obtained hj deducting the three previous amounts from the total. This table does not take into account the small quantities of heat lost with urine and fteces. It need hardly be remarked that the above is a mere illustra- tive experiment. Changes in the diet, in the atmospheric temperature, in the temperature of the food taken, in the activity of the sweat-glands, in the amount of moisture in the atmosphere, and in the amount of work done would con- siderably alter the above figures. Calorimetry. — Calorimeters employed in chemical operations are not suitable for experiments on living animals. An animal surrounded by ice or mercury, the melting and expansion of which respectively are measures of the amount of heat evolved, would be under such abnor- mal conditions that the results would be valueless. The apparatus most usually employed is the water calorimeter. This was first used by Lavoisier, and his apparatus as modified by Dulong is shown in fig. 104. The animal is placed in a metal chamber, surrounded by a water-jacket. There are tulles for the entrance and exit of the inspired and expired gases respectively. The heat given out by the animal warms the water in the jacket, and is measured by the rise of temperature observed in the water, of which the volume 3 I 850 GENERAL METABOLISM is also known. The air which passes out from the chamber goes through a long spiral tube, passing through the water-jacket, and thus the heat is abstracted from it and not lost. Pig. 104. — Dulong's Calorimeter : C, calorimeter, consisting of a vessel of cold water in which the chamber holding the animal is placed ; G', gasometer from which air is exijelled by a stream of water. The air enters the respiratory cliamber. G, gasometer receiving the gases of expiration and the excess of air. t, t', thermometers : a, a wheel for agitating the water. Observe the delivery-tube on the left is much twisted in the water-chamber, so as to give off its heat to the surrounding water. (From McKendrick's 'Physiology.') Rosenthal ' has iu%'ented an air-calorimeter, in which an air- jacket takes the place of the water-jacket of Dulong's apparatus. Regulation of the Temperature of Warm-blooded Animals We have seen that heat is produced by combustion processes, and lost in various ways. In order to maintain a normal temperature, both sides of the balance-sheet must be equal. This equalisation may be produced by the production of heat, adajjting itself to variations in discharge, or by the discharge of heat adapting itself to variations in production, or lastly, and more probably, both sets of processes may adapt themselves mutually to one another. We have, therefore, to consider (1) regulation by variations in loss and (2) regulation by variations in production. The following is a resume of our knowledge on these two points, as given in Professor Foster's text-book. ^ Regulation hy variations in loss. — The two means of loss susceptible of any amount of variation are the lungs and the skin. The more air that passes in and out of the lungs, the greater will be the loss in warming the expired air and in evaporating the water of respiration. In such animals as the dog, which perspire but little, respiration is a most important means of regulating the temperature ; and in these animals a close connection is observed between the production of heat 1 Arch.f. Physiol, u. Anaf.; j'hjjsiol. Ahth. 18Hi), p. 1. 2 P. 810 et seq. ANIMAL HEAT 851 and thfi respiratory activity.' Tlie great regulator, however, is un- doubtedly the skin, and this has a double action. In the first place, it regulates the loss of heat by its vaso-motor mechanism ; the more blood passing through the skin, the greater will be the loss of heat by conduction, radiation, and evaporation. Conversely, the loss of heat is diminished by anything that lessens the amount of blood in the skin, such as constriction of the cutaneous vessels, or dilatation of the splanchnic vascular area. In the second place, the special nerves of sweat-glands are called into action. Familiar instances of the com- bined action of these two sets of nerves are the reddening of the skin and sweating that occur after severe exercise, on a hot day, or in a hot-air or vapour bath, and the pallor of the skin and absence of sen- sible perspiration on the application of cold to the body. Jiegukitio7i by variations in production. — The rate of production of heat in a living body, as determined by calorimetry, depends on a variety of circumstances. It varies in different kinds of animals. The general rate of katabolism of a man is greater than that of a dog, and of a dog greater than that of a rabbit. Probably every species has a specific coefficient, and every individual a personal coefiicient of heat- production, which is the expression of the inborn qualities proper to the living substance of the species and individual. Another factor is the proportion of the bulk of the animal to its surface area, the struggle for existence raising the specific coefficient of the animals in which the ratio is high. Other important considerations are the relation of the intake of food to metabolic processes, and the amount of muscular work which is performed. These various influences are themselves regulated by the nervous system, and physiologists have long suspected that afferent impulses arising in the skin or elsewhere may, through the central nervous system, originate efferent impulses, the effect of which would be to increase or diminish the metabolism of the miiscles and other organs, and by that means increase or diminish respectively the amount of heat there generated. That such a metabolic or thermogenic nervous mechanism does exist in warm-blooded animals is supported by the following experimental evidence : — (1) Though in cold-blooded animals, a rise or fall of the surround- ing temperature causes respectively a rise and fall of their metabolic activity, in a warm-blooded animal the effect is just the reverse. "Warmth from the exterior demands a diminished production of heat in the intei'ior, and vice versa. 1 The panting of a dog when overheated is a familiar instance of this. A dog also, under the same circumstances, puts out its tongue, and loses heat from the evaporation that occurs from its surface. 3 I 2 852 GENERAL METABOLISM (2) That this is due to a reflex nervous impulse is supported by the fnct tliat a warm-blooded animal, when poisoned by curare, no longer manifests its normal behaA^our to extei-nal heat and cold, but is affected in the same way as a cold-blooded animal. Section of tlie medulla produces the same effects, as the nerve-channels, by which the impulses tra\el, are severed. "When curare is given, the reflex chain is broken at its muscular end, the poison exerting its influence on the end-plates, and causing a diminution of the chemical tonus of the muscles. The centre of this thermotaxic reflex mechanism must be situated somewhere alx»ve the spinal cord. (3) Various iiijuries caused by accident, or purposely produced by puncture, or cautery, or electrical stimulation of limited portions of the more centi-al portions of the brain, may give rise to great increase of temperature, not accompanied by other marked symptoms.