PRINCIPLES OF HUMAN PHYSIOLOGY PRINCIPLES OF HUMAN PHYSIOLOGY BY ERNEST H. STARLING C.M.G., F.R.S. M.D., HON. SC.D. (CAMBRIDGE AND DUBLIN), F.R.C.P. JODRELL PROFESSOR OF PHYSIOLOGY IN yNIVERSITY COLLEGE, LONDON THE CHAPTER ON THE SENSE ORGANS REVISED AND LARGELY REWRITTEN BY H. HARTRIDGE, M.A., M.B. CANTAB. THIRD EDITION With j?p Illustrations, 10 in Colour PHILADELPHIA LEA & FEBIGER 706 SANSOM STREET 1920 " Printed in Great Britain. : A '' r »»«••> * PREFACE TO THE THIRD EDITION EVEN during the five years of war, which have elapsed since the appear- ance of the last edition, physiology has continued to advance, and I have had, in revising this work, to introduce a number of alterations, especially in the latter half, in order to make the presentation of the material more in accord with our actual knowledge. The chief changes affect the section on Sense Organs, which has been revised and largely rewritten by Dr. H. Hart ridge, who is entirely responsible for the Section on Vision, which is quite new. The fifty pages increase in the size of the book is due entirely to the more adequate treatment of this subject which I have secured by Dr. Hartridge's co-operation, room having been found for the other additions to the work by corresponding omissions. In the preparation of this edition I have received valuable aid from my wife, who undertook not only the whole burden of proof correcting but also the arrangement of the index. I am also much indebted to many friends, known and unknown, who have pointed out mistakes and omissions in previous editions. I shall be glad to receive any suggestions as to points in which this text-book may be made more useful tq students. ERNEST H. STARLING. University College, London, March 1920. 4131 PREFACE TO THE FIRST EDITION PHYSIOLOGY, though dealing with the phenomena of living organisms, has to use the same tools, whether material or intellectual, as the sciences of physics and chemistry. Any advances which are made in these sciences not only increase our powers of attack upon physiological problems but at the same time alter the intellectual standpoint from which we view them. On the other hand, the investigation of the phenomena of living beings is continually attracting our attention and that of workers in the other branches of science to unexplored regions in physics and chemistry. This mutual stimulation and co-operation among the different sciences have as their result a continual modification of our attitude with regard to the fundamental problems of physiology. The present time has seemed to me, therefore, fitting for the production of a textbook which, while not neglecting the data of physiology, should lay special stress on the significance of these data, and attempt to weave them into a fabric representing the principles which are guiding physiologists and physicians of the present day in their endeavours to extend the bounds of the known and to increase their powers of control over the functions of living organisms. In a science such as physiology, based on so wide a discipline and with so diverse a technique, it is almost impossible for any one man to attain to a personal acquaintance with all its branches. In the present book I have therefore not hesitated to avail myself of the work of masters of the science in fields which I had not myself explored. Thus, in the physiology of the nervous system, which has been transformed and built up on a new basis by the researches of Sherrington, I have endeavoured to follow this author as closely as possible. I am also deeply sensible of my obligations to the writings of Tigerstedt, Leathes, and Lusk on general metabolism, of Abderhalden and Plimmer on physiological chemistry, of Bayliss on general physiology, as well as to various authors of articles in the Ergebmsse der Physiologic, in Nagel's Handbuch der Physiologic, and in Dr. L. E. Hill's Recent and Further Advances in Physiology. Although I have endeavoured to confine my demands on the previous knowledge of the student within the narrowest possible limits, I should recommend him in every case to read some primer on physiology in order to obtain a bird's eye view of the subject before beginning the study of this work. He will then be able to vary the order of chapters in this book according to the part of physiology which he is hearing about in his lectures or working at in his practical classes. Those of my readers who are entirely viii \ PREFACE unacquainted with physiology might do well on a first perusal to omit Book I., dealing with the general concepts of the science. I have deemed it a hopeless and indeed a useless task to give any full account of the multifarious methods employed in the experimental investi- gation of the different organs of the body. In most cases I have consigned to small type a description of one or two typical methods, which would suffice to show how the questions may be approached from the experimental side. Throughout the work I have sought to show that the only foundation for rational therapeutics is the proper understanding of the working of the healthy body. Until we know more about the physiology of nutrition, quacks will thrive and food faddists abound. Ignorance of physiology tends to make a medical man as credulous as his patients and almost as easily beguiled by the specious puffings of the advertising druggist. I trust therefore that the following pages will be found of value not only to the candidate for a university degree but also to the practitioner of medicine in equipping him for his struggle against the factors of disease. In the selection of diagrams for the illustration of this book I am especially indebted to Professor Schafer and to his publishers, Messrs. Longmans, for the permission to make use of a large number from Quain's Anatomy and from Schafer's Essentials of Histology. I must also express my obli- gation to Professor "Wilson for the use of certain figures from his admirable work on the cell, to the publishers of Cunningham's Anatomy, and to many physiological friends, especially to Dr. Mott and Dr. Gordon Holmes, for the use of original diagrams. The index was kindly made for me by Mr. Lovatt Evans. ERNEST H. STARLING. University College, London, May 1912. CONTENTS CHAPTER I INTRODUCTION PAG1 BOOK I GENERAL PHYSIOLOGY CHAPTER II THE STRUCTURAL BASIS OF THE BODY . 13 CHAPTER III THE MATERIAL BASIS OF THE BODY SECTION I. THE ELEMENTARY CONSTITUENTS OF LIVING CELLS . . .36 II. THE PROXIMATE CONSTITUENTS OF THE ANIMAL BODY . . 45 III. THE FATS 53 IV. THE CARBOHYDRATES 59 V. THE PROTEINS 71 VI. THE MECHANISM OF ORGANIC SYNTHESIS 107 CHAPTER IV THE ENERGETIC BASIS OF THE BODY I. THE ENERGY OF MOLECULES IN SOLUTION .... 121 II. THE PASSAGE OF WATER AND DISSOLVED SUBSTANCES ACROSS MEMBRANES 129 III. THE PROPERTIES OF COLLOIDS 137 IV. THE MECHANISM OF CHEMICAL CHANGES IN LIVING MATTER. FERMENTS 152 V. ELECTRICAL CHANGES IN LIVING TISSUES 169 BOOK II THE MECHANISMS OF MOVEMENT AND SENSATION CHAPTER V THE CONTRACTILE TISSUES I. THE STRUCTURE OF VOLUNTARY MUSCLE 177 II. THE EXCITATION OF MUSCLE 185 III. THE MECHANICAL CHANGES THAT A MUSCLE UNDERGOES WHEN IT CONTRACTS 194 CONTENTS CHAPTER V (continued) SECTION PAGE IV. THE CONDITIONS AFFECTING THE MECHANICAL RESPONSE OF A MUSCLE .205 V. THE CHEMICAL CHANGES IN MUSCLE 212 VI. THE PRODUCTION OP HEAT IN MUSCLE . 219 VII. ELECTRICAL CHANGES IN MUSCLE . . 224 VIII. THE INTIMATE NATURE OF MUSCULAR CONTRACTION . ' .234 IX. VOLUNTARY CONTRACTION . 239 X. OTHER FORMS OF CONTRACTILE TISSUE .... 243 CHAPTER VI NERVE FIBRES (CONDUCTING TISSUES) I. THE STRUCTURE OF NERVE FIBRES '..... 250 II. PROPAGATION ALONG NERVE FIBRES 253 III. EVENTS ACCOMPANYING THE PASSAGE OF A NERVOUS IMPULSE 256 IV. CONDITIONS AFFECTING THE PASSAGE OF A NERVOUS IMPULSE 258 V. THE EXCITATION OF NERVE FIBRES . 262 VI. THE CONDITIONS WHICH DETERMINE ELECTRICAL STIMULATION 270 VII. THE NEURO-MUSCULAR JUNCTION 275 VIII. POLARISATION PHENOMENA IN NERVE ..... 280 IX. THE NATURE OF THE EXCITATORY PROCESS . . . 284 CHAPTER VII THE CENTRAL NERVOUS SYSTEM I. THE EVOLUTION AND SIGNIFICANCE OF THE NERVOUS SYSTEM 288 II. THE NERVOUS SYSTEM OF VERTEBRATES . . . .297 III. GENERAL CHARACTERISTICS OF REFLEX ACTIONS . . . 303 IV. NATURE OF THE CONNECTION BETWEEN NEURONS . . . 307 V. FUNCTIONS OF THE NERVE CELL 312 THE SPINAL CORD VI. STRUCTURE OF THE SPINAL CORD 315 VII. THE SPINAL CORD AS A REFLEX CENTRE . . 322 VIII. THE MECHANISM OF CO-ORDINATED MOVEMEM . . 338 IX. TROPHIC FUNCTIONS OF THE CORD ..... 349 X. THE SPINAL CORD AS A CONDUCTOR 351 THE BRAIN XI. THE STRUCTURE OF THE BRAIN STEM 361 XII. THE FUNCTIONS OF THE BRAIN STEM 390 XIII. THE FUNCTIONS OF THE CEREBELLUM 395 X I V. VISUAL REFLEXES 405 XV. SUMMARY OF THE CONNECTIONS AND FUNCTIONS OF THE CRANIAL NERVES . 409 CONTENTS xi CHAPTER VII (continued) THE CEREBRAL HEMISPHERES SECTION PAGE XVI. GENERAL STRUCTURAL ARRANGEMENTS OF THE CEREBRUM . 415 XVII. THE FUNCTIONS OF THE CEREBRAL HEMISPHERES . -. 433' XVIII. THE NUTRITIVE AND VASCULAR ARRANGEMENTS OF THE CENTRAL NERVOUS SYSTEM . 461 THE AUTONOMIC SYSTEM XIX. THE VISCERAL OR AUTONOMIC NERVOUS SYSTEM . 466 CHAPTER VIII THE SENSE ORGANS PART I. INTRODUCTION ......... 478 II. VISION (BY H. HARTRIDGE) 486 SECTION 1. PROPERTIES OF LIGHT, COLOUR AND THE SPECTRUM . 486 2. ORBITAL CAVITY AND ITS CONTENTS .... 493 3. EYEBALL, ITS HISTOLOGY. PUPIL REFLEX . . 500 4. NUTRITION AND PROTECTION OF THE EYEBALL . . 514 5. OPTICAL MEDIA OF EYE, AND ACCOMMODATION . . 519 6. OPTICAL PROPERTIES AND DEFECTS OF THE EYES . 529 7. RETINA, ITS HISTOLOGY AND PHYSIOLOGY . . . 540 8. RESPONSE TO LIGHT AND COLOUR .... 555 9. SUBJECTIVE PHENOMENA OF VISION .... 566 10. DEFECTS OF VISION AND THEIR DETECTION . . 577 . 11. DUPLEX THEORY AND HYPOTHESES OF COLOUR VISION 583 12. BINOCULAR AND STEREOSCOPIC VISION . . . 588 III. HEARING .... 595 SECTION 1. PROPERTIES OF SOUND 595 2. THE EXTERNAL, MIDDLE AND INTERNAL EAR . •. 600 3. HYPOTHESIS OF AUDITION, AND APPRECIATION OF DIRECTION . . . . . . . .611 IV. VOICE AND SPEECH ... .618 V. CUTANEOUS SENSATIONS 626 VI. TASTE AND SMELL 639 VII. SENSATIONS OF MOVEMENT AND POSITION . . 646 VIII. LABYRINTHINE SENSATIONS 651 xii CONTENTS BOOK III THE MECHANISMS OF NUTRITION CHAPTER IX THE EXCHANGES OF MATTER AND ENERGY IN THE BODY (GENERAL METABOLISM) SECTION . PAGE I. METHODS EMPLOYED IN DETERMINING THE TOTAL EXCHANGES OF THE BODY . . 660 II. METABOLISM DURING STARVATION ..... 670 III. THE EFFECT OF FOOD ON METABOLISM . . 677 IV. THE EFFECT OF MUSCULAR WORK ON METABOLISM . 683 V. THE SIGNIFICANCE OF THE FOODSTUFFS . . . 688 VI. THE NORMAL DIET OF MAN 695 CHAPTER X THE PHYSIOLOGY OF DIGESTION CHANGES UNDERGONE BY THE FOODSTUFFS IN THE ALIMENTARY CANAL 703 I. DIGESTION IN THE MOUTH 706 II. THE PASSAGE OF FOOD FROM THE MOUTH TO THE STOMACH . 721 III. DIGESTION IN THE STOMACH 728 IV. THE MOVEMENTS OF THE STOMACH 742 V. THE PANCREATIC JUICE . . . . . . . 748 VI. THE LIVER AND BILE .... .759 VII. THE INTESTINAL JUICE . 764 VIII. FUNCTIONS OF THE LARGE INTESTINE 768 IX. MOVEMENTS OF THE INTESTINES 771 X. THE ABSORPTION OF THE FOODSTUFFS . . 779 XI. THE F^CES .799 CHAPTER XI THE HISTORY OF THE FOODSTUFFS I. PROTEIN METABOLISM 801 II. NUCLEIN OR PURINE METABOLISM 818 III. THE HISTORY OF FAT IN THE BODY 826 IV. THE METABOLISM OF CARBOHYDRATES . 839 CONTENTS xiii CHAPTER XII THE BLOOD SECTION PAGE GENERAL CHARACTERS OF THE BLOOD .... . 853 I. THE WHITE BLOOD CORPUSCLES . . . . . . 856 II. THE RED BLOOD CORPUSCLES 861 III. THE BLOOD PLATELETS 879 IV. THE COAGULATION OF THE BLOOD . . . . 882 V. THE QUANTITY AND COMPOSITION OF THE BLOOD IN MAN . 897 CHAPTER XIII THE PHYSIOLOGY OF THE CIRCULATION I. GENERAL FEATURES OF THE CIRCULATION .... 913 II. THE BLOOD PRESSURE AT DIFFERENT PARTS OF THE VASCULAR CIRCUIT 919 III. THE VELOCITY OF THE BLOOD AT DIFFERENT PARTS OF THE VASCULAR SYSTEM 931 IV. THE MECHANISM OF THE HEART PUMP . . . 935 V. THE FLOW OF BLOOD THROUGH THE ARTERIES . . . 962 VI. THE FLOW OF BLOOD IN THE VEINS 976 VII. THE PULMONARY CIRCULATION . . . . .979 VIII. THE CAUSATION OF THE HEART BEAT . . . . 982 IX. THE NERVOUS REGULATION OF THE HEART .... 1012 X. THE NERVOUS CONTROL OF THE BLOOD VESSELS . . . 1025 XI. THE CIRCULATORY CHANGES DURING MUSCULAR EXERCISE . 1051 XII. THE INFLUENCE ON THE CIRCULATION OF VARIATIONS IN THE TOTAL QUANTITY OF BLOOD' 1058 CHAPTER XIV LYMPH AND TISSUE FLUIDS . , . . . . 1061 CHAPTER XV THE DEFENCE OF THE ORGANISM AGAINST -INFECTION I. THE CELLULAR MECHANISMS OF DEFENCE .... 1070 II. THE CHEMICAL MECHANISMS OF DEFENCE . . . . 1079 CHAPTER X.VI RESPIRATION I. THE MECHANICS OF THE RESPIRATORY MOVEMENTS . . 1088 II. THE CHEMISTRY OF RESPIRATION . . . . . 1100 III. THE REGULATION OF THE RESPIRATORY MOVEMENTS . .1126 IV. THE EFFECTS ON RESPIRATION OF CHANGES IN THE AIR BREATHED 1148 V. THE MECHANISMS OF OXIDATION IN THE TISSUES . 1155 xiv CONTENTS CHAPTER XVII RENAL EXCRETION SECTION PAGE I. THE COMPOSITION AND CHARACTERS OF THE URINE . .1160 II. THE SECRETION OF URINE 1181 III. THE PHYSIOLOGY OF MICTURITION 1205 CHAPTER XVIII THE SKIN AND THE SKIN GLANDS . . . 1216 CHAPTER XIX THE TEMPERATURE OF THE BODY AND ITS REGULATION . 1219 CHAPTER XX THE DUCTLESS GLANDS . 1230 BOOK IV REPRODUCTION CHAPTER XXI THE PHYSIOLOGY OF REPRODUCTION SECTION I. THE ESSENTIAL FEATURES OF THE SEXUAL PROCESS . . 1251 II. DEVELOPMENT AND HEREDITY 1264 III. REPRODUCTION IN MAN 1269 IV. PREGNANCY AND PARTURITION 1282 V. THE SECRETION AND PROPERTIES OF MILK . . . 1289 INDEX . 1299 CHAPTER I INTRODUCTION PHYSIOLOGY in its widest sense signifies the study of the phenomena pre- sented by living organisms, the classification of these phenomena, and the recognition of their sequence arid relative significance. Such a range would include many studies which are not generally grouped under the term physiology, and would in fact correspond to the comprehensive science of biology. Thus the study of the relations of living beings to one another and to their surroundings is the special object of the science of cecology. The aims of physiology in its restricted sense are the description, analysis, and classification of the phenomena presented by the isolated organism, the allocation of every function to its appropriate organ, and the study of the conditions and mechanisms which determine each function. The fundamental phenomena of life are essentially identical throughout the whole series of living organisms. This continuity of function is the necessary correlation of the continuity of descent, which brings into relation all members of the animal and vegetable kingdoms. No living organism can therefore be regarded as outside the sphere of our investigations. The interest of mankind in this subject was, however, naturally awakened iri connection with his own body, and the science, growing up as ancillary and preliminary to medical studies, has always taken man as its chief type of study. In the present work the elucidation of the functions of man will also be our first concern, and this for two reasons. In the first place, in physiology, as in all other sciences, the motive of man's activity is his social instinct to increase the power of his race in the struggle for existence, by the acquisition of control, either over the external forces of nature, which may be turned to his own benefit, or over the factors, intrinsic and extrinsic, which tend to his enfeeblement or extirpation by disease and death. Consciously or unconsciously, all our researches on physiology, whether on the higher animals or on the lowest protozoa, have the welfare of man as their ultimate object. In the second place, the choice of the higher animals as our chief objects of study receives justification from the fact that whereas morphology, or the science of structure, must proceed from the lowest to the highest organisation, the science of function presents its problems in their simplest form in the most highly differentiated organisms. In the unicellular animal all the essential functions which we associate with Jiving beings are carried out, often simultaneously, in one little speck of proto- plasm. An analysis of these functions, the determination of their conditions 1 1 2 PHYSIOLOGY and mechanism, is obviously impossible under^uch circumstances. It is only when, as in the higher animals, one part of the living body is differentiated into an organ which has one function and one function only as the outlet for its activities, that it becomes possible to peer into the details of the function with some chance of discovering its ultimate mechanism. Our especial preoccupation with the physiology of man will not prevent our employment of examples from any part of the animal or vegetable kingdom, when light can be thrown by their study on fundamental physio- logical phenomena common to the whole of the living world. In many cases such a study will enable us to separate the essential features in a process from those which have been added as auxiliary, with increasing complexity of the structures concerned. What are the fundamental phenomena which are wrapt up in our con- ception of living beings ? When dealing with the higher animals, we are inclined to lay greater stress on the phenomena involving a discharge of energy. Thus we should say that a man was alive if his body were warm and if he were presenting spontaneous movements, such as those of respira- tion or of the heart. The life of a man in the ordinary sense of the term is made up of those movements which place him in relationship with his environ- ment. For the production of these movements, as for the maintenance of a constant body-temperature, a continual expenditure of energy is necessary. Experience teaches us that these movements come to an end in the absence of food or of oxygen, and that an increased call on the energies of the body must always be met by a corresponding increase in the air and in the food supplied. Two further processes must therefore be included among those making up our conception of life, viz. the function of assimilation (the taking in and digestion of food), and the function of respiration, in which oxygen is absorbed and carbon dioxide is excreted into the surrounding atmosphere. The substances which make up our foodstuffs are all capable of oxidation. Composed chiefly of carbon and hydrogen, with some oxygen, nitrogen, and sulphur, they yield on complete combustion carbon dioxide, water and small amounts of ammonia or allied bodies, and sulphates. In this process of oxidation there is liberation of heat. In the body a similar oxidation occurs, the products of oxidation being discharged into the surrounding medium. An amount of energy is thus set free which is available for the activities of the living organism. Before we can make any accurate in- vestigations of the conditions which determine these activities, we must know whether the two great laws of chemistry and physics, viz. the conser- vation of mass and the conservation of energy, hold good for the processes within the living body. The many experiments which have been made on this point have decided the question in the affirmative. Thousands of experiments have been made, both on man and on animals, in which the total income of the body, viz. food and oxygen, has been weighed, and compared with the total output, viz. carbon dioxide, water, and bodies allied to ammonia (urea, &c.). In every case complete equality has been INTRODUCTION obtained, and we can be certain that any matter found in the body must have been derived from without. There is no creation or destruction of matter in the body. The determination of the equation in the case of the total energy of the body is rather more difficult. We have, in the first place, to measure the total income and output of the body, and to determine the total heat which would be evolved by the oxidation of tb ?. foodstuffs taken into the carbon dioxide, water, &c., that are given out. We must then compare the figure so obtained with the actual output of energy by the body. The latter can be measured in terms of heat by placing the animal inside a calorimeter. Many practical difficulties arise in the performance of the experiment, in consequence of the necessity of providing the animal with a constant supply of air to breathe, and of allowing for the continual loss of water by evapora- tion which is going on at the surface of the animal. The first accurate experiments of this nature were made by Rubner. This observer deter- mined by means of the calorimeter the total heat loss of dogs. In the same animals the material income and output of the body were measured, and a calculation was made as to the amount of energy which would be set free in the body by the processes of oxidation involved in the change of material observed. The following Table represents a summary of Rubner's results : Dog. Condition of dog. Calculated heat production. Heat loss deter- mined calori- metrically. Duration of experiment. 1. 2. Fasting .... Cal. 259-3 545-6 Cal. 261-0 528-3 Days. 5 2 3. Fed with meat 329-9 333-9 1 4. Fed with fat . 302-0 299-1 5 5. Meat and fat ... 332-1 330-0 12 6. >» »» 311-6 331-0 8 7. ' Fed with meat 375-0 379-5 6 8. " ' 683-0 681-3 7 It will be seen that the average difference between the calculated and observed results amounts only to I'Ol per cent. — an amazing agreement considering the extreme difficulties of the experimental methods involved. The important deduction to be drawn from these observations is that the foodstuffs which are oxidised in the body develop in this process exactly the same amount of energy as when they are burnt up outside the body. From one aspect, therefore, the animal body may be looked upon as a machine for the transformation of the potential energy of the foodstuffs into kinetic energy, represented by the warmth and movements of the body as well as by other physical changes involved in vital processes. In the living organism we cannot, however, distinguish between the source of energy and the machinery, as we can in the case of our engines. When we endeavour to trace the foodstuffs after their entry into the body, we lose sight of 4 PHYSIOLOGY them at the point where they are built up to form apparently an integral part of the living framework. During activity there is a discharge of the products of oxidation of the foodstuffs from this living matter, which there- fore becomes reduced in mass. This reduction, or disintegration of the living matter, associated with activity, is always followed by a period of increased integration, during which the organism grows by the assimilation of more food. Our conception of life must therefore involve the idea of a constantly recurring cycle of processes, one of building up, repair, or inte- gration, and the other, associated with -activity, of destruction or disinte- gration. If the former process predominates, we obtain a steady increase in the mass of the organism, an increase which we speak of as growth, and in many cases, as in that of plants, it is this power of growth which we take as our criterion of the existence of life. In fact, the possession by the green parts of plants of the power of utilising the energies of the sun's rays for the integration of foodstuffs, such as starch, writh a high potential energy, is the necessary condition for the exfstence of all higher forms of life on this earth. Closely associated with the property of growth is the power possessed by all living organisms of repair, i.e. the replacement by newly formed healthy living material of parts which have been damaged by external events. The process of growth does not, in the individual, proceed indefinitely. At a certain stage in its life every organism divides, and a part or parts of its substance are thrown off to form new individuals, each of them endowed with the same properties as the parent organism, and destined to grow until they are indistinguishable from the organism whence they were derived. In the lowest forms of life, the unicellular organisms, these processes of growth and division may go on until brought to an end by some change in the environment which will not allow the necessary conditions of life, viz. assimilation and disintegration, to proceed. In all the higher forms, hovr- ever, after the process of reproduction has been completed, the parent organism begins to undergo decay, and the processes of assimilation and repair no longer keep pace with those of destruction, however favourable the environment, until finally death of the organism takes place. All these phenomena, viz. assimilation, respiration, activity associated with the discharge of energy, growth, reproduction, and death itself, are bound up in our conception of life. All have one feature in common, viz. they are subject to the statement that every living organism is endowed with the power of adaptation. Adaptation may indeed receive the definitk n which Herbert Spencer has applied to life — " the continuous adjustment of internal relations to external relations." A living organism may be regarded as a highly unstable system which tends to increase itself con- tinuously under the average of the conditions to which it is subject, but undergoes disintegration as a result of any variation from this average. It is evident that the sole condition for the survival of the organism is that any such act of disintegration shall result in so modifying the relation of tlie INTRODUCTION 5 system to the environment that it is once more restored to the average in which assimilation can be resumed. Every phase of activity in a living being must be not only a necessary sequence of some antecedent change in its environment, but must be so adapted to this change as to tend to its neu- tralisation, and so to the survival of the organism. This is what is meant by ' adaptation.' Not only does it involve the teleological conception that every normal activity must be for the good of the organism, but it must also apply to all the relations of living beings. It must therefore be the guiding principle, not only in physiology with its special preoccupation with the internal relations of the parts of the organism, but also in the other branches of biology, which treat of the relations of the living animal to its environment, and of the factors which determine its survival in the struggle for existence. The origin of new species and the succession of the different forms of life upon this earth depend on the varying perfection of the mechanisms of adaptation. We may imagine that the first step in the evolution of life was taken during the chaotic chemical interchanges which accompanied the cooling down of the molten mass forming the earth, when some compound was formed, probably with absorption of heat, endowed with the property of continuous polymerisation and growth at the expense of surrounding material. Such a substance could continue to exist only at the expense of the energy derived from the surrounding medium, and would undergo destruction with any stormy change in its environment. Out of the many such compounds which might have come into being, only such would survive in which the process of exothermic disintegration tended towards a condition of greater stability, so that the process would' come to an end spontaneously, and the organism or compound be enabled to await the more favourable conditions necessary for the continuance of its growth. With the con- tinued cooling of the earth, the new production of endothermic compounds would become rarer and rarer ; and in all probability the beginning of life, as we know it, was the formation of some complex substance, analogous to the present chlorophyll corpuscles, with the power of absorbing the newly penetrating sun's rays and utilising them for the endothermic forma^ tion of further unstable compounds. Once given an unstable system, such as we have imagined, the great principle laid down by Darwin, viz. survival of the fittest, will suffice to account for the production from it by evolution of the ever-increasing variety of living beings which have appeared in the later history of this globe. The ' adaptation,' i.e. the reactions of the primitive living material to changes in its environment, must become ever more and more complex, since only by means of increasing variety of reaction is it possible to provide for the stability of the system within greater and greater range of external conditions. The difference between higher and lower forms is therefore one of complexity of reaction, or of range of adaptation. In all the physiological processes which we shall study in the course of this work, adaptation will be found the constant and guiding quality. The 6 PHYSIOLOGY naked protoplasm of the plasmodium of Myxomycetes, if placed on a piece of wet blotting-paper, will crawl towards an infusion of dead leaves, or away from a solution of quinine. It is the same property of adaptation, the deciding factor in the struggle for existence, which impels the greatest thinkers of our time to spend long years of toil in the invention of the means for the offence and defence of their community, or for the protection of man- kind against disease and death. The same law which determines the down- ward growth of the root in plants is responsible for the existence to-day of all the sciences of which mankind is proud. This " adjustment of internal to external relations " is possible, however, only within strictly denned limits, limits which increase in extent with rise in the type of organism, and in the complexity of its powers of reaction. Some of these limiting conditions we shall have to study in the next chapter. Among the chief of them are temperature, and the presence of food material and of oxygen. At the present time the limits of temperature may be placed between 0° and 50° C. Many organisms, however, are killed by the alteration of only a few degrees in the temperature of their environment. Every shifting of a cold or warm current in the Atlantic, in consequence of storms on the surface, leads to the destruction of myriads of fish and other denizens of the sea. In the higher animals a greater stability in face of such changes has been accomplished by the development of a heat-regulating mechanism, so that, provided sufficient food is available, the temperature of the body is maintained at a constant level, which represents the optimum for the discharge of the normal functions of the constituent parts of the body. The presence of food material in the environment of the living organism is a necessary condition for its continued existence. In some cases. and this we must assume to be the primitive condition, the food material must be of a given character and form a constant constituent of the sur- rounding medium. In the higher forms, the development of a complex digestive system has enabled the organism to utilize many different kinds of food, while the storage of any excess of food as reserve material, either in the form of fats or carbohydrates, provides for a constant supply of focd to the constituent cells of the body, even when it is quite wanting in tin1 environment. Since plants depend for their food in the first place on the carbohydrates produced within the chlorophyll corpuscles out of tin1 atmo- spheric carbon dioxide by the energy of the sun's rays, necessary conditions for their existence will be sunlight and the presence of this gas in tin' surrounding atmosphere. One other necessary condition for the existence of life is the presence of water. Although this substance cannot furnish any energy to the complex molecules of which the living matter is composed, it is an essential con- stituent of all living matter, and takes part in all the < -haimes which deter- mine the transformations of matter and energy in the organism. This short summary of the chief characteristics of living beings would be incomplete without the mention of what is perhaps their distinctive feature, namely, organisation. Although little marked in the lowest members .INTRODUCTION 7 of the living kingdom, where we can detect only a speck of structureless material containing a few granules, of which one or more, in consequence of its reaction to stains, is distinguished by the name of a nucleus, in the higher members this organisation becomes more and more marked. This increased complexity of organisation, which we often speak of as histological differentiation, runs parallel with increasing range of power of adaptation, and with increasing efficiency of adaptive reactions attained by the setting apart of special structures (organs) for the performance of definite functions. This parallelism between the development of function and structure justifies us in the assumption generally, though often only tacitly, made by physio- logists, that the structure is the determining factor for the function. We might regard the histological differentiation as representing merely a con- tinuation of the increasing molecular complexity, which we assumed must accompany and determine every widening in the range of the adaptive power of the organism. . To sum up :— our objects in the study of physiology include the descrip- tion of the chief reactions of the body to changes in its environment, the analysis of these reactions into the simpler reactions of which they are made up, and the assignment to each differentiated structure of the organism its part in every reaction. We must determine the conditions under which each reaction takes place, so that we may learn to evoke any part of it at will by application of the appropriate stimulus, i.e. by effective change of environment. A reaction involves expenditure of energy, and this can be derived only from chemical change in the reacting organ, and ultimately from the dis- integration or oxidation of the foodstuffs. Our next task must be, there- fore, the analysis of the energetic and material changes, with a view to determining the whole sequence of events, from the occurrence of the external exciting change to the finished reaction, which will alter in tne direction of protection the relation of the organism to its environment. In short, it is the office of physiology to discover the routine sequence of events in the living organism under all manner of conditions. In attacking this problem our methods cannot differ fundamentally from those of the physicist and chemist. In every case our experiments will consist in the observation and measurement of movements of one kind or another which we shall interpret in terms of mass or energy. Physiology, if it could be completed, would therefore describe the how of every process in the body. It would state the sequence of events and would summarise these as so-called ' laws.' These laws would, however, no more explain the phenomena of life than does the ' law of gravitation ' explain the fact that two masses tend to move towards one another with uniform acceleration. Nor can we hope to explain physiological phenomena by reference to the laws of physics and chemistry, since these themselves are only expressions of sequences, and not explanations. With every growth in science, however, its generalisa- tions become wider and its laws summarise ever more extensive groups of phenomena.' We have no reason for asserting that, in the course of research, 8 PHYSIOLOGY we may not finally succeed in describing vital phenomena in the " conceptual shorthand " * used by the physicist, involving his ideal world of ether, atom, and molecule. At present we are far from such a consummation. The principle of adaptation is the only formula which will include all the pheno- mena of living beings, and it is difficult to see how this principle can be expressed by means of the concepts of the physicist. This difficulty, which must be felt with greater force the more deeply the physiologist endeavours to peer into the processes within the living cells, has led some, even at the present day, to the assumption of some special quality in living organisms which is designated as ' vital force ' or ' vital activity.' Such views are classified together under the term vitalism. From his beginning man has been accustomed to draw a sharp line of dis- tinction between those phenomena which by their constant occurrence seemed to him natural, and therefore explicable, and those phenomena of which he could not see the determining antecedent, and which were to him, therefore, anomic and capricious. To the latter he set up graven images, and not perceiving his own springs of action, endowed them with a self-determining personality such as he imagined himself to possess. This procedure, though possessing certain advantages in allowing him to perform his common duties free from the ever-lurking fear of supernatural interference, suffered from the great drawback that it fenced of! unknown phenomena as unknowable and not to be known. It has therefore acted as a continual check on the growth of man's knowledge and control of his environment. Such a graven image is vitalism. As a working hypothesis it must be sterile. Just as the hypothesis of special creation would impede all research into the relationships of animals and plants, so vitalism would stay the hand of the physiologist in his endeavours to determine the changes which occur within the living organism. In many cases, however, the terms ' vitalism ' and its antithesis ' mechanism ' are used unjustifiably. The production of energy within the body is due to the oxidation of the foodstuffs. In certain functions it is not yet fully established whether the changes involved take place at the expense of the energy, hydrostatic pressure or otherwise, of the fluids outside the cells, or whether energy is supplied to the process by the cells themselves at the expense of oxidative or other changes occurring in their living sub- stance. Both views are possible, but the adoption of either by a physiologist does not justify the statement that he is a ' vitalist,' ' neo-vitalist,' or * mechanist.' The office of the physiologist is the determination of the changes which occur in the living body and the establishment of the causal nexus (i.e. the routine of sequences) between them. For such a man to describe himself as a vitalist or mechanist is ;is ornnaiie to the subject as if he were to call himself a Trinitarian or a Plymouth Brother. Throughout this chapter we have assumed no n<>< vssary dividing line between the different classes of phenomena in the conceptual univrrsi', although in the present state of our knowledge we are far from being able to include the whole of them under the same general laws. It might be * Karl Pearson, "Grammar of Science," p. 328 et seq. (2nd ed.). INTRODUCTION 9 objected that in taking up this attitude we had left out of account one supreme fact, viz. the existence of consciousness in ourselves. As a com- parative and objective study, however, physiology is concerned, not with the study of consciousness, but with the conceptions in consciousness of the phenomena presented by living beings. Consciousness, in fact, we know only in ourselves. From the actions of other living beings similarly organised, we infer in them the existence of a similar consciousness. Again, from the fact that the reactions of the higher mammals are evidently determined, not by immediate impressions, but largely by stored-up impressions of past stimuli, we credit them also with a certain but lower degree of consciousness. As we descend the scale of animal life, evidence of the existence of consciousness, as we know it, rapidly diminishes and finally disappears, though it is im- possible to draw a sharp line between those animals which possess conscious- ness and those in which it is absent. That it is a necessary accompaniment of life is certainly not the case. A man is living though he is asleep, anaes- thetised, or stunned, and it would be absurd to speak of the consciousness of a cabbage. Consciousness is, in fact, connected with the possession of a highly developed central nervous system, and its activity is in proportion to the complexity of this system. Since the brain with all the other organs of the body is derived from a simple cell, the fertilised ovum, similar in its absence of differentiation to the lowest organisms, it might be argued that all types of life are endowed with something which is not consciousness, but which has the potentiality of developing into consciousness. To such a hypothetical property Lloyd Morgan has given the name ' metakinesis.' We have, however, no means of judging of the presence or absence of this hypo- thetical quality and still less of determining whether it is a property only of living substance, or is shared also by the atoms of so-called dead material. BOOK I GENERAL PHYSIOLOGY t CHAPTER II THE STRUCTURAL BASIS OF THE BODY THE CELL LL the higher animals and plantsawhen submitted to microscopic examina- tion, are seen to consist of structural units which are spoken of as cells. In each organ we find a mass of these cells closely resembling one another in all respects, and we may therefore regard the function of any organ as the sum of the functions of its constituent cells. Indeed, any given reaction of the whole body is the resultant of the reactions of the unlike cells of which the body is composed. The cell is therefore the physiological as well as the structural unit, and it is necessary to commence our study of the functions of the animal body with some consideration of the functions and reactions which are common to all the structural units. This composite structure is peculiar to the higher forms of life. Amongst the lower forms, both animal and vegetable, an immense number of organisms consist only of a single cell. In this cell are represented all the phenomena of life, all the adapted reactions which we associate with the life of the higher organisms. That the unicellular condition represents the more primitive stage from which the higher ^organisms have been evolved in the course of ages is indicated by the fact that every one of these higher organisms in the course of its development passes through a unicellular stage, namely, the fertilised ovurn. We may assume that the series of changes attending e development of the higher organism from the egg is a repetition in sum- ary of the changes which have determined the evolution of the species from the primitive unicellular type.