^ We thus see that the nervous system is intimately associated with the regulation of the temperature of the body. There is at least one — there may be several centres associated in this action. The centres receive afferent impulses from ^s"ithout : they send out efferent im- pulses by at least three sets of nerves : (1) the vaso-motor nenes, (2) the secretory nerves of the sweat-glands, (3) trophic or nutritional nerves. The fi^rst two sets of nei"ves, the vaso-motor and the secretory, affect the regulation oi temperature on the side of discharge ; the third set of nerves may be special nerve-fibres set apart for the regu- lation of chemical processes in the organs they supply ; or it may be that all nerves to muscles and other organs are capable of transmitting trophic impulses. The discussion as to whether there are or are not special trophic filjres Ls an interesting one ; but in whichever way this is finally settled it does not matter in the least in the present consideration. The fact remains that this third set of nerve-impulses affects the regulation of temperature on the side of production. Gaskell has gone so far as to consider that the impulses may be of two kiutls : the nerves which convey those impulses which produce building up of tissue, or anabolism, he calls anabolic 'nerves, while those that hiring about the reverse process he terms katabolic nerves. * See a recent paper by Hale Wliite, Journ. of Physiol, ii. 1. INDEX A Abetn, 133 Abrus prCT-atorius, 132, IM3 Absorptiometer, 284 Absorption, 327, 700 ; of carbohvdrates, 701 ; of proteids, 702 ; of fats, 704 ; the columnar epithelium during, 705 ; lymphoid tissue during, 705 Absorption spectra, 48. Sec also pig- ments Absorptive jtower of coloured sohi- tions, 52 Acetic acid, 70 ; and metaboHsm, 840 Acetone, 66, 70 — in blood and urine, 314 — in urine, 791 Acetj'lene, (JS — and haemoglobin, 282 Achromatin, 107 Achroo-destrin, 105, 627 Acid fermentation in urine, 767 — haematin, 288 — tide, 714, 733 Acidimetry, 16 Acidity of urine, estimation of, 798 Acids derived from glycols, 67, 71 — fatty, 65, 70, 490, 662 — free in body, 60 — of bile, 86, 680, 681 — of gastric juice, 636, 639, 640 Acrolein, 72 Acrylic acid series, 69, 72 Actiniochrome, 150 Actinosphajrium, digestion in, 193 Activity of reduction of oxyhtemoglo- bin, 285 Acute yellow atrophy of liver, 313, 552, 724, 727, 755 the bile in, 688 the urine in, 769 Adamkiewicz reaction, 121 Adelomorphic cells, 633 Adenine, 90, 203 Adenyl, 90 Adipocere, 427, 837 Adipose tissue, 71, 72, 468, 487 Adrenals, see Suprarenals Aerobic organisms, 164 Aerotonometer, 386 Age, influence of, on food, 608 ; on metabolism. 834 : on respiration, 372 ; on urine, 711, 723 Ague, 309, 555 — discharge of carbonic acid in, 373 — discharge of urea in, 841 Air-pump, mercurial, 30 Alanine, 83 Albumin in sweat, 820 — in urine, 780 Albuminates, class of, 128 Albuminoid degeneration, 144; of kid- ney, 559 ; of liver, 551, 688 Albuminoids, 143 Albuminose, 644: Albuminous glands, 620 Albumins, class of, 127 — derived, 128 Albuminuria, experimental, 781 ; in dis- ease, 782 ; physiological, 780 Albumoses, 129, 645 — ■ in cerebro-spinal fluid, 358 — in urine, 513,783 Alcaptonuria, 796 Alcohol, effect of, on resjjiration, 374 — in diet, 600 ^ — in intestine, 693 — in muscle, 425 — in stomach, 650 — metabolism, and, 840 Alcohols, 64, 69 Aldehydes, 65 — in proteids, 116 Aleurone grains, 132, 134 Alexis, St. Martin, 630 Alizarin, 109 Alkali-albumin, 128 Alkalimetry, 16 Alkaline fermentation in urine, 767 — haematin, 288 — tide, 714, 733 854 INDEX ALK Alkaloids, 160 — in urine, 766, 796 — in tiie intestine, 692, 694 Alkoxjhyr, G45 Allantoin. 90, 73fi — • formation of, from uric acid, 729; in urine, 725, 736 Allanturic acid, 90 Alloxan, 729 Alloxantin, 730 Allyl alcohol, 69 Alvergniat's pump, 32 ■ Amides, 81 Amido-acids, 72, 81 — as antecedents to urea, 726 ; in diges- tion, 661, 694 ; in urine, 769 Amido-benzene, 75 Amines, 80 Amitotic cell-division, 198 Ammonia in the body, 59 ; in pancreatic digestion, 661 — in urine, 764; in urine, estimation of, 806 Ammonium carbonate as an antecedent to urea, 727 as an antecedent to uric acid, 735 effect of, on liver glycogen, 548 — salts in body, 62 — sulphate, use of. for separation of peptone, 130, 645, 786 Amniotic fluid, 354 Amoeba, digestion in, 193 Amceboid movement, 1 87 Amphicreatinine, 179 Amphioxus, blood of, 218, 267 Amphipyrenin, 197 Ampho-deutero-albumose, 646 Ampho-i^eptone, 646 Amygdalin, 77, 109 Amyloid degeneration, see Albuminoid degeneration Amylolytic ferments, 158 Amylopsin, 656, 658 Amyloses, 92 Anabolic nerves, 520, 852 Anabolism, 206, 828 Anaemia, 298 Anaerobic organisms, 164 Analyser of polarising apparatus, 37 Analysis, gas, 28 — of organic compounds, ultimate, 19 — of urine, 798 — volumetric and gravimetric, 7 Analytic and synthetic processes in plants and animals, 210 Anethol, 78 Angelic acid, 72 Angstrom's measurements of wave- lengths, 50 Aniline, 75, 78 ASH Animal alkaloids, 169 — dextran, 108 — glucosides, 109 — gum, 108, 144, 480 in chlorosis, 3(XJ — — in mammarj- glands, 574 in stomach, 636 in urine, 757, 758 — heat, 847 ; regulation of, 850 — starch, see Glycogen — quinoidine, 173 Anisamic acid, 78 Anisotropous substances, 36 ; in muscle, 399, 401 — - — in protoplasm, 189 Antecedents to urea, 726 ; to uric acid, 735 Antedonin, 150 Anthropocholic acid, 88, 680 Anti-alburnid, 130, 646, 648 Anti-albumin, 645 Anti-alVjuminate, 646, 647 Anti-albumose, 129, 645 Anti-deutero-albumose, 646 Anti-humoralists, 297 Antilytic secretion, 619 Antimony poisoning, metabolism in, 840 Anti-peptone, 130, 645 Antipolar field of nucleus, 1 96 Apatite, 494 A-peptone, see Proteoses Aphidein, 150 Aplysiopurpurin, 150 Apnoea, 376 Apparatus and reagents, 6 Aqueous humour, 350 — vapour, tension of, 5 Arabin, 108 Arabinose, 108 Arachnida, blood of, 328 Areolar tissue, 467 Armadillo, exoskeleton of, 496 Aromatic oxyacids, 78 — substances, 68, 74 ; from proteids, 123; in intestine, 692 ; in urine, 738 Arsenic, effect of, on liver glycogen, 542 — poisoning, metabolism in, 840 Arterin, 382 Artificial butter, 586 — digestion of food, 611 — gastric juice, 637 ; estimation of activity of, 645 — human milk, 577 — pancreatic juice, 655 Asbestos cardboard, 12 — filters, 10 Ascites, 388 ; fluid of, 342' Ash, see Inorganic Substances, Salts, and Incineration Ash of filters, 12 INDKX 855 Asparagiiio, 8('>. \'M\ -— and im'tal>olism, SJ(); etrcci of, on urine, 75") Asparajrinic acid, tire Aspartic acid Aspartic- acid, 86 : in intestine, »!()1 Asphyxia. 'MCu 3'.tl Asymmetrical t'arlioii atoms, 4") Atlieroma, 4iM> Atmid-all>imiose. iL'it Atmosphere, composition of, iJG'J Atmospheric pressure in relation to respiration, ;i78 : in relation to urea, 839 Atoms of carbon, asymmetrical, 45 Atropine antagonistic to muscarine, 171*, oHO ; intiuence of, on salivary secretion, ()17, ()1S Attraction spheres, 202 Auto-intoxication, 694 Axis-cylinder, 521 B Bacillus anthracis, 153, 168, 175 — malaria;, 309 Bacteria, see Fermentation Balance-sheets of income and dis- charge in health, 832, 833 ; in in- anition, 834 : with abnormal diets, 837 ; in fever, 842 ; of heat, 849 Balsams of Tola and Peru, 78 Bantingism, 836 Barium chloride, alkaline solution of, 804 Baryta mixture, 801 Beaumont on the gastric juice, 630 Beef tea, 597 Bees' wax, formation of, 837 Benzene, 68, 74 Benzene-ring, 75 Benzoic acid, 77 : in relation to hippu- ric acid, 738 — aldehyde, 77 Benzoyl, 77 Benzyl alcohol, 77 Bernard on glycogen, 541, 546 Bernstein on muscular contraction, 435 Biaxal crystals, 272 Bile, (i68 ; secretion of, 668 ; influence of blood suppl_v on secretion of, 672, 673; methods of obtaining,674: quan- tity of, 674 ; intiuence of drugs on, 675 ; constituents of, 675 ; composition of human, 676 ; of dogs, 677 ; of other animals, 678 ; sulphur in, 678 ; iron in, 678 ; gases of, 392. 679 ; mucin of, 679 ; salts of, 679 : analysis of, 680; tests for, 681, 683; pigments of, 682 ; action on proteids, carbo- iiydratt!s, fats, 686; as a laxative and antiseptic, 687 ; fate of constituents of, 687 ; abnormal and pathological conditions of, 688 Bile acids and salts, 86, 679 — pigments in sweat, 820 ; in urine, 778 — resin, 681 — salts in urine, 779 Bilharzia hiematobia, 308, 772, 776 Biliary fistula, 674 -- urobilin. 685 Bilifidvin, 682 Bilifuscin, 684 Bilihumin, 684 Biliphtein, 682 Biliprasin, 684 Bilirubin, 682 Bilirubin-calcium, 689 — crystals in urine, 778 Biliverdin, 682, 683 Bird's nest edible, 486, 625 Bird's urine, 728, 780, 732, 734 Bismuth test for sugar, 96 in urine, 790 Biuret. 122, 722 — reaction, 122, 647 in urine, 786 Blisters, fluid from, 349 ; in gout, 352 Blood, 217 ; colour of, 218 ; specitio gravity, 218; reaction, taste, and odour, 219; coagulation, 220; quan- tity in body, 220 ; corpuscles of, 257 ; tests for, 295 ; in disease, 297 ; in aniemia, 298 ; in chlorosis, 298 ; in chronic anemia, 299 ; in pernicious anaemia, 300 ; in hajmogiobinuria, 302 ; in leucocj-tha^mia, 302 ; in Addison's disease, 303 ; in m>xoe- dema, 304 ; abnormal coagulation of, 305 ; in liiemophilia, scurvy, purpura, inflammation, 306 ; in rheumatism, g(jut, 307 ; parasites in, 308 ; in zymotic diseases, 308 ; in cholera, malaria, 309 ; in phthisis, septi- ciemia, and pytemia, 310 ; in heart diseases, lung diseases, liver diseases, jaundice, 311 ; in chotemia, acute yellowatrophy, phosphorus-poisoning, 313 ; in diabetes, diabetic coma, acetonjemia, 314 ; in lipiemia, Bright's disease, 315 ; of invertebrate animals, 316 ; of echinoderms, 318 ; of worms, 319; hjemocyanin in, 321 ; of molluscs, 323; of crustacaja, 324; of arachnida, 328 : of insects, 328 — corpuscles, see Blood — gases, 378 : collection of, 28 — in urine, 308, 770, 776 856 INDEX Blood pitrnu'Dts io sweat, 820 : in urine, 776 — quantity of. effect of, on metabolism, 839 — tablets, 261 Blue milk, 590 Blutplattchen, see Blood Tablets Bohr on carbonic acid haemoglobin, 888 Boiling, 12 Bojanus, organ of, 730 Bone, 468, 492 ; diseased, 510 Bonelleiu, 150. 214 Bottger's test for sugar. 9G, 790 l'>ojde's law. 383 Brain-sand. 496 Bread, 598, 609. (Ul Brieger's methods of isolating pto- maines, 176 Bright's disease, 782; blood in. 315 Brittle bones. 513 Bromohfematin, 291 Briicke on precipitation of proteids, 124 Brunner's glands, 6(;7 Buchanan on coagulation of blood. 243 Buff)' coat. 222 Burettes. 7 Bush tea, 601 Butter, 586 Butyric acid. 71, 585. 586 — fermentation, 103, 693 Cachexia stku"ipriva, .501 Cadaverine, 172. 173, 178 Caffeine, 601 Calcified cartilage, 494 Calcium carbona'e as a urinary deposit, 770 — oxalate, 71 ; in calculi. 774 : in urine, 751. 7(58, 770, 773 — salts in bodv, 62 : in bone, 493, 512, 513 — — physiological importance of, 256 and metabolism. 840 — urate, 510 Calcosphajrites. 495 <"alculi in gall-bladder. 689 — in urine, ?I Cement substance (if epithelia. 442 Centiarade scale, 5 Centinormal solutions, 6 Central cells of stomach, 633 INDEX b57 Central corpuscles. L02 CentrifujL'al niachirif, 17 Cephalopods. blood of, H-J2. H23 — liver of, 690 Cereals as food, o'.i? Cerebrin, oH'.i — in spleen, o'>i Cerebrospinal Huid, :Jo5 Cerotvl alcohol, 70 Cerumen, 823 Cetyl alcohol, 70 Cetylid, oM Chalk stones, 509 Charcoal stoves, 281 Charcot's crystals, HOI!, 563 Cheese, 5S!»" — fat in, N37 Chemical constitution uml circular polarisation, 44 Cliono-cholalic acid, 88, 680 Chinolin, 91 Chitin, 145. 454 — in invertebrate cartilage, 485 Chlorbenzene, 75 Chlorides of sodium and potassium, 01 — and metabolism, 840 — in pneumonia, 448 — in urine, 759 : estimation of, 800 Chlorocruorin, 816, 319, 320 Chlorohajmatin, see H:emin Chlorophane, 464 Chlorophanic acid, 213 Chlorophyll, 20>< ; in relation to spec- trum, 209 : cbemistFA- of, 21 1 ; spectro- scopic appearances. 212 ; in animals, 214; tests for, 215; functions, 216; corpuscles, 216 Chlorophyll-green. 212 Chloroph3llan, 213 Chloroplastin, 192, 205 Chlorosis, 299 — Egyptian. 30.S Choliemia, 313, 688 Cholagognes, 675 Cholalic'acid. 87. 680, 681 Choleic acid, 87, 680 Cholepyrrhin, 682 Cholera, stools in. 698 — the blood in, 309 ; the bile in, 688 Cholestencniia. 689 Cholesteric acid. 532 Cholesterin, 69. 531 — in lens, 565 : in sheep's wool, 822 — vegetable. 532 Cholic acid, gee Cholalic acid Choline. 179, 528. 529. 531, 692. 797 Cholo-hfematin, 684 Choloidic acid, 87 Chondrigen, 143, 483 Chondrin, 144, 482 Chondrin balls, 484 Chondri-glucose, 484 Chondroitic acid, 484, 4h5 Chondromucoid, 485 Chondrosin, 486 Choroid coat of eye, 564 Chorda saliva, 624 — tympani, 617 Chromatin. 145. 197 Chromidiosis, 80, 821 Chromogens. definition of. 1.^0 — in urine. 749. 751, 752 Chromophanes, 148. 464 Chromophvlls, 209 Chr\-sophvll, 214 Chyle, 332, 335 — gases of. 391 Chylous dropsy, 337. 345, 347. 34:t Ch3'luria, 308, 7t'5 Chvmosin. 581. 635 Cilia, 443 Cinnamene. 68 Circular iX)larisation, 40 and chemical constitution. 41 Circulating fluids for heart, 256 CiiThosis of the liver. 552. 735, 755 Citric acid, 72 Cladothrix, 164 Classification of proteids, 127 Coagulated proteids, 130 Coagulation of blcod, 220 : rapidity of, 222 ; theories of, 242 abnormal, 305 — of milk, 580 ; of muscle, 406 — of proteids, 117, 125 Coagulative ferments, 159 Cobric acid, 138 Coccus cacti, 150 Coccygeal glands, secretion of, 823 Cochineal, 150 Cocoa as food, 601 Coefficient of extinction, 51 Coelom in invertebrates, 317 Coffee and metabolism, 840 — as food, 601 CO-hicmochromogen, 290 CO-lia2moglobin. 281 : crystals, 281; spectrum of, 2s2 ; tests for, 281 Collagen, 143, 470 Collecting materials (blood, i:c.) for gas analvsis, 28 Collidine, 178 Collimator of spectroscope, 47 Colloid carbohydrates, precipitation by salts of, 106 — substance, 144 Colloids and crystalloids. 13, 120 Colostrum, 574, 576 Combining weights of elements, 5 Combustions, 20 858 INDEX Compensator of Soleil's saccharometer, 42 Complemental air, 371 CompoundH, organic and inorganic, in body, 57 Compressed air, utility of, in disease, 374 Conchiolin, 145, 45o Concretions, calcareous, in liver, 538 ; in lung, 560 ; in maninite, 590 ; in pan- creatic duct, 663 Condiments, 600 Cones of retina, 4G0 ; pigments of, 464 Conglutin, 134 Coniferin, 109 Connective tissues, 466 : cells of, 470 : white fibres of, 470 ; elastic fibres of, 473; ground substance of, 475; in disease, 497 Constituents cf tlie body, 57 Contraction of muscle, 431 Cooking of food. 610 Cooling after drying. 