* The general characteristics of the cell present important similarities, hether we are considering a cell which forms the whole of an organism or a ell which is but an infinitesimal part of a highly developed animal. The name ' cell ' was first applied by botanists to the structural units und by them in plant tissues, and involved therefore the idea of certain ualities which do not enter into our present conception of the term. A ction through the stem of a growing plant shows it to be made up of an xegation of cells in the etymological sense of the word, i.e. small sacs bounded by a wall of cellulose and containing cell sap. Immediately inside e cellulose wall is a thin layer, the primordial utricle, which encloses at one int a spherical or oval structure known as the nucleus. If the section be * This assumption is often spoken of as the * law of recapitulation,' 13 14 PHYSIOLOGY taken from the growing tip of a plant (Fig. 1), the cell sap is found to be wanting and the cells consist only of the substance known as protoplasm, which later on will forui the primordial utricle. This with a nucleus is enclosed in a delicate cellulose wall. The wall is not an <»ssrnt ial constituent, since it is absent from many vegetable cells at some period of their life and from animal cells generally. A better conception of the essentials of a cell can be obtained by the study FIG. 1. General view of cells in the growing root-tip of the onion, from a longitudinal section, enlarged 800 diameters. (WILSON.) a, non-dividing cells, with chromatin-network and deeply stained nucleoli ; &, nuclei preparing for division (spireme-stage) ; r, dividing cells showing mitotic figures ; e, pair of daughter-cells shortly after division. of a unicellular animal such as an amoeba (Fig. 2). This is an organism frequenting stagnant pools, of varying size (fromO'l to 0'3 mm. in diamctrr). apparently of a semi-fluid consistence. When first examined it is generally spherical, but in a short time begins to change its form, putting out processes known as pseudopodia. By shifting the distribution of its material among these processes, it is able to move about and also to ingest particles of food or pigment with which it may come in contact. Near its centre a differentiated portion can be distinguished which is known as the nucleus. The rest of the amoeba, the protoplasm or cytoplasm, often presents further differentiation into an outer clear layer and an inner finely granular substance. The latter may contain coarser granules, some of food material, others apparently formed in situ by the surrounding protoplasm, and often small vacuoles (' contractile vacuoles ') which are continually altering their size and serve to keep up a circulation of fluid in the interstices of the cytoplasm. In all cells, whether animal or vegetable, with whieh we are acquainted, this THE STRUCTURAL BASIS OF THE BODY 15 twofold structure is also found. So we may define a cell as a small mass of protoplasm containing a nucleus. Doubt has often been expressed whether a nucleus is to be regarded as essential to our conception of a cell. In many of the lowest forms of animals and plants, such as the Flagellata among the former and the CyanophycesB and Bacteria among the latter, no distinct nucleus can be demonstrated. In many of these forms the dimen- sions of the whole organism are too minute to allow of any definite statement being W V ' \ e FIG. 2. Amceba proteus, an animal consisting of a single naked cell, X280. (From SEDGWICK and WILSON'S Biology.) n, the nucleus ; wv, water- vacuoles ; cv, contractile vacuole ; fv, food-vacuole. lade as to the presence or absence of nuclear material. In the larger of them, how- ever, the cytoplasm of the cell contains numerous scattered granules which stain with dyes in the same way as do the nuclei of the cells of higher animals, and these granules possess the resistance to the action of certain digestive fluids which is typical of nuclei. They may therefore be taken as representing the nucleus in the higher forms. Even in the latter at certain stages, namely, during the division of the cell, the nucleus breaks up into discrete parts, and there is no reason for believing that such a scattered condition of the nuclear material may not last throughout the whole life of the cell. We have defined a cell as a small mass of protoplasm containing a nucleus, ince we shall have to use the term ' protoplasm ' on many occasions in the rse of this .work, we must have a definite conception of what we mean y it. The term is often used by histologists as implying a substance of certain definite chemical and staining characters. When employed by hysiologists it generally implies any material which we can, on a study of its Saviour to changes in its environment, regard as endowed with life. Huxley has defined it as " the physical basis of life." Though it may be con- venient to have a word such as protoplasm signifying simply ' living material,' t is important to remember that there is no such thing as a single substance protoplasm. The reactions of every cell as well as its organisation are the ultant of the molecular structure of the matter of which it is built up. e gross methods of the chemist show him that the composition of the 16 PHYSIOLOGY ' protoplasm ' of the muscle cell is entirely different from that of a leucocyte or white blood corpuscle. The finer methods of the physiologist show him that every sort of cell in the body has its own manner of life, its own pecu- liarities of reaction to uniform changes in its surroundings. No individual will react in exactly the same manner as another individual, even of the same species, and the reactions of the whole organism are but the sum of the Attraction-sphere enclosing two centrosonies Xucluus* Plasmosome or true nucleolus Chromatin network Linin network Karyosome or chromatin- nuclcolus Plastids Iyiii2 in the cytoplasm Vacuole Passive bodies (metaplasm or paraplasin) suspended in the cytoplasmic im sh- \vork FIG. 3. Diagram of a cell. Its basis consists of a nieshwork containing numerous minute granules (microtomes) and traversing a transparent ground -substance. (WILSON.) reactions of its constituent cells. There is not one protoplasm therefore, hut an infinity of protoplasms, and the use of the term can be justified only if we keep this fact in mind and use the word merely as a convenient abbreviation for any material endowed with life. Even in a single cell there is more than one kind of protoplasm. In its chemical characters, in its mode of life, and in its reactions, the nucleus differs widely from the cytoplasm. Both are necessary for the life of the cell and both must be considered, according to our present ideas, as ' living.' In the cytoplasm itself we find structures or substances which we must regard as on their way to protoplasm or as products of the breakdown of protoplasm ; but in many cases it is impossible to say whether a given material is to be regarded as lifeless or as reactive living matter. Even in a single cell we may have differentiation among its different parts, one part serving for the process of digestion while others are employed for the purpose of locomotion. Here again there must be chemical differences, and therefore different protoplasms. In this work the term protoplasm will be used in its broadest sense, namely, as the physical basis of living organisms. STRUCTURE OF THE CELL. In every cell can be distinguished the two parts — nucleus and cytoplasm. The nucleus is generally an oval or THE STRUCTURAL BASIS OF THE BODY 17 spherical body lying near the centre of the cell and bounded by a definite contour or nuclear membrane. In its interior it contains masses or filaments of a material known as chromatin, which are strung, so to speak, on a fine network of material known as linin. Besides the granules of chromatin, other masses are sometimes found which stain in a different manner and are called nucleoli. The material filling up the meshes of the network is the nuclear sap or nucleoplasm. The cytoplasm, which varies greatly in extent in different cells, varies also in its appearance, being sometimes homogeneous, sometimes alveolar, sometimes granular in structure. In it can be often distinguished differentiated parts which may be regarded as organs of the cell. Thus in the amoeba we have the contractile vacuoles already men- tioned. In the green parts of plants the cytoplasm contains green granules, the chloroplasts, whose special function it is to assimilate carbon dioxide from the atmosphere, and by means of the energy of the sun's rays to convert this into starch with the evolution of oxygen. Other parts of the plant have similar granules, the leucoplasts, whose office it is to build up sugar into starch, and it is possible that other kinds of these c plastids ' with special chemical functions are present in the cytoplasm of many cells. In addition to these cell organs, the cytoplasm may contain granules which represent stages in the metabolism of the cell and are either food material which is being assimilated or products of the disintegration of the protoplasm, formed either for the service of the cell itself or, in the case of the multi- cellular animals, for the service of other cells of the organism. Others of these granules may represent reserve material, i.e. excess of nourishment which has been put aside by the cell in an insoluble form, to serve for its subsequent needs in times of scarcity. THE PHYSICAL STRUCTURE OF PROTOPLASM. Owing to the close similarities which exist between the fundamental properties of all living organisms, histologists have sought to discover some corresponding uniform morphological organisation of the physical basis of these phenomena, namely, protoplasm. It is often impossible, even under the highest powers of the microscope, to make out any structure whatsoever in the cytoplasm, which is spoken of then as hyaline. In most cases examination of a cell, even unstained, shows some differentiation between a more or less regular framework or meshwork and a more fluid portion filling up its interstices, and these appearances are still more manifest when the cells have been fixed by various hardening fluids. All the results obtained in this manner must be regarded with some suspicion, since, as has been shown by Fischer and by Hardy, it is possible to imitate artificially the various structures, which have been assigned as characteristic of protoplasm, by hardening a homogeneous colloidal solution such as egg-white by different methods and with different agents. The theories of protoplasmic structure can be classified under three heads : 1. THE GRANULAR. THEORY OF ALTMANN. By the use of certain hardening reagents, a dense mass of spherical or rod-shaped granules may be demon- strated in almost all cells of the body (Fig. 4). These granules have been 2 18 PHYSIOLOGY regarded by Altmann as the elementary particles of life, and he locates in them the various vital functions, the sum of which makes up the life of the cell. According to Altmann these granules can arise only from the division of pre-existing granules, and he has formulated the phrase omne granulum e granule*, which is a further extension of Virchow's sentence omrtis cellula e cellula. It is probable that a number of different kinds of structures of vary- ing importance are included among Altmann's granules. In some cases they FIG. 4. Section of liver stained to show granules. (ALTMANN.) are the products of the activity of the cytoplasm and, as in secreting cells, will be later on cast out with water and salts as the specific secretion. In other cases they may be cell organs or jplastids with the special metabolic functions assigned to all granules by Altmann. In some cases no treatment whatever will display the existence of granules. 2. THE FIBRILLAK THEORY. By the employment of appropriate methods of hardening, it is easy in most cells to demonstrate a network or clusters of fibrils which form, so to speak, a denser part of the cell. This fibrillar network has been named the ' spongioplasm ' in contra-distinction to the structureless material rilling its meshes known as ' hyaloplasm.' A network is, however, one of the commonest pseudostructures produced in the coagu- lation of an albuminous fluid by any means whatever, and it is probable that in most cases the network which is seen in hardened cells is simply an artefact. Sometimes a large portion of the protoplasm may take a fibril] a r form which can be detected even in the unstained and unfixed cell, and UHMV is no doubt that, in certain phases at any rate, the fibrillar structure of the protoplasm is really present. 3. THE ALVEOLAR THEORY OF BUTSCHLI. This theory may be looked upon as corresponding morphologically to the granular theory of Altmann. If we imagine a hyaline protoplasm which is continually manufacturing metaplasmic products and storing them up in its protoplasm, these products ^yiU be deposited as spherules gradually increasing in size, so that the proto- THE STRUCTURAL BASIS OF THE BODY 19 FIG. 5. Diagram of a cell» highly magnified. (SCHAFER.) p, protoplasm, consisting of hyaloplasm and a network of spongioplasm, ex, exoplasm ; end, endoplasm, with distinct granules and vacuole s; c, double centrosome ; n, nu- cleus ; n', nucleolus. plasm between them will be converted into alveolar partitions between the droplets. In many an egg cell, where there is a growth of protoplasm from this building up of food into reserve materials, the development of such an alveolar structure can be followed in the living protoplasm, and such cells when mature show a marked alveolar structure whether ex- amined fresh or in the hardened and stained condition. Such a protoplasm would be prac- tically an emulsion of one fluid in another, and according to Butschli, artificial emulsions, made by mixing rancid oil with sodium carbonate solutions, may show under the microscope a very close resemblance to cell protoplasm (Fig. 6), and may even exhibit amoeboid changes of form in consequence of the diffusion currents set up at the surface of the drop between its contents and the surrounding water. Most histologists are in accord that none of the above theories can be regarded as applicable to all forms of protoplasm,but that during the life of a cell its protoplasm, as observed under the microscope, may be either hyaline and structureless or may present any of the structural modi- fications described above, according to its state of nutrition and the form in which its metabolic products are laid down in the cell. Of course it is possible that, even in the apparently hyaline protoplasm, a structural differentiation is still pre- sent, but is invisible owing to the minute size of its constituent parts or an identity of refractive index between the alveolar walls and their contents. The fact that; every chemical differentiation occurring within the colloidal mass will tend to cause differences of surface ten- sion, and therefore . formation of droplets, shows that an alveolar structure, i.e. one in which there is a large number of surfaces separat- ing heterogeneous mixtures inside the cells, must be of very common occurrence, even in cases where it is not detectable under the microscope. Such a structure must be present, at any rate, in those cases where, apart FIG. 6. A, protoplasm of an epidermal cell of the crayfish ; B, foam-like appearance of an emulsion of olive oil. (BUTSCHLI.) 20 PHYSIOLOGY from the existence of a solid cell wall, the cell presents a certain degree of rigidity and resistance to deforming stress. ULTRAMICROSCOPIC STRUCTURE OF PROTOPLASM. Since the study of the behaviour of the cell shows that it must possess a much more complex structure or organisation than that which is revealed by the microscope, one, that is to say, which permits of the spatial differentiation of the different chemical processes that may occur at one and the same time in the protoplasm, many theories have been put forward of an ultramicroscopic cell structure. Though Spencer in 1864 spoke of physiological units out of which protoplasm could be regarded as made up, and Darwin (1868) conceived ultramicroscopic particles — gemmules — which might be discharged from every cell in the body and, passing into the reproductive organs, serve as the material basis of heredity, the first elaborate conception of such a structure was worked out by Nageli (1884). According to Nageli all organised structures are made up of micellae, minute particles arranged in definite order and surrounded with water. For growth to take place it was necessary that the system should be in a condition of ' turgor,' which was determined by the amount of water between the micellae. These micellae arose in every case from the division of pre-existing micellae, and the vital properties of the protoplasm were to be regarded as the sum of the changes taking place in the individual micellae. Similar conceptions have been put forward by numerous other observers, each of whom has applied a different name to the elementary living particle, such as ' pangene,' ' plasome,' * biophor,' ' biogen-molecule,' and many others. The resemblance of these theories to that of Altmann is obvious, though the latter regarded the elementary particle as in many cases of microscopic size and capable of demonstration by appropriate methods of staining. That the cell possesses organs of smaller dimensions than itself, which may give rise to like organs by division, is shown by Schimper's observations on the plastids of plant cells. These apparently are not formed by a process of differentiation of the protoplasm, but are continuous from one generation to another and are reproduced by division. There is no doubt, however, that most of the granules to be observed in the cytoplasm are not of this character, but are elaborated by the general cytoplasm out of the foodstuffs which are supplied to it ; and though conceptions such as those of De Vries and Verworn are often of value as a means of describing certain phenomena in the life of the cell and have played a great part in the description of the phenomena of heredity, they cannot be regarded as having any serious justification in fact. At the present time our know- ledge of the properties of the colloidal and capillary systems, which must play so great a part in the organisation and reactions of living protoplasm, is much too meagre to justify weight being laid on any theory of the ultramicroscopic structure of protoplasm that can at present be put forward. One question which has been much discussed relates to the physical condition of protoplasm. Is it to be regarded as a viscous fluid or as a soft solid ? The perfect potential mobility of the protoplasm of many cells, as instanced by the flow of a substance of an amoeba into its pseudopodia, or the occurrence of rapid streaming movements in the threads of protoplasm found in many plants, e.g. the root hairs of tradescantia, indicates a fluid character for the protoplasm. Against such a character has been urged the fact that in protoplasm we may have shape, organisation, and power of resistance to deformation — qualities which are generally associated with the possession of solidity. It must be remembered, however, that the absence of resistance to deformation, which is characteristic of a liquid, applies only to the internal molecules, and that the surface of any liquid is in a condition of tension which not only limits deformation, but presents considerable resistance to any enlargement of the surface. Small water animals take * THE STKUCTURAL BASIS OF THE BODY 21 advantage of this resistance to run freely over the surface of water, although their specific gravity may be greater than that of water. The continued existence of protoplasm in a watery environment shows that not only must its composition be different from that of its environment, but that there must be a distinct surface separating the two. The superficial layers of the proto- plasm must therefore be in a condition of tension, and exercise pressure on the internal portions of the cell, which will tend to diminish the surface of the cell to the smallest possible extent, i.e. to bring it into the spherical form. This form is characteristic of free cells in their conditions of inactivity, and the smaller the mass of protoplasm, supposing it to be homogeneous, the greater will be the pressure exerted by its surface layer on its contents and the greater resistance will it present to deformation of the spherical form. A fluid drop, if suspended in a fluid with which it is immiscible, will present greater rigidity the smaller its dimensions. Almost any degree of rigidity can also be imparted to larger masses of fluid protoplasm if their interior has undergone chemical differentiation so as to be made up of two or more im- miscible fluids arranged as droplets within alveoli, as in Biitschli's theory. In such a case every droplet will present resistance to deformation and every surface will resist penetration or extension. The resistance of the surface in colloidal fluids is still further increased by a property common to all these fluids, namely, the aggregation in the surface of a greater concentra- tion of the dissolved substance than is present in the underlying fluid. If, for instance, we take a beaker containing egg-white diluted 100 times, and drop a steel magnetised needle on to the surface, it will float although it is much heavier than the fluid, in consequence of the resistance of the surface. If the needle be greasy the same thing will occur on a glass of water. In this case the needle will lie N. and S. On the albumen solution, however, the needle will lie in the position in which it has been dropped. The aggregation of the albumen molecules on the surface of the fluid is such that it is practi- cally solid and resists any turning of the needle. In consequence of the sur- face aggregation and solidification of the colloidal molecules, it is possible to throw out the greater part of the albumen in a solid form from a solution of this substance, if it be shaken up in a bottle with a little air so as to make a surface. As the fluid is shaken fresh surfaces are always being formed, and the albumen aggregating in each of these surfaces has not time to redissolve before a fresh aggregation occurs on a new surface, and the films thus pro- duced gradually collect to form a solid mass of insoluble protein. Proto- plasm may be regarded as essentially fluid in character, the form and rigidity which are acquired by most cells being due to chemical and physical differen- iation occurring in the fluid. THE SURFACE LAYER OF CELLS. Since it is by means of its surface layer that the organism enters into relation with its environment, this layer acquires a prime importance for the life of the cell, and we may there- fore consider here at greater length some of the properties of this layer, the Phsmahaut, as it has been called. The superficial layer of the protoplasm is not to be confounded with the 22 PHYSIOLOGY call wall. The latter, which plays a great part in the building up of vegetable tissues, is formed by a process of secretion from the living protoplasm and is situated altogether outside the superficial Plasmahaut. The cell wall differs considerably in its chemical composition from the protoplasm out of which it has been formed. In most plants it consists of cellulose, a substance belonging to the carbohydrate group, and with a composition represented by some multiple of the formula C6H1005. In other cells the wall may be built up from calcium carbonate or other lime salts, from silica, from chitin. In many cases it is perforated to allow the passage of communicating strands of protoplasm between adjacent cells. It is generally freely permeable to all kinds of solutions, and in this case plays no part in regulating the inter- changes of the cell with the environment. The superficial layer of protoplasm represents that part of the living substance which stands in immediate relationship to the environment. Every change in the latter can only influence the living cell through this layer, and it is through this layer that substances must pass on their way into the cell for assimilation, or out of the cell for excretion. The retention of an individuality by the cell must be determined by chemical and physical differences between this layer and the surrounding fluid. Since it differs from the rest of the protoplasm in the changes to which it is subject, it must also differ in its chemical composition, apart altogether from the factors which, as we saw above, determine molecular differences between the surface and the interior of any colloidal solution. On this account one must assume the existence of a definite boundary layer of the protoplasm, even where it is impossible to see any differentiation between this layer and the deeper parts under the highest powers of the microscope. A (living) cell, which leads its life in a liquid environment, must take up the greater part of its food material in the form of solution, and it is the permeability of the superficial protoplasm which will determine the passage of food substances from the surrounding medium into the body of the cell. The immiscibility of the protoplasm with the surrounding fluid shows that the permeability of the membrane must be a limited one. The qualitative permeability can be easily studied in vegetable cells. These present within a cellulose wall a thin layer of protoplasm (the primordial utricle), enclosing a cell sap. If the root hairs of tradescantia be immersed in a 10 per cent. solution of glucose or in a 2 to 3 per cent, solution of salt, a process of plasmolysis takes place. The cell sap diminishes in amount by the diffusion of water outwards so that the primordial utricle shrinks (Kig. 7). On im- mersing the cells in distilled water, water passes into the cell sap until the further expansion of the protoplasmic layer is prevented by the tension of the surrounding cell walls. This behaviour can be explained only on the assumption that the protoplasm is impermeable both to sugar and to salt. but is freely permeable tu molecules of water, i.e. it behaves as a semi- permeable membrane. Similar experiments can be made on animal cells. The most convenient for ihis purpose are the red blood corpuscles. These also shrink when immersed in salt solutions with a greater molecular con- THE STKUCTUKAL BASIS OF THE BODY 23 centration than would correspond to the plasma of the blood from which the corpuscles were derived, whereas if placed in weak salt solutions or distilled water they swell upland burst, discharging their haemoglobin in solution into the surrounding fluid. By comparison of various salts it is found that the strength of each salt solution which is just necessary to cause plasmolysis or haemolysis, as the case may be, is determined entirely by its molecular concentration, i,e. a decinormal solution of sodium chloride will be equivalent 1234 FIG. 7. Vegetable cells, showing varying degrees of plasmolysis. (DE VEIES.) in its effect on the cells to a decinormal solution of potassium nitrate or of potassium chloride. The impermeability of the plasma skin does not apply to all dissolved substances. Overton has found that, whereas this layer is practically impermeable to salts, sugars, and ammo-acids, it permits the easy passage of monatomic alcohols, aldehydes, alkaloids, &c. All these substances are more soluble in ether, oil, and similar media than they are in water. The passage of dissolved substances through a membrane wetted by the solvent depends on the solubility of these substances in the membrane, and Overton therefore concludes that the superficial layer of protoplasmic cells must itself partake of a ' lipoid ' character, and that cholesterin and lecithin probably enter largely into its composition. Thus only those aniline dyes which are soluble in a mixture of melted lecithin and cholesterin have the property of penetrating the living cell, and only these dyes, such as methylene blue, neutral red, can be used for intra vitam staining. For the same reason substances which have the power of dissolving lecithin and cholesterin, such as ether or bile salts, also act as hsemolytic agents, i.e. they cause a destruction of the red blood cells by dissolving the superficial layer which is necessary for their preservation from the solvent effects of the surrounding fluid. The semi-permeability of the plasma skin can be altered by changes in the saline concentration or other factors of the surrounding medium. Over- ton has shown that, whereas a 7 per cent, solution of saccharose produces plasmolysis in living cells, no plasmolysis is observed if they are treated with a solution containing 3 per cent, methyl alcohol plus 7 per cent, cane sugar. The superficial layer, therefore, is able to dissolve a mixture of methyl alcohol and cane sugar, although it has no solvent power on cane sugar in 24 PHYSIOLOGY pure watery solutions. It is possible that, in order to serve the nutrient needs of the cells, more extensive changes may take place in the permeability of the surface layer under limited conditions of time and space. There is no doubt, for instance, that dextrose, to which the surface layer is apparently impermeable, can yet serve as a very efficient food for the cell, and one might ascribe the fact that the cell assimilates only the food which it requires and no more, to such limited changes in permeability. An important factor in the process of assimilation, at any rate by lowly organised cells, must be the relative solubility of the absorbed substances in the cell and its surrounding medium respectively. When a watery solution of iodine is shaken up with chloroform, the latter sinks to the bottom, carrying with it the greater part of the iodine. If a watery solution of organic acid be shaken with ether, the latter fluid will extract the greater quantity of the acid. In no case will the extraction be complete, but there will be a definite ratio between the amount dissolved by the ether and the amount dissolved by the water, the so-called ' coefficient of partage,' depending on the different solubilities of the dissolved substance in the two menstrua. In the same way a mass of protoplasm will tend to absorb from the surrounding medium and to con- centrate in itself all those substances which are more soluble in the colloidal system of the protoplasm than in the surrounding fluid, and this process of absorption may be carried to a very large extent, if the dissolved substances meet in the cell with any products of protoplasmic activity with which they form insoluble compounds so that they are removed from the sphere of action. It is probably by such a process as this that we may account for the accumulation of calcium or silicon in such large quantities in connection with the bodies of various minute organisms. Whereas assimilation by a living cell is ultimately conditioned by the permeability of the surface protoplasm, its form is determined by the tension of this layer. If the tension is uniform at all parts of the surface the form of the cell will be spherical. Any diminution of the surface tensibn at one point must tend to cause a bulging of the fluid contents at this point, just as on distending a rubber tube with one weak spot in its wall this suddenly gives way with the production of a large balloon, which rapidly extends in size and ruptures unless the pressure be diminished. Diminution of the sur- face tension at one point of the cell will be attended by a contraction of all the rest of the surface and a driving out of the contents through the weak part. This process will not as a rule result in destruction of the cell ; the resulting protrusion will be limited by the distortion of the internal alveolar structure of the protoplasm caused by any alteration of the spherical form of the cell. Change of form in living structures thus depends ultimately on alterations in surface tension, return to normal being effected by the elastic reaction of the structural arrangement of the protoplasm. This point we shall have to consider more fully when dealing with muscular contraction. At present it is sufficient to see how any slight alteration in the chemical environment, such as might be due to the presence of a particle of foodstuff, may cause local variations in the surface tension of the plasma -skin and thus THE STRUCTURAL BASIS OF THE BODY 25 result in the protrusion of pseudopodia and the ingestion of the food particle. VITAL PHENOMENA OF CELLS. A. Assimilation. The activity of every living being, whether uni- or multi-cellular, can be regarded as com- pounded of two phases, assimilation and dissimilation. By assimilation we mean the building up of the living substance at the expense of material )btained from the external world. In this process substances are formed of high potential energy, and this energy can be obtained only at the expense either of energy imparted to the system at the moment of assimilation, as, e.g. in the assimilation of carbon from carbon dioxide under the influence of the sun's rays, or of energy contained in the food-stuffs themselves. In all living organisms, except those provided with chlorophyll corpuscles, it is the latter method which is adopted, and a food-stuff therefore connotes some substance which can be taken in by the cell and can serve it as a source of chemical energy. The evolution of energy, which is required for the movements and other vital activities of the cell, is derived from a disintegra- tion or dissimilation of the protoplasm and is generally associated with the process of oxidation. In assimilation, besides the building up of living protoplasm, there may also be a synthesis of more complex from less complex compounds, without their necessary entry into the structure of the living molecule. In the absence of any definite criteria by which we may judge as to the living or non-living condition of parts of the cell, it is a little dangerous to draw any hard-and-fast distinction between these two sets of processes. Assimilation requires the ingestion of food into the organism, and in the second place its digestion, i.e. its solution in the juices of the cells. These two processes are succeeded, through stages which we cannot trace, by an actual growth in the living material. In naked cells ingestion may occur either at any part of the surface, as in the amoeba, or at a specialised portion, so-called ' mouth,' as in many of the infusoria. Digestion is apparently effected in most cases by the production and secretion around the ingested food particle of solutions containing ferments, i.e. agents which have the power of hydrolysing the different foodstuffs and rendering them soluble. In the vast majority of living organisms the energy for their activities is derived from the oxidation, ultimately of the foodstuffs, but immediately of molecules attached to the living protoplasm. A necessary condition, therefore, for the life of these cells is the presence of oxygen in the surround- ing medium, from which it is taken up in the molecular form. We may therefore speak of an assimilation of oxygen ; but it is still a matter of dispute whether the oxygen is built up as such in the living molecule (so-called intra-molecular oxygen) to be utilised for the formation of carbon dioxide when a discharge of energy is necessary, or whether it is taken in only at the moment when the combustion of the carbon and hydrogen constituents of the food or protoplasm is necessary for the supply of energy. However this may be, products are formed as a result of this oxidation which are of no further value to the cell and are therefore excreted, i.e. turned out of the cell. The chief of these are the products of oxidation of carbon and hydrogen, namely, 26 PHYSIOLOGY carbon dioxide and water. There are also many substances resulting from the oxidation of the nitrogenous portions of the protoplasm, which have to be excreted in the solid or dissolved form. Although the assimilation of oxygen is so general a quality of living protoplasm, the presence of this gas, at any rate in the free form, does not seem to be necessary for all kinds of life. Thus a number of the bacteria are known which are anaerobic, i.e. exist only in the absence of oxygen. Examples of such are bacillus tetanus, and the bacillus of malignant oedema. In order to cultivate them it is necessary to dis- place all the air in the cultivating vessels by means of a current of hydrogen. It has been supposed that the ultimate source of the energy of these organisms is also derived from a process of oxidation, and that they differ from other organisms in being able to utilise for this purpose oxygen which is built up into the structure of their food sub- stances. It is possible, however, that these organisms derive the energy for the building up of their protoplasm, for their movements, &c., not from a process of oxidation at all, but from processes of disintegration of the substances which they utilise as food. It is by such means that in all probability the intestinal worms, fairly highly organised animals, are able to exist in the intestine in a medium containing no oxygen, but rich in carbon dioxide. Here they are plentifully supplied with foodstuffs and can afford to adopt a wasteful method of nutrition, in which only a small fraction of the energy is obtained which would be produced by a total oxidation of the food. B. The Phenomena of Dissimilation. The activities of a living cell or organism can be regarded in every case as dependent originally on en- vironmental change, and are adapted to this change, i.e. are of such a nature that they tend to preserve the organism intact, to favour its growth, or pre- vent its destruction. The property of reacting in such a manner to changes in the environment is fundamental to all protoplasm and is spoken of as excitability, and the change which will influence an organism and cause a corresponding adaptive change in it is known as a stimulus. Stimuli may be of various kinds. Thus mechanical, thermal, chemical, electrical changes, light, and so on, may act as stimuli. The reactions which they evoke involve in every case chemical changes in the protoplasm, i.e. changes in the metabol- ism of the cell. Sometimes this change may be assimilatory in character, leading to an increased growth of the protoplasm, or at any rate to a cessation of dissimilation. In such a case the stimulus is spoken of as inhibitory, because it diminishes or prevents the output of energy by the organism. The frequent result of a stimulus is an increased output of energy, which may appear in the form of movement, in the form of heat, or as chemical change. A common feature of all dissimilatory changes evoked by the application of a stimulus is that the energy of the reaction is always many times greater than the energy represented by the stimulus, the excess, of course, being supplied at the expense of the potential energy of the food material which has been stored up in or built up into the living protoplasm. This dispropor- tion between stimulus and reaction can be well illustrated on an excitatory 1 issue such as muscle. Thus in one experiment the gastrocnemius muscle of a frog was loaded with a weight of 48 gms. The nerve running to the muscle was placed on a hard surface and a weight of half a irraimne was allowed to fall upon it from a height of 10 mm. The muscle contracted in response to this mechanical stimulus applied to the nerve and raised the weight 3*8 mm. THE STRUCTURAL BASIS OF THE BODY 27 In this case the work performed by the muscle was 48 X 3*8 = 182 '4 grm. mm., while the potential energy of the stimulus represented only 0*5 X 10 '0 = 5'0 grm. mm. Thus the work performed by the muscle was thirty- six times larger than the energy of the stimulus applied to the nerve. In the case of unicellular organisms, definite classes of motor reaction to stimulus have been described. The ordinary retraction of a unicellular organism, such as the vorticella, in response to a touch is called thigmotaxis. Certain cells are influenced by gravity, tending to rise or fall in the surrounding medium according to the conditions which favour their existence. A similar sensitiveness to gravity is observed in the growing parts of plants, where the root always grows downwards and the stem up- wards. This reaction to gravity is known as geotaxis, which is distinguished as * nega- tive ' or ' positive ' respectively, according as the plant grows hi opposition or in obedi- ence to the gravitational attraction. If growing plants be placed on the rim of a wheel and rotated so that the centrifugal force is greater than that of gravity, the stems all grow towards the centre of the wheel while the rootlets grow outwards. In the same way the reaction of micro-organisms to light is known as phototaxis, some organisms seeking the light while others shun it. Among the primitive reactions of cells perhaps the most important in the life of higher animals are those grouped under the term chemiotaxis. The fertilisation of the ovum in the prothallus of ferns is effected by the penetration of the antherozoids produced in the male organs at some little distance from the female organs. It was shown by Pfeffer that the movement of the antherozoids towards the ova is effected in response to a chemical stimulus, probably malic acid, since he found that antherozoids suspended in a fluid will always swim towards any locality where there is a gre&ter concentration of this acid. In the same way aerobic bacteria are attracted by the presence of oxygen. If such bacteria are present in a solution with an alga, on exposure of the fluid to light there is an evolution of oxygen by the green alga, and a consequent congregation of the bacteria round the seat of production of the oxygen. The movements of the white corpuscles of the blood of the higher animals are also largely determined by their chemical sensibility, and various substances can be divided into (a) those which exercise positive and (&) those which exercise negative chemiotactic influence on the leucocytes. Thus the intro- duction under the skin of an animal of a capillary tube containing a solution of sub- stances of the first class, such as peptone, tissue extracts, or the chemical products of certain bacteria, leads to an accumulation within the tube of leucocytes which pass to it from all the surrounding tissues. Other substances, such as quinine, exert a nega- tive chemiotaxis. Tubes filled with these, after introduction into the subcutaneous tissue of a mammal, will be found many hours later to contain no leucocytes at all. THE RELATIONS OF THE NUCLEUS TO THE CYTOPLASM. The universal existence in living cells of a differentiated nucleus indicates that the life cycle of assimilation and dissimilation must depend on an interaction between the nucleus and cytoplasm, and that each plays a distinct part in the sum of the changes which make up the life of the cell. The different staining reactions of nucleus and cytoplasm suggest a corresponding difference in their chemical composition, a suggestion which is confirmed by analysis. In the building up of protoplasm proteins play an important part. They are not present, however, as simple proteins, but built up with other complex bodies to form conjugated proteins. Whereas in the cytoplasm these conjugated proteins consist chiefly of compounds of protein and lecithin, in the nucleus the chief constituents belong to the class of nucleo-proteins. The nucleo- proteins are of varying composition, and are distinguished chiefly by the 28 PHYSIOLOGY large amount of phosphorus in their molecule. A nucleo-protein can be broken down into nuclein and protein. Nuclein can be broken down into nucleic acid and a protein-like substance, protamine. Nuclei differ among each other and at different periods of their existence or in different conditions of activity according to the greater or less amount of protein which is Fia. 8. Nucleated and non-nucleated fragments of Amaba. (WILSON after HOFER.) A, B. An Amoeba divided} into nucleated and non-nucleated halves, five minutes after the operation. C, D. The two halves after eight days, each containing a con- tractile vacuole. combined with the nuclein. The latter seems to be the essential constituent of cell nuclei and to be present in only small quantities in the cytoplasm. The properties and reactions of these bodies will be dealt with at greater length in the next chapter. In order to appreciate the part played by the nucleus in the ordinary cell processes, we must study the behaviour of cells or parts of cells deprived of a nucleus and compare it with that of similar cells or parts of cells still containing a nucleus. By means of a fine needle it is possible to divide the larger protozoa into two pieces, one with and one without a nucleus. Hofer, experimenting on the amoeba, foimd that the fragment containing the nucleus quickly regenerated the missing part and pursued a normal existence. On the other hand, the non-nucleated fragments showed no signs of regeneration. They might, indeed, live as long as fourteen days after the operation (Fig. 8). THE STRUCTURAL BASIS OF THE BODY 29 Their movements continued for "a short time and then ceased, though the pulsations of the contractile vesicle were but little affected. The power of digestion of food was completely lost. Other observers have shown that Stentor, an infusorium which possesses a fragmented nucleus, may be broken up into fragments of all sizes. Nucleated fragments as small as one-twenty-seventh the volume of the entire animal are still capable of a FIG. 9. Regeneration in the unicellular animal Stentor. (From GRUBEK after BALBIANI.) A. Animal divided into three pieces, each containing a fragment of the nucleus. B. The three fragments shortly afterwards. C. The three fragments after twenty-four hours, each regenerated to a perfect animal. regeneration. The wound quickly heals and the special organs — the mouth, with its surrounding cilia, and the contractile vacuole — are regenerated, but all non-nucleated fragments quickly perish (Fig. 9). Many similar observations have shown that the non-nucleated cytoplasm, though it may survive for some time and perform normal movements in ponse to stimuli, such as those of ingestion of food particles, loses entirely e power of digestion, secretion, and growth. In animals possessing a shell, a small secretion of the lime salts may occur on the surface, but this process apidly comes to an end as the store of material in the cytoplasm is exhausted, vegetable cells it is possible to break up the protoplasm by means of lasmolysis into nucleated and non-nucleated parts. The nucleated part uickly forms a new cell wall. The non-nucleated part is unable to effect is formation, and soon dies unless it is in connection with an adjacent 11 containing a nucleus by means of fine threads of protoplasm which s through pores in the intercellular septa (Fig. 10). In the higher animals res the 30 PHYSIOLOGY we have, in the case of the nerve-cell, an* example of the necessity of the nucleus for growth. Here division of the nerve fibre causes degeneration of the whole fibre separated from the cell containing the nucleus, and regenera- tion of the fibre, when it occurs, is effected by a down-growth of that part of the fibre which is still in connection with the nucleus. All these facts show A m .,..- D FIG. 10. Formation of membranes by protoplasmic fragments of plasmolysed cells. (WILSON after TOWNSEND.) A. Plasmolysed cell, leaf -hair of Cucurbita, showing protoplasmic balls connected by strands. B. Calyx-hair of Gaillardia ; nucleated fragment with membrane, non- nucleated one naked. C. Root-hair of Marchantia ; all the fragments, connected by protoplasmic strands, have formed membranes. Z). Leaf -hair of Cucurbita ; non- nucleated fragment, with membrane, connected with nucleated fragment of adjoining cell. that the power of morphological as well as of chemical synthesis depends on the presence of a nucleus. On this account the nucleus, as we shall learn later on, must be regarded as the especial organ of inheritance. The trans- mission of the paternal qualities from one generation to the nexl is effected by the entrance simply of the nuclear material of the male cell, the spermato- zoon, into the ovum. In Ilie words of Claude Berna'rd,," the functional phenomena in which there is expenditure of energy have their seat in the protoplasm of the cell (i.e. the cytoplasm). The nucleus is an apparat us for organic synthesis, an instrument <>l production, the germ of the cell." Similar conclusions may. be drawn from a study of the changes in the nucleus which accompany different phases in the activity of the whole cell. THE STRUCTURAL BASIS OF THE BODY 31 Thus in growing plant-cells the nucleus is always situated at the point of most rapid growth. In the formation of epidermal cells the nucleus moves towards the outer wall and remains closely applied to it so long as it is growing in thickness. When this growth is finished the nucleus moves to another part of the cell. In the formation of root hairs the outgrowth always takes place in the immediate neighbourhood of the nucleus, which is carried forward and remains near the tip of the growing hair. The active growth of cyto- plasm, which accompanies the activity of secreting cells, is always associated with changes in the position and in the size of the nucleus. Where the nutritive activity of the cell is very intense, as in the silk glands of various lepidopterous larvae, the nucleus is found to be very large and much branched (Fig. 11) so as to present the greatest possible extent of surface through Flo 1L Branohfld ' nucleu8 from which interchanges can go on between the spinning gland of butterfly nucleus and cytoplasm. The important changes which the nucleus undergoes in the process of cell division we shall have to consider more fully in the later chapters of this work. In the function of assimilation it is natural to assume that it is those constituents of the nucleus which are peculiar to it both morpho- logically and chemically, namely, the chromatin filaments, which are most directly concerned. This assumption receives support from the changes which have been observed to occur in these filaments during various phases of nutritive activity of the cell. The staining powers of chromatin are in direct proportion to the amount of nuclein it contains. In the eggs of the shark it has been shown that the chromosomes undergo characteristic changes during the entire growing period of the egg. At first they are small and stain deeply with ordinary nuclear dyes, but during the period of growth they undergo a great increase in size and at the same time lose their staining capacity,* their surface being increased by the development of long threads which grow out in every direction from the central axis. As the egg approaches its full size, the chromosomes diminish in size and are finally reduced to minute intensely staining bodies which take part in the first division of the egg preparatory to its fertilisation (Fig. 12). We must conclude that whereas the processes -of destructive metabolism or dissimila- tion, which determine the activity of the cell, have their immediate seat in the cytoplasm, the processes of constructive metabolism which lead to the „ formation of new material, to the chemical and morphological building up of the cell, are carried out in or by the intermediation of the nucleus. HISTOLOGICAL DIFFERENTIATION OF CELLS. Even within the limits of a single cell, differentiation of structure can take place by the setting apart of distinct portions of the cell for isolated functions. Thus in an organism such as vorticella the cell ia shaped somewhat like a wine-glass, * Riickerfc, cited by Wilson. 32 PHYSIOLOGY the stem being composed of a spiral contractile fibre which has the function of withdrawing the rest of the organism when necessary towards its point of attachment. The main portion of the cell presents at its free extremity a part which is the seat of ingestion of food, and is therefore spoken of as the ' mouth.' This is surrounded by a circle of cilia whose function it is to set up currents in the surrounding fluid and so favour the passage of food parti- cles towards the mouth. Food when ingested at this end passes only a FIG. 12. Chromosomes of the germinal reside in the shark Pristiurus, at different periods drawn to the same scale. (RUCKERT.) A. At the period of maximal size and minimal staining-capacity (egg 3 mm. in diameter). B. Later period (egg 13 mm. in diameter). C. At the close of ovarian life, of minimal size and maximal staining-power. short distance into the body of the vorticella. Here fluid is secreted around it which serves for its digestion. This portion of the cell may therefore be regarded as the alimentary canal or stomach. The indigestible residue of the food is excreted in close proximity to the mouth. In addition to these organs we have the usual differentiation of the protoplasm into an external and internal layer, and the development within the protoplasm of contractile vacuoles which serve to keep up a circulation of fluid and therefore to pass the products of digestion through all parts of the cell body. Within the limits of the single cell which forms the vorticella we may therefore speak of organs for contraction, for digestion, for circulation, and so on. The organs which are thus formed in unicellular animals or plants can be divided into two classes, namely (1) temporary organs, which are formed out THE STRUCTURAL BASIS OF THE BODY 33 of a common structural basis and can therefore be replaced at any time by the cytoplasm if destroyed. Examples of such organs are the cilia, the commonest motor apparatus of unicellular organisms ; the pseudopodia, which, as we have seen, can be made and destroyed at will ; the mouth of animals such as Volvox or Vorticella ; and the stinging cells or nectocysts, which surround the mouth of many of these animals and serve to paralyse or kill the smaller living organisms brought by the cilia within reach in order that they may serve as food. In contradistinction to these organs are (2) a number of others which must be regarded as permanent. These cannot be formed by differentiation from the cytoplasm of the cell, but are derived by the division of pre-existing organs of the same character, and are therefore transmitted from one generation to another. As examples of such cell-organs may perhaps be mentioned the nucleus, with its chromo- somes, and the plastids, of which the chloroplasts of vegetable cells are the most conspicuous. Certain cell organs may fall into either class. Thus the contractile vacuoles are sometimes derived by the division of the pre- existing vacuoles in a previous generation, at other times are certainly formed out of the common cytoplasm. The centrosome, a small particle generally situated in the cytoplasm, which plays an important part in cell-division, is generally derived by the division of a pre-existing centrosome, but under certain conditions and in some organisms can be developed in situ in the cytoplasm itself. The possibility of histological differentiation and of the adaptation of structure to definite functions becomes much more pronounced as we pass from the unicellular to the multicellular organisms or metazoa. The lowest of the metazoa, such as the sponges, consist of little more than an aggregation or colony of cells. All the cells are still bathed with the outer fluid, and any differentiation of structure or function seems to be entirely conditioned by the position of the cell. In the coelenterata the differentiation is already much more marked. The hydra, one of the simplest of the group, consists of a sac formed of two layers of cells and attached by a stalk to some firm basis. Round the mouth of the sac is a circle of tentacles. The inner layer, or hypoblast, represents the digestive and assimilatory layer, while the epiblast, or outer layer, is modified for the purposes of protection, of reception of stimuli, and of motor reaction. In the jelly-fish the differentiation of the outer layers leads to the formation of the first trace of a nervous system, i.e. a system fitted especially for the reception of stimuli and for their trans- mission to the reactive tissues, namely, the muscles. In all these classes of animals the external medium of every cell forming the organism is the sea- water or other medium in which they live. This can penetrate through the interstices between the cells, and every cell is there- fore exposed to all the possible variations which may occur in the composition of the surrounding medium. A great step in evolution was accomplished with the formation of the coelomata, the class to which all the higher animals belong. In these, by the formation of a body cavity containing fluid, an Kternal medium is provided for all the working cells of the body. The com- 3 34 PHYSIOLOGY position of this internal medium is maintained constant by the activity of the cells in contact with it, and the stress of sudden changes in the chemical com- position of the surrounding medium is borne entirely by the outer protective layer of epiblast cells. These are rendered more or less impermeable by the secretion on their surfaces of a cuticular layer, and only such of the con- stituents of the surrounding medium are allowed to enter the organism as can be utilised by it for building up its living protoplasm. Out of the ccelom is later on formed a circulatory system which, by the circulation of the ccelo- mic fluid or of blood through the whole body, can procure a still more per- fect uniformity in the chemical conditions to which every cell is exposed. It is not till much later that the organism achieves an independence of external conditions of temperature. In the mammalia, by means of the reactive nervous system, the heat produced in every vital activity by the chemical changes of combustion and disintegration is so balanced against the heat lost through the external surface to the environment that the temperature of the internal fluid is maintained practically constant. One of the main results of the differentiation of function and structure is therefore a gradual setting free of the majority of the cells of the body from the influence of variations in the environment ; and in the highest type of all animals, in man, this inde- pendence of external conditions is carried to a much further extent by conscious adaptations, such as the use of clothes, dwellings, artificial heating, and so on. The differentiation of the cells which compose the organs of the body is determined in the first place by the different conditions to which they are exposed in virtue of their positions in the course of development. All the higher animals may be considered as built in the form of a tube, the external surface of which is modified for the purpose of defence and for adaptation to changes in the environment. From this layer there are developed not only the protective cuticle, but also the organs of motor reaction, namely, the special senses and the nervous system. The internal surface of the tube is modified for purposes of alimentation. From it are developed all those structures which serve for the digestion of the food-stuffs, for their absorption into the common circulating fluid, for their elaboration after absorption, and their preparation for utilisation by other cells of the body. Between these two surfaces are situated the supporting tissues of the body as well as the organs for the conversion of the potential energy of the body into motion and work, namely, the muscles. Here also is the ccelum or body cavity, repre- sented in the higher animals by the pleural and peritoneal cavities. The alimentary canal projects for a considerable part of its course into this coelum, being attached to the body wall only by one side. From the ccelom is also developed the blood vascular system, surrounded by contractile and con- nective cells which maintain a constant circulation of the blood throughout the body. By this differentiation the body becomes divided into a number of organs, each of which is composed of like cells, modified for a common function and bound together by connective tissue, the latter serving also to carry the blood-vessels which convey the common medium for the working THE STRUCTURAL BASIS OF THE BODY 35 cells. In the study of physiology our task consists, firstly, in the description of the special part taken by each organ in the general functions of the body, and, secondly, in the determination of the limiting conditions of such func- tions and of the physical and chemical factors which determine them. Finally, we have to endeavour to form a complete conception of the chain of events concerned in the discharge of each function and of their causal nexus. In the foregoing lines we have compared the higher animal to a colony of cells, and we often speak of an isolated cell of the body as if it were an independent elementary organism. A better term for such an aggregation of cells as presented by the higher animals is not however ' cell colony,' but * cell state,' since, just as in the state politic, no cell is independent of the activities of the others, but the autonomy of each is merged into the life of the whole. With increasing differentiation there is increasing division of func- tion among the various members of the state, and each therefore becomes less and less fitted for an independent existence or for the discharge of all its vital functions. The more highly civilised a man becomes and the greater his specialisation in the work of the community, the smaller chance would he have of existing on a desert island. Thus the life of the organism is essen- tially composed of and determined by the reciprocal actions of the single elementary parts. It is evident that, if the process of specialisation has gone far enough, a discussion whether each unit has or has not an independent life is beside the mark, since it cannot possibly exist apart from the activities of the other cells. Of late years -histologists have brought forward evidence which seems to imply that an actual structural interaction exists, in addition to the functional dependence which is a necessary resultant of specialisation. Even in the case of plant cells with their thick cellulose walls, fine bridges of protoplasm can be made out passing from one cell to another through pores in the cellulose wall. In animals protoplasmic bridges are known to exist joining up adjacent cells in unstriated muscle, epithelium and cartilage cells, and in some nerve-cells. The conclusion has therefore been drawn that the morphological unit is not the cell, but the whole organism, and that the division of the common cytoplasm into cells is merely a question of size and convenience. There can be no doubt that the determining factor in the division of cells is their growth : the cell divides because it grows. With increased mass of living substance it is necessary to provide for increase of surface both of cytoplasm and of nucleus. Whether all the tissues of the higher animals remain in structural continuity by protoplasmic bridges, &c., must be to us a matter of indifference, since all that is necessary for the interdependent working of the different cells of the body is a functional continuity, and this in the higher animals is effected by the presence of a common circulating fluid and a reactive nervous system connected by conducting strands with all the cells of the body. CHAPTER III THE MATERIAL BASIS OF THE BODY SECTION I THE ELEMENTARY CONSTITUENTS OF PROTOPLASM THE material basis of which living organisms are built up is derived from the surrounding medium, and the elements which compose the framework of the body must therefore be identical with those found in the earth's crust. Not all the elements are so utilised in the formation of living matter. Every living organism without exception contains the following elements : carbon, hydrogen, oxygen, nitrogen, sulphur, phosphorus, chlorine, potassium, sodium, calcium, magnesium, and iron. In addition to these twelve elements others are found in certain organisms, sometimes to a large extent, but it is not known how far they are necessary to the proper development of these organisms, and it is certain that they do not form an integral constituent of all organisms. Of these elements we may mention especially silicon, iodine, fluorine, bromine, aluminium, manganese, and copper. Dealing with the first class, which includes those essential to all forms of life, we find that their relative proportions in living organisms have little or no relation to their proportions in the environment of the organisms. Their presence, however, in the latter is a necessary condition of life. In the case of plants which have a fixed habitat and cannot move in search of food, the growth of the plant is limited by the amount of the necessary element which is present in smallest quantities in the surrounding medium. This is what is meant by the agriculturist's ' Law of the Minimum. ' Of the elements derived from the earth's crust, those present in the smallest amounts in most soils are potassium, nitrogen, and phosphorus. The growth of a crop in any given soil is determined by the amount of that one of these three substances which is present in smallest quantities, and the aim of agriculture is to supply to every soil the ingredient thus present in minimal amount. Carbon forms the greater part by weight of the solid constituents of living protoplasm. The proximate constituents of living organisms are practically all carbon compounds, so that organic chemistry, which was originally the chemistry of substances produced by the agency of living organisms, has come to be synonymous with the chemistry of carbon com- 36 THE ELEMENTARY CONSTITUENTS OF PROTOPLASM 37 pounds. The carbon compounds which make up the living cell are com- bustible, i.e. they can unite with oxygen to form carbon dioxide with the evolution of heat. In the inorganic world practically all the carbon occurs in a completely oxidised form, namely, carbon dioxide. A small amount, 4 parts in 10,000, is present in the atmosphere, while vast quantities are buried in the crust of the earth as carbonates of the alkaline earths, &c., in the form of chalk and limestone. In this condition the carbon dioxide is practically removed from the life cycle, the whole of the carbon contained in the tissue of living beings, whether plant or animal, being derived from the minute proportion of carbon dioxide present in the atmosphere. The energy for the conversion of carbon dioxide into the oxidisable forms with high potential energy, which make up the tissues of plants and animals, is furnished by the sun's rays. The machine for the conversion of the radiant energy into the potential chemical energy of the carbon compounds is represented by the chlorophyll corpuscles in the green parts of plants. In these corpuscles, under the influence of the sun's rays, the carbon dioxide of the atmosphere, together with water, is converted into carbohydrates, viz. starch (C6H1005), and the oxygen liberated in the process is set free into the surrounding atmosphere. 6C02 + 5H20 = C6H1005 + 602. In this process a large amount of energy is absorbed, an energy which can be set free later by the oxidation of the starch to carbon dioxide. In the oxidation of one gramme of starch about 4500 calories are evolved, and this represents also the measure of the solar energy which must be absorbed by the chlorophyll corpuscle in the process of formation of starch from the carbon dioxide of the atmosphere. By this means the world of life is pro- vided with a source of energy. At the expense of the energy of the starch further synthetic processes are carried out. By the oxidation of a part of the carbohydrates, sufficient energy may be supplied to deoxidise other portions of the carbohydrates with the production of fats. Thus 3C6H1206-802-C18H3602 (Glucose) (Stearic acid) The potential energy of a fat is still greater than that of a carbohydrate, one gramme of fat giving on complete combustion to carbonic acid and water as much as 9000 calories. By the introduction of ammonia groups (NH2) into the molecules of fatty acids, amino-acids may be produced, from which the complex proteins are built up to form the chief constituents of the living protoplasm. The synthesis of carbon compounds from the inert carbon dioxide of the atmosphere can be effected only by chlorophyll corpuscles. All animals take in carbon, hydrogen, nitrogen, oxygen, and sulphur in the form of the carbohydrates, fats, and proteins which have been built up in the living plants. In the animal organism these food-stuffs serve as sources of energy. They undergo a gradual oxidation, and finally leave the body in the form of 38 -PHYSIOLOGY carbon dioxide, water, ammonia or some related compound, and sulphates. A sharp distinction has therefore often been drawn between the metabolism of plants and animals, plants being regarded as essentially assimilatoiy in character while animals are dissimilatory, utilising the stores of energy which have been accumulated by the plant. There is however no definite line of demarcation. Although, generally speaking, the green plant breaks up carbon dioxide, giving off oxygen and storing up carbon compounds, and the animal taking in carbon compounds oxidises them with the help of the oxygen of the atmosphere to carbon dioxide, which is redischarged into the surrounding medium and is available for further assimilation by plants, yet this process of respiration is common to all living organisms, whether plants or animals. In the green plant it may be masked by the assimilatory process occurring under the influence of the sun's rays, but in the dark all parts of the plant, and in the light all parts which are free from chlorophyll, display a process of respiration, i.e. they are constantly taking up oxygen from the atmosphere and using it for the oxidation of carbon compounds in their tissues, with the production of carbon dioxide. The sum total of the processes of life tend therefore to maintain a constant proportion of carbon dioxide and oxygen in the atmosphere, the decomposition of carbon dioxide by the green plants being balanced by the oxidation of the carbon compounds and the continual discharge of carbon dioxide by animals. It is not certain however that this balance will be maintained throughout all time. As Bunge has pointed out, there are cosmic factors at work which are apparently tending to cause a constant diminution in the quantity of carbon dioxide in the atmosphere, which alone is of value to the plant. One of these factors is the variable affinity of the silica and carbon dioxide respectively for the chief bases of the earth's crust. At a high temperature silica can displace carbon dioxide from its compounds. Thus chalk heated with silica will give rise to calcium silicate with the evolu- tion of carbon dioxide. At an early geological epoch therefore, it is probable that the greater part of the silica was present in combination with bases and that the proportion of carbon dioxide in the atmosphere was very much higher than it is now. At temperatures at present ruling on the earth's surface carbon dioxide is a stronger acid than silica. The action of water charged with carbon dioxide oh a silicate is to cause its gradual decomposi- tion with the formation of carbonate and silica. Both these products, being insoluble, are deposited as part of the earth's crust, the silica in the form of sandstone, the carbonate as chalk or limestone. The carbon dioxide is being constantly removed by water from the atmosphere and being locked up in this way in the earth's crust, the process of separation of calcium car- bonate being aided to a marked extent by the agency of living organisms themselves. The whole of the extensive deposits of limestone and chalk have been separated from the sea-water by the action of living organisms. With the cooling of the earth's crust which is supposed to be going on, the discharge of carbon dioxide by volcanoes must get less and less, so that one can conceive a time when the whole of the carbon dioxide will be bound up THE ELEMENTARY CONSTITUENTS OF PROTOPLASM 39 with bases in the earth's crust, and life, without any source of carbon, must become extinct. Hydrogen exists almost exclusively in the form of water. In this form it is taken up by plants and animals, with the exception of a small proportion absorbed in the form of ammonia. In this form too it is discharged by living organisms. Oxygen is the only element which, in all the higher organisms at any rate, is taken up in the free state. It forms one-fifth of the atmosphere and, as the oxides of the various metals, a considerable fraction of the earth's crust. It takes a position apart from the other focd-stuffs in that its presence is the essential condition for the utilisation of their potential energy. In the living cells it combines with the oxidisable compounds formed by the agency of the living protoplasm, with the production of carbon dioxide and water, and the evolution of energy. This process is spoken of as respira- tion. Like the three elements we have already considered, nitrogen is also derived directly or indirectly from the surrounding atmosphere. In conse- quence of its feeble combining power for other elements and the instability of its compounds, very little nitrogen is to be found in the combined state in the earth's crust, whereas it constitutes four-fifths of the atmospheric gases. It can be taken up by most plants only in the form of ammonia, nitrites, or nitrates. To animals these compounds are useless, and their only source of nitrogen is the protein which has been built up by the agency of the plant cell. Since nitrogen in the free state is useless to nearly all living organisms, the existence of life must depend on the amount of com- bined nitrogen which is available. In view oj: the small tendency presented by this element to enter into combination, it becomes interesting to inquire into the source of the combined nitrogen which is the common capital of the living kingdom. There are certain cosmic factors which result in the pro- duction of combined nitrogen. The passage of electric sparks or of the silent discharge through moist air leads to the production of ammonium nitrite. N2 + 2H20 = NH4N02. Every thunderstorm therefore will result in the production of small quan- tities of ammonium nitrite, which will be washed down with the rain and serve as a source of combined nitrogen to the soil. Every decaying vegetable or animal tissue serves as a source of ammonia, so that from various causes the soil may contain nitrogen in the form of ammonia or of ammonium nitrite. These forms .of combined nitrogen are not however suitable for all classes of plants. Most moulds can assimilate ammonia as ammonium carbonate or as amino-acids or amines, provided that they are supplied at the same time with sugar, the oxidation of which will serve them as a source of energy. Some moulds, many of the higher plants, and especially the Graminese, which include the food-producing cereals, require their nitrogen in the condition of nitrates. It is necessary therefore that the ammonia or nitrites in the soil shall be converted into this highly oxidised form. This 40 PHYSIOLOGY conversion is effected by a group of micro-organisms. There are a number of bacteria (bacterium nitrosomonas) which have the power of converting ammonia into nitrites. Others (bacterium nitro- monasj convert nitrites into nitrates. If sewage matter rich in ammonia is allowed to percolate through a cylinder packed with coke and the process be continued for several weeks, it is found after a time that in its passage through the filter the fluid has lost its ammonia and contains the whole of its nitrogen in the form of nitrate. If the cylinder be tapped (Fig. 13) half-way down, say at K, the fluid will be found to contain, not nitrates, but nitrites. In this conversion the two kinds of microbes men- tioned above are concerned. At the top of the cylinder the nitrous bacterium is present, in the bottom of the cylinder the nitrate bacterium is present. The conversion of ammonia into nitrates by the agency of bacteria has been made the basis of a method of treatment of sewage which is now very largely employed. These different bacteria play an important part in all soils in preparing them for the cultivation of crops. Is the total capital of combined nitrogen, which is worked over by these bacteria and utilised by the whole living world, confined to the small quanti- ties produced by atmospheric discharges ? Of late years definite evidence has been brought forwrard that such is not the case and that organisms exist which can utilise and bring into combination the free atmospheric nitrogen itself. Thus certain soils have been found to undergo a gradual enriching in nitrogen although no nitrogenous manure has been applied to them. Winogradsky has shown that this fixation of nitrogen by soils is effected by a distinct micro-organism,which may be isolated by growing it on gelatinous silica free from any trace of combined nitrogen, so that the organism has to procure its entire nitrogen from the atmo- sphere. Under such conditions the numerous other micro-organisms of the soil die of nitrogen starva- tion, and only the microbe survives which is able to utilise free nitrogen. This organism, which he called clostridium pasteurianum, grows well on sugar solution if free from ammonia and enriches the solution with combined nitrogen. It is anaerobic, i.e. only grows in the absence of oxygen. In the soil, where oxygen is constantly present, it occurs associated in a sort of symbiosis with two species of bacteria which are aerobic and protect it from FIG. 13. Arrangement for studying the nitrifica- tion of sewage. (Miss H. CHICK.) THE ELEMENTARY CONSTITUENTS OF PROTOPLASM 41 the surrounding oxygen. The mechanism by which this organism is able to fix free nitrogen, and the nature of the first product of the assimilation are not yet ascertained. Such an assimilation will serve to the organism as a source of energy, since the application of heat is necessary for the dissociation either of ammonium nitrite or of nitrous acid into nitrogen and water, as is seen from the following equation ; HN02Aq. + 308 Cal. = H + N + 0, + Aq. NH4N02Aq. + 602 Cal. = 2N + 4H + 20 + Aq. In addition to this spontaneous fixation of nitrogen by humus, a method has long been known to farmers by which the fertility of a soil can be in- creased without the application of nitrogenous manures. If a plot of land is to be left fallow .it is a very usual custom to sow it with some leguminous crop such as sain- foin. Careful experiments by Boussingault, Lawes and Gilbert, and others, have shown that the growth of almost any leguminous crop in a soil poor in nitrogen may result not only in the production of a crop containing much com- bined nitrogen, but also in an actual increase of nitrogen in the soil from which the crop is taken. It was then shown by the last two observers, as well as by Schloesing and Laurent, that the power of a leguminous crop to enrich the soil with nitrogen was dependent on the presence on the roots of certain small nodules which had been described long before by Malpighi (Fig. 14). They showed also that the production of these nodules took place only as a result of infection. Beans grown in sterilised sand produced a plant free from nodules, which however grew very scantily unless nitrogenous manure were added to the sand. Such a crop derived the nitrogen for its growth from the added nitrogen, the total amount of which in the soil was there- fore t diminished by the crop. If however the sterilised sand were treated with an infusion of root nodules from another plant without the addition of any combined vetch with nod- nitrogen at all, the beans developed nodules on their roots ules. and grew luxuriantly, and at the termination of their growth the soil was richer in nitrogen than at the commencement. On microscopic examination the protoplasm which makes up these nodules is found to be swarming with small rods (Fig. 15), and it was shown by Beyerinck that these rods are bacteria and can be cultivated in media apart altogether from the plant. We have thus an example of a class of bac- teria which, like those of humus, are able to assimilate the free nitrogen of the atmosphere, but, unlike them, can only effect this assimilation in a condition of symbiosis, i.e. living in the growing tissues of a leguminous plant. Similar nodules have been described on the roots of other plants which can grow in a 42 PHYSIOLOGY soil free from combined nitrogen, e.y. conifers, but it is in the leguminoeffi that their presence is most widespread. The source of the combined nitrogen, which can be built up by plants into proteins and utilised in this form by animals, is thus not only the ammonium nitrite produced by the agency of electric discharges in the FIG. 15. Section of a root nodule of Dorychnium. (Vi ILLKMIN.) a, cortical tissue ; b, cells containing bacteria. atmosphere, but also the free nitrogen of the atmosphere assimilated by various types of bacteria. Sulphur is found in all soils in the form of sulphates, generally of lime. As sulphates it is taken up by plants. In the plant cell a process of deoxida- tion takes place at the expense of the energy derived either from the starch or, in the case of bacteria, from other ingredients of their food-supply. It is built up, together with nitrogen, carbon, and hydrogen, to form sulphur derivatives and ammo-acids such as cystine, and these, together with <.thcr amino-acids, are synthetised to form proteins. Practically the whole ol tin- sulphur taken in by animals is in the form of proteins. It shares the oxida- tion of the protein molecule in the animal body which it leaves in the foini « .f sulphates. The output of sulphates by an animal can t herel'ore be regarded, like the nitrogen output, as an index of the protein metabolism. It is returned to the soil in the form in which it was taken by the plant, and the cycle can be continuously repeated. Iran, although forming but a minute proportion of the material basis of living organisms (the whole body of man contains only six grammes), is nevertheless indispensable for the maintenance of life. It is necessary, for instance, in two important functions, viz. the formation of chlorophyll in the green plant and the respiratory process in the higher animals. Although iron forms no part of the chlorophyll molecule, plants grown in the absence of this THE ELEMENTARY CONSTITUENTS OF PROTOPLASM 43 substance remain etiolated, but form chlorophyll if the smallest trace of iron is added to the soil in which they are growing or even if the leaves are washed with a very dilute solution of an iron salt. In animals iron forms an essential constituent of haemoglobin, the red colouring-matter of the blood, whose office it is to carry oxygen from the lungs to the tissues. It is probable too that the minute traces of iron in protoplasm exercise an important function in the processes of oxidation which are continually going on. Even in the inorganic world iron plays the part of an oxygen carrier. In the earth's crust it occurs as ferrous salts and as ferric oxide. The ferrous silicate, for instance, may be decomposed by water containing carbon dioxide into silica and ferrous carbonate ; the latter then absorbs oxygen from the atmosphere, liberating carbon dioxide and forming ferric oxide. In the presence of decomposing organic matter, the ferric oxide parts with its oxygen to oxidise the organic substances and is converted once more into ferrous carbonate, and this may be decomposed by the oxygen of the air as before. In the presence of sulphates and decomposing organic matter, ferrous sulphate, which is first formed, undergoes deoxidation to ferrous sulphide, and this may again be oxidised to sulphates and ferric salts on exposure to the atmosphere, so that both the sulphur and the iron act as oxygen carriers between the atmosphere and the organic matter. Iron is obtained by plants from the soil as ferrous or ferric salts. In the protoplasm it is built up into highly complex organic compounds, and in this form is taken up by animals. It is probable that the main requirements of the animal for iron, which are very small, may be satisfied entirely at the expense 'of these organic compounds, but there can be little doubt that the animal can, if need be, also utilise the iron salts present in its food. The animal proceeds extremely economically with its supply of iron. Any excess of iron above that needed to supply the iron lost to the body is excreted almost entirely with the faeces in the form of sulphide. In the soil this undergoes oxidation and returns once more to the form in which it was originally taken up by the plant. Phosphorus is absorbed by the plant as phosphates. In the cell proto- plasm it is built up with fatty acids and other organic radicals to form com- plex compounds such as lecithin, a phosphorised fat, and nuclein, a com- bination of phosphorus with nitrogenous bases of great variety. Both leci- thin and nuclein are essential constituents of living protoplasm. Practically the whole of the phosphorus income of animals is represented by these lecithin and nuclein compounds. After absorption into the animal body they are broken down by processes of dissociation and oxidation, with the pro- duction, as a final result, of phosphates, which are excreted with the urine or faeces and return to the soil. Chlorine, potassium, sodium, calcium, and magnesium are taken up by the plants in the form of salts. Although playing an essential part in all vital processes, they do not seem to be built up into organic combination with the protein, and other constituents of the cell protoplasm. They are therefore taken up also by animals in the form of salts, and as such are again excreted with the urine. 