11 Copper in birds' feathers, sfe Turacin ; in body, 62 ; in blood, 321 ; in gall- stones, 689 ; in liver, 538 Corals, 456 Cornea, 470 Cornein, 145, 456 Corpora amylacea, 496 — lutea, 564 Corpuscles of blood, 257 ; gases of, 381, 388 Correction of volume of gases for pressure, &c. 34 Crayfish, gastric juice of, 650 Cream, 71, 73, 575, 586, 590 Creatine, 84 — influence of, in urea formation, 726, 727 — in muscle, 418 ; in urine, 737 Creatinine, 84 — estimation of, 813 — in muscle, 421 ; in urine, 737 Cresol, 77 Cresol-sulphate of potassium in urine, 743 Cretinism, 501 Crop of birds, secretion of, 592 Crotonic acid, 72 Cruso-creatinine, 179 Crusta petrosa, 495 — phlogistica, 222 Crustacea, blood of, 324 ; colour of, 324; specific gravity, reaction, constituents of, 325 ; extractives of, coagulation of, 326 ; corpuscles of, 327 ; salts of, 325, 327 Cryptophanic acid, 91 Crystallin, 128, 565 Crystalline proteids, 120, 133, 594 Crystalloids and colloids, 13, 120 Crystals, optical characters of, 271 Cultivation of bacteria, 1 62 Cumarin, 78 Cutaneous respiration, 394 — secretions, 818 Cysn-alcohols, 116, 410 Cyanamide, 420 — as an antecedent to urea, 727 Cyan-h?ematin, 291 Cyanhydrins, 116 Cyanicacid as an antecedent to urea, 727 Cyanogen in proteids, 116, 122 Cyanophyll, 214 Cyanosis, 311 Cyanuric acid, 722 Cynurenic acid in urine, 91, 758 Cynurin, 91 Cystein in urine, 769 Cystic kidney, 353 Cystin, 86 — in urine, 768, 770; in calculi, 774, 775 ; in sweat, 827 Cystinnria, diamines in, 174, 769, 797 Cytochylema, 192 Cytochyma, 192 Cytohyaloplasma, 192 Cytoplasm, 191 D Dalton-Henry law, 383 Damoluric acid, 713 Dark lines of solar spectrum, 46 Daughter nuclei, 201 Decantation, 10 Decinormal solutions, 6 Decomposition of proteids, 1)3, 694 Delomorphic cells, 633 Denis on coagulation of blood, 243 Densimetric metliod of estimating pro- teids, 126, 816 Density of water, 5 Dentine, 469, 495 Deoxycholic acid, 88 Deposits in urine, 766 ; of chemical nature, 767 ; of anatomical nature, 770 Derived albumins, 128 Dermoid cysts, 822 Deutero-albumose, 129, 646 Deutei'o-elastose, 475 Deutero-proteose in urine, 784, 786 Dextrane, 109 Dextrin, 104, 105 — in muscle, 425 ; digestion of, 6i:7 Dextro-rotatorj' substances, 44 Dextrose, 94 INDEX 859 Dextrose, estimation of, 97 — in blood, 252. 547, titUi — in normal urine, 7")(> in diabetic urine, 7S'.> — in sweat, 820 — in urine, estimation oi', SI-1 Diabetes, cataract in, 565 — metabolism in, 842 — the blood in, 314 ; the urine in, 788 — urea formation in, 724, 72(5 Diabetic coma, 314 Dialysers, 13 Dialysis, 13 Diamines in urine, 174, 797. See also Ptomaines, Putrescine, Cadaverine Diarrhoea, 698 Diastase, 628 — l)ancreatic, 656 Diatomic alcohols, 67 Diazo-reaction in urine, 797 Diet, 602 — effect of, on metabolism, 836 Diffusion of fluids, 13 — of gases within the lungs, 370 — • through living and dead membranes, 15, 701 Digestibility of food, 609 Digestion in intestines, 652 ; in mouth, 626 ; in rliizopods, 193 ; in stomach, ()43 Digestive juices, action of (summary), 612 Digital in, 109 Dimetliyl-benzene, 75 Dimethylxanthine, 601 Dimorphism in crj^stals, 272 Dioxindole, 79 Diox)'cholesteric acid, 532 Dippel's oil, 113 Diptera, blood of, 328 Direct cell-division, 198 Direct-vision spectroscope, 48 Disdiaclasts, 402, 436 Dispirem, 201 Dissociation, 384 ; tension of, 385 ; of oxyh;emoglobin, 385 Distearyl-lecithin, 529 Distillation, 13 Diuretics, 711 Dobie's line, 398, 402 Dolium galea, sulphuric acid in, (iO Double refraction, 36 of muscle, 402 Dropsical fluids, 338 gases of, 392 Dropsy, causes of, 332 Drugs, influence of, on bile secretion, 675 on urea formation, 724 — in milk, 589 — in sweat, 820 EQU Drugs in urine, 7(;(; Drying, 1 1 — in vacuo, 12 Dutrocliefs eiidosmomcter, 14 Dumas' method of estimating nitrogen, 22 Dyaster, 201 Dys-albumose, 646 Dysentery, stools in, 698 Dyslysin, 87, 681 Dyspepsia, 650 Dyspeptone, 644 Dyspnoea, 376 E Ear, tLe, 566 Echinochronie, 319 Echinoderms, blood of, 318 Edible bird's-nest, 486, 625 Egg-albumin, 127, 594 Eggs as food, 592 ; respiration in, 394 ; shells of, 592 ; white of, 593 ; yolk of, 594 Egyptian chlorosis, 308 Elastic fibres, 473 — tissue, 467 Elastin, 145, 473 — peptone, 475 Elastoses, 145, 475 Electrical organs, 439, 449 — changes on muscular acti(jn, 434; on salivary secretion, 618 Eleidin, 145, 452 Elementary analysis, 19 Elements found in bod}', symbols and combining weights of, 5 EUagic acid, 699 Embolism, 306 Emulsin, 159 Emulsion, 489 — pancreatic, 662 Emydin, 594 Enamel, 469, 495 Enchondromata, 497 Endemic h:eniaturia, 308 Endolymph, 351 Endosmometer, 14 Endosmotic equivalent, 14 Endothelium, 442 Enterochlorophyll, 215 Enzymes, see Unorganised ferments Epiblast, 184 Epicritical increase of urea, 842 Epithelium, 441 ; pavement, 442 ; columnar, ciliated, 443 ; secreting, 448 ; compound, 451 ; of retina, 455; in urine, 771 Equatorial stage in cell-division, 200 Equivalent, endosmotic, 14 860 INDEX Erucic acid, 72 Eructation?;, fJoO Erythrite, 08 Erythrodextrin, lOo, eJL'T Erythrogranulose, 1U4 Erythrophyll, 214 Ethane, 64 Ethene lactic acid, 410 Ethereal sulphates in mine, 74o. TOO; estimation of. 803 Etherification, 156 Ethers, 6o Eihidene lactic acid, 410 Ethyl. (U — alcohol, 09 — chloride, 74 Ethvl-diacetic acid in blo(xl and urine, 3i4, 701, 792 Ethyleniinine, o6ii Etiolin, 214 Eucalin, 108 Eudiometer, 82 Euglena viridis, 109 Evaporation, 12 Exchange of material, 827 ; effect of various external conditions in, 839 Exercise, effect of, on liver glycogen, 541 ; on muscle, 431 ; on respiration, 375 ; on urine, 435 Exostoses, 497 Expired air, composition of, 3G9 Exsiccators, 11 Extinction, co-efiicient of, 51 Extirjmtion of pancreas, 558, 663, 789 ; of liver, 727, 735 ; of kidneys, 725 Extraction of gases from blood, muscle, ice. 30 Extractives of muscle, plasma, serum, urine, ice. See Muscle, Plasma, Serum, Urine, ice Extraordinary ray. 36 F.^CES. 695 Faiirenheit scale, 5 Fat, 65, 72. 487 — as food, 572 — grannies in white corpuscles, 259 — effects of, on metabolism. sHt! ■ — in milk, 585 — in muscle, 427 — in the liver cells, 550 — in the blood, 315, 706 — in urine, 795 — in urine, estimation of, 817 — of sheep's wool, 822 — origin of, in body, 837 — putrefaction of, in intestine, 693 Fatigue of muscle, 432 Fatty acids. 65, 542 ^ in sebum, 822 in sweat, 820 in urine, 755 Fattj' degeneration of liver, the bile in, 088 — liver, 542 Faulhorn, ascent of, 436 Feathers, 452, 454 Fehling's test for sugar. 95, 98 : in urine, 790, 814 Fellic acid, 88, 680 Ferment coagulation, 125 Fermentation, general description of, 151 ; unorganised ferments, 158 ; classification, 158 ; organised fer- ments, 161: classification, 163; con- ditions under which ferments act, 167. &<;also Putrefaction, Digestion, Coagulation. Fermentation test for sugar, 96, 97, 790 Fermentations, acid and alkaline in urine, 767 — of sugar, 95 Ferments, 146 — in blood, 239; in milk. 580.; in muscle, 411, 415 — in plants, 137 — in urine, 757 Fever, effect of, on liver glycogen, — metabolism in, 841 — respiration in. 373 — the bile in, 688 — urea formation in, 724, 841 Fibres of connective tissue, 470 ; umscle, 398 ; of nerve, 519 : nucleus, 196 Fibi-in, 231 ; preparation. 