44 PHYSIOLOGY Little is known about the significance, if any, of the other elements which I have mentioned as occasional constituents of living beings. Silicon, which is of universal distribution, is assimilated as silica, probably in colloidal solution, and is distributed in minute quantities through all plant and animal tissues. It forms a very large percentage of the mineral basis of grasses, but even here it does not seem to be indispensable, since these will grow in a medium devoid of silica as luxuriantly as under normal conditions. Fluorine is found in the enamel of the teeth and in minute traces in other tissues of the body. Bromine, though present in quantity in some seaweeds, appears to play no part in the economy of higher animals. Iodine is found in large quantities in many seaweeds and is present as an organic iodine compound in the skeleton of certain horny sponges. An organic iodine compound is also found in the thyroid gland of the higher animals, and may possibly be the active principle by means of which these glands are able to affect the nutrition of the whole body. Iodine, therefore, would seem to be an essential constituent of the higher animals. Aluminium is found in large quantities in certain lycopods. Whether it is essential to their growth is not known. Copper is certainly not a necessary constituent of a large number of plants and animals. In one class, the cephalopods, it appears to take the part of iron in the formation of a blood pigment. The haemocyanine, which was described by Fredericq, plays the same part in the blood of cephalopods that is played by haemoglobin in the blood of vertebrates. When oxidised it is of a blue colour, but gives off its oxygen and is reduced to a colourless compound on exposure to a vacuum. Among these elementary constituents of the body, a definite line of demarcation can be drawn between the carbon and hydrogen on the one hand and all the other constituents on the other. The first two elements are built up in a deoxidised form into the living structure of the protoplasmic molecule. The products of their complete oxidation are volatile, namrly. carbon dioxide and water, and leave the body in these forms. The nitrogen set free by the breaking down of the proteins will pass off as free nitrogen or as ammonia. The sulphuric acid formed by the oxidation of the sulphur combines with the basis to form non-volatile salts. We may therefore divide the ultimate constituents of the body into those which are combustible and are driven off on heating, and those which are left behind as the ash. SECTION II THE PROXIMATE CONSTITUENTS OF THE ANIMAL BODY of the enormous variety of the proximate constituents of living organisms, they are all members or derivatives of three classes of compounds. Since living organisms form the entire food of the animal kingdom, a study of these proximate constituents includes the study of all the food-stuffs. These classes are : (a) Proteins, containing the elements carbon, hydrogen, nitrogen, oxygen, and sulphur ; in some cases also phosphorus. (6) Fats, containing carbon, hydrogen, and oxygen. (c) Carbohydrates, containing carbon, hydrogen, and oxygen, the two latter elements being present in the proportions in which they form water. THE CHIEF TYPES OF ORGANIC COMPOUNDS OCCURRING IN THE ANIMAL BODY The full consideration of the various modifications undergone by these three classes of food-stuffs in the body, especially if we include the by-products occurring both in plants and in animal metabolism, involves a wide knowledge of organic chemistry which indeed at its origin was simply the chemistry of the products of living (i.e. organised) beings. The most important substances with which we shall have to deal belong to a comparatively restricted number of groups. For the convenience of the reader a short summary of the relationships of these groups to one another and to the hydrocarbons is given here. THE HYDROCARBONS (FATTY SERIES). These form a continuous homologous series, and may be saturated or unsaturated. Examples of the saturated series are CH4 methane C2H6 ethane C3H8 propane C4H10 butane, and so on, the general formula for the group being CnH2n + 2. These paraffins, the lower members of which are gaseous, while the higher members form the petroleum ether, the heavy petroleums, vaseline, and the paraffin wax with which we are all familiar, are entirely inert in the animal body. If taken with the food they pass through the alimentary canal unchanged. In order to render them accessible to the action of the living cell they must first undergo oxidation. The unsaturated hydrocarbons have the general formulae C^H^, CnH2o_2> .4, &c. 45 46 PHYSIOLOGY Examples of the first two groups are ethylene CH2 II CH2 and acetylene CH CH Derivatives of all these groups occur in the body. THE ALCOHOLS. The first product of the oxidation of hydrocarbons is the series of bodies known as the alcohols. Examples of these are : CH3OH methyl alcohol C2H6OH ethyl C3H7OH propyl C4H9OH butyl C5HUOH amyl C6H13OH capryl „ and so on, the general formula for the group being CuH2n + iOH. In all these alcohols the OH group is, so to speak, more mobile than the other atoms connected with the carbons, and can therefore be replaced by other substances or groups with comparative ease. In this respect therefore an alcohol can be compared to water HOH or to alkaline hydroxide NaOH or KOH. The best-known example of the group is ethyl alcohol, the ordinary product of fermentation of sugar. In these alcohols the H of the OH group can be replaced by Na. Thus, water with metallic sodium gives sodium hydroxide and hydrogen as follows : 2HOH + 2Na = 2NaOH + H2. In the same way alcohol treated with metallic sodium gives off hydrogen, and the remaining fluid contains sodium ethylate, thus : 2C2H5OH + 2Na = 2C2H5ONa + H2 (sodium ethylate) On the other hand, the OH group may be replaced by acid radicals. Thus, if ethyl alcohol be treated with phosphorus pentachloride, ethyl chloride is1 formed together with phosphorus oxychloride and hydrochloric acid. Thus : Et.OH + PC15 = POC13 + Ha 4- Et.Cl (ethyl chloride) With concentrated sulphuric acid the reaction is similar to that which obtains between sodium hydrate and this acid, and we have formed ethyl hydrogen sulphate and water. Thus: Et.OH + H2SO4 - Et.HSO4 + HOH If alcohol be warmed with acetic acid and strong sulphuric acid, among the products of the reaction is ethyl acetate, which is volatile, and therefore passes off. Thus : Et.OH + HC2H302 = Et.C2H302 + HOH. These compounds of the hydrocarbon group of the alcohol, such as methyl, ethyl, propyl, &c., with an acid, in which the ethyl takes the part of a base, are known as esters. An ester treated with an alkali is decomposed with the formation of an alkaline salt of the acid, and the corresponding alcohol which, being volatile, is given off on warming the mixture. Thus : Et.C2H302 + NaHO = NaC2H302 + Et.OH. (ethyl acetate) (potassium acetate) (alcohol) This process of decomposition of an ester with the formation of the alkaline salt of an acid is often spoken of as saponification, i.e. soap formation, though the term ' soap ' PROXIMATE CONSTITUENTS OF THE ANIMAL BODY 47 is applied only to the compounds of alkalies with the higher fatty acids. The series of alcohols we have just dealt with containing one OH group replaceable by metals or acid radicals are known as monatomic alcohols. If in the molecule of the paraffin two or more atoms of hydrogen have been replaced by the group OH, we speak of diatomic or polyatomic alcohols. Thus, derived from the paraffin propane C3H8 we may have the monatomic alcohol C3H7OH, propyl alcohol, or the triatomic alcohol C3H5(OH)3, which is known as glycerin, or glycerol. Other alcohols of physiological importance are cholesterol and cetyl alcohol. Cho- lesterol is a monatomic alcohol with the formula C27H45OH. It is very complex in structure, and belongs to the aromatic series. Recent work points to an affinity of cholesterol with the terpenes, which have hitherto been found only as the product of the metabolism of plant cells. Cholesterol is a constant constituent of protoplasm. It occurs in large quantities in the medullary sheath of nerves; it is a normal con- stituent of bile and may form concretions (biliary calculi) in the gall bladder. In combination with fatty acids it is an important constituent of sebum and of wool fat. CH3 I Another alcohol — cetyl alcohol — Ci6H340 = (CH2)14 occurs in the feather glands of I CH2OH the duck and forms an important constituent of the wax, spermaceti, obtained from a cavity in the skull of the sperm whale. ALDEHYDES. By oxidation of any of the alcohols wre obtain another group of compounds — the aldehydes. From ethyl alcohol, for instance, by warming with potas- sium bichromate and dilute sulphuric acid, ethyl aldehyde is produced and given off. In H /H . I these aldehydes the group C -H is converted into the group C = O, and it is the ' I OH | possession of this group which determines the aldehyde character of any compound, as well as the reactions which are typical of this class of compounds. Some of the typical reactions of aldehydes may be here shortly summarised : (1) They act as reducing agents, the CHO group being converted into the group COOH, which is distinctive of an acid. We may therefore say that on oxidation aldehydes are converted into the corresponding fatty acids as follows : CH3 CH3 I +0= | CHO COOH (ethyl aldehyde) (acetic acid) On account of the ease with which this oxidation takes place, aldehydes act as strong reducing agents. Warmed with an alkaline solution of cupric hydrate, they take up oxygen, reducing the cupric to a red precipitate of cuprous hydrate. If warmed with an ammoniacal solution of silver (i.e. silver nitrate solution to which ammonia has been added until the precipitate first formed is just redissolved), they reduce the silver nitrate with the formation of a mirror of metallic silver on the surface of the glass vessel in which they are heated. (2) On warming with phenyl hydrazine, they give the typical compounds, hydra- zones and osazones, which are also given by the sugars and will be mentioned in connection with these bodies. (3) They also form addition products. With ammonia, they yield the group of compounds known as aldehyde ammonia. Thus : CH3 CH3 | + NH3 = | NH2 CHO C^H NOH 48 PHYSIOLOGY With sodium hydrogen sulphite the following reaction takes place : CH3 CH3 | + NaHSO CHO SO3Na These compounds of aldehydes with sodium sulphite can be readily obtained in a crystalline form and furnish a convenient means of separating the aldehydes from their solutions. (4) All the aldehydes possess a strong tendency towards polymerisation. Ethyl or acetic aldehyde treated with strong sulphuric acid gives the compound paraldehyde. Thus: 3C2H40 C6H1203. (acetic aldehyde) (paraldehyde) If warmed with strong potash the polymerisation occurs to a still further extent with the formation of resinous substances of unknown composition, but at any rate of a very high molecular weight, the so-called ' aldehyde resin.' Formic or methyl aldehyde, CH20, may in the same way undergo polymerisation with the formation of a mixture of substances belonging to the group of sugars, namely, the hexoses, as follows : 6CH2O = C6H1206. This formation of sugar from formic aldehyde probably plays an important part in the assimilation of the carbon from the carbonic acid of the atmosphere by the green parts of plants. ACIDS. By the oxidation of the group CHO of the aldehydes we obtain the group COOH, which is characteristic of an organic acid. Thus, formic aldehyde on oxidation gives the compound HCOOH, formic acid. Ethyl or acetic aldehyde, CH3CHO, with an atom of oxygen, gives the compound CH3COOH, acetic acid. CHS CH3 I +0= | CHO COOH. Since these acids are derived from the paraffins a whole series of them exists corre- sponding to the series of paraffins, and known as the fatty acids. Examples of this group are : Formic acid Acetic acid Propionic acid Butyric acid HCOOH CH3 CH3 CH3 I I I COOH CH2 CH2 I I COOH CH2 I COOH. In addition to these fatty acids, there are also unsaturated acids, derived from the unsaturated hydrocarbons. DERIVATIVES OF THE FATTY ACIDS AMINO- ACIDS are derived from the fatty acids by the replacement of one atom of hydrogen by the group NH2. Thus from propionic acid we may have: CH2NH2 CH3 I I CH2 or CH.NH2 I I COOH COOH. 13 « The second form, the a-amino acid, is the only one which occurs in the body. PKOXIMATE CONSTITUENTS OF THE ANIMAL BODY 49 OXY ACIDS are formed by the replacement of one H atom by the group OH. Thus: CH3 I CHOH is oxypropionic acid or lactic acid. I COOH KETO-ACIDS. Oxyacids are formed by the oxidation of the group CH2 or CH3 If at the same time the H2 group be removed by oxidation a keto-acid may be formed. This is probably the manner in which such acids arise in the body, though it is more usual to regard a keto-acid as the result of oxidation of a ketone. Thus : CO CO CO I I I CH3 CH2.OH • COOH (acetone) (pyruvic acid — a keto-acid) ACID" AMIDES are formed from a fatty acid by replacing the — OH of the — COOH group by — NH2, e.g. : CH3 CH3 . | from | CO.NH2 COOH. (acetamide) (acetic acid) AMINES. These may be regarded as formed from ammonia NH3 by replacing one or more of the H atoms by an organic radical. Thus we may have : CH3 CH3 /CH3 N^H N(CH3 N^CH3 XH XH XCH3 (methylamine) (dimethylamine) (trimethylamine) Under the action of living organisms primary amines may be formed from a-amino acids by a process of decarboxylation. Thus : CH3 CH3 I I CH.NH2 - C02 = CH2.NH2 COOH (a-amino-propionic' acid) (ethylamine) AROMATIC COMPOUNDS These all contain a nucleus, made up of six carbon atoms, which is extremely stable, so that processes of oxidation, reduction, &c., can be carried out in the compound without destruction of the nucleus. The simplest aromatic compound is benzene C6H6. It behaves as a saturated compound. It is represented as a hexagon with a hydrogen atom at each angle. H H 50 PHYSIOLOGY All the hydrogen atoms are of equal value. They may be replaced by other groups, such as OH, Cl, NH2, or by more complex groups belonging to the fatty series, e.g. CH3, C2H5, &c. Monosubstitution derivatives, exist only in one form : C6H6.X Disubstitution compounds exist in three forms, according to the relative position of the substituted H atoms. These are known as the ortho, meta, and para compounds, and have the formulae : H The following are some of the most important monosubstitution derivatives of benzene : Nitrobenzene C6H5.N02. Aniline C6H5.NH2. Benzene sulphonic acid C6H6.SO3H. Phenol C6H5.OH. Toluene C6H5.CH3. . Benzyl alcohol C6H6.CH2OH. Benzylaldehyde C6H5.CHO. Benzoic acid C6H6.COOH. Of the disubstitution compounds, we need mention only the following : The dihydroxybenzenes : Pyrocatechin or catechol Resorcinol Hydroquinone OH OH OH .....-• i 10H ortho- meta- OH 1 \X)OH. Salicylic acid (o -hydro xybenzoic acid) C6H / Tyrosin (parahydroxyphenyl alanino) : OH CH2.CH(NH2)COOH. 0 Examples of trisubstitution derivatives of benzene are : OH Pyrogallol PROXIMATE CONSTITUENTS OF THE ANIMAL BODY 51 OH Homogentisic acid CHL.COOH Adrenaline OH CH.OH I CH2.NH(CH3) OH Picric acid OPTICAL ACTIVITY Most of the compounds produced by the agency of living organisms exhibit optical activity, i.e. have the property of rotating the plane of polarised light either to the right or to the left. / In an ordinary wave of light the vibrations of the waves take place in all planes perpendicular to the direction of its propagation. When such a ray is passed through a Nicol's prism (made of Iceland spar) it emerges as a plane polarised beam, i.e. waves in one plane only are transmitted. Another Nicol's prism will allow such a ray to pass only if it is parallel to the first prism. If it is rotated through a right angle, no light will pass. A Nicol's prism may thus be used to determine the plane of polarisation of any beam of light. In the polarimeter two Nicol's prisms mounted parallel to one another are employed. One of them (the polariser) is fixed ; the other (the analyser) can be rotated round FIG. 16. Diagram of polarimeter. B, polariser ; D, analyser ; O, tube containing solution under examination. the axis of the beam of light passing through the first. When both prisms are parallel light passes through the analyser. On interposing a solution of an optically active substance between the two prisms, the plane of polarisation of the beam is rotated, so that the light passing through the analyser is diminished. The light may be brought to its original intensity by rotating the analyser either to the right (clockwise) or to the left. In this way the direction and degree of the optical activity may be determined. Optical activity is connected with the molecular arrangement of the substance exhibiting this property, and depends on the presence of one or more ' asymmetric carbon atoms ' in the molecule. CH3 CH3 Thus in lactic acid H.COH, or in alanine HC.NH2, the middle carbon atom is \ \ COOH COOH asymmetric, i.e. it is unequally loaded on the four sides. 52 PHYSIOLOGY We can imagine such a carbon atom as occupying the interior of a tetrahedron. B R3 K3 Fig. 17 In this tetrahedron, if we represent the four groups combining with the carbon by Rx, R2, R3, R4, they can be arranged either as in A or B. It is evident that no amount of turning about will convert the tetrahedron A into tetrahedron B, but that, if we hold A before a mirror, its image in the mirror will be represented by B. One arrange- ment is therefore the mirror image of the other, and a compound containing one such carbon atom will be capable of existing in two forms, namely, one form corresponding to A, the other form corresponding to B. It is found that the unequal loading of the carbon atom, which is present in such an asymmetric arrangement, causes the com- pound containing the asymmetric carbon to have an action on polarised light. One of the varieties will rotate polarised light to the right, while its mirror image ivill rotate polarised light to the left. A mixture of equal parts of the two compounds will rotate equally to left and right, i.e. will have no action on polarised light. The variety rotating to the right is dextrorotatory, and the other laevorotatory,* while the mixture of the two is known as the racemic or inactive variety. The three forms are said to be stereoisomeric, and are distinguished as the d, I, and t forms respectively. If two asymmetric carbon atoms are present in a compound, we may have four stereoisomers ; 'and generally if there are n asymmetric atoms in a molecule, there will be 2W possible stereoisomers. These will not all be necessarily optically active, since the dextrorotation due to one asymmetric carbon atom may be exactly neutralised by the laevorotation due to another, so that ' internal compensation ' takes place and the substance is optically inactive. Thus in tartaric acid four forms are known, namely, d, I, racemic or », and meso tartaric, also inactive, in which internal compensation occurs. These four varieties may be represented as follows : COOH COOH COOH HCOH HOCH HCOH HOCH HCOH HCOH COOH COOH COOH rf-tartaric acid Z-tartaric acid mesotartaric acid inactive tartaric acid Several methods may be employed to separate the racemic form into its two optically active components. One of these methods, first employed by Pasteur, is to grow moulds in the solution. One of the optical isomers is destroyed, leaving the other unchanged. Another method is the fractional crystallisation of the salts with alkaloids, e.g. strychnine in the case of lactic acid. * The 'specific rotatory power of a substance is equal to the number of degrees through which the plane of polarisation is rotated when it passes through a 100 per cent, solution of the substance in a tube 1 decimetre long. Thus polarised light passing through such a tube of 10 per cent, glucose solution would show a rotation of 5-25 degrees, i.e. its specific rotatory power is -f 52-5. SECTION III THE FATS THESE substances are widely distributed throughout the animal and vege- table kingdoms. In the higher animals they are the main constituents of the fatty or adipose tissue lying under the skin and between the muscles, and often forming large accumulations around the viscera. In the marrow of bones they may amount to 96 per cent. They also occur in fine particles distributed through the protoplasm of cells and probably also in combination with the other constituents which make up protoplasm. Large amounts are also found in certain members of the vegetable kingdom, as, for instance, in the fatty seeds and nuts, e.g. linseed, olives, Brazil nuts. CHEMISTRY OF THE FATS The fats are esters of glycerol and the fatty acids. Glycerol is a trihydric or triatomic alcohol and can therefore form esters with one, two, or three of its hydroxyl groups ; thus with acetic acid the following compounds are known : (1) (2) CH2OH CH2OH I I CHOH CH— 0— OC.CH3 I I CH20— OC.CH3 CH2OH a-monacetin /3-monacetin monoglycerides (3) (4) (5) CH2— 0— OC.CH3 CH2OH CH2— 0— OC.CH3 I ' I I CHOH CH— 0— OC.CH3 CH— 0— OC.CH3 I I I CHa— 0— OC.CH3 CH2— 0— OC.CH3 CH2— 0— OC.CH3 a, a diacetin a, /3 diacetin triacetin x .- ' triglyceride diglycendea In these compounds the phenomenon of isomerism occurs owing to the presence of primary and secondary alcohol groups in glycerol. In the case 53 54 PHYSIOLOGY of the diglycerides and the triglycerides mixed esters, in which the fatty acid radical varies, are possible : (6) (7) CH2— 0— OC.CH8 CH2OH I I CHOH CH— O— OC.CH3 I I CH2— 0— OC.CH2.CH3 CH2— 0— OC.CH2CH3 CH2— O— OC.CH3 I CH— 0— OC.CH2.CH3 I CH2— 0— OC.CH2.CH2.CH3 The glyceryl esters which compose the fatty material of living matter — whether animal or plant — are mainly triglycerides, the monoglycerides and diglycerides being seldom found in -nature. The natural fat is usually found to consist of a mixture of triglycerides ; these triglycerides, instead of being mixed esters as in formula (8), are generally simple esters as in formula (5). The differences in the composition of the natural fats depend therefore on the variety of the fatty acid radical combined with the glycerol. The fatty acids which enter into the composition of the triglycerides belong to two main homologous series : A. The saturated fatty acids, namely : Formic acid, H.COOH Acetic acid, CH3.COOH Propionic acid, CH3.CH2.COOH Butyric acid, CH3.CH2.CH2.COOH Valerianic acid, CH3.(CH2)3.COOH Caproic acid, CH3.(CH2)4.COOH Caprylic acid, CH3.(CH2)6.COOH Capric acid, CH3(CH2)8.COOH Laurie acid, CH3(CH2)10.COOH Myristic acid, CH3(CH2)12.COOH Palmitic acid, CH3(CH2)14.COOH Stearic acid, CH3(CH2)16.COOH Arachidic acid, CH3(CH2)18.COOH Behenic acid, CH3(CH2)20.COOH Lignoceric acid, CH3(CH2)22.COOH B. The unsaturated fatty acids, namely : (1) Acrylic series, e.g. oleic acid (CnH2n.202) (2) Linoleic series, e.g. linoleic acid (CnH2n.402) (3) Linolenic aeriee e.g. linolenic acid (CnH2n.602) Of the long list of fatty acids given above only a few occur to any extent a, THE FATS 55 in the animal body. In milk, although the greater part of the fat consists of the triglycerides of oleic, palmitic, and stearic acids, other members of the series given above are present in small amounts. On the other hand, the adipose tissue, strictly so called, consists almost exclusively of the fats de- rived from the fatty acids, palmitic, stearic, and oleic, i.e. tripalmitin, tri- stearin, and triolein. The great differences in the appearance of the fat of different animals are due to the varying amounts in the relative quantities of these three fats which may be present. While triolein is liquid at 0° C., tri- stearin and tripalmitin are solid at the temperature of the body. According to the relative amounts of these three substances therefore, we may have a fat which like mutton suet is solid at the body temperature, or a fat con- taining much olein which is still fluid and runs away when the body is opened after death, even when it has already cooled. PROPERTIES OF THE FATS. The fats are colourless substances evoid of smell. They are insoluble in water, in which they float. They are soluble in warm absolute alcohol, but separate out into crystalline form on cooling. They are easily soluble in ether. If they are strongly heated with potassium bisulphate they give of! pungent vapours of acrolein derived from the decomposition of the glycerin of their molecule. C3H5(OH)3 - 2H20 = C3H40 If they are heated with water or steam or submitted to the action of certain ferments, they undergo hydrolysis, taking up three molecules of water, and are split into three molecules of fatty acid and one molecule of glycerin, e.g., C3H5(C16H3102)3 + 3H20 - 3HC16H3102 + C3H5(OH)3 (neutral fat — tripalmitin) (palmitic acid) (glycerin) This process may occur spontaneously when fat is left exposed to the air. Fat which has been artificially split in this way is said to be rancid. Most natural fats generally contain a small amount of fatty acid which gives them an acid reaction. On boiling a neutral fat for a long time with an aqueous solution of potassium or sodium hydrate, or better still with an alcoholic solution of potassium or sodium ethylate, the fat undergoes saponification, giving the alkaline salt of a fatty acid and glycerin. The former compound is spoken of as a soap. In water the soaps form a sort of pseudo-solution on heating which sets to a solid jelly on cooling. From a dilute watery solution the soap can be thrown down in the solid form by the addition of neutral salts. Fats are insoluble in and non-miscible with water. If shaken up with water the droplets rapidly run together and rise to the surface, forming a continuous layer of the oil or fat. The same thing happens if an absolutely neutral fat be shaken up with a dilute solution of sodium carbonate. If however the fat be slightly rancid, i.e. if fatty acid be present, the latter combines with the alkali with the expulsion of C02 to form a soap. The presence of soap in colloidal solution in the water at once diminishes or abolishes the surface tension between the neutral fat and the water. Like many other colloidal 56 PHYSIOLOGY solutions, a soap solution presents the phenomenon of surface aggregation, i.e. the concentration of the soap at the surface is increased to such an extent as to form practically a solid pellicle of molecular dimensions on the surface of the fluid. The same pellicle formation occurs at the surface of every oil globule, so that on shaking up rancid oil with dilute sodium carbonate, the whole of the oil is broken up into fine droplets, which show no tendency to run together again and remain in suspension in the water. The suspension of fine oil droplets, which has the appearance of milk, is spoken of as an emulsion. It can be at once destroyed by adding acid. This decomposes the soap, setting free the fatty acids, which are insoluble in the water. The pellicle around each globule is destroyed, and the globules run together as neutral oil would in pure water. In order to characterise any given animal fat or mixture of fats the following reactions are made use of: (1) The ' acid number ' of the fat, i.e. its content in free fatty acids, is determined by titrating it in ethyl alcohol solution — with alcoholic solution of potash, using phenolphthalein as an indicator. (2) The ' saponification number.' This represents the number of milligrammes of potassium hydrate necessary to saponify completely one gramme of fat. (3) The percentage of volatile fatty acids is determined by saponifying the fat, then treating it with a mineral acid to set free the fatty acids and distilling over the volatile acids in a current of steam. (4) _fThe iodine number is the amount of iodine which is taken up by a given weight of fat. It is a measure of the amount of unsaturated fatty acid present, i.e. in ordinary fat, oleic acid. Besides the glycerides, a certain number of substances occur in the body derived, not from a combination of fatty acids with glycerol, but from a formation of esters of the fatty acids and other alcohols, e.g. cholesterol or cetyl alcohol. Thus, spermaceti is a mixture of cetyl palmitate with small quantities of other fats. The fatty secretion of the sebaceous glands in man and the higher animals, which furnishes the natural oil of hair, wool, and feathers, consists of cholesterol esters with small traces of glycerides. Lanoline, which is purified wool fat, consists almost entirely of cholesteryl stearate and palmitate. These cholesterol fats are attacked with extreme difficulty by ferments or micro-organisms. It is probably on this account that they are manufactured in the body for protective purposes. So far as we know, when once formed, they are incapable of further transformation in the body. They are not appreciably altered by the digestive ferments of the alimentary canal, and the cholesterol is said to pass through the latter unaltered.* Cholesterol is also found in combination with fatty acids in every living cell. Whenever protoplasmic structures are extracted with boiling ether, a certain amount of cholesterol is present with the fats which are so extracted. In view of the great stability of this substance when exposed to the ordinary mechanisms of chemical change in the body, it seems probable that the part played by cholesterol is that of a framework or * According to Gardner, cholesterol may be absorbed from the intestine. I THE FATS 57 skeleton, in the interstices of which the more labile constituents of the protoplasm can undergo the constant cycle of changes which make up the phenomena of life. PHOSPHOLIPINES OR PFOSPHATIDES The fats form the chief constituent of the deposited and reserve fat throughout the animal kingdom and are also contained in the protoplasm of the living cell. The chief fatty constituents of protoplasm differ from the above fats in the following particulars : they contain phosphoric acid and an amine. On this account they have been called phosphorised fats. Thudi- chum, who isolated various compounds of this nature from brain, suggested the term phosphatides as a general name for them. The term lipoid has also been used, but it includes all the substances composing a cell which are soluble in ether, e.g. cholesterol, cetyl alcohol, and the fats. Leathes has suggested the term phospholipine for those compounds, for it denotes that the com- pound is partly fat (lip), that it contains phosphorus, as well as a nitrogenous basic radical (ine). The phospholipines comprise the substances lecithin, cephalin, cuorine, sphingomyeline. In brain and other tissues similar com- pounds, which contain no phosphorus, occur, and in the place of glycerol we may find galactose. Leathes has proposed calling these compounds lipines and galactolipines. Lecithin, the chief phospholipine, is an ester compounded of two fatty acid radicals, phosphoric acid, glycerol, and the amine, choline. The various lecithins may be distinguished, according as they contain different fatty acid radicals, as oleyl-lecithin, steaTyl-lecithin. The following formula represents distearyl-lecithin : CH2— 0— OC.(CH2)16CH3 I CH— 0— OC.(CH2)16CH3 I CH2-0V .0 O.CH2.CH2.N(CH3)3 I OH On warming with baryta water lecithin is broken down into fatty acid, glycerophosphoric acid, and choline. The. latter base, which is trimethyl- (C2H4OH oxethyl-ammonium hydrate, N \ (CH3)3 must be distinguished from (OH (C2H3 neurine, N UCH3)3 which is trimethyl- vinyl-ammonium hydrate, and is (OH much more poisonous than choline. Choline forms a salt with hydrochloric acid which, with platinum chloride, yields a double salt of characteristic crystalline form, insoluble in absolute alcohol. The universal distribution of lecithin seems to indicate that it plays an important part in the metabolic 58 PHYSIOLOGY processes of the cell. There is no doubt that it may serve, inter alia, as a source of the phosphorus required for building up the complex nucleo-proteins of cell nuclei. It seems to represent an intermediate stage in the utilisation of neutral fats by protoplasm, and its occurrence in the brain as a constituent of more complex molecules, which contain also a carbohydrate nucleus (galactosides, such as cerebrin), might be interpreted as indicating some share also in the metabolism of carbohydrates. Lecithin may be extracted from tissues by boiling with absolute alcohol. On cooling the alcoholic extract in a freezing mixture, the lecithin separates out as granules or semi-crystalline masses. When dried in vacuo, it forms a waxy mass, which melts at 40° to 50° C. In water it swells up. to form a paste which, under the microscope, is seen to consist of oily drops or threads, the so-called myelin droplets. In a large excess of water it forms an emulsion or a colloidal solution. Its power of taking up water on the one hand, and its solubility in alcohol and similar media on the other, give it an intermediate position between the water-soluble crystalloids and the insoluble fats, and enable it to play an important part both as a vehicle of nutritive substances and as a constituent of the lipoid membrane, which bounds and determines the osmotic relationships of all living cells. The phospholipines are provisionally classified according to the proportions of N and P in their molecule, as follows : (a) Mono-amino-monophosphatides, N : P= 1 : 1 (including lecithin and cephalin). (6) Diamino-mono-phosphatides, N : P = 2 : 1 (e.g. sphingomyelin). (c) Mono-amino-diphosphatides, N : P = 1 : 2 (e.g. cuorin, a lipine extracted from heart muscle by Erlandsen). (d) Diamino-diphosphatides, N : P*= 2 : 2. (e) Triamino-monophosphatides, N : P =3:1 (an example has been reported as occurring in egg yolk). All these bodies (except cuorin) are obtained by the extraction of the brain or of nerve fibres. Many also occur in egg yolk. The galacto-lipines include two substances extracted from the brain, viz. phrenosin and kerasin. Both these on decomposition yield galactose, a nitrogenous base called sphingosine and a fatty acid. We know little or nothing of their significance. SECTION IV THE CARBOHYDRATES THE carbohydrates are a group of bodies of wide distribution and great importance in both the vegetable and animal kingdoms. In plants the first product of assimilation of carbon is a carbohydrate, and in animals these substances form one of the most important sources of energy. They consist of the elements carbon, hydrogen, and oxygen, the two last-named being almost invariably in the proportions necessary to form water. It is on this account that the term carbohydrate has been given to the group. Their general formula might be expressed CnH2nOn. Certain derivatives of the group, obtained by the substitution of methyl and other radicals for a hydrogen atom, though necessarily classified with carbohydrates on account of their reactions, do not conform to this general formula, e.g. rhamnose, C6H1205. All the carbohydrates which are of importance in the animal economy contain six carbon atoms or a multiple of this number. Analogous substances however can be prepared containing less or more than this number of carbon atoms. A series of compounds exist which contain in their molecule 2, 3, 4, 5, 6, 7, 8, 9 carbon atoms, and are termed dioses, trioses, tetroses, pentoses, hexoses, heptoses, and so on ; the termination ' ose ' with the Greek numeral prefixed, indicating the number of carbon atoms, gives them a distinct designation. These are all oxidation products of polyatomic alcohols, being either ketones or aldehydes of these alcohols. Thus from COH I jlycerol we may obtain glyceryl aldehyde CHOH and dioxy acetone I )H2OH CH2OH I ). Both these substances behave as sugars and belong to the group of I [2OH -ioses. They are generally obtained together and are called glycerose. CH2OH 'rom the hexatomic alcohol (CHOH)4 we may obtain either the aldehyde I CH2OH 59 60 PHYSIOLOGY CH2OH I COH CO I I (CHOH)4 or the ketone (CHOH)3. These .two oxidation products of the I I CH2OH CH2OH polyatomic alcohols are known as aldoses and ketoses respectively. All these compounds are distinguished by the termination ' ose.' It is convenient to call those compounds containing six carbon atoms the sugars, because it is to this group that the natural sugars belong. Stereoisomerism in the Sugars. It will be noticed that of the six carbon CH2OH I atoms contained in the sugar molecule, e.g. the aldose (CHOH)4, four are COH asymmetric, i.e. their four combining affinities are saturated with groups of different kinds, viz. several carbon atoms, one H atom, and one OH group . C I H— C— OH They must therefore present many stereoisomeric forms. If n represent the number of asymmetric carbon atoms in a compound, the possible number of stereoisomers is 2n. Thus an aldehexose with four asymmetric carbon atoms (CHOH)4 must present 24 isomers, i.e. sixteen isomeric compounds, so that there must be sixteen sugars all possessing the formula CH2OH(CHOH)4COH, iri'addition to the inactive sugars obtained by a mixture of two oppositely active members of this group. Of the sixteen possible sugars of this formula, as many as fourteen have been found or have been artificially prepared. Only a small number are however of any physiological importance. These include the aldoses, glucose, mannose, and galactose, and the ketose, fructose or levulose. All the other sugars are unassimilable by the animal cell and are not manufactured by plants. Since these sugars can be divided into the optically active and the inactive varieties, an obvious mode of designation would be to represent them as d-, 1-, and i- varieties respectively, i.e. dextro-rotatory, Isevo-rotatory, and inactive. On Fischer's suggestion however, this mode of nomenclature has been altered in favour of representing, by the letter prefixed, not the optical qualities of the substance in question, but its relation to other substances-, especially glucose. Thus, d-fructose means that fructose is the ketose corre- sponding to the dextro-rotatory glucose, d-fructose itself being Isevo-rotatory, though its active asymmetric C atoms are identically arranged with those in glucose. With this limitation one may say that it is only the d-hexoses of a THE CARBOHYDRATES 61 particular form which are assimilable, and therefore of physiological im- portance. The small differences in the configuration of .the four d-sugars can be readily seen if their graphic formulse be compared : CHO I H.C.OH . I HO.C.H I H.C.OH I H.C.OH I CH2OH d-glucose CHO I HO.C.H I HO.C.H I H.C.OH H.C.OH I CH2OH d-mannose CH,OH CO I HO.C.H I CHO H.C.OH HO.C.H H.C.OH HO.C.H 1 1 H.C.OH H.C.OH 1 1 CH2OH CH2OH d-fructose d-galactose THE PENTOSES. C6H1005 x These bodies occur largely in plants in the form of complex polysaccharides, the pentosanes, which give pentoses on hydrolysis with acids. Two forms of pentose have been found in the animal body, namely, i-arabinose, which has been isolated from the urine in cases of pentosuria, and 1- xylose (or d-ribose, Levene), which occurs built up into the nucleic acid molecule of the pancreas and perhaps other organs. The pentoses can apparently be utilised by herbivora as food-stuffs. We know nothing as to the part they play in the animal body or as to the causation of the rare condition of pentosuria. Since however they are reducing substances and the presence of pentose in urine might therefore lead to a suspicion of diabetes, it is necessary to mention the tests by which the presence of pentoses may be detected. The two following are the chief tests for pentoses : (1) The solution supposed to contain a pentose is mixed with an equal volume of concentrated hydrochloric acid. To the mixture is added a small quantity of solid orcin and the whole is heated. If pentose is present the solution becomes at first reddish-blue and later bluish-green. The colour can be extracted on shaking the fluid with amyl alcohol, the solution, on spectroscopic examination, showing an absorp- tion band between C and D. (2) Instead of adding orcin, we may add phloroglucin to the mixture of hydrochloric acid and pentose. The solution on heating becomes first cherry red and then cloudy. On shaking with amyl alcohol a red solution is obtained which shows a band between D and E. THE HEXOSES AND THEIR DERIVATIVES The most important of the carbohydrates belong to this class and are either hexoses or formed by a combination of two or more hexose molecules. They are divided into three main groups : (1) Monosaccharides, with the formula C6Hi206, examples of which ?,re glucose, fructose, &c. (2) Disaccharides, which are derived from two molecules of a monosac- charide with the elimination of a molecule of water, as follows : 2C6H1206-H20=C12H32On. (Examples, maltose and cane sugar.) 62 PHYSIOLOGY (3) PolysaccJtarides, composed of three or more molecules of a mono- saccharide. The number of molecules which are associated in the com- pounds of this group may be very large. We can regard their general formation as represented by the following equation : nC6H1206 -nH20 = (C6H1005)n. (Examples, starch, dextrin, &c.) THE MONOSACCHARIDES Only four hexoses out of the large number which have been synthetised are assimilable by the animal body. These are mannose, glucose, galactose, and fructose, the three former being aldoses, while the last is a ketose. All of them are derivatives of d-glucose. They may be synthetised in several ways. The most interesting, because it probably represents the mechanism of synthesis of hexoses in plants, is the formation from formaldehyde. In alkaline solutions formaldehyde polymerises with the formation of a mixture of hexoses known as acrose. From this mixture a-acrose can be isolated by the formation of its osazone and the reconversion of this osazone into sugar. It is found to be identical with i-fructose. If a solution of this i-fructose be treated with yeast, d-fructose is fermented, leaving l--fructose behind. For the preparation of d-fruetose it is necessary to convert the inactive sugar into the corresponding acid, mannonic acid. This with strychnine or morphia forms salts which can be separated into the d- and 1- groups by fractional crystallisation. From the d- modification d-mannose can be obtained, and this by conversion into the osazone and reconversion into a sugar gives d-fructose. All the monosaccharides, however many carbon atoms they contain, present certain general reactions determined by their chemical composition. (a) Like ordinary aldehydes and ketones; the sugars act as strongly reducing sub- stances, and, like aldehydes, reduce ammoniacal solution of silver to metallic silver, and many of the higher oxides of metals to lower oxides. On this behaviour is founded the commonest of all the tests for the presence of reducing sugar— Trommer's test. (6) On oxidising a monosaccharide the COH group becomes converted to COOH. Thus glucose on oxidation gives gluconic acid : COH(CHOH)4CH2OH + 0 = COOH(CHOH)4CH2OH. On further oxidation the end group CH2OH also is affected, a^nd we obtain a dibasic acid. Thus glucose gives saccharic acid. (c) By means of nascent hydrogen the monosaccharides can be reduced to the corresponding polyatomic alcohol. Thus the three hexoses, glucose, fructose, and galactose give the corresponding three alcohols, sorbite, mannite, and dulcite C6H14O6. (d) Another important general reaction of the monosaccharides depending on the COH or the CO group is the reaction with phenyl hydra/inr. On warming a solu- tion of sugar with a solution of phenyl hydrazine in acetic acid, the following reactions take place. The first reaction results in the production of a hydrazone : CH2OH(CHOH)3CHOHCHO + H-jN.NH.CeHg = CH2OH(CHOH)3CHOH.CH : N.NH.C6H5 -f H2O. The hydrazone then reacts with another molecule of phenyl hydrazinc with the pro- duction of an osazone : THE CARBOHYDRATES 63 CH2(OH)(CHOH)3CHOH.CH : N.NH.C6H5 + H2N.NHC6H5 *= CH2OH(CHOH)3C.CHN.NH.C6H5 II N.NH.C6H5 + H20 + H2. The hydrogen formed in this reaction acts upon a second molecule of phenyl hydrazine, splitting it into aniline and ammonia. On this account it is always necessary to have an excess of phenyl hydrazine in the operation. The osazones form well-defined crystalline products which are generally yellowish in colour and differ in their melting-point and in their crystalline form. They are therefore of extreme value in the separation and identification of different carbohy- drates. They can be also used for the artificial preparation of certain sugars. Under the influence of acetic acid and zinc dust they form osamines, which on treatment with nitrous acid are reconverted into the corresponding sugar, generally a ketose. GLUCOSE, DEXTROSE or GRAPE SUGAR, is the chief constituent of the sugar of fruits, especially of grapes. It occurs in the body as the end- product of the digestion of starch. When pure it forms white crystals which melt at 100° C., and lose the one molecule of water of crystallisation at 110° C. It is easily soluble in water, and the solution shows bi-rotation. Its final specific rotatory power at 20° C. is 52 *74. TESTS FOR GLUCOSE. Trommer's test depends on the power possessed in common with the other sugars of reducing cupric hydrate to cuprous oxide. The sugar solution is made alkaline with caustic potash or soda, and a few drops of copper sulphate solution added. On heating the blue solution thus obtained to boiling, it turns yellow, and a yellowish-red precipitate of cuprous hydrate is produced. This test is generally performed with Fehling's solution, which consists of an alkaline solution of cupric hydrate in Rochelle salt. The proportions in making the solutions are so arranged that 10 c.c. of Fehling's solution are completely reduced by -05 gramme glucose. This reaction is made use of for the quantitative determination of glucose in solution. The determination may be carried out either volumetrically, as in Fehling*s or Pavy's method, or gravimetrically, as in Allihn's method. Moore's Test. A solution of glucose treated with a little strong caustic potash or soda and warmed, becomes first yellow and then gradually dark brown, and gives off a smell of caramel. With ordinary yeast, glucose solutions ferment readily, giving off CO2, and form alcohol with small traces of amyl alcohol, glycerin, and succinic acid. With phenyl hydrazine glucose gives well-marked needles of glucosazone. These are precipitated when the liquid is still hot, the precipitate being increased as the liquid cools. The crystals form bundles of fine yellow needles which are almost in- soluble in water, but are soluble in boiling alcohol. When purified by recrystallisation they melt at 204-205° C. On treating a watery solution of glucose with benzoyl chloride and caustic soda and shaking till the smell of benzoyl chloride has disappeared, an insoluble precipitate is produced of the benzoic ester of glucose. This method has been often used for isolating glucose from fluids in which it occurs in minute quantities. Molisch's Test. On treating 0-5 c.c. of dilute glucose solution with one drop of a 10 per cent, alcoholic solution of a-naphthol, and then pouring 1 c.c. of concentrated sulphuric acid gradually down the side of the tube, a purple ring is produced at the junction of the two fluids, which on shaking spreads over the whole fluid. This reaction depends on the formation of furfurol from the glucose. tin order to identify glucose in a normal fluid, the following tests may be applied, 3r removing any protein which may be present : (1) Reduction of cupric hydrate or Fehling's solution. 64 PHYSIOLOGY (2) Estimation of reducing power of solution. (3) Estimation of rotatory power of solution on polarised light. (4) Formation of osazone crystals with phenyl hydrazine. These crystals must come down while the fluid is still hot. They must be purified and their melting-point taken. A determination by combustion of their nitrogen content will give direct information whether the sugar is a monosaccharide or disaccharide. (5) The solution is made acid and boiled for some time. It is then made up to its former volume and its reducing power and effect on polarised light once more taken, In the case of a disaccharide, which would be converted into monosaccharide by boiling in acid solution, these two readings would be altered, whereas neither the rotatory power nor the reducing power of glucose would undergo any change. (6) Fermentation with ordinary yeast. A positive result would exclude glycuronic acid. D-FRUCTOSE, or L^EVULOSE, occurs mixed with dextrose in honey and in fruit sugar. It is also, with glucose, formed by the digestion or inver- sion of cane sugar. It is crystallisable with difficulty. Its watery solution is Isevo-rotatory, and reduces Fehling's solution somewhat less strongly than glucose, its reducing power being 92, if we take that of glucose as 100. It ferments readily with yeast ; with phenyl hydrazine it gives the same osazone as is formed from glucose. GALACTOSE is formed by the digestion or hydrolysis of milk sugar or lactose. It is also obtained on hydrolysing cerebrin, a constituent of the brain, with dilute mineral acids, and by the hydrolysis of certain vegetable gums. It is much less soluble in water than glucose. It is dextro-rotatory and shows marked bi-rotation. With ordinary yeast it ferments but ex- tremely slowly. One species of yeast is known, namely, saccharomyces apicu- latus which, while fermenting d-fructose and glucose, has no effect on galac- tose. This yeast can therefore be used to isolate galactose from a mixture of the monosaccharides. It reduces Fehling's solution, its reducing power being somewhat less than that of glucose. Yeasts can be trained to ferment galactose. MANNOSE. Mannose, though an assimilable sugar, is of such rare occurrence in our food-stuffs that it plays practically no part in animal physiology. It is dextro- rotatory, reduces Fehling's solution, ferments easily with ordinary yeast, and gives an osazone which is identical with that derived from glucose. DERIVATIVES OF THE HEXOSES Two derivatives of glucose are of considerable physiological importance, namely, glucosamine and glycuronic acid. Glucosamine, C6H13N06, has the structural formula : CH2OH I (CH.OH)3 I CH.NH2 I CHO \ THE CARBOHYDRATES 65 It is obtained from chitin, which forms the exoskeleton of large numbers of the inver- tebrata, by boiling this with concentrated hydrochloric acid. It is stated to have been obtained as a, decomposition product of certain proteins and their derivatives, such as the mucins. It is of special interest as affording an intermediate product between the carbohydrates and the oxy-amino acids which can be obtained by the disintegration of proteins. In solution it is dextro-rotatory, reduces Fehling's solu- tion, and gives an osazone resembling that derived from glucose. n GLYCURONIC ACID. C6H10O7, may be regarded as one of the first results of oxidation of the glucose molecule. The group which has undergone oxidation is not the readily oxidisable CHO group, but the CH2OH group at the other end of the molecule. The formula of this acid is therefore : COOH I (CH.OH)4 I CHO. In the free state it does not occur in the animal body. It is constantly found in the urine after administration of certain drugs such as phenol, camphor, or chloral, and then occurs as a conjugated acid with these substances. These conjugated acids are lee vo -rotatory, though the free acid is dextro-rotatory. In the free state it reduces Fehling's solution and gives an osazone which is not sufficiently characteristic to dis- tinguish from glucosazone. It does not undergo fermentation with yeast. This test is therefore the best means of distinguishing the acid in urine from glucose. THE FORMATION OF GLUCOSIDES The graphic formulae given on p. 61 do not explain all the possible modes of arrange- ment of the groups of the sugar molecules. Many of these sugars, when dissolved in water, present the phenomenon known as multi-rotation. If their rotatory power be taken immediately after solution, it is found to be greater or less than the rotatory power taken some hours or days later. Glucose, for instance, immediately after solu- tion, has a high specific rotatory power, which diminishes rapidly if the solution be boiled, and more slowly if it be allowed to stand. Finally, the specific rotatory power becomes constant at + 52-5° D. This change in rotatory power seems to be associated with a change in the arrangement of the groups, the aldose for example assuming, »y the shifting of a mobile oxygen atom, what is known as a lactone arrangement. Thus glucose COH(CHOH)2CHOH.CHOH.CH2OH becomes CHOH.(CHOH)2.CH.CHOH.CH2OH This change in the arrangement of the molecule renders a further stereoisomerism jible, owing to the fact that now the end group which was formerly COH becomes H I O— C— OH I C that now there are five instead of four asymmetric carbon atoms. The two isomers 5 66 PHYSIOLOGY of glucose, which are thus rendered possible, are represented by the following structural formulae : H— C— OH OH— C— H or HCOH CH,OH HCOH I CH2OH In these molecules the OH attached to tbe end group can be replaced by other radicals, including other sugar molecules. In this way we get the formation of glucosides. Thus, if glucose be dissolved in methyl alcohol and be treated with hydrochloric acid, we obtain a and ft methyl glucosides, the formulae of which would be represented as follows : H— 0-OCH3 CH30— C— H r HCOH I \Q I HOCH /u HOCH HCOH I CH2OH Instead of methyl we might insert other groups, and even other hexose groups, such as glucose or galactose, obtaining the two sugars maltose and lactose, which may thus be regarded as glucosides — maltose as the a-glucoside of glucose, lactose as the /3-galac- toside of glucose. The mode of combination of the two hexose groups to form these disaccharides may be represented as follows : H H OH H H CH2OH— C — C — C — C — C glucose rest OH V HOH 0 HO H HO HO OHC — C — C — C — C — CH2 glucose rest H OH H H O maltose. H OH II CH2OH — C— C— C— C— -C galactose rest OH H H OH HO H HO HO OHC — C — C — C — C — CH2 glucose rest H OH H H lactose ucv ! THE CARBOHYDRATES 67 A very large number of glucosides occur as plant products. Among these we may mention amygdalin, salicin, phloridzin, indican, &c. THE DISACCHARIDES The disaccharides are formed by the union of two molecules of mono- saccharides with the elimination of one molecule of water, and can be re- garded, according to the manner in which the molecules are combined, as glucosides, galactosides, &c. On hydrolysis, e.g. on heating with acids, they take up one molecule of water and are split up into the corresponding mono- saccharides. Thus cane sugar gives equal parts of glucose and fructose, maltose gives equal parts of glucose and glucose, while milk sugar or lactose gives equal parts of glucose and galactose. CANE SUGAR, sometimes known as saccharose, is widely distributed throughout the vegetable kingdom, and forms an important article of diet. It has no reducing power on Fehling's solution. It is strongly dextro-rota- tory and has a specific rotatory power of + 66 '5°. On hydrolysis it is converted into equal molecules of glucose and fructose. Owing to the fact that fructose rotates polarised light more strongly to the left than glucose does to the right, the mixture of the two monosaccharides so obtained is Isevo-rotatory. On .this account the change from free cane sugar to the mixture of monosaccharides is known as inversion, and the mixture is often spoken of as ' invert sugar.' The, term ' inversion ' has since been loosely applied to the process of hydrolysis itself, so that we often speak of the inversion of maltose or of lactose, meaning thereby the hydrolysis of these sugars with the production of their constituent monosaccharides. With east, cane sugar first undergoes inversion by a special ferment present in the yeast (invertase), and the mixture of fructose and glucose is then fermented. MALTOSE is formed during the hydrolysis of starch by acids or by digestive ferments, and is also the chief sugar in germinating barley or malt. It is strongly dextro-rotatory, ferments easily with yeast, and reduces Fehling's solution ; its reducing power is about 70 per cent, of that of glucose. With phenyl hydrazine it gives phenyl maltosazone, which forms definite yellow crystals with a melting-point of 206° C. MILK SUGAR or LACTOSE is found only as a constituent of milk. It forms colourless rod-like crystals, which are much less soluble in water than are the two other disaccharides. On account of this solubility it is much less sweet than either cane sugar or maltose. It is dextro-rotatory and shows bi-rotation. It is not fermented by ordinary yeast. Before fermen- tation can occur the lactose must be split by the agency of acids or by a :erment, lactase, which occurs in the animal body and in certain moulds, into .e monosaccharides glucose and galactose. Lactose reduces Fehling's solution and gives with phenyl hydrazine lactosazone, which is easily soluble hot water and therefore does not come down until the fluid is cold. THE POLYSACCHARIDES These play an important part throughout the whole vegetable kingdom, where all the supporting tissues of the plants, their protective substances, 68 PHYSIOLOGY and many of their reserve materials consist of members of this group. In the animal body, where the supporting tissues are composed chiefly of deriva- tives of proteins, the sole significance of polysaccharides lies in their value as food-stuffs. In plants, anhydrides both of hexoses and pentoses occur in bewildering variety. Here however we may confine our attention to those members of the group of polysaccharides which are important as food-stuffs. STARCH (C6H1008) is present in large quantities in nearly all vegetable foods, and is an important constituent of the cereals, from which flour and bread are derived, as well as of tubers, such as the potato. In the plant cells it occurs as concentrically striated grains within minute protoplasmic structures — the amyloplasts, the office of which it is to manufacture starch from the glucose present in the cell sap. When freed, by breaking up the cells and washing with water, it forms a white powder consisting of micro- scopic grains, each of which presents the characteristic concentric stria tion. It is insoluble in cold water. In hot water the grains swell up and burst, forming a thick paste, which sets to a jelly on cooling. This semi- solution, as well as the original starch-grains, gives an intense blue colour on the addition of iodine. On treating starch with cold alkalies or cold dilute acid, it is converted into a soluble modification, the so-called soluble starch or amylodextrin, which also gives a blue colour with iodine. This modification is also produced as the first stage of the action of diastatic ferments upon starch. On boiling with dilute acids, starch is converted first into a mixture of dextrins, then into maltose, and finally into glucose. On acting upon starch with various ferments, such as the diastase which may be extracted from malt or germinating barley, or with the amylase occurring in saliva or pancreatic juice, it undergoes hydrolysis, the final result of the action being a mixture of four parts of maltose to one part of dextrin. As to the inter- mediate stages in this reaction opinions are still divided. The first product is soluble starch, amylodextrin, giving a blue colour with iodine. This breaks up into a reducing sugar, and another dextrin, erythrodextrin, which gives a red colour with iodine ; and this dextrin, on further hydrolysis, yields reducing sugar and achroodextrin, which is not coloured by the addition of iodine. Thus there are a series of successive hydrolytic decom- positions of the molecule, each resulting in the splitting off of a molecule of sugar and the production of a lower dextrin. The DEXTRINS are ill-defined bodies which are difficult to separate. They are amorphous white powders, easily soluble in water, forming solutions which, when concentrated, are thick and adhesive. They are insoluble in alcohol and ether. With cupric hydrate and caustic alkali they form blue solutions, which reduc.e slightly on boiling. They are not precipitated by saturation with ammonium sulphate. On boiling with dilute acids, they are converted entirely into glucose. The changes undergone by starch during its hydrolysis by means of diastase have been used by Brown and his co-workers as a method of arriving at some idea of the size and structure "of "the starch molecule. Proceeding from the discovery that the end-products of this reaction consisted of 81 per cent, maltose and 19 per cent, dextrin, THE CARBOHYDRATES 69 they concluded that starch must consist of five dextrin-like groups, four of which are arranged symmetrically round the fifth. At each stage one of these groups is split off and {CHO ) ,p VI 23. ^ [• one molecule of water being taken up. The malto-dextrin group is then changed into maltose by the further assimilation of two molecules of water. The central dextrin-like group is attacked with great difficulty by the ferment, and therefore remains at the end of the reaction as achroodextrin. The malto-dextrin, the penultimate stage in the action of diastase, can be regarded as formed by the condensation of three molecules of maltose attached by the oxygen of two CHO groups, so that one CHO group remains free and determines the reducing power of the malto-dextrin molecule. Its formula may therefore be represented as follows : >.C12H21O10 < yC12H2009 ° CO.CH2.NH8 (3) NH2.CH2.C(OH) = N.CH2.COOH THE PROTEINS N.CH..CO C(OH)CH2.NHS (2) and (4) being the intramolecular form of the formulae (1) and (3). (3) and (4) are sometimes spoken of as the enolic form. If we consider that perhaps some hundred of the amino-acid groups may go to making up a single protein molecule, it is possible to form some conception of the enormous variability in reaction possible to such a compound. THE CONSTITUTION OF DIFFERENT PROTEINS All the proximate constituents of proteins, so far as we know, are amino- acids. Of these the following have been isolated, namely, glycine, alanine, amino-valerianic acid, leucine, isoleucine, proline, oxyproline, serine, phenyl Serum albumin a If \n "U o o> W6£ (3 5 | 1 o c I 1 Gelatin Keratin (from horse 1 hair) 1 Glycine . 0 0 3-8 0-9 0 0 0 _ 16-5 4-7 Alanine 2-7 8-1 3-6 2-7 1-5 4-2 — — 0-8 1-5 Serine 0-6 — 0-33 0-12 0-5 0-6 7-8 — 0-4 0-6 Amino-valeri- - anic acid — — present 0-3 7-2 — 4-3 — 1-0 0-9 Leucine . 20-0 7-1 20-9 6-0 9-35 29-0 0 — 2-1 7-1 Proline 1-0 2-25 1-7 2-4 6-70 2-3 11-0 — 5-2 3-4 Oxyproline — — 2-0 — 0-23 1-0 — — 3-0 — Glutamic acid . 7-7 8-0 6-3 36-5 15-55 1-7 — — 0-88 3-7 Aspartic acid 3-1 1-5 4-5 1-3 ,1-39 4-4 — — 0-56 0-3 Phenylalanine . 3-1 4-4 24 2-6 3-2 4-2 — . — 0-4 0 Tyrosine . 2-1 1-1 2-1 2-4 4-5 1-5 — — 0 3-2 Tryptophane . present present present 1-0 1-50 present — — 0 — Cystine 2-3 0-2 0-25 0-45 ? 0-3 — — — 10+ Lysine — 2-15 1-0 0 5-95 4-3 0 12-0 2-75 1-1 Arginine . — 2-14 11-7 3-4 3-81 5-4 87-4 58-2 7-62 4-5 Histidine . — — 1-1 1-7 2-5 11-0 0 12-9 0-4 0-6 alanine, glutamic acid, aspartic acid, tyrosine, tryptophane, cystine, lysine histidine, arginine, and ' di-amino-trioxydodecoic ' acid. The question now arises whether all the different varieties of protein owe their peculiarities to the presence of different amino-acids or whether the greater number of the amino-acids above mentioned are present in all pro- teins, the differences between the latter being determined by differences in the arrangement and relative amounts of their proximate constituents. A large number of analyses of different proteins have been made by Abderhalden, Osborne, and others, utilising the methods for the isolation of amino-acids devised by Fischer. The constitution of some representative proteins as -determined in this way is given in the Table above. 90 PHYSIOLOGY These results show that all the proteins contain a very considerable proportion of the total number of amino-acids which have as yet been isolated from acid digests of proteins. The differences in various proteins cannot therefore be determined by qualitative differences in their constituent molecules, but must depend on the relative amounts of the amino-acids which are present and on their arrangement in the whole molecule. As regards relative amounts of amino-acids we find very striking differences, i Thus glutamic acid, which forms 8 per cent, of egg albumin and only 1 '7 per cent, of globin (derived from haemoglobin), amounts to 36 '5 per cent, in gliadin, the protein extracted from wheat flour. Striking differences are also notice- able in the relative proportions of the diamino-acids and bases, the so-called hexone bases. Whereas in casein they form about 12 per cent, of the total molecule, in globin they form about 20 per cent. ; and in the protamines, salmine and sturine, about 85 per cent, of the total molecule consists of these bodies. On this account the two last-named bodies have a strongly basic character. From these figures it is evident also that certain of the amino- acids must occur many times over in the protein molecule. Thus in globin, if we assume the presence of one tyrosine molecule, there must be at least thirty-two leucine and ten histidine molecules. On these data the molecular weight of haemoglobin would come out at about 14,000, a figure which agrees with that derived from a study of the amounts of sulphur and iron respec- tively in its molecule. THE DISTRIBUTION OF NITROGEN IN THE PROTEIN MOLECULE Attempts have been made to differentiate among the proteins by a method which, while less laborious than the isolation and recognition of the individual amino-acids, may yet throw some light on the manner in which the nitrogen is combined within the molecule, and on the relative amounts of the different classes of nitrogen groups which may be present. One method, which was devised by Hausmann, is carried out as follows. One gramme of the protein is dissociated by boiling with strong hydrochloric acid. The nitrogen, which has been split off as ammonia and is present in the solution as ammonium chloride, is then distilled off with magnesia and received into decinormal acid, where its amount can be determined by titration. This nitrogen is variously designated as amide nitrogen, ammonia nitrogen, or easily displaceable nitrogen. The remaining fluid, freed from ammonia, is precipitated with phosphotungstic acid. By this means all the diamino-aci< I s and bases are thrown down. The nitrogen in the precipitate is determined by Kjeldahl's method and is called diamino- or basic nitrogen. In the remaining fluid, which contains mono-amino-acids, the total nitrogen, the mono-amino-nitrogen, is determined by Kjeldahl's method. Table I., p. 91, gives some of the results obtained in this manner, and shows that there are considerable differences in the distribution of the different kinds of nitrogen among the various classes of proteins. The method is however only a rough one as compared with the separation of the individual maino-acids. THE PROTEINS TABLE I. 91 Group Protein Source N per cent. Amide N Amino N Basic N Humin * N Protamines ( Salmine j Sturine Salmon-roe Sturgeon -roe 0 0 87-8 83-7 Histones Histone Thyinus ' — 3-3 38-7 Albumins \ and phospho- [ Ovalbumin 1 Caseinogen Egg-white Milk 15-51 • 15-62 8-64 10-36 68-13 66-00 21-27 22-34 1-87 1-34 proteins j Globulins j Globulin ( Edestin Wheat Hemp seed 18-39 18-64 7-72 10-08 53-40 57-83 37-10 31-70 1-52 0-64 Alcohol - soluble proteins )Zein i Gliadin Maize i Wheat and rye 16-13 17-66 18-40 23-78 77-56 70-27 3-03 5-54 0-99 0-79 / Prot- Witte's Albumoses albumose 1 Hetero- v albumose peptone Witte's peptone — 7-14 6-45 68-17 57-4 25-42 38-93 — TABLE II. — DISTRIBUTION OF THE NITROGEN IN VARIOUS PROTEINS (VAN SLYKE) Gliadin Edestin Hair (dog) Gelatin Fibrin Hsemo- cyanin 0 x haemo- globin Ammonia N . 25-52 9-99 10-05 2-25 8-32 5-95 5-24 Melanine N 0-86 1-98 7-42 0-07 3-17 1-65 3-60 Cystine N 1-25 1-49 ! 6-60 0 0-99 0-80 0 ? Arginine N 5-71 27-05 15-33 14-70 13-86 15-73 7-70 Histidine N . 5-20 5-75 3-48 4-48 4-83 13-23 12-70 Lysine N 0-75 3-86 5-37 6-3^ 11-51 8-49 10-90 Amino N of the filtrate 51-98 47-55 47-50 56-30 54-30 51-30 57-00 Non-amino N of the ; filtrate (proline, oxyproline, ^ tryptophane) 8-50 1-70 3-10 14-90 2-70 3-80 2-90 Sum 99-77 99-37 99-85 99-02 99-58 100-95 100-00 An improved means of determining the distribution of nitrogen in the protein molecule has been devised by Van Slyke. Some of his results are given in Table II., above. * When a protein is boiled for a long time with strong acid, a black precipitate may occur which contains nitrogen. This is known as humin nitrogen. 92 PHYSIOLOGY TESTS FOR PROTEIN A. COLOUR REACTIONS OF THE PROTEINS These are of importance since in many cases they are an indication of the nature of the groups present in the protein molecule. (1) THE BIURET REACTION. When a solution of a protein is made strongly alkaline with caustic potash or soda, and dilute copper sulphate added drop by drop, a colour varying from pink to violet is produced. In the case of the proteoses and peptones (the hydra ted proteins) the colour is pink ; in the case of the coagulable proteins, violet. According to Schiff this colour is given by all compounds containing the following groups : ,CO.NH2 CO 2<; X .NH2 CO.NH2 CO— NHa I CO— NH2 and the group I I (NH2)C-CO— NH— C I I We have already seen that this grouping is typical of the protein molecule. (2) THE XANTHO-PROTEIC REACTION. On adding strong nitric acid to a solution of protein and boiling, a yellow colour is produced which turns to a deep orange when excess of caustic alkali or ammonia is added. The production of this reaction points to the existence of benzene derivatives in the protein molecule, and it is therefore a general test for the presence of aromatic groups. (3) MILLON'S REACTION. Millon's reagent is a solution of mercuric nitrate in water containing free nitrous acid. On adding a few drops of this to a protein solu- tion a white precipitate is produced which turns a brick-red colour on boiling. It depends on the presence in the protein of a hydroxy-derivative of benzene, and is determined in the protein by the tyrosine, which is oxyphenylalanine. (4) SULPHUR REACTION. On warming a solution of protein with caustic soda in the presence of lead acetate, a black colour is produced owing to the precipi- tation of lead sulphide. The depth of coloration gives a rough indication of the amount of sulphur in the protein under investigation. (5) THE HOPKINS-ADAMKIEWICZ REACTION. It was stated by Adam- kiewicz that on the addition of acetic acid and concentrated sulphuric acid to protein, a violet colour was produced. Hopkins and Cole showed that the success of this reaction depended on the presence of glyoxylic acid CHO.COOH as an impurity in the acetic acid used. The test is therefore performed now as follows : Glyorylic acid is prepared by the action of sodium amalgam on a solution of oxalic acid. A few drops of this solution are added to the solution of protein, and strong sulphuric acid poured down the side of the tube. A bluish violet colour is produced at the junction of the two fluids. This reaction is due to the presence in the protein of tryptophane. The so-called Liebermann's reaction has been shown by Cole to be essentially a modification of the above, and is due also to the presence of tryptophane. In this test the protein is precipitated by alcohol, washed with ether, and heated with con- centrated hydrochloric acid, when a blue colour is produced, glyoxylic acid being derived from the alcohol and ether. THE PKOTEINS 93 (6) REACTIONS INDICATING THE PRESENCE OF CARBOHYDRATES. Molisch's test is applied as follows. A few drops of alcoholic solution of a-naphthol and then strong sulphuric acid are added to a protein solution. A violet colour is produced, which on addition of alcohol, ether, or potash turns yellow. The reaction is determined by the presence, either as an impurity or a constituent part of the mole- cule, of a carbohydrate radical which, under the influence of strong sulphuric acid, is converted into furfurol. The furfurol gives the colour reaction with the a-naphthol. Another test for the carbohydrate radical is the orcin reaction. A small quantity of the dried albumin is added to 5 c.c. of fuming hydrochloric acid, and the mixture is then warmed. When the albumin is nearly all dissolved, a little solid orcin is added on the point of a knife, and then a drop of ferric chloride solution. After warming this mixture for some minutes, a green colour is produced which is soluble in amyl alcohol and gives a definite absorption spectrum. B. METALLIC SALTS The following metallic salts form double insoluble compounds with proteins, and therefore cause a double precipitation when added to solutions of these bodies : ferric chloride, copper sulphate, mercuric chloride, lead acetate, zinc acetate. C. ALKALOIDAL REACTIONS Proteins, like the polypeptides and the amino-acids of which they are composed, may function either as weak acids or as weak bases, according as they are treated with bases or acid radicals respectively. In the presence of _ strong acids therefore, proteins act like organic bases, and are thrown down in an insoluble form by the various alkaloidal precipitants. With certain proteins, such as the protamines, where there is a preponderance of basic groups, it is not necessary to add mineral acid in order to ensure the precipitation. The following are the principal alkaloidal precipitants which may be employed : (a) Phosphotungstic acid. (b) Phosphomolybdic acid. (c) Tannic acid. (d) Potassium mercuric iodide. (e) Acetic acid and potassium ferrocyanide. (/) Trichloracetic acid. (In order to precipitate all the coagulable proteins from a solution, it is treated with an equal volume of 10 per cent, trichloracetic acid, well shaken and filtered.) (g) Metaphosphoric acid. (h) Salicyl-sulphonic acid. These two latter are generally employed in a 5 per cent, solution. (i) Picric acid. A mixture of picric and citric acids is largely employed, under the name of Esbach's reagent, as a precipitant for coagulable proteins in the urine. D. TESTS DEPENDING ON THE COLLOIDAL CHARACTER OF THE PROTEIN (1) HEAT COAGULATION. On boiling proteins in a very slightly acid solution some are coagulated and form an insoluble white precipitate. This test is applicable to albumins, globulins, and under certain conditions to the derived albumins. In order that the separation of protein in this way may be complete, it is necessary to provide for the presence of neutral salts and also for the maintenance of a slight acidity. The best method of carrying out this test therefore is to boil the protein in slightly alkaline or neutral solution after the addition of 2-5 per cent, of sodium chloride or sodium sulphate. While the solution is in active ebullition, 1 per cent, acetic acid is added drop by drop until the reaction is just acid to litmus. By this means a nearly perfect separation of all the coagulable proteins may be effected. 94 PHYSIOLOGY (2) HELLER'S TEST- On pouring a solution of protein carefully down the side of a test-tube containing strong nitric acid so as to form a layer on the top, a white layer of coagulated protein is produced at the junction of the two fluids. A similar coagulative effect is given by other strong mineral acids. (3) PRECIPITATION BY NEUTRAL SALTS. On addition of a neutral salt in excess to a colloidal solution, the relation between the solvent and the particles which are in suspension or pseudo -solution is altered. It is therefore possible in many cases by the addition of neutral salts to separate out the dissolved colloid without otherwise altering its characters in any way, so that, on collecting the precipitate and separating the salt carried down with it, it can be dissolved again by adding water. Some classes of proteins can be salted out very readily, while others require a much higher concentration of salt before they are precipitated. The salts which are generally employed for salting out proteins have been divided by Schryver into three classes : Class I. Class II. Class III. Sodium chloride. Potassium acetate. Ammonium sulphate. Sodium sulphate. Calcium chloride. Zinc sulphate. Sodium acetate. Calcium nitrate. Sodium nitrate. Magnesium sulphate. The two calcium salts are however rarely employed, as they tend to render the precipitated protein insoluble. The salts of the first class require much higher concentration for the precipitation of the proteins than those of the second, and these than those of the third. Since the degree of concentration of any salt necessary for the precipitation of any particular protein is characteristic for this body, it is possible to employ a fractional process of salt precipitation in order to separate mixtures of proteins into their components. Owing however to the tenacity with which different colloids adhere to one another, it is difficult, even after many repetitions of the process of fractional salting out, to obtain products which can be regarded as free from admixture. For the purpose of fractional precipitation the salts most frequently employed are those of the third class, namely, ammonium sulphate and zinc sulphate.* We shall have to deal with results obtained by this method when treating of the separation of albumoses and peptones. The precipitability of different proteins with neutral salts serves also as the basis of the ordinary classification of these bodies. THE CLASSIFICATION OF PROTEINS It is possible that in the future, when we know all the disintegration products of the various proteins and the manner in which they are arranged in the molecule, the classification of these bodies will be based on their con- stitution. At the present time it is obviously impossible to make any classi- fication on such a basis, since the necessary knowledge is wanting, and we have therefore to use a purely artificial classification, such as that adopted by the Chemical and Physiological Societies in 1907, based chiefly on t In- solubilities of the various proteins in water and salt solutions. We shall here only indicate the characters of the main groups into which proteins are conventionally divided, and leave the closer study of the individual proteins to be dealt with in connection with the organs or tissues in which they are found. (1) THE PROT AMINES. These occur in the body only in combination with other groups. They are obtained from the ripe spermatozoa of certain THE PROTEINS 95 fishes, where they are in combination with nucleic acid. They are charac- terised by the very large amount of bases contained in their molecule, amounting to 85 per cent, of the total substance. It was formerly thought by Kossel that the protamines contained only diamino-acids and bases, but it has been shown later that a small proportion of mono-ammo-acids may also be obtained from their disintegration (v. Table, p. 98). On account of their constitution they possess strongly basic characters and form well- marked salts, e.g. sulphates and chlorides, as well as double salts with platinum chloride. They contain no sulphur and do not coagulate on heating. (2) HISTONES. This class of proteins, like the protamines, only occurs in combination with other groups, such for instance as nuclein and ha3matin. They may be obtained from red blood-corpuscles, where they form the globin part of the haemoglobin molecule, or from the leucocytes of the thymus gland, or from the spermatozoa of fishes. The histones are precipitated from their watery solutions by addition of ammonia, but are soluble in excess of this reagent. In the presence of salts they are coagulated on boiling. With cold nitric acid they give a precipitate which dissolves on warming, but is thrown down again on cooling. The most characteristic feature of this class of bodies is however the high proportion of diamino-acids and bases contained in their molecule. (3) ALBUMINS. These are soluble in pure water and are precipitated by complete saturation with ammonium sulphate, zinc sulphate, or sodio- magnesium sulphate. EGG ALBUMEN forms the greater part of the white of egg. It gives the ordinary protein tests, coagulates on heating at about 75° C., and is precipitated from its solutions if shaken with a drop of dilute acetic acid in excess of ether. It is laevo-rotatory, its specific rotatory power being -35'5°. SERUM ALBUMEN occurs in large quantities in the blood plasma, serum, lymph, and tissue fluids of the body. It coagulates at 75° C., and is dis- tinguished from egg albumen by its greater specific rotatory power, —56°, and by the fact tha*t it is not precipitated by ether and sulphuric acid. Some vegetable proteins belong to this class, e.g. the leucosin of wheat. (4) GLOBULINS. These bodies are insoluble in pure water and require the presence of a certain amount of neutral salt to dissolve them. They are precipitated from their solutions by complete saturation with magnesium sulphate or by half-saturation with ammonium sulphate. The chief members of this class are : CRYSTALLIN, obtained from the crystalline lens by passing a stream of carbon dioxide through an aqueous extract of this body. SERUM GLOBULIN or PARAGLOBULIN, a constituent of blood plasma and blood serum. FIBRINOGEN, which occurs in blood plasma and is converted into fibrin when the blood clots. PARAMYOSINOGEN, a normal constituent of muscle, 96 PHYSIOLOGY Midway between these two groups may be placed the muscle protein, myosin (or myosinogen), which, though soluble in pure water, resembles the class of globulins in the ease with which it is precipitated by the addition of neutral salts. In addition to the members of the globulins named above and derived from the animal body, proteins allied to this class form an important con- stituent of plants, and are found in large quantities in many seeds used as articles of food. These are vegetable globulins. Prominent members of the group are the edestins, which may be obtained from hemp seeds, cotton seeds, and sunflower seeds, zein from maize, kgumin from beans. (5) GLIADINS, contained in cereals, and soluble in alcohol. (6) GLUTELINS, proteins also obtained from cereals and soluble in weak alkalies. (7) DERIVATIVES OF PROTEINS. A. METAPROTEINS. These may be regarded as compounds of the protein molecule or of part of the molecule with acid or basic radicals. ACID ALBUMIN, or acid metaprotein, is formed by the action of warm dilute acids or of strong acids in the cold on any of the preceding bodies. If a weak alkali be added so as nearly to neutralise the solution of acid metaprotein, this latter is precipitated. If the precipitate be suspended in water and heated, it is coagulated and becomes insoluble in dilute acids or alkalies. ALKALI ALBUMIN, or alkaline metaprotein, is formed by the action of strong caustic potash on white of egg or on any other protein, or by adding alkali in excess to a solution of acid metaprotein. It is precipitated oh nsutralisation of its solution. In close association with this group may be included the proteins as they occur hi combination with the metallic salts, such as copper sulphate. On splitting off the copper moiety from these compounds, the protein left is practically free from ash, and behaves in many respects like an albuminate, being insoluble in absolutely pure water, but easily dissolved by the addition of a trace of free acid or alkali. A group of protein derivatives described by Hopkins is produced by the action of the free halogens on protein solutions. We get in this way two definite classes of compounds. One class, which contains the largest percentage o^ halogen, is obtained by treating a protein solution with chlorine, bromine, or iodine, dissolving up the resultant precipitate in alcohol and pouring the alcoholic solution into ether, when Ilio halogen compound is thrown down as a fine white precipitate. By dissolving this precipitate in weak soda and precipitating with acid, we obtain a series of compounds containing only about one-third as much of the halogen as is contained in the first precipitate, suggesting that the halogen forms both substitution and additive com- pounds with the protein molecule. Albumins, globulins, and metaproteins are often associated together as the coagulable proteins, since they may be thrown down entirely from their solution on boiling in slightly acid medium in the presence of neutral salts. B. HYDRATED PROTEINS. When proteins are subjected to the action of superheated water or steam, or heated with acids, or acted on at the body temperature by certain ferments, e.g. pepsin, trypsin, or papain, they undergo a change which is attended by the addition of a number of THE PROTEINS 97 molecules of water to the protein molecule (hydrolysis). This action, when carried to its end, results in the production of the amino-acids which we have already dealt with. These hydrolytic changes proceed by a series of stages, so that the intermediate products still present many of the protein reactions. The hydrated proteins are divided into two groups, proteoses and peptones. The formation of these intermediate products is especially marked with the proteolytic ferments. Pepsin with hydrochloric acid, the ferment of the gastric juice, for example, only breaks down the protein molecule as far as the proteoses and peptones. Trypsin also gives rise to both proteoses and pep- tones as intermediate products. The action of these ferments on proteins is in fact closely analogous to the action of diastase on the great polysaccharide molecule of starch. In this case, as intermediate products we have first dextrins of various complexity, secondly maltose, and finally, if the ferment maltase be also present, dextrose. The monotony of the starch molecule determines a great similarity of composition between its various disintegra- tion products. It may be regarded as an anhydride of many (100 or more) molecules of a hexose, and the intermediate stages in this hydrolysis are also hexoses and their anhydrides. The protein molecule is distinguished by the variety of the groups which enter into its formation, and this heterogeneous character of the molecule renders possible a much greater variety of inter, mediate products than we find in the starches. Thus a protein molecule may consist of the groups, A, B, C, D, E, F, G, H, &c. When hydrolysis occurs it may result in the immediate splitting off, say, of part of group A, while the residue breaks up into a series of proteoses whose composition may be represented as ABF, ABC, DFG, BDEF, &c. With further hydrolysis these groups are broken into still smaller ones, and the penultimate stages of the hydrolysis will be polypeptides similar to those which have been synthetised by Fischer from the ultimate products of protein hydrolysis. No sharp dividing line can be drawn between the proteoses, peptones, and poly- peptides. Of the last group we have already seen that the higher members give.the biuret reaction as well as the other protein reactions, if the necessary groups, e.g. tyrosine, tryptophane, are present in the molecule. The prote- oses and peptones are however ill-defined bodies. We have at present no satisfactory means of isolating the different members of these groups and, obtaining them in a state of chemical purity. Their classification is there- fore, like that of the proteins generally, a conventional one, depending on their solubilities and their precipitability by neutral salts, especially ammo- nium sulphate. Both proteoses and peptones give the xanthoproteic and Millon's reactions common to all proteins, and, like these, are precipitated by such reagents as mercuric chloride, potassio-mercuric iodide, or phospho- tungstic acid. On adding excess of caustic potash and a drop of dilute copper sulphate to solutions of either of these classes of bodies, a pink colour is produced which deepens to a violet on addition of more copper (the biuret reaction). Their solutions can be boiled without undergoing coagulation. Many of them may be thrown down from their solutions by absolute alcohol, 7 98 PHYSIOLOGY but are not rendered insoluble even by prolonged standing under the alcohol. The characters of the different members of these groups will be considered at greater length when dealing with the changes undergone by the proteins during the process of digestion. At present we may merely summarise the distinguishing features of these two classes. (a) PROTEOSES, e.g. albumose from albumin, caseose from casein, elastose from elastin. All of these are precipitated from their solutions on saturation with ammonium sulphate. In the presence of a neutral salt they give a precipitate on the addition of nitric acid. This precipitate is dissolved on heating the solution, but reappears on cooling. All, with the exception of heteroalbumose, are soluble in pure water, and all are soluble in weak salt solutions or dilute acids or alkalies. They are slightly diffusible through animal membranes. (6) PEPTONES, e.y. fibrin peptone, gluten peptone. These are all soluble in pure water, diffuse fairly readily through animal membranes, but other- wise give the same reactions as albumoses. From the latter class peptones are distinguished by the fact that they are not precipitated on saturation of their solutions either in acid or alkaline reaction with ammonium sulphate or any other neutral salt. Many of them are soluble in alcohol. (8) THE PHOSPHOPROTEINS. In this class may be grouped a number of substances of very diverse properties, which however resemble one another in containing phosphorus as an integral part of their molecule. When subjected to digestion with pepsin and hydrochloric acid they are dissolved, but a small quantity of a phosphorus-containing complex may remain behind undissolved. This residue has been called paranuclein or pseudonuclein. It is in reality derived from nucleoprotein, which is present in thje phosphoprotein as impurity and should be called simply nuclein. The phosphoproteins have markedly acid characters. They are insoluble in pure water, easily soluble in alkalies and ammonia from which the original body is thrown down again on addition of acid. Their solutions in alkali are not coagulated by heating. To this class belong caseinogen, the chief protein of milk, vitellin, the main protein in the yolk of egg, and the vitellins in the eggs of fishes and frogs. The vitellins are generally associated with a large amount of lecithin. The phosphoproteins differ from the nucleoproteins, which also contain phosphorus, in the facts that they are readily decomposed by caustic alkali with the liberation of phosphoric acid, and do not contain purine bases. The phosphorus of the nucleoproteins is not split off by alkali (1 per cent.), and on hydrolysis the nucleic acid constituent gives rise to purine bases. (<>) CONJUGATED PROTEINS, Various complex bodies which play an important part in building up cells and in the various processes of the body make up this group of compounds. They resemble one another only in the fact that in each of them a protein radical is combined with some other body, often spoken of as the prosthetic group.