231 ; varie- ties of, 232 ; solubilities of, 2;i3 ; estimation of. 233 ; factors, 234 ; di- gestion of, 643 Fibrin-ferment, 239 FiVjrin-network, 221 Fibrinogen, 234 ; preparation, 234 : properties of, 235 ; estimation of, 236; coagulation by heat of, 218; in urine, 780, 795 Fibrinogen-poisoning, 332 FiVjrinoplastic substance, .see Serum globulin Fibrocartilage, 482 Fibroin, 145, 456 Fibrous tissue, 467 Fick and Wislicenus, ascent of Faul- horn by, 436 Filaria sanguinis hominis, 308, 349, 772, 795 Filter-pump, 9 Filtration, 10 Fishes, re.spiration of, 395 ;12 of of INDEX 801 Fistulii-liiks and tiall-bladflor bile, 67(5 Fistula, hiliarv, 674; gastric, (ii?7 ; in- testinal, S7 Fleischl's bitnionieter, 2815 Flour, 135 ; as food, 598 Fluorine in bone, 493, 494 : iii urine, 7rinciples of, 56Vt ; the principal food-stuffs (milk, egg-s, meat, A:c.), 572 ; heat value of, 60<), 848. Si'e aliio Digestion Foot-and-mouth disease, 590 Formic acid, 70 Fornmhv, empirical and const itutiimal, 26 Fossil l>oiies. 49 i Fractional distillation, 13 — heat coagulation, 119 Fruuenhofer's lines, 46 Funnel, hot-water, 10 Furfuraldehyde reaction, 88, 682 Fuscin, 149," 458 Fusible calculus, 774 Gadinine, 179 Galactoscope, 590 Galactose, 100, 103. 757 — from cerebrin, 534 Gall-bladder bile and fistula bile, 676 — secretion of, 689 Gallic acid, 78 Gall-stones, 689 Gas analysis, 28 Gases of 'bile, 392, 679 — of blood, 378 : of blood-serum, 380 ; of blood-corpuscles, 381 : changes during circulation, 382 ; with different pres- sures, 383 : tension of, 383 ; of chyle, lymph, effusions, 391 ; of pus, 392 — of intestine, 692 — of milk, 578 — of muscle, 428 ; of saliva, 392, 626 ; of secretions, 392 — of stomach, 651 — of urine, 764 Gastric fistulas, 637 — juice, 630 : physiology of secre- tion of, 631 : cells which secrete, 633 ; composition of, 637 ; methods of obtaining, 637 ; artificial, 637 ; acids of, 638 ; ferments of, 641 : action of 643 ; pathological condi- tions of. 649 : of crayfish, 650 : as a germicide, 691 GME Gastropods, blood of, 322, 323 Geissler's speciHc-10 Gower's hBemocytometer, 262 — h;emoglobinometer, 283 Graham on dialysis, 13 Granulose, see Starch Grape sugar, see Dextrose Gravimetric analysis, 7 Green glands, 727, 736 — vegetables as food, 600 Greenwood, Miss, on vacuoles, l'.)3 Grey and white matter, 514 Ground substance of connective tissue, 475 Guanine, 90 ; in urine, 736 Guarana, 601 Guinea-pig's blood-crystals, 270 Gum, animal, see Animal gum Gummose, 480 Gums, 108 H H^MATIX, 288 — in urine, 776 — reduced, 289 Hjematoblasts, 262 Hfematogen, 300, 588 Hematogenous jaundice, 311 Hiematoidin, 293, 682 Hjematolin, 292 Hsematoporphyrin, 292, 294 — in urine, 751 Hsematoporphyroidin, 292 Hsematoscope of Hermann, 48 Hajmatozoa, 308 Htematuria, endemic, 308. See also Blood in urine Hsemerythrin, 316, 320, 321 Hsemerythrogen, 321 Haimin, 289, 290, 294 Hseraochromogen, 289 Hiemocyanin, 63, 147, 321 Haemocytomet er, 262 Haemoglobin, 147, 267 — distribution of, 267 ; preparation of crystals of, 268 ; of different animals, 270 ; crystallography of, 270 — water of crystallisation of, 273 ; compounds of, 274 ; estimation of, 282 ; composition of, 286 ; decom- position of, 287 ; in bile, 685 ; in muscles, 417 ; in worms' blood, 319 ; in urine, 776 — action of gastric juice on, 648 — crystals, 268 ; in septic diseases, 315 Hsemoglobinsemia, 302 Haemoglobinometer. 283 Hemoglobinuria. 302, 776; paroxysmal, 777 ' Hajmolyniph, 316 Hsemometer, 283 Haemophilia, 306 Haenioplasmodium malarie, 310 Kaemosiderin, 293 Hairs. 452 Halitus sanguinis, 219 Hammarsten on coagulation of blood, 227, 244 Hamster's blood crystals, 270 Haycraft's method of estimating uric acid, 807 Heat-coagulation, 117 Heat-value of foods, 606, 848 Heintz's method of estimating uric acid, 807 Hemi-albumin, 645 Hemi-albumose, 129, 645 — in urine, 513, 785 Hemi-coUin, 473 Hemi-deutero-albumose, 646 Hemi-elastin. 475 Hemihedry of tartaric acid, 44 Hemi-peptone, 130, 645 Hemp-seed calculus. 774 Henry-Dalton law, 383 Hepatin, 551 Hepatisation, 561 Hepatogenous jaundice, 311 He pa to -globulin, 539 Hermann on gases of muscle, 428 — on muscular contraction, 434 Hermann's htematoscope, 48 Hetero-albumose, 129, 646 — in urine, 513, 785 Hetero-globulose in urine, 784 Hewson on coagulation of blood, 242 Hexatomic alcohols, 68 Hibernation, 372, 831 Hippomelanin, 499 Hippuric acid, 77 — estimation of, 809 ■ — in urine, 738 Histohaematins, 147, 417 Histon, 205 Hofmeister's method of precipitating proteids, 124 ; on absorption, 702 ; on crystalline egg-albumin, 594 Homoiothermal animals. 847 Hoppe-Seyler on classification of fer- ments, 165 ; on haemoglobin, 273, 298, 292 ; on lecithin, 526 " Horny material, see Keratin Horsley on myxoedema, 505 Hot-air oven, 11 Hot-baths, influence of. on urea forma- tion, 724 INDEX 803 IH'M Hiiniin, it.") Humoral )iathol(i<,'y, 297 Humour, aqueous, ',\')0 Humous suhstanci's. ;)5, H'.t, 7.")2, T'.Mi Hyalins, ]15, 186 Hyalogens, 145. 486 Hydatid cysts, 354 Hydrates of oxyhienioglobin, 273 Hydrobilirubin", ()84. d'M, Hydrocele fluid, 348 Hydrocephalus, see Cerebrospinal II aid Hydrochloric acid in stouiach, forma- tion of, 63G ; detection of, (!39 ; esti- mation of, G40 Hydrochloride of h:ematin, sec lla'iiiin Hydrocollidine, 178 Hydrocyanic acid and ha3iiioglobin, 2S2 Hydrogen, estimation of, 20, 21 — in intestine, 693 Hydrogen-peroxide in the body, 59 — in urine, 764 Hydrolymph, 316 Hydrolysis, 160 Hydrometer, 15 Hydronephrosis, 353 Hydroparacumaric acid. 78 in urine, 740 Hydrophyr, 644 Hydroquinone, 76. 77 — in urine, 745 Hydrothorax, 346 Hydroxybutyric acid in urine. 791, 792 Hymatomelanic acid, 95 Hyo-cholalic acid, 88, 680 Hyo-glycocholic acid, 680 Hyo-taurocholic acid, 680 Hyperpnoea, 376 Hypoblast, 1S4 Hypoxanthine, 90 — in muscle, 421 - — in urine, 736 Hysterical flatulence, 693 1 Iceland spar, 3(i Ichthin, 594 Ichthulin, 594 Ichthyosis, 566 Icterus, nee Jaundice Imides, 90 Inanition, effect of, on liver glycogen, 541 ; effect of, on muscle glycogen, 423 — loss of water in, 58, 59 ; metabolism in, 834 ; temperature in, 835 — respiration in, 374 Incineration, 12 Index of refraction, 45 Indican of vegetables, 78 ISO Indican of urine, 79, 713, 75t; Indiffusibility of pmteids, 120 Indiglucnn, 79 Indigo and derivatives, 78: iu calculi, 774 ; in sweat. 820 — in urine, 79, 744. 776 Indigo-carmine test for sugar, 96 Indigo-white as a reducing agent, 380 Indigogt^n. 78 Tndigotin, 7K Indole. 78, 79 Indole-producing organisms, ()94 Indoxyl glycuronate, 794 — sulphate of jjotassium, 79 in urine, 743 Inflammation, the blood in, 306 Injection, nattn-al. of liver, 669 Ink gland of sei)ia, 69(» Inogen, 434 Inorganic constituents in plasma and serum, 254 ; investigation of, 255 ; physiological importance of, 256 ; in food, 571; in muscle, 427; nerve, 517 ; organs, 535; urine, 759 — compounds in body, 57, 58 Inosic acid, nee Inosinic acid Inosinic acid, 90, 422 Inosite, 100: in muscle, 425 — in urine, 757. 