* * By the Germans the term ' proteid ' is often applied to this group. In English however the term ' proteid ' has been generally used for the simple protein known THE PROTEINS 99 (a) CHROMOPROTEINS. Of this class, consisting of a colouring-matter combined with a protein, the most important is hcemoglobin. This substance, which is the red colouring-matter of the red corpuscles of the blocd and plays an important part in the processes of respiration, acting as an oxygen carrier from the lungs to the tissues, is composed of the protein, globin, united with an iron-containing body, haematin. Oxyheemoglobin contains from 4-5 per cent, hsematin (C32H32N404Fe). It is easily crystallisable, and its physical and chemical characters have therefore been more precisely determined than is the case with most other members of the group of conjugated proteins. We shall have to deal more fully with its properties in the chapters on Blood and Respiration. (b) THE NUCLEOPROTEINS. These are formed by the combination of a phosphorised organic acid, nucleic acid, with a protein which may belong to any of the classes we have enumerated above. Some of the best-marked members of this group consist of compounds of nucleic acid with basic histones or protamines. The combination between protein and the prosthetic group seems to take place in two stages. If a nucleoprotein be subjected to gastric digestion a large amount of the protein goes into solution as proteose or peptone, leaving an insoluble remainder. This precipitate is not however nucleic acid, but still contains a protein group, the compound being spoken of as nuclein. From the latter nucleic acid can be split off by heating with strong acids or other means. The nucleoproteins are soluble in water and salt solutions, and are easily soluble in dilute alkalies. They have acid characters and are precipitated by the addition of acids. The nucleins, on the other hand, are insoluble in water and salt solutions, but are easily dissolved by dilute alkalies. The nucleins and nucleoproteins form the chief and invariable constituent of cell nuclei. They may be therefore prepared from the most diverse organs. The heads of the spermatozoa of the salmon consist entirely of nuclein. Miescher and Schmiedeberg found that the nuclein obtained from this source contained 60*5 per cent, nucleic acid and 35*56 protamine, and was in fact a nucleate of protamine. The nuclein derived from the spermatozoa of echinoderms has been found to be a com- pound of nucleic acid and histone. From organs rich in cells, such as the thymus and the pancreas, and from nucleated red blood-corpuscles, nucleo- proteins may be obtained which can be broken down into nuclein and protein, the nuclein again being composed of a protein residue with nucleic acid. As first extracted from the animal cell the nucleoproteins are associated with a considerable proportion of lecithin, and in this labile compound form the ' tissue fibrinogen ' of Wooldridge. To prepare this substance an organ rich in cells, such as the thymus, is minced and extracted with water or normal salt solution. After separa- ting the cells by means of the centrifuge, the clear fluid is decanted off and acidified with acetic acid. A precipitate is produced consisting of ' tissue fibrinogen.' This substance is soluble in excess of acid and is easily soluble in alkalies. All the tissue to the Germans as ' Eiweisskorper.' On account of the confusion which has risen from this double use of the term ' proteid,' I have attempted to avoid it altogether in this volume. 100 PHYSIOLOGY fibrinogens are highly unstable bodies and undergo changes in the mere act of pre- cipitation and re-solution. When injected into the blood they cause intravascular clotting. On digestion with gastric juice they yield a precipitate of nuclein, and this precipitate contains a large proportion of the lecithin present hi the original substance. In the nucleoproteins nucleic acid is combined with proteins in two degrees, a large portion of the protein being separable by gastric digestion, while the remainder needs stronger reagents for its dissociation. The relation of the two portions of the nucleo- protein may be represented therefore by the following schema : Nucleo-protein Protein Nuclein Protein Nucleic acid (generally histone or protamine) By various means, all of which involve hydrolysis, the nucleic acid may be broken up into its proximate constituents. These differ according to the source of the nucleic acid. Whatever the source, the disintegration products belong to closely allied groups of substances. These may be grouped as follows : (1) Phosphoric Acid. The proportion of phosphorus varies within but narrow limits in the different nucleic acids, the average being about 10 per cent. It is probable that the phosphoric acid represents, so to speak, the combining medium for the groups contained in the nucleic acid molecule, as is the case with the various groups which make up the lecithin molecule. (2) The Purine Bases. Among the products of disintegration of nucleic acid we find constantly one of the bases adenine, C5H6N6, and guanine (C5H5N50). These substances, with the products of their oxidation, xan- thine, C6H4N402, hypoxanthine, C5H4N4O, have long been known to be closely allied to uric acid, C5H4N403, but their true relationships have only been thoroughly known since the researches of Fischer on this group. According to Fischer they can be all regarded as derivatives of the body purine, 1N=«CH I I 2HC 5C— NH7 Each group in this purine ring is generally designated with a number indicated in the structural formula, in order that it may be possible to represent the position of any substituted groups in its derivatives. Uric acid itself is 2-6-8-trioxypurine with the following formula : HN— CO I I OC C— NH. I II >co THE PROTEINS 101 It can be synthetised by fusing together in a sealed tube trichlorolactamide and urea. Thus : NH2 CONH2 I I CO + CHOH + NH2 I I >co Th NH9 CCL NH ' NH— CO I I CO C— NH + NH4C1. + 2HC1 I II >co NH— C— NH' e relation of xanthine, hypoxanthine, guanine. and adenine to uric acid is shown by the following formulse : NH— CO II CO C— NH I II >co HN -- CO II CO— C— NH NH— C— NH Uric acid 2 -6-8-trioxypurine HN— CO N = C.NH2 II II HC C— NH. HC C— NH N_C _N Hypoxanthine 6-oxypurine HN - C^-N Xanthine 2-6-dioxypurine NH— CO II NH2C C— NH N— C— N Adenine 6-amino-purine N— C— N Guanine 2-amino 6-oxypurine Closely allied to this group of bodies are the chief constituents of tea, coffee, and cocoa, namely caffeine, which is trimethyl dioxypurine, and theobromine, which is dimethyl dioxypurine. From the structural formulse given it will be seen that the purine radical contains two nuclei. The nucleus N— C I I C C I I N— C is spoken of as the pyrimidine nucleus, pyrimidine having the formula I I 2HC 6CH I II 3N— 4CH The other is the radical which we have met with' already in histidine, a disintegration product of proteins, namely iminazol : HC— NH HO-N Besides the purine bases proper, we find among the disintegration products of nucleic acid a series of bases derived from the pyrimidine ring. These are uracil, thymine, and cytosine. URACIL is 2-6-dioxypyrimidine, 102 PHYSIOLOGY NH— CO I I CO— CH THYMINE is 5-methyl uracil, NH— CH NH— CO I I CO C.CH3 I II NH— CH while CYTOSINE is 6-amino-2-oxypyrimidine, N =C.NH2 I I CO CH I II NH— CH Besides these two groups of nitrogenous compounds derived from the purine and pyrimidine rings, many nucleic acids yield on hydrolysis a carbo- hydrate. Thus Hammarsten has isolated a pentose from the nucleo- proteins of the pancreas. It is supposed that the nucleic acid of the thymus gland contains a hexose, since it is possible to split off from it Isevulinic acid, which is one of the first products of the decomposition of a hexose. The complex constitution of the nucleic acids and nucleoproteins may be rendered clearer from the following schema : on digestion yields Nucleo-protein nuclein proteoses and peptones dissolved in alkali and precipitated with hydrochloric acid yields nucleic acid hydrolysed yields acid derivatives of protein, histones or protamines phosphoric acid I reducing sugar pentose or hexpse purine bases pyrimi adenine urac guanine thyn cytosinc It must not be imagined, however, that all these disintegration products are present in all nucleic acids. Thus the nucleic acid derived from the pancreas, the so-called guanylic acid, yields of the purine bases only guanine, and of the pyrimidine bases only thy mine and uracil, and every variety is met with as we analyse the nucleic acids of different origin. The fact that nucleic acid is a characteristic and necessary constituent of all nuclei adds interest to the divergence of its constituent radicals from those which dis- tinguish the proteins of the cell protoplasm. Further importance is lent t<> THE PROTEINS 103 this section of the chemistry of the body by the close relationship which we shall have to study later between the nuclein metabolism of the body and the production and excretion of uric acid. The researches of Levene have thrown light on the manner in which these different groups are bound together to form nucleic acid. In the acid obtained from the thynms the carbohydrate group Hexose is joined to a nitrogenous ring compound, forming what is termed a ' nucleoside.' Four of these nucleosides, in thymic acid, join with four molecules of phosphoric acid to form a ' tetra-nucleotide.' The formula provisionally assigned to thymic acid is therefore as follows : HO, HO = PO- - HO C5H4N50 guanine group O =PO -C6H1004 (/ I C6H802 -- C5H5N202 thymine group O HO C6H802 C4H4N30 cytosine group O = PO C6H1004-C5H4N5 adenine group HO Other nucleic acids are simpler in constitution and may be composed of only one or two nucleotide groups. Thus the inosinic acid of muscle is a mono-nucleotide, con- sisting of phosphoric acid linked by a pentose group with hypoxanthine. The defi- nition of a nucleotide would thus be a compound in which a carbohydrate group links a phosphoric acid group with a purine or a pyrimidine group. Nucleic acids are simple or compound nucleotides. The pentose in inosinic acid is d-Ribose. The same pentose occurs in yeast nucleic acid. The nucleic acid of the pancreas, also called Guanylic acid, consists of phosphoric acid linked with guanine by a molecule of d-Ribose. (c) THE GLYCOPROTEJNS. In the glycoproteins the prosthetic group is represented by a carbohydrate radical, generally containing nitrogen, such as glucosamine or galactosamine. They are split into their two constituents, protein and carbohydrate radical, on prolonged boiling with dilute mineral acids or by the action of alkalies. They may be divided into the two main groups of mucins and mucoids. The mucins play a large part in the animal kingdom as protective agents. They form the slimy secretion which covers the inner surface of the mucous membranes and the outer surface of many marine animals, and is secreted either by the goblet cells of the epithelium or by special groups of cells collected together to form a mucous gland. They may be precipitated from their solutions or semi-solutions by the addition of acfds, and after precipita- tion need the addition of alkalies for their re-solution. They are not coagu- 104 PHYSIOLOGY lable by heat. The presence of their protein moiety causes them to give the various typical protein tests, such as the xanthoproteic, Millon's, the biuret reaction, and so on. Prolonged boiling with acids splits the molecule, with the production of acid metaprotein and albumoses and glucosamine. From the mucin of frogs' eggs a similar treatment results in the production of galactosamine. With the raucins may be classified certain bodies which have been derived from ovarian cysts, namely, pseudomucin and paramucin. Pseudomucin occurs as a con- stituent of the colloid material from ovarian tumours. It forms slimy solutions which do not coagulate by heat and are not precipitated by acetic acid. It is precipitated by alcohol, the precipitate being soluble in water even after standing a long time under the alcohol. On boiling with acid it gives a reducing substance. Paramucin differs from the above in reducing Fehling's solution before boiling with acids. Otherwise it resembles pseudomucin. Leathes, in investigating this body, isolated from it a reducing substance which apparently was an amino -derivative of a disaccharide, perhaps in combination with glycuronic acid. The mucoids include a number of substances wrhich may be extracted from various tissues by the action of weak alkalies, e.g. from tendons, bone, and cartilage. The best studied example of this group is the chondromucoid which, with collagen, forms the ground substance of cartilage. Chondro- mucoid is especially rich in sulphur and gives protein by long treatment with weak alkali. On boiling for a short time with acid it is decomposed into sulphuric acid and chondroitin, and this latter, on further action of the acid, is converted into a substance chondrosin, which is certainly an ammo- derivative of a polysaccharide containing the elements of glycuronic acid and an amino-disaccharide. Chondroitin-sulphuric acid occurs not only in cartilage but also in bone, yellow elastic tissue, white fibrous tissue, and as a constant constituent of the lardacein or amyloid substance which occurs as a deposit in the middle coat of the blood-vessels as the result of syphilis or long-continued suppuration, and gives rise to the condition known as ' lar- daceous disease.' Another example of this class of mucoids is ovomucoid, which is a constituent of egg-white. In order to prepare ovomucoid the globulin and albumin are precipitated by boiling diluted egg-white. From the filtrate ovomucoid can then be thrown down by alcohol. A similar body has been prepared from blood serum. Both these mucoids yield a large amount of reducing substance on hydrolysis. Thus from 100 grm. of ovo- mucoid it is possible to prepare 30 grm. of glucosamine. (10) THE ALBUMINOIDS OR SCLERO-PROTEINS. Under this heading are grouped a number of diverse substances which play an important part in building up the framework of the body. Their value as skeletal tissue seems to be determined by their insoluble character. On this account it is practically impossible to speak of purifying them. In every case we can simply take the residue of a skeletal tissue which is left after extraction of the soluble constituents. When broken down by the action of strong acids, they yield a series of disintegration products which are included among those we have already studied as the disintegration products of proteins. Their difference from the proteins which are employed in metabolism for their THE PROTEINS 105 nutritive value is caused either ^by the absence of certain groups common to all the nutritive proteins, by the presence of an excess of one or two groups, or by the presence of certain polypep tides which present considerable resist- ance to the action of digestive ferments. This class plays the part in the animal economy which in the vegetable kingdom is filled by the anhydrides of the hexoses and pentoses, e.g. the celluloses, lignin, the pentosanes, &c. Collagen forms the main constituent of white fibrous tissue and the ground substance of bone and cartilage. It is insoluble in water, hot or cold, and in trypsin. Under the action of acids or when subjected to prolonged boiling with water, especially under pressure, it is converted into gelatin, which is soluble in hot water, forming a colloidal solution liquid at high temperatures, but setting to a jelly when cold. When subjected to acid hydrolysis it gives a series of amino-acids from which tyrosine and tryptophane are wanting. On this account gelatin does not give any reaction either with Millon's reagent or with glyoxylic acid. On the other hand, there is a preponderance of such groups as glycine and phenylalanine, and it is probable that glycine, phenylalanine, and leucine are joined together, perhaps with other amino- acids, to form a polypeptide which is not attacked by digestive ferments, and therefore determines the resistance of the original collagen molecule to solu- tion. Gelatin is precipitated by tannic acid, but not by acetic acid. It is dissolved with hydrolysis by gastric juice or by pancreatic juice, whereas collagen, its anhydride, is unaffected by the latter. On prolonged boiling in water it is converted into a modification which does not form a jelly on cooling. Under the action of formaldehyde it is converted into an insoluble modification which does not melt on warming. Reticulin. This name has been applied to the tissue which forms the supporting network of adenoid tissue, and has also been described in the spleen, the mucous mem- brane of the intestine, liver, and kidneys. It differs from collagen in resisting digestion by gastric juice, and also in containing phosphorus in organic combination. According to Halliburton there is no essential difference between reticulin and collagen. The keratins are produced by the modification of epithelial cells and form the horny layer of the skin as well as the main substance of hairs, wool, nails, hoofs, horns, and feathers. They are distinguished by their insolubility in water, dilute acids or alkalies, and in the higher animals pass through the alimentary canal unchanged. Although differing in their elementary composition, according to the tissue from which they are pre- pared, they are all distinguished by the very large amount of sulphur present in their molecule. The greater part of this sulphur is in the form of cystine, of which as much as 10 per cent, can be extracted from keratin. They also yield, on acid hydrolysis, tyrosine in larger quantities than is the case with the ordinary proteins. N euro keratin, which forms the basis of the neuroglial framework of the central nervous system, must be grouped by its general behaviour as well as by its origin with the keratins. It resembles the other members of this class in its insolubility and in its high content in sulphur. It is extracted from nervous tissues by boiling these with alcohol and ether and then submitting 106 PHYSIOLOGY the tissue to prolonged tryptic digestion, which leaves the neurokeratin unaffected. Elastin is a constant constituent of the connective tissues, where it foims the elastic fibres. In some localities, as in the ligamentum nuchse, practically Fibroin of silk Klastin Krratin from horn Keratin from horsehair K'lTSltill from tVjitllrrs < it-hit in Glycine .... 36-0 25-75 0-45 4-7 2-6 1 <;-.-> Alanine .... 21-0 6-6 1-6 1-5 1-8 n-s Amino-valerianic acid 0-0 1-0 4-5 0-9 0-5 1-0 I'roline .... present 1-7 r>--2 Leucine .... 1-5 21-4 15-3 7-1 8-0 2-1 Phenylalanine . 1-5 3-9 1-9 0-0 0-0 0-4 Glutamic acid. 0-0 0-8 17-2 3-7 2-3 0-88 Aspartic acid . present present 2-5 0-3 1-1 0-56 Cystine .... — — 7-5 — — Serine . . . • 1-6 1-1 0-6 0-4 0-4 Tyrosine. 10-5 0-34 3-6 3-2 3-6 0-C Tryptophane . — — — — — 0-0 Lysine .... traces — 0-2 1-1 — 2-75 Arginine . . 1-0 0-3 2-7 4-5 — 7-62 Histidine small amount — — 0-6 — 0-4 Oxyproline ~ 3-0 the whole tissue is made up of these fibres. Elastin is insoluble in water, alcohol, or ether, or in dilute acids and alkalies. It is slowly dissolved on prolonged treatment with gastric juice, but is practically unaffected in the alimentary canal. It gives the xanthoproteic and Millon's tests. Other members of this group are fibroin, which forms the main substance of silk, spongin, the horny framework of sponges, conchiolin, the ground substance of shells, and perhaps the amyloid substance or lardacein which we have already mentioned in connection with the mucoids. All these sclero -proteins present considerable differ- ences in their qualitative and quantitative composition in amino-acids. Their proxi- matte composition is shown in the Table given above (Abderhalden). We have finally to mention a miscellaneous collection of bodies which are allied to the proteins and are distinguished by their extreme insolubility. They are often designated as albumoids. Of their composition we know practically nothing. Under this name are grouped such substances as those forming the membrana propria of glands, the sarcolemma of striated muscle, the albumoid of the crystalline lens, the ground substance of the chorda dorsalis, the organic basis of fish scales, and many similar Mil.staiiees. In every case the substance is characterised necessarily according to its place of origin, little or nothing being kimun as to its chemical composition. SECTION VI THE MECHANISM OF ORGANIC SYNTHESIS THE ASSIMILATION OF CARBON THE building up of protoplasm from the material which is available at the earth's surface must be an endothermic process. The food presented to the plant contains the necessary elements, but as a rule in a state of complete oxidation. The energy of the living plant, as of animals, is derived almost entirely from the oxidation of its constituents. The building up of un- organised into organised material must therefore be effected at the expense of energy supplied from without. The source of this energy is the sun's rays. The machine for the conversion of solar radiant energy into the chemical potential energy of protoplasm is the green leaf. Here a deoxidation of the carbon dioxide of the atmosphere takes place, with the production of carbo- hydrates, generally in the form of starch. The formation of starch must be regarded as the first act in the life-cycle, since this substance serves as a source of energy to the already formed protoplasm in its work of building up all the other constituents of the living cell. It is the solar energy captured by the green leaf which is utilised by all plants devoid of chlorophyll, as well as by the whole animal kingdom. There are one or two exceptions to this statement. Thus the bacterium nitro- somonas, described by Winogradsky, grows on a medium devoid of all organic con- stituents, and derives the energy for its constructional activity from that set free in the conversion of ammonia into nitrites. The sulphur bacteria apparently derive their energy from the decomposition of hydrogen sulphide and the liberation of sulphur. The fundamental importance of this process of assimilation for the whole of physiology justifies some account of the researches which have been directed to the elucidation of its mechanism. The production of oxygen by the green plant was discovered by Priestley in 1772, and a few years later Ingenhaus showed that this production occurred only in the light and was effected only by green plants. De Saussure (1804) pointed out that the essential process concerned was a setting free of the oxygen from the carbon dioxide of the atmosphere, and recognised that the co-operation of water was also necessary. Mohl in 1851 observed the formation of starch grains in the chlorophyll corpuscles, and regarded these as the first products of assimilation. The organs of carbon dioxide assimilation are the chloroplasts. These, which are responsible for the green colour of plants, are generally small oval bodies embedded in the cytoplasm, but sometimes, as in spirogyra, may have the form of spiral bands. In a plant which has been kept for some time in the dark, or in an atmosphere free from carbon dioxide, they present 107 108 PHYSIOLOGY no enclosed granules. Within three to five minutes after exposure to light in the presence of carbon dioxide, starch granules make their appearance within them, and grow rapidly, assuming the typical laminated structure. Engelmann has pointed out a means by which it can be proved that the chloroplasts carry out this process without the co-operation of the rest of the cytoplasm. Certain bacteria have a great avidity for oxygen and present movements only in the presence of this gas. If a filament of spirogyra be placed in a suspension of these bacteria and be examined under a microscope, the bacteria will be seen to congregate in the immediate neighbourhood of the chlorophyll bands. The same phenomenon is observed in the case of chlorophyll corpuscles isolated by breaking up the cells in which they were contained. These corpuscles therefore take up carbon dioxide and water, and form carbohydrate and oxygen, as follows : n(6C02 + 5H20) = (C*H100.)n + n(602) The whole structure of the green leaf is directed to the furthering of this process. Its cells contain chlorophyll corpuscles, which change their position according to the intensity of the illumination. A free supply of air to all the cells is provided by means of the stomata on the under surface of the leaf. Horace Brown has shown that the rate at which carbon dioxide diffuses through such fine openings is as great as if the whole leaf were an absorbing surface. We get therefore optimum absorption of, carbon dioxide by the leaf, with the maximum protection of the absorbing tissue and the necessary limitation of loss of water by transpiration. In view of the very small amount of carbon dioxide in the atmosphere, the extent of the assimilatory process is remarkable. One square metre of leaf of the catalpa can lay on 1 grm. of solid per hour, using up for this pur- pose 784 com. carbon dioxide. The rapidity of assimilation is increased within limits by increasing the intensity of the light falling on the plant, though an over-stimulation of the process is prevented by the movements of the chloroplasts just mentioned. It is also increased by raising the per- centage of carbon dioxide in the atmosphere supplied to the leaf. The optimum percentage of carbon dioxide will of course vary with the other conditions of the leaf. In certain experiments Kreusler found the optimum to be about 1 per cent. Taking the amount of assimilation in normal air with •03 per cent, carbon dioxide at 100, the assimilation in an atmosphere con- taining 1 per cent, was 237, and was not increased by raising the percentage of carbon dioxide to 7 per cent. Owing to the decomposition of the organic matter of the soil, the percentage of carbon dioxide near the ground is always greater than in the higher strata of the atmosphere — a fact which is taken advantage of by the low-growing plants and herbage. Other necessary con- ditions of assimilation are the presence of water and the maintenance of a certain external temperature. The absorption of the sun's rays by the leaf raises the temperature of the latter above that of the surrounding medium, and so quickens the process of assimilation. The assimilation of carbon dioxide, the formation of starch, and the THE MECHANISM OF ORGANIC SYNTHESIS 109 evolution of oxygen will go on in the isolated chloroplast. In the absence of chlorophyll, as in an etiolated leaf, the formation of starch will take place if the plant be supplied with a sugar such as glucose, and this conversion represents the main function of the leucoplasts present in all the cells of the reserve organs of plants. In the absence of chlorophyll no decomposition of carbon dioxide takes place, so that this pigment is evidently essential for the utilisation of the sun's energy. Chlorophyll may be extracted from leaves by means of absolute alcohol. A solution is thus obtained which is green by transmitted and red by reflected light, i.e. chlorophyll is a fluorescent substance. It presents four absorption bands, the chief being an intense black band between Fraunhofer's lines B and C. If the chlorophyll is the means of conversion of the solar into chemical energy, the conversion must take place at the expense of the light which is absorbed by the pigment. One would expect therefore the process of assimilation to be most' pro- nounced in those parts of the spectrum corresponding to the absorption bands — an expectation which has been realised by experiment. As to the exact chemical changes effected by these absorbed rays physio- logists are still undecided. There can be no doubt that an early product of the process is a hexose, which is rapidly converted into cane sugar or into starch. It was suggested by Baeyer in 1870 that carbon dioxide was reduced to formaldehyde, which later by condensation yielded sugar. We know that formaldehyde easily polymerises to form a mixture of hexoses, but until recently no evidence had been brought forward of its presence as an intermediate product in the assimilatory process. For most plants, indeed, formaldehyde is extremely poisonous, though certain algse, as well as the water-plant, Elodea. can stand a solution containing -001 per cent, formaldehyde. Bokorny stated that spirogyra could form starch out of such derivatives of formaldehyde as sodium oxymethyl-sulphonate, or from methylal. The difficulty in these cases is that possibly a spontaneous formation of sugar from the formaldehyde had taken place in the solution and that the plants were using up the sugar rather than the formaldehyde as the source of their starch. One must assume, with Timiriazeff, that the function of chlorophyll in the process of assimilation is that of a sensitiser. Just as the addition of eosin to the emulsion used for coating photographic plates will render these sensitive to the red and green parts of the spectrum, i.e. will excite change in the silver salt when light from these parts of the spectrum falls upon it, so the chlorophyll serves as a means by which the absorbed solar energy can be utilised for the production of chemical change in the chloroplast. Attempts have been made to imitate this process outside the plant. Thus Bach passed a stream of carbon dioxide through a 1-5 per cent, solution of a fluorescent substance, uranium acetate, in sunlight. As a result there was a precipitate of uranium oxide and peroxide, with the formation of traces of formaldehyde. Usher and Priestley, on treating a solution of carbon dioxide with 1-5 per cent, uranium "acetate or sulphate in bright sunlight, obtained uranium peroxide and formic acid, but no formaldehyde. The formation of peroxides 110 PHYSIOLOGY in these condition* suggests that the first change in the chloroplast may be as follows : C02 + 3H20 - 2H202 4- CH20 Such a reaction must be regarded as reversible since the hydrogen per- oxide first formed would tend to oxidise the formaldehyde again. Moreover it would have a destructive influence on the chlorophyll itself, which is easily oxidised. In order therefore that the reaction should go on in one direction only, i.e. that of assimilation, means must be present in the chlorophyll cor- puscles for the removal of both hydrogen peroxide and formaldehyde as soon as they are formed. The removal of the hydrogen peroxide can be effected by a catalase, which is fairly widely distributed in plants and has been shown by the last-named authors to be present in the chloroplasts. In order to demonstrate the production of the first result of assimilation, i.e. formalde- hyde, the further stages in its conversion must be stopped by killing the plant and the catalase it contains. They therefore placed leaves, which had been boiled, in water saturated with carbon dioxide and exposed them to bright sunlight. The leaves were bleached by the oxidation of the chlorophyll, and some substance of an aldehydic nature was produced, as shown by the red colour obtained on placing them in rosaniline, previously decolorised with sulphurous acid. Two proofs were brought forward that this substance was formaldehyde : (a) Some of the bleached leaves were soaked for twelve hours in aniline water. The chloroplasts under the microscope were seen to 'contain crystals resembling methylene aniline. (b) The leaves were distilled in a current of steam. The distillate was shown to contain formaldehyde by the formation of methylene aniline crystals on treatment with aniline, and by the preparation from it of the characteristic tetrabrome derivative of hexamethylenetetramine. Usher and Priestley conclude that the first products of the photolysis of carbonic acid are hydrogen peroxide and formaldehyde. Both these substances are rapidly removed from the reaction. The hydrogen peroxide is broken up by the catalase into water and oxygen which is turned out by the plant. The formaldehyde is at once polymerised in the protoplasm of the chloroplast with the formation first of a hexose and then of starch. The formaldehyde, if not removed in this way, destroys the catalase. The hydrogen peroxide, if not broken up by the catalase, destroys the chlorophyll. The relations between the various factors in this process may be dia- grammatically expressed thus : THE MECHANISM OF ORGANIC SYNTHESIS 111 Carbon dioxide -f- Water I r (// not removed, destroys}^ CHLOROPHYLL •f Hydrogen peroxide + Formaldehyde / (// not removed, poisons) * / ENZYME LIVING PROTOPLASM \ \ Oxygen Carbohydrates In thus reducing certain of the stages in the assimilation of carbon to phenomena which can be imitated outside the living organism, we have made considerable strides in the ' understanding ' of the process. The stage for which the vitality of the chloroplast is absolutely essential is the formation of starch from formaldehyde. Outside the body, our polymerisation of formaldehyde results in the formation of a mixture of sugars which are optic- ally inactive. The same process, in the living cell, leads to the production of optically active sugars which are connected stereochemically and mutually convertible one into the other, e.g. fructose and glucose. The derivatives of protoplasm, containing asymmetric carbon atoms, are in the same way optically active, and it seems that the asymmetry of the protoplasmic molecule conditions a corresponding asymmetry in the substance which it builds on to itself. The protoplasm furnishes, so to speak, a mould in which polymerisation of formaldehyde can result only in the production of sugars of certain definite stereochemical configurations. Few, if any, chemical reactions are pure. Nearly all are attended with by-reactions, so that the yield of end product never attains 100 per cent, of the theoretical yield. Even if the above mechanism be regarded as the chief one, it is probable that side reactions take place at the same time, so that we may have the formation of substances such as glyoxylic acid and other derivatives of the fatty acid series. Such by-products might play an im- portant part in the other synthetic activities of the cell, and especially in the formation of fats and proteins. THE FORMATION OF PROTEINS Our knowledge of the mechanism by which proteins are synthetised in plants is still more incomplete than that of the synthesis of carbohydrates, and we are reduced in most cases to a discussion of the possible ways in which, from our knowledge of the chemical behaviour of the constituents of the protein molecule, we might conceive of its formation. We can at any rate state the problems which have to be solved and study the conditions under which the synthesis of protein is possible in plants and in animals. We know that plants are independent of any organic food for building up their various constituents, whether carbohydrate, protein, or fat, pro- vided only that they possess chlorophyll corpuscles and so are able to utilise 112 PHYSIOLOGY the energy of the sun's rays. Most plants will grow in the dark if supplied with sugar and with combined nitrogen either in the form of ammonia or of nitrates. The higher plants are especially dependent on the presence of nitrogen in the latter form, and it is on this account that the nitrifying bac- teria of the soil acquire so great an importance for agriculture. From the carbon dioxide of the atmosphere or from the hexose formed by the assimila- tion of carbon, and from nitrogen, in the form either of ammonia or nitrates, together with inorganic sulphates, the plant cell is able to build up all the various types of protein which are distributed throughout the vegetable kingdom. Our study of the disintegration products of proteins has shown that this class of bodies contains a large number of the most diverse groups, having as a common character the possession of nitrogen in their molecule, generally as an NH2 or NH group. These disintegration products can be 'classified as follows : (a) Open chain amino-acids. (6) Heterocyclic compounds, including : (1) Pyrrol derivatives. (2) Pyrimidine derivatives. (3) Iminazol derivatives. These two last groups co-exist in all the purine compounds. (c) Benzene derivatives. (d) Indol derivatives. The first step in the synthesis of proteins is probably the formation of these constituent groups. Just as in digestion the protein molecule is taken to pieces with the formation of the different amino-acids, so in the synthetic action of protoplasm the reverse process of dehydration occurs, resulting in a coupling up of the different groups, as has been effected by Fischer in the case of the polypeptides. Wherever transport of protein from one part of the organism to another is necessary the protein is carried, not in its original form, but in the hydrolysed condition of amino-acids. Thus the germination of seeds which contain rich stores of protein is accompanied by a liberation of proteolytic ferments within the cells of the seeds, and the breakdown of the reserve protein into its constituent amino-acids. As amino-acids it is transported into the growing tip and leaves of the seedling, analysis of the latter showing a very large percentage of nitrogen in the form of amino-acids. This is especially the case if the synthetic functions of the growing tip are hindered by interference with assimilation, as, e.g. by keeping the plant in the dark. Under these circumstances, asparagine may form as much as 25 per cent, of the total dried weight of the seedling. In animals the greater part of the protein of the food is broken down into its constituent amino- acids in the intestine. These are absorbed and probably carried to the different organs of the body, where they are resynthetised, generally in different proportions from those of the original protein, into the protein specific for the organ or tissue. The same process of hydrolysis and subsequent synthesis occurs whenever the transport of protein is neces- sary from one organ to another. We shall later on have to discuss the THE MECHANISM OF ORGANIC SYNTHESIS 113 possibility of synthesis of the different amino-acids in animals. We need therefore at present deal only with the possible methods by which, from the glucose or substances produced in the assimilation