789 Inotagmata, 189, 435 Insects, blood of, 328 Intensity of respiration, 395 Interpolation curves, 50 Intestinal catarrh, 699 — fistula, 664 — juice, 664 ; action of, 665 Intestines, digestion in, 652 ; propor- tion of. to body. 653 Intravascular clotting, 305 Inulin, 109 Inversion of cane sugar, 99, 102 Invertebrate animals, blood of, 267, 316; cartilage of, 485 ; liver of, 690; urine of, 728 Invertin in intestinal juice, 665 Inversive ferments, 158 lodohfematin, 291 Iron, estimation of. 25, 500 — in anffimia, medicinal use of, 289 — in bile and liver cells, 678 — in body, 62 — in milk, 585, 587 — in the liver, 551 ; in the spleen, 554 — in urine, 764 Isatin, 78 Isatyde, 79 Isethionic acid, 85 Isocliolesterin, 532, 822 Isomeride of urea, 81, 721 Isomerism, 66, 74 864 INDEX Isotropic crystals, 271 Isotropous substances, ?ifi in muscle, 401 Janthinin, 150 Jaundice, 311, 688 — stools in, 6'.t9 — urine in, 778 Jecorin, 551 Jelly-like connective tissue, -467 Jequirity, see .Abrus Johnson's method of estimatina- dex- trose, it9 Joint-diseases. 498 Karyokixesis, 11H5, 198, 199 Karj'omitosis, see Karyokinesis Katabolic nerves, 520, 852 Katabolism, 206, 828 Kephalines, 524 Kephir, 579, 585, 587 Kerasene, 534 Keratin, 145, 452 Ketones, 66 Kidney, composition of, 559 — in gout, 510 — structure and secretion of, 709 Kjeldahl's method of estimating nitro- gen, 23 ; in urine, 807 Knapp's method of estimating dextrose, 99 Koch on microbes and diseases, 309 Kola-nut, 601 Koumiss, 579, 585, 586, 587 Krause's membrane, 399 Kr3-ptophanic acid in urine, 758 Kiihne on digestion, 645, 654, 657 ; on muscle, 405 ; on retinal pigments, 461, 464 Lacmoid, 16 Lactalbumin, 5S3 Lacteals, 332 Lactic acid, 71 and metabolism, 840 in muscle, 409, 425, 433 — ■ — in nervous tissue, 516 in rickets, 511 ; in osteomalacia, 512 • fermentation, 103 in stomach, 636, 639, 640 LIK Lactic anliydride, 426 Lactide, 420 Lactog-lobulin, 583 Lactometer. 15 Lacto-protein, 584 Lactose, 102, 586 : in urine, 757, 789 Lfevorotatory substances. 44 Lamellibranchs, blood of, 323 Lardacein, 144 Latex, 133, 135 Latham's theory of proteids, 1 1 6 Laurent's polar i meter, 42 Lea's absorptiometer, 284 Leatlier, 472 Le Bel on circular polarisation, 45 Lecithin, 73, 524, 526, 531 — in muscle. 422; digestion of, 531, 662, 692, 797 Lecithines, 524 Leech extract, influence of, on blood, 226, 229 Legumin, 134 Leguminous plants as food, 599 Lens, crystalline, 565 Lepidoptera, blood of, 328 Leptocephalus, blood of, 218, 267 Leptothrix, 164 Leucfemia, see Leucocj^thtemia Leuceines, 115 Leucic acid, 71 Leucine, 82 — as an antecedent to urea, 726, 845 — in pancreatic digestion, 660 ; in urine, 769. 770 Leucines, 115 Leucocytes, see White blood corpuscles Leucocythfemia, 302 — metabolism in, 843 — the spleen in. 555 — uric acid in, 733 Leucocytosis, 302 — in inflammation, 307 Leucomaines, 169 Levulose, 99 — in urine, 110, 789 Lichnin, 109 Lieberkiihn's jelly, 128 Liebermann's reaction, 121 Liebig's condenser, 13 — • extract, 597 — method of ultimate analysis, 20 of estimating chlorides, 801 urea, 722, 810 Ligamentum nuchaj, 474 Light, influence of, on metabolism, 211 — polarisation of, 36 Lime in urine, estimation of, 805 Limpet, liver of, 690 Limulus, blood of, 328 Lines of Frauenhofer, 46 t.w. INDEX 865 T.inin, lit? Lipaciduria, ~rA\ Lipieinia, •ilii Lipochriii, 4r)lt Lipot'hroines, 148, 25:5, 465 Lipoiuata. 497 Liquor sanguinis, sec Plasma Lithofellic acid. G99 Litmus, 16 Liver, the, 5H6 : chemical composition of, 537 ; proteids of, 538 ; glycogen of, 5i{9 ; fat of, 550; extractives of, 550; inorganic constituents of, 551 ; pathological conditions of, 552 — the blood in diseases of, 311 — urea formation in diseases of, 724, 726 — extirpation of, 424, 727, 735, 755 — of invertebrates, 690 — secretion of, (i(;s Living test-tube, 224 Loew's theorj' of proteids, 1 1 6 Ludwig's circulating fluid, 257 — method of estimating uric acid, 808 Lung, composition of, 560 Lunge's nitrometer, 33 Lupino-toxin, 137 Lutein, 148,293, 564 Luxus consumption, 843 Lymph, 331, 333 — cells of, 258 — gases of, 391 — plasma, 331 Lymphadenoma, 302, 556 Lymphatic glands, composition of, 555 Lymphoid tissue, 468 M Magnksia in urine, estimation of, 806 — mixture (Salkowski's), 808 Magnesium salts in body, 62 Malaria, 309 Malic acid, 72 Malpighian glomeruli, 70t» : tubes of insects, 727 Malto-dextrin, 106 Maltose, 103, 627 — in muscle, 425 — inversion of, 665 Mammary glands, 572 : di-eases of, 590 Mannite, 68 — aldehydes of, 93 Mannitic acid, 94 Margaiin, 487 Margarine, 586 Marienbad cure, 375 Marrow, 802, 468 MIL Marrow, fat of, 496 Marsh gas, see Methane Mate, 601 Measures and weights, 3 Meat as food, 596 ; salted, 597 ; smoked, 597 — eflf(!Cts of, on metabolism, 836 Meconium, 697 Medico-legal detection of blood, 295 Medullary sheath, 520 Medullic acid, 496 Meibomian glands, secretion of, 823 Melan;cmia, 453 Melanin, 149, 453, 499 Melanosis in insects' blood, 330 Melanotic sarcoma, 149, 499 Melanuria, 501, 752 Melicyl alcohol, 70 Melitose, 108 MeHzitose, 108 Mercurial air-pump, 30 Mercuric cyanide method of estimating sugar, 99 — nitrate method of estimating urea. 810 Mesoblast, 184 Mesoxalic acid, 729 Meta-aromatic compounds, 76 Metabolism, 827 — in cells, 206 ; in muscle, 435, 436 ; in salivary glands, 618 Metacresol in urine, 743 Metakinesis, 201 Metalbumin, see Pseudo-mucin Metals in borly, 62 Metapeptone, 644 Metaxin, 205 Methjcmoglobin, 278: crystals of, 279; spectrum of, 280 — in bile, 685 — in urine, 776 Methane, 64, 69; in intestine, 693 Methyl, 64 Methylbenzene, 75 Methylglj'cocine, see Sarcosine Methylhydantoin, 84 Methylindole, see Skatole Methyl-orange, 16 Methyl-uramine, 84 ^Metric system, 3 Metschnikofi' on leucocytes, 706 Microcheinical reactions, 36 Micrococcus, 164 — ureic, 81, 722, 767 Micro-organisms, classification of, 163 Microscope, 36 — polarising, 38 Microsomes, 192 Microspectroscope, 49 Milk, 572; microscopical appearances 3e b6 6 INDEX MIL of, 574 ; reaction, specific gravity, amount secreted, composition, 575 artificial human, 577 ; cow's, 578 gases of, 578 ; other animals', 579 proteids of, 580 ; coagulation of, 580 fats of, 585; sugar of, 586; extrac- tives of, 58(1; siilts of, 587; preser- vation of, 588; in disease, 589; analy- sis of, 590; uterine, 592 Milk-curdling ferment of pancreas, 659 Milk-sugar, xee Lactose Millon's reaction, 121 — reagent, 121 Mitom, 192 Mitosis, see Karyokincsis Model to illustrate polarisation of light, 36 Moisture in the air, "69 Molecular weights, 26 — — of carbohydrates, 9:5 Molken-protein, see Whey proteid Mollities ossium, sec Osteomalacia Molluscs, blood of, 323 Monaster stage in cell-division, 200 Monatomic alcohols, 65 Monobasic acids, 65 Moore's test for sugar, 96 Moulds, action of, on optically active substances, 45 Mucedin, 136 Mucic acid, 94 Mucin, 144 — in bile, 679 ; in connective tissues, 476 ; in saliva, 622, 623 — in myxoedematous tissues, 502 — ■ in urine, 758 Mucinogen, 621 Mucous glands, 620 — membrane of mouth, secretion of, 625 Mucus, 444 — in urine, 771. See also Sputum Mulberry calculus, 774 Mulder's theory of proteids, 114 Murcx, 150 Murexide test. 89, 718, 729, 730 Muscarine, 179 Muscle, 398; microscoiDic study of, 398; chemical composition of, 405 ; plasma and serum of, 406 ; clot of, 407 ; for- mation of acid in, 409 ; ferments in, 412; proteids of, 412 ; rigor mortis, 415; pigments of, 417; extractives of, 418 ; ash of, 427 ; gases of, 428 ; respiration of, 429 ; contraction of, 430 ; fatigue of, 433 : theories of con- traction of, 434 ; effect on urine of contraction of, 435 I\I uscle-plasma and muscle serum, 406 Muscular activity and respiration, 374 NIT Muscular respiration, 429 investigation of, 29 Musculin, 413 Mycose, 108 Mydaleine, 178 Myelin, 520 Myelines, 524 Myelogenic leucocythaimia, 302 Myeloidin, 459, 460, 520 Myo-albumose, 413, 415 Myoglobulin, 413, 415 Myohajmatin, 417 Myolemma, see Sarcolemma Myoproteose, 415 Myoryctes Weissmanni, 400 Myosin, 407 — in cornea, 470 Myosin-ferment, 412 Myo-sinogen, 413 Myosinoses, 129, 648 Jlyronate of potassium, 109 Mj'rosin, 159 Mytilotoxine, 172 Myxoedema, 304, 501, 557; in animals, 505 N Nails, 451 Naphthalene, 81 Naphthylamine, 81 Nascent hj'drogen, 158. 749 — oxygen, 158, 749 Necrosis, 513 Nencki and Sieber on ha?matin, 294 Neossidin, 486 Neossin, 486 Nephridia, 727 Nephrozj-mase, 758 Nerve-cells, 518 Nerve-libres, 519; development of, 521; degeneration of, 522 Nerves of kidney, 710 ; of stomach, 632 ; of salivary glands, 617 ; of sweat glands, 818 ; trophic, 852 Nervous tissues, 514 ; reaction of, 514 ; composition of, 516; proteids cf, 523; phosphorised constituents of, 524 Neuridine, 178 Neurilemma, 520 Neurine, 172, 179, 530 Neurochitin, 520 Neuroglia, 514 Neurokeratin, 452, 460, 514, 520 Nicol's prism, 37 Nitric acid in urine, 764 — oxide hiBuioglobin, 282 Nitro-aerial particles, 434 Nitro-benzene, 75 Nitro-derivatives of proteids, 114 INDEX 867 Nitrogoii, discliarjic in metabolism, 8i50 — estimation of, 22 — in intestine, ()9I5 — in urine, estimation of, 807 — tests for, 1!) Nitrogenous bases in plants, i;)(! Nitrometer, Lunge's, I?!) Normal acids and alkaline solutions, 17 — solutions, () North on work and urea, 1.57 Nuclear matrix, 198 — membrane. 198 Nuclein, 145, 202. 259 — artilicial, 204 — iron compounds, 551 — in milk, 585 — of spermatozoa, 562 Nucleo-albumins, 145, 205, 445 — in bile, t;79 — in synovia, 351 — in white corpuscles, 260 Nucleoli, 195, 197, 207 Nucleo-plasm, 191 Nucleus, 194 : resting, 195 ; dividing, 198 ; constituents of, 202 ; functions of, 207 — of white blood-copuscles, 258 O Obesity, 836 Odontogen, 469 S Ptyalinogen, 621 Ptyalose, 627 Pump, mercurial air, 30 — Sprengel's filter, 9 Punicin, 150 Purgatives, 698 Purple, Tyrian, 150 Purpura, 150 — ha3morrhagica, 306 Purpurate of ammonia, sec Murexide Purjiurin, 751 Pus, 361 — cor]3uscles of, 362 — gases of, 393 — in urine, 770 — pigments in, 364 — serum of, 364 Putrefaction in the stomach, 650 — in the intestines, 691 ; of pi'oteids, 114. See also Fermentation Putrescine, 172, 173, 178 Pyferaia, 306, 310 Pyenometer, 15 Pyloric glands, 633 P3'oc3'anin, 364 Pyoxanthose, 364 Pyreuin, 197 Pyrenoids, 207 Pyrocatechin, 77 — in cerebro-siDinal fluid, .35 — in urine, 743, 745, 796 — isomerides of, 76 870 I^'DEX I'YK Pyrogallic acid, 7« Pyrogallol propionic acid, 700 Q QUADRUEATES, 732 Quartz, action of, on light, iO Quinoidine, animal, 173 Quotient, proteid, 335, 341 — in urine, 782 — respiratory, 309, 830 B Racemic acid, action of, on polarised light, 44 Rachitis, see Rickets Raffinose, 109 Rancidity of fats, 73 Raoult's investigations on freezing- points, 92 Raynaud's disease, 311 Reaction, determination of, 1*> Reagents and apparatus, 6 Reaumur scale, 5 Red blood coipuscles, enumeration of, 262 ; specific gravity, shape, and size, 263 ; action of reagents on, 263 ; nucleus of, 264 ; origin of, 261 ; com- position of, 265 ; proteids of stroma of, 266 ; heemoglobin of, 267 Red marrow in anaemia, 302 Red muscles, 404 Reduced hsematin, 289 Reducing substances in blood, 391, 433 Reduction and oxidation of bile pig- ments, 683 Refraction, double, 36 Refractive index, 45 Regeneration of serum-albumin, 703 Regnault and Reiset's method of inves- tigating gases of respiration, :}67 Rennet, 635, 642 — in urine, 758 — preparation of, 580 Rennin, 5S1 Reserve air, 370 Residual air, 370 Resorcin, 76 Respiration, 365 ; methods of in\esti- gating gases of, 366 ; frequency. 371 ; effect of external circumstances on, 372 ; gases of the blood in relation to, 378 ; of tissues, 390 ; cutaneous, 393 ; foetal, 394 ; in eggs, 394 ; in fishes, 394 ; intensity of, 394 Respiratory oxygen, 274 — pigments, 147 — quotient, 369, 830 Respired air, poisonous effscts of, 377 Reticulum in protoplasm, 191 Retiform tissue, 468 Retina, 456 ; hexagonal pigment cells of, 457 ; rods and cones of, 460 Rheumatism, 307 Rhizopods, digestion in, 193 Rhodophane, 464 Rhodopsin, see Visual purple Rice-water stools, 698 Rickets, 510 Rigor mortis, 411, 415 Ringer on inorganic constituents of blood, 256 ■ — on urea discharge in fever, 841 Rods and cones, 460 pigments of, 460 Rose's method of incineration, 12 Rosolic acid, 16 Rotation of polarised light, 40 by carbohvdrates, 92 ; by pro- teids, 120 " Ruberythric acid, 109 Saccharic acid, 94 Saccharimeters, 41 Saccharomj'ces, 151 Saccharoses, 92 Saccharumic acid, 94 Sachsse's method of estimating dex- trose, 99 Saft Kaniilchen, 475 Salicin, 109 Salicylic acid, 77 Salicyluric acid, 74() Saliiio-sulphureous particles, 434 Saliva, 616; secretion of, 616; struc- ture of cells that secrete, 619 ; com- position of, 622 ; action of, 626 ; in- fluence of reaction on, 628 ; gases of, 392, 624, 626 Salivary glands, composition of, 558 Salkowski on aromatic substances froni proteids, 123; on ethereal sulphates, 803 — on the proteid tests, 123 Salts as food, 571 — inorganic, see Inorganic salts Saponification, 489, 492, 662 Saprin, 178 Sarcina, 164 Sarcolactic acid, 410, 425, 433; in lymph glands, 261; in thj-mus, 556; in thy- roid, 557 ; in urine, 755 ' Sarcolemma, 145, 399, 405 Sarcomata, 149, 497 ; pigments of mela- notic, 499 INDEX 871 Sarcosine, 84, 420 Scales of fishes, 495 Scarlet fever transmitted by milk, 500 JSchwann on cells, 185 Schirt's test for uric acid, 718, 72!» Schizomycctes, 163 Schizoneura lanuginosa, 108 Schmidt on coagulation of blood, 24:! Schiitzenbergcr's theory of protcids, 115 Sclerotic, 470 Scrofula in lymphatic glands, 550 Scum of boiled milk, 583 Scurvy, 30G Scvbaliu, 699 Scyllite, 108 Sea-water, gases of, 395 Sebaceous cysts, 822 Sebum, 821 Secretory- nerves of salivary glands, 617 of sweat glands, 818 Selmi on alkaloids, 171 Semen, composition of, 5G2 Semi-glutin, 473 Sepia, ink of, 690 Sepiaic acid, 690 Sepsin, 172, 311 Septic diseases, haemoglobin crystals in, 315 Seplic:emia, 310 Serous glands, 620 Serpent's urine, 728, 730, 732, 734 Serum, general characters of, 230; pro- teids of, 231, 247, 249 Serum-albumin, 245 ; preparation of, 245 : quantity of in serum, 247 ; co- agulation by heat, 118, 248 ; in cold- blooded animals, 248 ; in urine, 782 Serum-casein, see Serum-globulin Serum -globulin, 236 : preparation of, 236 ; estimation of, 238 ; sources of, 238 ; quantity in serum, 247 ; coagu- lation by heat, 248 ; in urine, 783 Serum-lutein, 253 Sieber and Isencki on haematin, 294 Silicic acid in urine, 764 Silicon in the bodv, 63 Silk, 145, 456 Silkworms, 108 Silver test for sugar, 96 Sister threads, 200 Skatole, 79, 80; in intestine, 660, 692, 695 Skatole-pigment, 80 — in urine, 745 Skatoxyl glycuronate, 794 Skatoxyl sulphate of potassium, 80 in urine, 744 Skein stage of cell-division, 200 Skeletins, 145, 454 Skin, the, 566 — secretions of, 818 Smegma preputii, 822 Smoky urine, 776 Snakes, blood of, 226 — poison of, 138. 