of carbon and from the ammonia or nitrates derived from the soil, the plant is able to make the different groups which go to the building up of the protein molecule. All the amino-acids contain the NH2 group in the a position. We can therefore consider them as formed by the interaction of an a-oxyacid and ammonia. Thus : CH3 CH3 ! I CH.OH + NH3 CH.NH2 + H2O I I COOH COOH lactic acid alanine This particular example, namely, the formation of 'alanine, may occur at the expense of the glucose produced as the first product of assimilation of carbon dioxide. If a solution of glucose together with lime be exposed to sunlight for a considerable time it undergoes decomposition with the forma- tion of lactic acid. Thus : C6H1206 2C3H603 glucose lactic acid This change of glucose to lactic acid under the catalytic influence of the alkaline calcium hydrate probably occurs by means of a shifting of the elements of the water, a process which in many long chains seems to occur with considerable facility, and is dependent on the spatial configuration of the molecule involved. Thus the change of sugar to lactic acid is readily effected by means of many micro-organisms in the case of glucose, fructose, and mannose, but with considerable difficulty in the case of galactose. In the three former sugars the atoms round the two middle carbon atoms of the chain are disposed thus : I I OH.C.H H.C.OH I or | H.C.OH OH.C.H I I When either of these arrangements reacts with water, thus : CH2OH CH2OH I I CHOH CHOH I I OH.C.H OH COH + H2O HCOH H CHoOH I I CHOH CHOH I I COH COH 114 PHYSIOLOGY we obtain two molecules of gly eerie aldehyde, which then by a further shifting of the OH and H groups becomes CH3 I CH.OH I COOH lactic acid Lactic acid with ammonia and some dehydrating agent will give amino- propionic acid or alanine. The formation of the higher amino-acids in- volves a process of reduction of the sugar first formed in the chlorophyll granules. It is possible however that the starting-point for the amino-acid synthesis may be, not a hexose itself, but some other substances, formed, so to speak, as by-products in the assimilation of sugar from carbon dioxide. We have seen reason to believe that the first result of the action of the sun's rays within the chlorophyll corpuscle is formaldehyde. This substance in the presence of calcium carbonate when exposed to the light gives a mixture of glyceryl aldehyde and dihydroxyacetone. If we can assume that acetone is formed from the latter by a process of reduction, we might possibly derive leucine from an interaction of this substance with lactic acid and ammonia. Thus: CH3 CH3 CH3 CH3 \CH/ I I I CO + CH.OH + NH3 + H2 = CH2 -f 2H2O I I I CH3 COOH CH.NH2 I COOH As an intermediate product in the synthesis of starch, glyoxylic acid CHO has been described as occurring in the green parts of plants. This COOH substance with ammonia gives formyl gly cine, and by the splitting off of formic acid, glycine or amino-acetic acid. Why nitrates are necessary for certain forms of plants is not at present understood. In the proteins nitrogen always occurs in an unoxidised form as NH or NH2, and the nitrates taken up from the soil must therefore undergo reduction before they can be built into the protein molecule. It is supposed that they may pass through a series of reductions, namely : HN03 HN02 HNO H2N— OH nitric acid nitrous acid hyponitrous acid hydroxylamine and that the latter substance then reacts with formaldehyde or other sub- stance derived from the carbon dioxide assimilation to form amino-roin- pounds. In general we may say that the probable mechanism of formation of arnino-acids is the production of a-oxyacids, which then react with ammonia to form the amino-acids of the protein molecule ; but of the THE MECHANISM OF ORGANIC SYNTHESIS 115 exact steps in this process we are at present ignorant. Knoop's work would point to the ketonic acids as formftig one step, and as interacting with ammonia, with simultaneous reduction, to form amino-acids. The pyrrol ring which occurs in proline and in oxyproline may possibly be derived from an open chain amino-acid, and it has in fact been suggested that the proline found in the products of the acid digestion of proteins is derived from ornithine by a process of condensation with the loss of ammonia. Thus: CH2NH2.CH2.CH2.CH.NH2COOH becomes CH2.CH2.CH2.CH.COOH NH or, as it is generally written : CH2— CH2 I I CH2 CH.COOH V NH Its pre-existence in the protein molecule is however practically assured, and it plays an important part in the building up both of chlorophyll and of haematin, the prosthetic group of haemoglobin. CH— NH Iminazol || CH— N occurs in histidine (which is iminazol alanine), and can be formed fairly readily by the action of certain catalytic agents on a mixture of glucose and ammonia. Thus, if a solution of glucose with ammonia and zinc oxide be exposed to light, methyl iminazol is formed in large quantities. Windaus and Knoop imagined that in this process glyceric aldehyde and formaldehyde are first formed, and that these then interact with ammonia to form methyl iminazol. CH3 C — NH II >H CH— N It is interesting to note that, if we attach to this compound carbamide or urea, we obtain a body belonging to the class of purines. Xanthine, for instance, would have a formula NH— CO I I CO C— NH I H NH— CH— N Thus by the action of simple catalytic agencies on sugar and ammonia we can obtain the iminazol nucleus, and by easy transitions pass through 116 PHYSIOLOGY this to the purine nucleus with its contained ring, the pyrimidine nucleus, found in the bases cytosine, uracil, &*., which occur in the nucleins. With regard to the formation of the aromatic constituents of the protein molecule, i.e. those containing the benzene and indol rings, we have at present very little indication even of the lines along which it might be possible to prosecute our researches. It has been suggested that inosite may represent some stage in the formation of the benzene ring from the open chain found in the carbohydrates. Inosite has the same formula as glucose, namely, C6H120'6, but is a saturated ring compound : CHOH CHOHi^CHOH CHOH \/ CHOH CHOH and may be expected to be formed as a result of polymerisation of formalde- hyde. We have no evidence however of the possibility of such a formation, and the relations of this substance with the benzene compounds are by no means intimate. It is of such universal occurrence, both in plants and animals, that it is difficult to refrain from the suspicion that it may play some part as an intermediate stage between the fatty and the aromatic series. Since plants are able to manufacture all these varied substances out of the products of assimilation of carbon and ammonia or nitrates, they must also find no difficulty in transforming one amino-acid into another, and we know that most plants can procure their nitrogen from a solution of a single amino-acid as well as from a nutrient fluid containing the nitrogen in the form of ammonia. In animals the power of transforming one amino-acid into another, of one group into another, is probably strictly limited. So far as we know, nearly all the amino-acids utilised in the building up of the animal proteins are derived directly from those contained in the food. On the other hand, we have evidence in the animal body of synthesis of the purine bodies, and therefore of the pyrimidine and iminazol rings. The hen's egg at the beginning of incubation contains very little nuclein, nearly the whole of its phosphorus being present in the form of phosphoproteins and lecithin. As incubation proceeds these substances disappear, their place being taken by the nucleins which form the chief constituent of the nuclei of the developing chiek. In the same way the ovaries and testes of the salmon are formed during their sojourn in fresh water at the expense of the skeletal muscles, especially those of the back. Here again there is a transformation of a tissue poor in purine bases into a tissue which consists almost exclusively of nucleins and protamines. Whether in this case there is a direct conversion of the mono-amino-acids of the muscle proteins into the diamino-acids and bases typical of protamines, we do not know. It is more probable that only diamino-acids and bases previously existing in the muscle are utilised for the THE MECHANISM OF ORGANIC SYNTHESIS 117 formation of the generative glands, the other amino-acids being oxidised and utilised for the ordinary energy requirements of the animal. THE SYNTHESIS OF FATS In some plants fat globules have been stated to appear as the first products of the assimilation of carbon dioxide under the influence of sunlight, but there is no doubt that as a rule the formation of fats as reserve material in seeds or fruits occurs at the expense of carbohydrates. 'In the higher animals too, although a certain amount of the fat of the body is derived from the fat taken up with the food, the organism can also manufacture neutral fat out of the carbohydrates presented to it in its food. The problem therefore of the synthesis of the fats is the problem of the conversion of a sugar such as glucose into glycerin and the fatty acids. Although this conversion is apparently so easily effected by the living organism, it is one which from the chemical standpoint involves considerable difficulties. On account of the fact that the higher fatty acids consist largely of oleic and -stearic acids, i.e. acids containing eighteen carbon atoms in their chain, it has been thought that the synthesis might be brought about by the linking together of three molecules of a hexose. Such a change, would involve a series of difficult chemical transformations. For instance, no less than sixteen out of the eighteen oxygen atoms present in the three glucose molecules would have to be dislodged in order to convert the chain into stearic acid. Moreover, although these two acids contain a multiple of six carbon atoms, a whole array of fats are found both in plants and animals which could not be derived by a simple aggregation of glucose molecules ; and it is worthy of note that, of all the fatty acids which occur in nature, all those with more than five carbon atoms contain an even number of carbon atoms. Thus in milk, in addition to the three common fats, tristearin, tripalmitin, and triolein, we find the glycerides of caproic, caprylic, capric, lauric, and myristic acids, i.e. acids with 6, 8, 10, 12, and 14 carbon atoms. In all cases these acids are the normal acids with straight unbranched chains. It seems probable that in the transformation of carbohydrate into fatty acid the latter is built up, not by six carbon atoms, but by two carbon atoms at a time. It has been suggested by Magnus Levy and by Leathes that the transformation may occur by way of lactic acid. We have seen already that glucose and the sugars of analogous composition may be converted under the influence either of sunlight or of micro-organisms into lactic acid. Lactic acid breaks down with readiness into aldehyde and formic acid. CH3 CH3 I I CHOH = CHO + H I I COOH COOH Aldehyde undergoes condensation to form aldol. 118 PHYSIOLOGY CHS CH3 2| I CHO CHOH I CH2 I CHO aldehyde aldol Aldol reacts with water and undergoes a shifting of its OH and H groups, in a manner with which we are already familiar as occurring in the conversion of glucose into lactic acid, forming butyric acid. We may represent the reaction in the following way, placing the water molecules opposite those groups of the aldol molecule with which they react : CH3 I H HO CH H CH2 I OH O C H OH gives 2H2O CH3 I CH2 I CH2 I COOH It will be seen that although water must enter into the reaction there is no addition of water to the aldol in order to form the butyric acid. It has been suggested that similar reactions might account for the forma- tion of the higher fatty acids, in which case one molecule of acetic aldehyde would be added to the fatty acid in order to build up the acid which is next highest in the series. Although certain of the higher acids have been pre- pared in this way, proof is still wanting that a continuous series of SVM-- theses may be effected by the continuous addition of aldehyde. Such a hypothesis is however more probable than the direct conversion of three molecules of sugar into one molecule of stearic acid. The latter change would be associated with a very great absorption of energy, whereas a con- tinuous building up of fatty acids, by the addition of aldehyde obtained through lactic acid from the disintegration of hexose molecules, requires only a small expenditure of energy, which could be obtained by the combustion of the formic acid formed as a by-product in the process. If we suppose that the synthesis of the higher fatty acids from sugar is carried out in this way. the energy equations would he as follows (Leathes ) : 1 g. mol. glucose 677-2 cals. H 2 g. mnls. aldehyde + 2 g. mols. formic acid. 2 + 275-5 +2 X 151-7 = 6744 cals. THE MECHANISM OF ORGANIC SYNTHESIS 119 2 g. mols. aldehyde j J 1 g. mol. aldol j fl g. mol. butyric acid. 551 cals. ( 546-8 cals. j -\ 517-8 cals. Or, tracing the same change on as far as palmitic acid : 4 g. mols. glucose | f 1 g. mol. palmitic acid + 8 g. mols. formic acid. 2708 cals. J j 2362 cals. + 494 cals. = 2856 cals, In the first stage of the synthesis, the reaction leading to butyric acid, the net result would be, supposing the formic acid to be oxidised, that some 160 calories or nearly 25 per cent, of the whole energy, would be rendered avail- able for other purposes. In the latter stages leading to palmitic acid some of the energy derived from the oxidation of the formic acid would be required for effecting the synthesis, and only about 12-5 per cent, of the original amount contained in the sugar would be set free. It is worth noting that in the butyric fermentation of sugar by micro-organisms there is a production first of lactic acid, and this substance then disappears to give place to butyric acid. At the same time carbonic acid and hydrogen are evolved, both gases being derived from the decomposition of the formic acid. In the process a certain amount of caproic acid is always produced, and the crude butyric acid of fermentation is used as the source from which commercial caproic acid is derived. Attempts to produce the higher fatty acids by the condensation of successive molecules of aldehyde have so far resulted only in the production of branched chains of carbon atoms, whereas the normal fatty acids of the body are straight chains ; though Raper has shown that the normal caproic acid may be formed by the condensa- tion of aldol with itself. Miss Smedley has suggested that a more probable line of synthesis lies through pyruvic acid. Pyruvic acid, which may be produced in the body from lactic acid, and so from carbohydrate, is fermented by yeast with the production of acetaldehyde and carbon dioxide, by means of a ferment carboxylase. If we assume the existence of a similar ferment in the cells of the body, it would split this acid into aldehyde and CO2. Aldehyde however combines with a molecule of pyruvic acid to form a higher keto =acid, which might either be oxidised to the fatty acid containing one carjbon atom less, or might be again transformed by enzymes into an aldehyde capable of reacting with another molecule of pyruvic acid. These changes are represented in the following equations: CH3CO.COOH = CH3CHO + CO2 CH3CHO + CH3CO.COOH = CH3CHOH.CH2.CO.COOH CH3CHOH.CH2.CO.COOH + O == CH3CHOH.CH2COOH + C02 /3-oxyacids would thus be a normal stage in the building up as well as in the breaking down of fatty acids. The glycerin which enters into the formation of the ordinary neutral fats can be synthetised by both plants and animals, and there is every ground for believing that it, like the fatty acids, may be derived from carbohydrates. We have already seen that in the conversion of glucose into lactic acid the first step is the formation of glyceric aldehyde, 120 PHYSIOLOGY CH2OH CH2OH I I CHOH CHOH I I CHOH CHO CHOH CH2OH I I CHOH CHOH I I CHO CHO and it is easy to understand how by a process of reduction the aldehyde is converted into the corresponding alcohol, namely, glycerin. The synthesis of the neutral fat from glycerin and fatty acid is a change which can be accomplished by many ferments. It is one involving practically no absorp- tion or expenditure of energy. The change is a reversible one, and we find both in plants and animals that a hydrolysis of neutral fat into fatty acid and glycerin always occurs when a transport of the fat is required, while the laying down of fat as a store of energy is always preceded by a resynthesis of the neutral fat. We shall have occasion to deal in greater detail with these questions when we have to discuss the formation and fate of the fat in the animal body. m E s CHAPTER IV THE ENERGETIC BASIS OF THE BODY SECTION I THE ENERGY OF MOLECULES IN SOLUTION EVERY vital act involves at the same time a transformation of the material basis of the living cell and a transformation of energy. The ultimate source of the energies displayed by the animal organism is the chemical energy of the substances taken in as food. In all the changes undergone by either matter or energy in the body there is neither destruction nor creation. The living organism may therefore be regarded in one sense as a :~~achine, that is to say, a system for the conversion of one form of energy to another. Thus the steam-engine converts the potential energy of over- neated steam into mechanical work ; a gas-engine the chemical energy of an explosive mixture of gases into heat and mechanical energy ; in a battery ere is a transformation of chemical into electrical energy ; in a dynamo, of mechanical into electrical energy, and so on. In the living cell the chemical energy of the food may undergo conversion into any of the other forms mentioned above, i.e. heat, work, electrical difference of potential, or it may be used for the production of other chemical substances possessing perhaps as much potential energy as or more than the food-stuffs themselves. Protoplasm, which is the seat of all these changes in both plants and animals, is active only within fairly narrow limits of temperature, approxi- mately between 5° and 40° C. In consistence it is slimy and wet, water forming from 70 to 95 per cent, of its bulk. No substance introduced into the protoplasm has any influence on it, unless it be soluble, and the first stage in the preparation of food-stuffs for assimilation always consists in a process of solution. The sole source of energy to the body being that con- veyed with the food, it follows that all the energy with which we have to 1 is the energy of molecules in watery solution, the playground of whose tivities is a jelly-like mass of colloidal material, heterogeneous yet struc- turally continuous. It is important therefore at the outset to inquire into ;he nature of this energy and the methods by which it may be measured. OSMOTIC PRESSURE. If we place two gas jars together, mouth to mouth, as in Fig. 19, the upper jar containing hydrogen and the lower jar me heavier gas, such as oxygen or carbon dioxide, within a very short time 121 • *v a t ' n I 122 PHYSIOLOGY H CO- the gases will have become intimately mixed, and each jar will contain an equal amount of both gases. We say that each gas has diffused into the other, and ascribe the diffusion to the movement of the gaseous molecules. In closed vessels the rapidly moving molecules are continually impinging on the walls of the vessels and rebounding, and it is this bombardment by the gaseous molecules which is responsible for the pressure exerted by a gas on its containing walls. If we double the amount of gas in a given space, we double the amount of molecules which strike a unit area of the wall in unit time, and therefore double the pressure exerted by the gas on the vessel wall. In this way we may explain the law of Boyle that the pressure of a gas is inversely proportional to its volume, or the product of pressure and volume at a given temperature is a constant, PV=C, or since the energy of the molecules is proportionate to the absolute temperature, PV = RT, the familiar gas equation. The molecules of substances in solution behave, within the limits of the solution, in a manner precisely similar to the free molecules of a gas. Thus, if a vessel be half filled with a 10 per cent, solution of sugar and be then filled up by carefully pouring distilled water, so as to form a distinct layer on the heavier sugar solution, the sugar at once begins to move upwards into the distilled water. In consequence of the resistance offered to the movement of the sugar molecules through the water, this process of diffusion is slow, but if the vessel be left undisturbed and free from any agitation for two or three months, the sugar will be found to spread gradually throughout the liquid, so that at the end of this time all parts of the fluid contain a uniform amount of sugar. This process of diffusion, like that of gases, must be ascribed to a con- tinuous translatory movement of the dissolved molecules. Since the mole- cules possess mass and are endowed with a velocity, it is evident that they can exercise a pressure on any membrane or dividing surface which tends to hinder their free passage within the limits of the solvent. Thus if we take a pig's bladder containing a 20 per cent, solution of dextrose and immerse it in distilled water, water will pass in and distend the bladder to such an extent that it may burst from the rise of pressure in its interior. This swelling of the bladder is due to the fact that the molecules of sugar pass through it only with difficulty, and therefore in their passage outwards towards the confines of the water exert a pressure on the walls, driving t hem apart and so causing a distension of the bladder. It is impossible however by this means to obtain the full osmotic pressure due to the pressure exerted by the, suizar molecules, since the bladder wall itself is not absolutely im- permeable to sugar. If we imagine the sugar solution confined in a cylinder and covered with a layer of distilled water, the movement of the sugar molecules will cause them to wander from the lower to the upper part, and FIG. 19. THE ENERGY OF MOLECULES IN SOLUTION 123 : : FIG. 20. this process of diffusion will cease only when the concentration has become the same in all parts of the solution. Supposing however the two fluids are separated by a piston, p (Fig. 20), which is ' semi-permeable/ i.e. allows free passage to water, but not to the dissolved sugar, the molecules of sugar will now exert a pressure on the piston similar to that exerted on the walls of the containing vessel, and will tend to drive it upwards. The force which it is necessary to apply to the piston to prevent its upward movement will be the measure of the osmotic pressure of the sugar in the solution. If the piston be pressed down with a greater force, the sugar molecules alone are pressed together, since water can pass freely through the surface of the piston, and the sugar solution is therefore rendered more concentrated. Since force must be applied to the piston in order to press it down, work is done in the process, so that the concentration of any solution volves the performance of an amount of work determined by e initial and final osmotic pressures of the solution. If, on e other hand, a weight be applied to the piston which is less than the osmotic pressure exerted by the sugar solution, e piston with its weight will be moved upwards, and the lution will undergo dilution until its osmotic pressure exactly balances the weight on the piston. We see that the osmotic pressure of a solution represents a certain amount of potential energy, which can be utilised in an osmotic machine, such as at represented in the diagram, for the performance of work. THE MEASUREMENT OF OSMOTIC PRESSURE. By a method iffering but little from the one just sketched out, Pfeffer succeeded directly in measuring the osmotic pressure of certain solutions. For this purpose Pfeffer took advantage of the fact, discovered by Traube, that various pre- cipitates, if deposited in the form of membranes, were impermeable to the substances producing them as well as to some .other dissolved substances, though allowing a free passage of water. Thus, if a drop of a concentrated solution of potassium ferrocyanide suspended to a glass rod be introduced carefully into a more dilute solution of copper sulphate, it will be observed that at the junction of the drop and the surrounding fluid there is a brown membranous precipitate of copper ferrocyanide. In consequence of the greater concentration of the fluid in the drop, a constant passage of water takes place from without inwards through the membrane, and the drop therefore grows continually in size, sometimes sending out branches as a result slight currents in the fluid set up by accidental vibrations. Sugar intro- uced into such a drop, although quickening its rate of growth,. does not pass out into the surrounding copper sulphate solution, nor is there any passage of copper sulphate inwards or potassium ferrocyanide outwards. Pfeffer con- ceived the idea of depositing such a semi-permeable membrane within the €'erstices of a clay cell. Strengthened in this way, it is able to afford a istance to pressure, and therefore to permit of the contained fluid reaching full osmotic pressure. For this purpose a porous jar carefully cleansed and containing a solution of sugar mixed with a little copper sulphate is tn< i 124 PHYSIOLOGY dipped into a weak solution of potassium ferrocyanide. A semi-permeable membrane of copper ferrocyanide is thus produced in the pores of the filter, and this, while allowing the passing of water, is impermeable to the sugar. The tube is then fitted with a cork provided. with a closed mercurial mano- . meter and is immersed in distilled water, when it is found that water passes into the cell until the pressure within the latter is equal to the osmotic pressure of the dissolved substances. By this means Pfeffer obtained the following results with a 1 per cent, solution of cane sugar at different tem- peratures : Pressure in atmospheres Temp. °C. Calculated 6-8 Atm. 0-664 Atm. 0-665 13-7 0-691 0-681 22-0 0-721 0-701 32-0 0-716 0-725 36-0 0-746 0-735 It is always possible to calculate the pressure of a gas when its nature, its mass, and its volume are known. By Avogadro's hypothesis, equal volumes of gases at the same pressure contain equal numbers of molecules. On this account the molecular weight of any gas/can be reckoned directly from its density. The figures obtained by Pfeffer show that the same laws apply to the osmotic pressure of substances in solution as to the pressure of gases in their free state. It is therefore possible to reckon the osmotic pressure which would be exerted by 1 per cent, sugar in solution at a given temperature. This calculation is carried out as follows : A gramme molecule of any gas at 0° C. and 760 mm. Hg has a volume of 22-4 litres, therefore 342 grammes of cane sugar (the molecular weight of C12H220U = 342), if it could be converted into a gas at 0° C. and 760 mm. Hg, would have a volume of 22-4 litres. One gramme of sugar therefore at 22-4 the same temperature and pressure would have a volume of—— litres =65-5 c.c. In 348 Pfeffer's experiment the gramme of sugar was dissolved in 100 grammes of water, making a total volume at 0° C. of 106-6 c.c. The gaseous pressure of the sugar molecules in this solution will therefore amount to • 0-651 atmosphere. At a temperature of 6-8 the pressure would be 0.667 atmosphere, as against the observed 0'664 atmosphere. Pfeffer's method is difficult to carry out and is not applicable to all dissolved substances, since the cupric ferrocyanide membrane is permeable for many substances, such as potassium nitrate or hydrochloric acid. Other indirect methods have therefore been applied to the comparison of the osmotic pressures of different solutions. THE ENERGY OF MOLECULES IN SOLUTION 125 DETERMINATION OF OSMOTIC PRESSURE BY PLASMOLYSIS. Solutions which have the same osmotic pressure are spoken of as isosmotic or isotonic. The method of plasmolysis, which we owe to the botanist De Vries, consists essentially in the comparison of the osmotic pressure of solutions with that of the cell sap of certain plant cells, and depends on the fact that the ' primordial utricle,' the layer of protoplasm enclosing the cell sap, while freely permeable to water, is impermeable to a large number of salts and other crystalloids, such as sugar. It is therefore, so far as concerns these substances, * semi-permeable.' The cells which have been most used for this purpose are the cuticular cells on the mid-rib of the lower surface of the leaves of tradescantia discolor. If some of these cells are brought into a concentrated salt solution, which is * hypertonic ' as compared with the cell sap, water passes out of the cell into the salt solution, until the contents of the cell attain a molecular concentration equal to that of the surrounding medium. The protoplasmic layer therefore shrinks, leaving a space between it and the cell wall (Fig. 7, p. 23). If the outer solution has a smaller molecular concentration than the cell sap, water passes into the cell and causes here a rise of pressure which simply presses the protoplasm still more closely against the cell wall. If we determine the concentration of the salt solution at which the shrinkage of the proto- plasm, the plasmolysis, just occurs, and another smaller concentration at which plas- molysis is absent, we know that the concentration of the cell sap lies between those of the two salt solutions. Thus, if plasmolysis occurs in a solution containing 0-6 per cent, sodium chloride and is absent in a solution containing O59 per cent, of the same salt, the concentration of the cell sap must be about equivalent to a 0*595 per cent. NaCl solution. Solutions of different salts, in which plasmolysis just occurs, must also be isotonic with one another. Thus a 1-01 per cent, solution of KN03 is found to be isotonic with a O58 per cent. NaCl solution. DETERMINATION BY HAMBURGER'S BLOOD-CORPUSCLE METHOD. The limiting external layer of red blood corpuscles resembles the primordial utricle of plant cells in being impermeable to a number of dissolved substances. If therefore it be placed in a solution of smaller concentration than the corpuscle contents, it will swell up and, since it has no supporting cell wall, the increase in size will go on until the corpuscle bursts, and its contained red colouring-matter, haemoglobin, passes into solution in the surrounding fluid. If the corpuscles be then allowed to settle or be centrifuged, the fact that haemolysis has occurred is shown by the red colour of the clear supernatant fluid. With a given sample of blood, the concentration of a potassium nitrate solution is found at which the first traces of haemolysis occur. In order to determine the osmotic pressure of a solution, say, of sugar or of sodium chloride, these are also added in various dilutions to blood corpuscles until we get solutions in which haemolysis just occurs. These solutions will then be isotonic with the first determined potassium nitrate solutions. As an example of this method may be adduced the following results : Concentration Concentration of the -solution of the solution in which the in which the Mean blood corpuscles blood corpuscles ! concentration do not lose begin to lose haemoglobin hemoglobin Per cent. Per cent. Per cent. Potassium nitrate 1-04 0-96 1-00 Sodium chloride 0-60 0-56 0-585 Cane sugar 6-29 5-63 5-96 Potassium iodide 1-71 1-57 1-64 Sodium iodide . 1-54 1-47 1-505 Potassium bromide . 1-22 1-13 1-17 126 PHYSIOLOGY OSMOTIC PRESSURE OF ELECTROLYTES. It will be noticed in the last Table that the isotonic solutions of different salts contain these salts in the proportion of their molecular weights, i.e. each solution contains the same number of molecules of dissolved salt. For the term isotonic we might therefore employ equimolecular. When however these salts are compared with solutions of sugar, it is found that the osmotic pressures of the salt solutions are double or nearly double those of equimolecular solu- tions of sugar. The osmotic pressure of a sugar solution is equal to the pressure which its molecules would exert if they occupied the same space in a gaseous form. A dilute salt solution therefore acts as if every one of its molecules were doubled. This deviation of salt solutions from solutions of sugar is bound up with the power of the former to conduct an electric current. A sugar solution conducts electricity little better than pure water. On the S 6 7 FIG. 21. Diagram to illustrate Barger's method of determining osmotic pressure. The upper figure shows the capillary tube with nine alter- nate drops of cane sugar and the substance under investigation. other hand, the smallest trace of salt added to distilled water enormously increases its conducting power. As Arrhenius has shown. 1 his increase of the osmotic pressure of a salt solution is determined by the dissociation which all these salts undergo in watery solution. A dilute solution of sodium chloride contains, not the molecule NaCl, but an equal number of the ions Na and Cl, Na carrying a positive charge while the Cl ions carry a negative charge. As regards osmotic pressure and various other properties, each of these charged ions acts as a whole molecule. It is the existence of these ions which confers on the salt solution the power of conducting electricity- — a power the exercise of which is attended with a dissociation (an electrolysis) of the salt into its constituent ions, the electro-positive ion being deposited at the negative pole while the electro-negative ion is deposited at the positive pole. The molecular weight of NaCl is 58 '5. The molecular weight of glucose is 180. If there were no dissociation, a 0'58 per cent, solution of NaCl would be isotonic or isosmotic with a 1*8 per cent, solution of glucose. On account of the ionic dissociation or ionisation, it is actually isosmotic with a glucose solution of about 3 '5 per cent. THE ENERGY OF MOLECULES IN SOLUTION 127 INDIRECT METHODS OF MEASURING OSMOTIC PRESSURE. Equi- molecular solutions have the same osmotic pressures. Since the osmotic pressure of a solution is therefore directly dependent on the number of molecules it contains in unit space, any method which will give us informa- tion as to the number of molecules present will also enable us to determine the osmotic pressure. Other properties of solutions which, like the osmotic pressure, are functions of the number of molecules present, are vapour-tension, boiling-point, freezing- point. The presence of a substance in solution in water diminishes its vapour-tension at any given temperature, raises its boiling-point, and depresses its freezing-point, and the extent of the deviation from distilled water is proportional to the number of dissolved molecules present. The determination of the rise of boiling-point, though much employed by chemists, is of very little value in physiology, owing to the fact that nearly all the fluids of the body are seriously modified in character by a rise of tem- perature to 100° C. On the other hand, Barger has suggested an ingenious method in which the altera- tion of vapour-tension is made the basis of a method for determining the osmotic pressure of small quan- ities of fluids at ordinary temperatures. And this method may find important applications in phy- siology. BARGER'S METHOD. Drops of the fluid, the vapour- tension of which it is desired to ascertain, are drawn up into a tube (1-5 mm. in diameter), so as to alternate with small drops of cane sugar solution of known content (Fig. 21). Water in a state of [vapour will pass from the solution of c which the vapour-tension is the higher. By observing the edge of a drop under a magnification of 65 diams., it can be easily seen whether it has grown or diminished in size. If the edge of the drop remains stationary, it shows that the vapour- tension and the osmotic pressure of the two fluids equal. A series of trials- is made with different strengths )f salt solution until this equality is established. In lis method only minimal quantities of material are re- quired, and the determination of the aqueous tension is le at ordinary temperatures. The method however, which is of greatest value physiology, is the measurement of the depression of freezing-point. e depression of freezing-point can be converted directly into osmotic >ressure by multiplying the depression of freezing-point observed by the ictor 122 '7. Thus a 1 per cent, solution of sodium chloride with A = 0'61 ill have an osmotic pressure of 0'61 X 1227 = 74'847 metres of water. The determination is carried out in a Beckmann's apparatus with a thermometer ?ading to n\0-° C. (Fig. 22). A solution freezes at a lower temperature than pure water, FIG. 22. Beckmann's apparatus for determi- nation of freezing-point. 128 PHYSIOLOGY and the depression of freezing-point is proportional to the number of molecules present. Thus the freezing-point of a 1 per cent, solution of NaCl is — O61° C. The depression of freezing-point is generally represented by the Greek letter A. This method has the advantage that the fluids are in most cases hi no wise altered by the process of freez- ing, and it can be applied to solutions containing coagulable proteins which would be irretrievably altered by any considerable rise of temperature. Every substance in solution possesses therefore a certain amount of potential energy in the form of osmotic pressure. This pressure is inde- pendent of the nature of the substance dissolved and is determined merely by its molecular concentration. It can be used as a driving force for the movement by diffusion of the molecules themselves, or, by the use of appropriate mechanisms or ' machines,' for the performance of mechanical work, or, as will be seen later, for the production of electrical differences of potential. In addition to this osmotic or volume energy every molecule in solution can be regarded as endowed with a chemical energy, which is dependent not only on the number of molecules present, but also on the nature of the molecules. In the case of electrolytes and of substances which are susceptible of ionisation, the potential or intensity of the chemical energy of each molecule is capable of measurement. On the other hand, the chemical energy of a substance such as glucose cannot be definitely expressed apart from consideration of the conditions under which it is present. If we take the whole course of transformations undergone by glucose in the body, we may speak of it as having a potential energy, which is measured by the total heat energy given out by this substance on its complete com- bustion with oxygen to carbon dioxide and water. In the intermediate changes which it undergoes during its metabolism in the cells of the body, this energy is probably set free by degrees, but its chemical energy in any given phase cannot be measured unless the conditions and the end results of the chemical changes which it is undergoing are known. This chemical energy may be utilised for the production of heat, for the performance of chemical work in the building up of other substances, or by the multiplica- tion of the number of molecules in a solution, for the production of increased osmotic pressure, which in its turn may be converted into the energy of movement either of masses or of molecules. B«1 = SECTION II THE PASSAGE OF WATER AND DISSOLVED SUBSTANCES ACROSS MEMBRANES E have already seen that if, in a solution, the concentration of the dis- solved substance or solute is not uniform, there is a movement of the sub- stance from the place of higher to the place of lower concentration, and his movement proceeds until the concentration is equal throughout the fluid. This movement of dissolved substances through a fluid is spoken of as diffusion, and is analogous in all respects with the process by which the inter- ixture of gases is attained. The movement in the case of dissolved sub- stances, as of gases, takes place from the region of higher to the region of lower (osmotic) pressure. It can therefore be ascribed to differences of pressure, or rather to the factor which we regarded as responsible for the production of the pressure, viz. the movement of the molecules themselves. The rate of diffusion is not the same for all substances. In gases the rate varies inversely as the square root of the density of the gas. Thus hydrogen (density =• 1) diffuses four times as rapidly as oxygen (density = 16). We find similar differences between the 'rates of diffusion of dissolved substances — differ- ences which also are determined in all probability by the weight and size of the individual molecules, although the relation between molecular weight and rate of diffusion is not so simple as the ratio between these two quantities in gases. The diffusibility of a substance is given by its diffusion coefficient. The amount of dissolved substance, which diffuses in a unit of time across a given area of fluid, is proportional to the difference between the osmotic pressures at two cross-sections of the column of fluid at an in- finitesimally small distance apart. If we take a cylindrical mass of solution which is one centimetre long and has a sectional area of one square centimetre (Fig. 23), and maintain a constant difference of concentration between A and B =1, the diffusion coefficient is the amount of substance which diffuses in a unit of time from A to B. Thus the statement that the A ision coefficient of urea is 0 -810 / cm . it 7 -5° C. denotes that if A be con- FIG. 23. tinually filled with a 1 per cent, solution of urea, while in B a constant current of distilled water is I kept up so as to maintain the concentration at zero, in the course of a = 130 PHYSIOLOGY day 0-810 gramme of urea will pass from A to B through the cylinder of one centimetre in length and one square centimetre in cross-section. The deter- mination of these diffusion coefficients presents many difficulties. The task is however rendered easier by the fact, first ascertained by Graham, that diffusion of salts occurs as rapidly through a solid jelly of gelatin or agar-agar as through water. It is therefore possible to make the plug in the diagram solid by the admixture of one of these two substances, and to maintain a con- stant concentration on the two sides of it by the circulation of fluid without affecting the rate of diffusion through the cylinder by setting up accidental currents. More important from the physiological point of view than diffusion through fluids is the exchange of fluids (water and dissolved substances), which may take place across membranes. Such processes are of constant occurrence in all parts of the body and are concerned in such functions as the formation and absorption of lymph, the absorption from and secretion into the intestines, absorption from serous cavities, and so on. In many of these functions we shall have to consider later whether the transference across the membrane is determined solely by the nature and concentration of the fluids on the two sides of it or is effected by the active intervention, involving the expenditure of energy, on the part of living cells forming constituent elements of the membrane itself. It is worth our while there- fore to consider at some greater length the purely physical factors which may be concerned in the passage of water and dissolved substances across membranes. In the case of fluids containing only one substance in solution, the exchange across the membrane will be determined .entirely by the osmotic pressures. Thus, if two watery solutions, with the same osmotic pressure, are separated by a membrane through which diffusion can take place,' no change in volume occurs on either side of the membrane. If the solutions on either side of the membrane are of unequal osmotic pressure, water passes from the side where the pressure is lower to the side where it is higher, and there is a simultaneous passage of the solute from the side of greater to the side of less concentration. If however the solutions on the two sides contain dissimilar substances, with different diffusion coefficients, the conditions are more complicated, and may tend even to produce a movement of fluid in apparent opposition to the difference of osmotic pressure. Under these circumstances the. nature of the membrane itself is all-important. We may therefore shortly consider the various modes in which interchanges may take place across membranes of varying permeability. We .shall see that the close analogy which exists between substances in solution and gases, when dealing with ' semi- permeable ' membranes, is also borne out by experiment when used to predict the behaviour of solutions separated by such permeable membranes as occur in the body. The simplest case is that in which two fluids are separated by a perfect semi-permeable membrane that permits the passage of water but is absolutely m 8 FIG. 24. PASSAGE OF WATEK AND DISSOLVED SUBSTANCES 131 impermeable to dissolved substances. In this case the transference of water from one side to the other depends entirely on the difference of osmotic pressure between the two sides. If we suppose two vessels, A and B (Fig. 24), separated by such a mem- brane, A containing a solution of a and B a solution of ft water will pass from A to B so long as the osmetic pressure of ft is greater than the osmotic pressure of the solution of a. If B be subjected to a hydrostatic pressure greater than the osmotic difference between the two fluids, water will pass from B to A until the force causing filtration or transudation (the hydrostatic pressure) is equal to the force causing absorption into B (the difference of osmotic pressures). Under no circum- stances will there be any transference of salt or dissolved substance between e two sides. Such semi-permeable- embranes as this, however, rarely cur in the body over any extent of ace. The external layer of the cell protoplasm may resemble the rotoplasmic pellicle of plant cells in possessing this ' semi-permeability ' ; but in nearly all cases where we have a membrane made up of a number E cells, it can be shown to permit the free passage of at any rate a large nber of dissolved substances. Let us now consider what will occur when the two solutions A and B separated by a membrane which permits the free passage of salts and ber. If the osmotic pressure of B be higher than A at the commencement of the experiment, the force tending to move water from A to B will be equal to this osmotic difference. But there is at the same time set up a diffusion of tne dissolved substances from B to A and from A to B. The result of this diffusion must be that there is no longer a sudden drop of osmotic pressure from B to A, and the result of the primary osmotic difference on the move- ment of water will be minimised in proportion to the freedom of diffusion which takes place through the membrane. Now let us take a case in which A and B represent equimolecular and iso tonic solutions of a and ft. It is evident that the movement of water into A will vary as Ap — Bp * — 0. But diffusion also occurs of a into B and of ft into A. Now the amount of sub- stance diffusing from a solution is proportional to the concentration, and therefore to its osmotic pressure, as well as to its diffusion coefficient. Hence the amount of a diffusing into B will vary as A p. ok (when k is the diffusion coefficient). In the same way the amount of ft diffusing into A will vary as ~Bp. /3k'. Hence, if ok is greater than ftk', i.e. if a is more diffusible than ft, the initial result must be that a greater number of molecules of a will pass into B than of ft into A. The solutions on the two sides of the membrane will thus be no longer equimolecular, but the total number of molecules of a + ft in B will be greater than the number of molecules of a + ft in. A, and this differ- * Ap = osmotic pressure of A,.