625 Soaps in blood, 251 : in faeces, 695. See also Saponification Soda in urine, estimation of. 805 Sodio-magnesium sulphate, 246 Sodium salts in body, 61 Soleil's saccharimeter, 41 Solidists, 297 Solids and water, estimation of, 18 — in urine, estimation of, 798 Solutions, normal, decinormal, &c., 6 Sorbin, 108 Soups, 597 Specific gravity bottle, 15 determination of, 15 of blood, 218, of milk, 575, 590 ; of urine, 715 — rotatory power, 43 Spectrophotometer, 50, 284 Spectrophotometric estimation of iron, 500 Spectropolarimeter, 53 Spectroscopes, 45, 275 Spectrum, 46 Spermatin, 145, 563 Spermatozoa, composition of, 562 ; in urine, 771 Spermine, 563 Spiders" excrements, guanine in, 736 Spiders' webs, 456 Spina bifida, sec Cerebro-spinal fluid Spindle in cell -division, 200 Spirem stage of cell-division, 200 Spirillum, 164 Spirographin, 486 Spirometer, 371 Spleen, the, 553: chemical composition, 554 ; functions of, 555 — does it liberate hjemoglobin ? 673 Spongin, 145, 456 SprengelV filter-pump, 9 Sputum, 447 Squirrel's blood-crystals, 270 Standard solutions, 6, 7 Starch, 104. digestion of, by saliva, 627 ; by pancreatic juice, 661 — in the stomach, digestion of, 628 — animal, see Glycogen Starvation, sec Inanition Stas-Otto process, 175 Steapsin, 656, 659 Stearic acid, 71, 491 Stearin, 490 Steatolytic ferments, 158 Stentorin, 150 872 INDEX StercobiliD, Gs4, 696 Sterilisation, 162 St. Martin, Alexis, 630 Stoffwechsel, 82'.t Stokes's reagent. 276 Sublingual saliva, 625 Submaxillary trland, 617, 621 : saliva, 628 Substantia hvalina, 192 — opaca, 192 Succinic acid. 72 — and metabolism, 8iO — in urine, 755 Succus entericus, 664 Sucroses, 92 Sugar in normal blood, 252 : estimation of, 253 ; in urine, 789, 814. See also Dextrose, Diabetes Sugars, 94 Sulphates in the body, (>?, — in iirine, 760, estimation of, 802 — ethereal in urine, 740. 803 Sulph-indigotate of soda as a means of natural injection, 669 Sulpho-cyanic acid, 91 in urine, 737 Sulphonates in urine, 740 Sulphur, estimation of, 24 — in bile, 678, 681 ; in proteids, 114 — tests for, 19 — excretion of, in epidermal scales. 819 Sulphuretted hydrogen in the body, 59 in the intestine, 693 in \irine, 764 Sulphuric acid iu gasteropod saliva, 60. See also Sulphates Supplemental air, 370 Suprarenal body, composition of, 557 in Addison's disease, 303 Surgical fever, 305 Sweat, secretion of, 818 : composition of, 819 ; reaction of, 819 : in disease, 821 Swimming-bladder, gases of, 396 Symbols of elements. 5 Sympathetic saliva, 625 Synanthrose, 108 Synaptase, 159 Synovia, 351 Synthetic and analytic processes in plants and animals, 210 •Syntonin, 128 T.ENIA ECHINOCOCCUS in uriup, ho.ik- lets of, 772 Tannin, 109 — in tea, 601 Tannin -red, 95 Tapetum, 458 Tartar, 622 Tartaric acid, 72 action of, on polarised light. 44 and metabolism, 840 Tartronic acid, 69 Taurine, 85 — in muscle, 422 — preparation of. from bile, 681 Taurocholic acid. 87, 680, 681 Taurylic acid, 713 Tea as food. 601 Tears, 823 Temperature of animals, 847 — of air, effect of. on metabolism, 839 : — of bodv. effect of. on metabolism, 839 Tenotomy, 425 Tension of aqueous vapour, 5 — of the blood gases, 383 Terebenthene, 68 Testis, composition of, 561 Tetanine. 173, 179 Tetra-methyl-benzene, 75, 78 Tetramethylparaphenylene-diamine, 59, 641 Tetratomic alcohols, 68 Tetronerythrin, 148 Theine, 601 Theobromine, 601 Thermometric scales, 5 Thermotaxic nervous mechanism, 849 Thiocyanic acid in saliva, 622, 623 ; in urine, 737 Thiry's fistula, 664 Tlirombosis, 305 Tliymol, 78 Thymus, composition of, 556 Thyroid, composition of, 557 ; cysts of, 557 — function of, 507 — in mvxoedema, 557 Tidal air, 371 Tissue respiration, 390 Titration, 8 Toluene. 75 Tonka-bean, 78 Tooth- stones, 623 T.iphi, 509 Tortoiseshell, 495 Tonilae, 151 ; in stomach, 650 ; in urine, 81, 722, 767 Toxines. 168, 173 Trehalose, 108 Triatomic alcohols, 68 Tri-bromo-phenol, 76 Tributyrin. 73 Tricaprin. 73 Tricaproin, 73 INDHX 873 TKI Trii'iijuyliii, 7."> Trimargiirin, 7;$ 'I'rimctliylaniinc, 80 Tri-mctliyl bonzene, 75, 78 'rrimetliylxant Iiine, fiOl Tri-nitro-phenol, 76 Triolein, 73 'rril)aliaitin, 72 Tiil>lK'nyl-rosanilin in milk, 500 Tiit;tearin, 72 Trivulerin, 73 Trommer's test for sugar, !t5, 790 Trophif nerves, 850 — - of salivarj' glands, 018 Trypsin, ()55, 65(3, 657 — in urine, 758 Trypsinogen, G55 Tryptones, 659 Tryptophan, 661 Tubercle bacillus in milk, 590 — in lymphatic glands, 556 ; in lung, 561 " Tunicates, test of, 107 Tunicin, 107, 456 Turacin, 63, 150, 454 Tvphoid fever, gastric juice in, 650 -"- — the bile in, 688 ; stools in, 699 Tvphotoxine, 173, 179 Tyrein, 580 Tvrian ])iirple, 150 Tyrosine, 78, 83 ■ — as an antecedent to urea, 726 — in pancreatic digestion, 660 — in urine, 769, 770 Tyrotoxicon, 172 U Ulmin, 95 Ultimate analysis of organic com- pounds, 19 Ultra-red raj's, 46 Ultra-violet rays, 46 Uniaxal crystals, 271 Unorganised ferments, 146, 158 Urccniia. 315 — the bile in, 688 ; stools in, 699 Urates, 89, 730 — deposit of, in gout, 509 — deposit of, in urine, 768, 770 Urea, 81, 717, 720; preparation and properties of, 721 ; compounds of, 722 ; quantity of, in urine, 723 ; for- mation of, 725 — effect of muscular contraction on, 436 — estimation of, 810 — in muscle, 422 — in blood, estimation of, ^51 VAC Ureometers, 8 1 2 Urethan in urine, 758 Uric acid, 88, 727 ; preparation and ]>r()p<'rf i('s of, 728; decompositions of, 729 : compounds of, 730; quantity of, in urine, 733 : formation of, 734 — as an antecedent to urea, 726 — deposit of in urine, 707, 770 — estimation of, 807 •==^ in blood, estimation of, 252 — in leucocythitimia, discharge of, 843 — in muscle, 421 Urine, secretion of, 709 ; general cha- racters, quantity, and colour of, 711 ; transparency, turbidity, odour of, 7 13; reaction of, 714 ; specific gravity and constituents of,715 ; quantitative com- position of, 715, examination of for normal constituents, 716; aromatic substances in, 738 ; pijiinents of, 746; other organic constituents of, 754, inorganic constituents of, 759 : ab- normal and pathological, 765 ; drugs in, 76() ; deposits in, 76() ; blood in, 776 ; bile in, 778 ; proteids in, 780 ; in diabetes, 788 ; glycuronic acid in, 793 ; fats in, 795 ; pyrocatechin in, 796; alkaloids in, 796; diazo-reactiou in, 797 ; quantitative analysis of, 798. See also Metabolism Urinometer, 15 Urobilin, 312, 684, 697; biliary, 685 — in pernicious anajmia, 552 — normal, 747 ; pathological or febrile, 750 Urobilinoidin, 751 Urocanic acid, 91 — in urine, 758 Urocanin, 91 Urochloralic acid, 794 Urochrome, 751 Uroerythrin, 751 Urofuscohajmatin, 750 Urohajmatoporphyrin, 292, 750 Uroleucic acid in urine, 796 Uromelanin, 752 Uronitrotoluol, 794 Uropittin, 752 Urorosein, 751 Urorubin, 751 Urorubrohiematin, 750 Urostealith, 774 Uterine milk, 592 Vaccines, 168 Vacuoles, contents of, 193 — excretory, 194 874 INDEX Valerianic acid, 71 Vanilin, 109 Van 't Hoff on circular polarisation, io Vapour, tension of aqueous, 5 Vami-shing the skin, 394, 821 Varrentrapp and Will's method of esti- matine nitrogen, 22 Vegetable acids as foofl, 572 — foods, 597 — proteids. 131 — paraglobulins, 1 34 Vegetarianism. 599 Vella's fistula, 664 Vernix caseo5F(;/eA'5 by charles murchison, m.d., LL.D., F.R.S., cp^c, Felloiu of the Royal College 0/ Physicians ; late Physician a»J Lecturer on the Princip es and Practice 0/ Medicine., St. Thomas's Hospital. 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