&c. 132 PHYSIOLOGY ence will be most marked in the layers of fluid nearest the membrane. The result therefore of the unequal diffusion of the two substances is to upset the previous equality of osmotic pressures. The layer of fluid on the B side of the membrane will have an osmotic pressure greater than the layer of fluid in immediate contact with the A side of the membrane, and there will thus be a movement of water from A to B. Hence if we have two equimolecular and isotonic solutions of different substances separated by a membrane permeable to the solutes, there will be an initial movement of fluid towards the side of the less diffusible substance. We have an exact parallel to this in Graham's familiar experiment, in which a porous pot filled with hydrogen is connected by a vertical tube with a vessel of mercury. In consequence of the more rapid diffusion outwards of the hydrogen than of atmospheric air inwards, the pressure within the pot sinks below that of the surrounding atmosphere and the mercury rises several inches in the tube. We must therefore conclude that, even when the two solutions on either side of the membrane are isotonic, there may be a movement of fluid from one side to the other with a performance of work in the process. In fact, osmosis may occur from a fluid having a higher towards a fluid having a lower osmotic pressure. If, for example, equimolecular solutions of sodium chloride and glucose be separated by a peritoneal membrane, the osmotic flow will take place from the fluid having the higher osmotic pressure — sodium chloride.* We might compare with this experiment the results of separating hydrogen at one atmosphere's pressure from oxygen at two atmospheres' pressure by means of a plate of graphite. In this case the initial result will be a still further increase of pressure on the oxygen side of the diaphragm — a movement of gas against pressure taking place in consequence of the greater diffusion velocity of hydrogen. So far we have considered only the behaviour of solutions when separated by a membrane, the permeability of which to salts is comparable to that of water ; so that the passage of salts through the membrane depends merely on the diffusion rates of the salts. There can be no doubt however that we might get analogous movements of fluid against total osmotic pressure determined, not by the diffusibility of the salts, but by the permeability of the membrane for the salts — a permeability which may depend on a state of solution or attraction existing between membrane and salts. We have an analogue to such a condition of things in the- passage of gases through an india-rubber sheet. If two bottles, one containing carbonic acid, the other hydrogen, be separated by a sheet of india-rubber, carbon dioxide passes into the hydrogen bottle more quickly than hydrogen can pass out into the carbon dioxide bottle, so that a difference of pressure is created, and the rubber bulges into the carbon dioxide bottle. We might, in the same way, conceive of a membrane which permitted the passage of dextrose more * In consequence of ionic dissociation of the sodium chloride, a decinormal solution of this salt will have an osmotic pressure nearly twice as great as that of a similar solution of the non -ionised glucose. PASSAGE OF WATER AND DISSOLVED SUBSTANCES 133 easily than that of urea. The importance of the membrane in determining the direction of the osmotic passage of fluid is well illustrated by Raoult's experiments. When alcohol and ether were separated by an animal mem- brane, alcohol passed into the ether, whereas if vulcanite were employed for the diaphragm, the osmotic flow was in the reverse direction, and an enormous pressure was set up on the alcohol side of the diaphragm.* The next point to be considered is the passage of a dissolved substance across membranes, in consequence of differences in the partial pressure of the substance in question on the two sides of the membrane. Stress has been laid by Heidenhain and others on the fact that in the peritoneal cavity, as well as from the intestine, salt may be taken up from fluids containing a smaller percentage of this substance than does the blood plasma, and m they regard this absorption as point- ing indubitably to an active inter- vention of living cells in the process. ^ o This argument requires examination. Let us suppose the two vessels A and B (Fig. 25) to be separated by a mem- brane which offers free passage to water FIG. 25. and a difficult passage to salts. Let A contain 0-5 per cent, salt solution and B a solution iso tonic with a 1 per cent. NaCl, but containing only 0-65 per cent, of this salt, the rest of its osmotic tension being due to other dissolved substances. If the membrane were absolutely * semi-permeable,' water would pass from A to B until the two fluids were isotonic, i.e. until A contained 1 per cent. NaCl (we may regard volume B as infinitely great to simplify the argument). If however the membrane permitted passage of the dissolved substances, the course of events might be as follows : At first water would pass out of A, and salt would diffuse in until the percentage of NaCl in A was equal to that in B. There would now be an equal partial pressure of NaCl on the two sides of the membrane, but the total osmotic pressure of B would still be higher than A. Water would therefore still continue to pass, from A to B more rapidly than te other ingredients of B could pass into A. As soon however as more water passed out from A, the percentage of NaCl in A would be raised above that in B. The extent to which this occurs will depend on the impermeability of the membrane. As the NaCl in A reaches a certain concentration it will pass over into B, and this will go on until equilibrium is established between and B. Extending this argument to the conditions obtaining in the living body, we may conclude that neither the raising of the percentage of a * Here we have a possible clue to the explanation of some phenomena of cell activity, to which the term * vital ' is often assigned. In the swimming-bladder of fishes, for instance, we find a gas which is extremely rich in oxygen, and the oxygen is said to have been secreted by the cells lining the bladder. It is, however, possible that the processes here may be analogous to Graham's atmolysis, and that the bladder may represent a perfected form of Graham's india-rubber bag. 134 PHYSIOLOGY salt in any fluid above that of the same salt in the plasma, nor the passage of a salt from a hypotonic fluid into the blood plasma, can afford in itself any proof of an active intervention of cells in the process. In the case of the pleura, for example, we seem to have a membrane which is very imperfectly semi-pprmpable. It is permeable to salts, but presents rather more resist- ance to their passage onan to the passage of water. Hence on injecting 0*5 per cent. NaCl solution into the pleural cavity, water passes from the pleural fluid into the blood, until the percentage of sodium chloride in the fluid is raised perceptibly above that in the blood plasma. The limit of the resistance of the pleural membrane to the passage of salt is however soon reached, and then salt passes from pleural fluid into blood ; but in every case this passage is from a region of higher to a region of lower partial pressure. Hence at a certain stage of the experiment we find a higher percentage of salt in the pleura than in the blood-vessels, although the total amount of salt in the pleural fluid is less than that originally put in, or, in other words, salt has been absorbed. We have already seen that the effective osmotic pressure of a substance, i.e. its power of attracting water across a membrane, varies inversely as its diffusibility, or as the permeability of the membrane to it. What then will be the effect if on one side of the membrane we place some substance in solution to which the membrane is impermeable ? We will suppose that A and B both contain 1 per cent. NaCl, but that B contains in addition some substance x to which the membrane is im- permeable. Since the osmotic pressure of B is higher, by tlie partial pressure of x, than that of A, fluid will pass from A to B by osmosis. But the conse- quence of this passage of water will be to concentrate the NaCl in A, so that the partial pressure of this salt in A is greater than in B. NaCl will therefore « diffuse from A to B, with the result that the former difference of total osmotic pressure will be re-established. Hence there will be a continual passage of both water and salt from A to B, until B has absorbed the whole of A. This result will be only delayed if the osmotic pressure of A is at first higher than B, in consequence of a greater concentration of NaCl in A. There may be at first a flow of fluid from B to A, but as soon as the NaCl concentration on the two sides has become the same by. diffusion, the power of x to attract water from the other side will make itself felt, and this a/ttrac- tion will be proportional to the osmotic pressure of x. We shall have occasion to discuss a specific instance of this case when dealing with the mechanism of absorption of fluid by the blood-vessels from the connective tissue spaces. A more familiar example is afforded by the process known as dialysis. Many animal membranes, all of which are colloidal in character, and others such as vegetable parchment, while freely permeable to salts, are impermeable to dissolved colloids. If therefore a fluid containing both colloids and crystalloids in solution, e.g. blood-serum, be enclosed in a tube of vegetable parchment, which is hung up in a large bulk of distilled water (Fig. 2G), all the salts diffuse out, and if this be frequently changed, we obtain finally a fluid within the dialyser free from salts and other crystalloid substances, but containing the whole of the colloidal proteins originally present. PASSAGE OF WATEK AND DISSOLVED SUBSTANCES 135 Thus the transference of fluids and dissolved substances across membranes is determined not only by the osmotic pressure of the solutions, but also by the diffusion coefficient of the solutes and the permeability of the membrane. This permeability may be of the same character as the permeability of water, in which case the rates of passage of the dissolved substances across the FIG. 26. Dialyser. consisting of a tube of parchment paper immersed in a vessel through which a constant stream of sterile distilled water can be passed. (WROBLESKI.) membrane vary as«their diffusibilities, and are therefore probably some func- tion of their molecular weights. On the other hand, the membrane may exhibit a certain attraction for, or power of dissolving, some of the solutes to the exclusion of others, in which case there will be no relation between the diffusibilities and the rates of passage of the dissolved substances. Bayliss has drawn attention to certain other factors which may determine permanent equality of distribution of a salt on the two sides of a membrane permeable to the salt. Congo red, which is a compound of an indiffusible colloid acid with sodium, be placed an osmometer which is immersed in water, a certain osmotic pressure is developed, adding sodium chloride either to the inner or outer fluid, there is a fall in the osmotic pressure if time be allowed for equilibrium to be established. At this point it is found that the outer fluid, which is free from dye, contains a larger percentage of sodium chloride than the inner solution of dye. This difference is permanent and is more marked the greater the concentration of the dye salt. In the following Table is given the concentrations of the two fluids with different percentages of salt. The numbers indicate the litres to which each gramme molecule of the salt is diluted. Apparently 136 PHYSIOLOGY Chlorine Dye Inside Outside 30 52 30 30 465 73-6 30 <5500 180 100 32-9 29-5 the difference depends on the fact that the non -dissociated salt must be equal on the two sides of the membrane and that the dissociation is much impeded on the inner side on account of the presence there of another salt of sodium. A sodium salt of any other indiffusible substance, e.g. of a protein such as caseinogen, would behave in a precisely similar fashion. SECTION III THE PROPERTIES OF COLLOIDS ALTHOUGH the chemical changes involved in the various vital phenomena occur between substances in watery solution, the solution in every cas; is bound up within the meshes or adsorbed by the surfaces of a heterogeneous mass of colloids. The complex chemical molecules which make up protoplasm itself are all colloidal in character. The participation of colloids in chemical reactions introduces conditions and modes of reaction differing widely from those which have been studied in watery solutions. Our knowledge of these conditions is still very imperfect, but the important part played by colloids in the processes of life renders it necessary to discuss in some detail their properties and modes of interaction. The term colloid, from *oXX?7, -glue, was first introduced by Thomas Graham, Professor of Chemistry at University College from 1836 to 1855. Graham divided all substances into two classes, viz. crystalloids, including such substances as salt, sugar, urea, which could be crystallised with ease, diffused rapidly through water, and were capable of diffusing through animal membranes ; and colloids, which included substances such as gelatin or glue, gum, egg-albumin, starch and dextrin, were non-crystallisable, formed gummy masses when their solutions were evaporated to dryness, diffused with extreme slowness through water, and would not pass through animal membranes. The process of dialysis was therefore introduced by Graham for the separation of crystalloids from colloids. Although the broad dis- tinction drawn by Graham between colloids and crystalloids still holds good, some of the criteria by which he distinguished the two classes are no longer strictly applicable. For instance, it has been shown that many typical colloidal substances, such as haemoglobin, can be obtained in a crystalline form. On the other hand, all gradations exist between substances, such as egg-albumin, which are practically indiffusible, and those, such as common It, which are very diffusible. Graham pointed out that colloids exist under TO conditions : (1) In a state of solution or pseudo-solution, in which they form sols, and ire distinguished as hydrosols, when the solvent is water ; and (2) In a solid state, in which a relatively small amount of the colloid its with a large amount of a fluid, such as water, to form a jelly. This solid form is known as a gel. The most familiar instance is the jelly which t;s obtained on dissolving a little gelatin in hot water and allowing the mixture 137 138 PHYSIOLOGY to cool. Such a jelly is known as a hydrogel. In many of these gels the water can be replaced by other fluids, such as alcohol, without any alteration in the appearance of the solid, which is then known as an alcogel. An example of an alcogel is the jelly which can be made by dissolving soap in warm alcohol and allowing the mixture to cool. A number of these colloidal substances can be shown on purely chemical grounds to consist of monstrous molecules. Thus the molecular weight of haemoglobin is at least 16,000, and one must ascribe similar high molecular weights to such substances as egg-albumin and globulin. Still greater must be the molecular size of such substances as the cell proteins, which may be made up of more than one type of protein built up with various nucleins, with lecithin and cholesterin, to "form a gigantic complex, to which it would probably not be an exaggeration to ascribe a molecular weight of over 100,000. This chemical complexity is not however ^, necessary condition of the colloidal state, as is shown by the existence of colloidal silica, of colloidal ferric hydrate and alumina, and even of colloidal metals. On neutralising a weak solution of sodium silicate or water-glass by means of HC1, we obtain a solution which contains sodium chloride and silicic acid. On dialysing this mixture for some days against distilled water, the whole of the NaCl passes out, and a solution of silicic acid or colloidal silica is left in the dialyser. This solution can be concentrated over sulphuric acid. When concentrated to a syrupy consistence it becomes extremely unstable. The addition of a minute trace of sodium chloride or other electrolyte to the solution causes it to set at once to a solid jelly (gelatinous silica), the change being accompanied by an appreciable rise of temperature. The change is irreversible, in that it is not possible to bring the silicic acid into solution again by removal of the electrolyte by means of dialysis. If however it be allowed to stand with weak alkali for some time, it gradually passes into solution. Analogous methods are employed for the preparation of colloidal Fe203 and A12O3. Of special interest are the colloidal solutions of the metals. Faraday long ago pointed out that, on treating a weak solution of gold chloride with phosphorus, it underwent reduction with the formation of metallic gold. The gold was not precipitated, but remained in suspension or pseudo- solution, giving a deep red * or a blue liquid, according to the con- ditions under which the reaction was effected. This solution was homo- geneous in that it could be filtered without change, and could be kept for months without deposition of the gold. The latter was however thrown down on addition of mere traces of impurity, though greater stability could be conferred on the solution by adding to it a little ' jelly,' i.e. a weak solution of gelatin. In 1899 Bredig showed how similar hydrosols might be prepared from a number of different metals, viz. by the passage of a small arc or electric sparks between metallic terminals submerged in distilled water. If, for example, the terminals be of platinum, the passage of the current is seen to be accompanied by the giving off of brown clouds, which spread into the surrounding fluid. These clouds consist of particles of platinum * Ruby glass is a colloidal ' solid ' solution of gold in a mixture of silicates. THE PROPERTIES OF COLLOIDS 139 of all sizes. The larger settle at the bottom of the vessel, the smaller — which are ultra-microscopic in size, i.e. from 5 /X/A to 40^* — remain in sus- pension, and we obtain a brown fluid which can be filtered through paper or even through a Berkefeld filter without losing its colour. It may be kept for months without any deposit taking place. The addition of minute traces of electrolytes precipitates the platinum particles, leaving a colourless fluid. We shall have to return later on to the consideration of the behaviour of these metallic sols. Colloidal solutions or sols may be divided into two classes, emulsoids and suspensoids, according as they may be regarded as suspensions of liquid in liquid or as suspensions of solid particles. Most protein solutions are emulsoids, while the metallic sols belong to the class of suspensoids. Dilute egg-white is an emulsoid, but if it be boiled, although no visible precipitation is produced, the fine particles are coagulated and it behaves as a suspensoid. PROPERTIES OF GELS. A typical hydrogel is the firm mass in which a solution of gelatin sets on cooling. It is clear, hyaline, apparently structure- less, and possesses considerable elasticity, i.e. resistance to deforming force. It may be regarded as formed by the separation of the warm pseudo-solution of gelatin into two phases : first a solid phase, rich in gelatin and forming a tissue or meshwork, in the interstices of which is embedded the second phase, consisting of a very weak solution of gelatin. If the process be observed under the microscope, according to Hardy minute drops first appear which, as they enlarge, touch one another and form networks. In stronger solutions the first structures to make their appearance consist, not of the more con- centrated phase, but of droplets of the dilute solution of gelatin ; the stronger solution collects round these drops and solidifies to a honeycomb structure. In many cases the more fluid part of the gel is practically pure water. In such a case immersion in alcohol causes a diffusion outwards of the water, which is replaced by alcohol with the formation of an alcogel. In a dry atmosphere the gel loses water and becomes shrivelled and dry, but in some cases, e.g. gelafin, it can resume its former size and characters on immersion in water. Other gels, such as silicic acid or ferric hydrate, lose the power of swelling up after drying. The change in them is therefore irreversible. A gel adheres to the last traces of water with extreme tenacity. In consequence of its structure, it presents an enormous extent of surface on which adsorption can take place. At this surface the vapour-tension of fluids is diminished, as well as the osmotic pressure of dissolved substances. On this account gelatin must be heated for many hours at a temperature of 120° C. in order to be thoroughly dried. When dry, it, as well as other solid colloids, can exert a considerable amount of energy when caused to swell up by moistening. This energy was made use of by the ancient Egyptians in the quarrying of their stone blocks by the insertion of wedges million * One /A is one-thousandth of a millimetre ; one /A/A is one-thousandth /A, i.e. one- th of a millimetre. HO PHYSIOLOGY of wood ; water was poured on the wood, and the swelling of the wedges split the rock in the desired direction.* On account of the extent of surface it is practically impossible to wash out the inorganic constituents from a gel. The diminution of the osmotic pressure of many dissolved substances at surfaces causes the concentration at the surface of the solid phase to be greater than that in the surrounding medium. Thus if dry gelatin be immersed in a salt solution it will swell up, but the solution which it absorbs will be more concentrated than the solution in which it is immersed, so that the proportion of salt in the latter will be diminished. When however equilibrium is established between a gel and the surrounding fluid, it is found to present no appreciable resistance to the passage of dissolved crystalloids. Thus salt or sugar diffuses through a column of solid gelatin as if the latter were pure water. On the other hand, gels are practically impermeable to other colloids in solution. This impermeability is made use of in the separation of crystalloids from colloids by dialysis, membranes used in this process being generally irreversible and heterogeneous gels (i.e. vegetable parchment, animal membranes). Other gels, such as tannate of gelatin or copper ferrocyanide, are not only impermeable to colloids, but also to many crystalloid substances. These membranes therefore were used by Pfeffer for the determination of the osmotic pressure of such crystalloids as cane sugar. PROPERTIES OF HYDROSOLS. Substances such as dextrin or egg- albumin may be dissolved in water in almost any concentration. If a solution of egg-albumin be concentrated at> a low temperature, it become^ more and more viscous and finally solid. ' But there is no distinct point at which the fluid passes into the solid condition. Such solutions are known as hydrosols. Much discussion has arisen whether they are to be regarded as true solutions or as pseudo-solutions or suspensions. The chief criterion of a true solution is its homogeneity. In a solution the molecules of the solute are equally diffused throughout the molecules of the solvent, and it is impossible, without the application of energy, to separate one from the other. Thus filtration, gravitation leave the composition of the solution unchanged. It is true that, by the employment of certain kinds of mem- branes, e.g. the semi-permeable copper ferrocyanide membrane, it is possible to separate solute from solvent, but in this case the force required to effect the filtration is enormous and grows with every increase in the strength of the solution. The measure of the force required is the osmotic pressure of the solution, and it 'becomes natural therefore to regard the possession of an osmotic pressure as a distinguishing criterion of a true solution. Is there any evidence that colloidal solutions also display an osmotic pressure ? I have shown that it is possible to determine the osmotic pressure of colloidal solutions directly, taking advantage of the fact that colloidal mem* * According to Rodewald, the maximal pressure with which dry starch attracts water amounts to 2073 kilo, per sq. cm. THE PROPERTIES OF COLLOIDS 141 branes, while permitting the passage of water and salts, are impermeable to colloids in solution. The method originally adopted was as follows : In order to determine the osmotic pressure of the colloidal constituents of blood-serum, 150 c.c. of clear filtered serum are filtered under a pressure of 30-40 atmospheres through a porous cell which has been previously soaked with gelatin. The first 10-20 c.c. of filtrate, which contain the water squeezed out of the meshes of the gelatin and have also lost salt in consequence of absorption by the gelatin, are rejected. The filtration is allowed to go on for another twenty-four hours, when about 75 c.c. of a clear colourless filtrate are obtained, per- fectly free from all traces of protein, but possessing practically the same freezing-point as the original serum. (Although the colloids, if they possess an osmotic pressure, must also cause a depression of the freezing-point, any such depression would be within the errors of observation, since a pressure of 45 mm. Hg would correspond only to 0-005° C.) The concentrated serum left behind in the filter is then put into the osmo- meter, the filtrate being used as the inner fluid. The construction of the osmometer is shown in the diagram (Fig. 2^). The tube BB is made of silver gauze, connected at each end to a tube of solid silver. Round the gauze is wrapped a piece of peritoneal membrane, as in making a cigirette. This is painted all over with a solution of gelatin (10 per cent.) and then a second layer of membrane applied. Fine thread is now twisted many times round the tube to , A* FIG. 27. revent any disturbance of the membranes, and the whole tube is soaked for half an hour in a warm solution of gelatin. In this way one obtains an even layer of gelatin between two layers of peritoneal membrane and supported by the wire gauze. The tube so prepared is placed within a wide tube, AA, which is provided with two tubules at the top. One of these, O, is for filling the outer tube ; the other is fitted with a mercurial manometer, M. Two small reservoirs, CC, are connected with the outer ends of BB, by means of rubber tubes. The whole apparatus is placed in a wooden cradle, DD, pivoted at X, and provided with a cover so that it may be filled with fluids at different temperatures if necessary. The colloid solution is placed in AA, and the reservoirs, CC, and inner tube, BB, are filled with the filtrate, i.e. with a salt solution approximately or absolutely isotonic with the colloid solution. The apparatus is then made to rock continuously for days or weeks by means of a motor. In this way the fluid on the two sides of the membrane is continually renewed, and the attainment of an osmotic equilibrium facilitated. With this apparatus I found that the colloids in blood-serum, containing from 7 to 8 per cent, proteins, had an osmotic pressure of 25 to 30 mm. Hg, which would correspond to a molecular weight of about 30,000. 142 PHYSIOLOGY A more convenient form of osmometer has been devised by B. Moore, using parchment paper as the membrane. With this osmometer, the existence of an osmotic pressure in colloidal solutions has been definitely established both by Moore in the case of haemoglobin, proteins, and soaps, and by Bayliss in the case of colloidal dyes, such as Congo red. The osmotic pressure of haemoglobin was found y Hiifner to correspond to a molecular weight of about 16,CCO, i.e. a molecular weight already deduced from its composition and its combining powers with oxygen. Often however the osmotic pressure is yery much smaller than would be expected from the molecular weight of the substance, owing to the fact that colloids in solution may be in many different conditions of aggregation. Thus the molecule of colloidal silica must be many, probably thousands of times larger than the molecule as represented by H2Si03. The osmotic pressure being proportional to the number of molecules in a given volume of solution, the larger the aggregate the smaller would be tne total number of molecules, and the smaller therefore the osmotic pressure of the solution. It is in consequence of the huge size of the molecular aggregates that colloidal solutions, such as starch or glycogen, and probably globulin, display no appreciable osmotic pressure. We cannot divide colloidal solutions into two classes, viz. those which form true solutions and present a feeble osmotic pressure, and those which form only suspensions and therefore exert no osmotic pressure. In inorganic colloids, such as arsenious sulphide, Picton and Linder have shown that all grades exist between true solutions and suspensions. With increasing aggregation of the molecules, the suspension becomes coarser and coarser until finally the sulphide separates in the form of a precipitate. The measurement of the osmotic pressure of the colloids of serum points to their having a molecular weight of about 30,000. Chemical evidence shows that haemoglobin has a molecular weight of about 16,000, and we have every reason to believe that the much more complex molecules forming the cell proteins may have molecular weights of many times this amount. When however we arrive at molecular weights of these dimensions, the disproportion between the size of the molecules and those of the solvent, water, becomes so great that a homogeneous distribution of the two sub- stances, solute and solvent, is no longer possible. The size of a molecule of water has been reckoned to be -7 x 10— 8 mm. A molecule 10,000 times as large would have a diameter of -7 x 10 — 4 mm. — -07^, a size just within the limits of microscopic vision. Long before molecules attained such a size they would no longer react according to the laws which have been derived from the study of the behaviour of the almost perfect gases, but would possess the properties of matter in mass. They have a surface of measurable extent, and their relations to the molecules of water or solvent will be determined by the laws of adsorption at surfaces rather than by the laws of interaction of molecules. As a matter of fact we find that such solutions present an amazing mixture of properties, some of which betray them as mechanical suspensions, while others partake of the nature of the p THE PROPERTIES OF COLLOIDS 143 chemical reactions such as those studied in the simpler compounds usually dealt with by the chemist. OPTICAL" BEHAVIOUR OF HYDROSOLS. Nearly all colloidal solu- tions present what is known as the Faraday- Tyndall phenomenon. When a beam of light is passed through an optically homogeneous fluid, the course of the beam is invisible. A beam of sunlight falling into a dark room is rendered visible by impinging on and illuminating the dust particles in its course. Each of these particles, being illuminated, acts as a centre of dis- persion of the light, so that the course of the beam is apparent to a person standing on one side of it. Tyndall showed that, if the particles were sufficiently minute, the light dispersed by them at right angles to the beam was polarised. This can be easily tested by looking at the beam through a Nicol's prism. If the prism be slowly rotated, it will be found that, while at one position the light is bright, in the position at right angles to this it becomes dim or is extinguished. The production of the Tyndall pheno- menon may therefore be regarded as a test for the presence of ultra-micro- scopic particles, varying in size from 5 to 50 jnu. The phenomenon is perhaps too sensitive to be taken as a proof that a fluid presenting it is a suspension rather than a solution. It is shown, for instance, by solutions of many bodies of high molecular weight, such as raffindse (a tri-saccharide) or the alkaloid brucine (Bayliss). A particle having a diameter less than half the wave-length of light, i.e. about 300 /C or -3 JLL, cannot be clearly distinguished under any power of the microscope. The fact that an ultra- microscopic particle may serve as a centre for dispersal of light may be used for rendering such particles visible under the microscope. For this purpose a strong beam of light is passed in the plane of the stage of the microscope through a cell containing the hydrosol, which is then examined under a high power. On examining with this apparatus a dilute gold sol, we see a swarm of dancing points of light, ' like gnats in the sunlight,' which move rapidly in all directions, rendering it almost impossible to count their number in the field. The coarser particles present slight oscillations similar to those long known as the Brown- ian movements. The smallest particles which can be seen show a combined movement, consisting of a translatory movement, in which the particle passes rapidly across the field in one-sixth to one-eighth of a second, and a r$ovement of oscillation of much shorter period. The representation of the course of such a particle is given in Fig. 28. The size of the smallest particles seen in this way may amount to -005 //. Not all colloidal solutions show these particles in the ultra-microscope. In some cases this is due simply to the small size of the particles, and the addition of any substance, which causes aggregation and therefore increase in the size of the particles, will bring them into view. In others the absence of optical inhomogeneity may be due to the coincidence of the refractive indices of the two phases of the hydrosol, or to the absence of any surface tension and therefore dividing surfaces between the two hases. 144 PHYSIOLOGY ELECTRICAL PROPERTIES OF COLLOIDS In the case of many hydrosols the ultra-microscopic particles of which they are composed carry an electric charge which, according to the nature of the solution, may be either positive or negative. On this account, the particles move if placed in an electric field, and the direction of their move- ment reveals the nature of their change. Thus colloidal ferric hydrate is electro-positive and travels from anode to cathode. Silicic acid, in the presence of a trace of alkali, is electro-negative, and the same is true of a hydrosol of gold. When a current is passed through these hydrosols, the colloidal particles travel to the anode, where they are precipitated. In certain colloids the charge varies according to the conditions under which they are brought into solution. If, for instance, egg-white be diluted ten times with distilled water, filtered and boiled, no precipitate occurs, but FIG. 28. Movements of two particles of india-rubber latex in colloidal solution, recorded by cinematograph and ultra-microscope. (HENBL) we obtain a colloidal suspension of albumin. When thoroughly dialysed, this protein is insoluble in pure water, but is soluble in traces of either acid or alkali. In acid so'ution the protein particles carry a positive charge, whereas in alkaline solution their charge is negative. The charged condi- tion of these particles must play a considerable part in keeping them asunder and therefore in preventing their aggregation and precipitation. This is shown by the fact that any agency which will tend to discharge them will cause precipitation and coagulation. Among such agencies is the passage of a constant current, just mentioned. To the same action is due the coagulative or precipitating effects of neutral salts. Thus any of the colloids we have mentioned, ferric hydrate, silica, gold, or boiled albumen, are thrown down by the addition of traces of neutral salts, and it is found that in this process they carry with them some of the ion with the opposite charge to that of the colloidal particle. Thus, in the precipitation of the THE PROPERTIES OF COLLOIDS 145 electro-positive ferric hydrate the acid ion of the salt is the determining factor, the coagulative power increasing rapidly with the valency of the acid.. On the other hand, in the precipitation of a gold sol the electro-positive ion is the effective agent, and here again the coagulative effect is enormously increased by a rise in valency. This is shown in the following Tables, where it will be seen that in the coagulation of gold, barium chloride with the divalent Ba", is seven times as powerful as K2S04 containing the univalent K'. On the other hand, in the precipitation of the electro-positive ferric hydrate, K2S04 with a divalent SO/', is 400 times as effective as BaCL. AMOUNT OF SALT NECESSARY TO PRECIPITATE COLLOIDAL SOLUTIONS To coagulate Gold To coagulate Fe203 K2SO4 1 g. mol. in 4,000,000 c.c. MgS04 „ „ „ 4,000,000 „ BaCl2 „ „ „ 10,000 „ BaCl2 1 g. mol. in 500,000 c.c. NaCl „ „ „ 72,000 „ K2S04 „ „ „ 75,000 „ NaCl „ „ „ 30,000 „ The presence of a charge is not however a necessary condition for the stability of a colloidal solution. Thus the proteins of serum, globulin in a weak saline solution, or gelatin, present no drift when exposed to a strong electric field. In such cases one must assume the stability of the solution to be determined by the absence of any surface tension between the two phases in the solution, or between the particles of solute and solvent. Thus no force is present tending to cause aggregation of the particles. The charged condition of a colloidal particle makes it behave in an electric field in much the same way as a charged ion of an electrolyte, and this similarity extends also to its chemical behaviour, so that we have a class of compounds formed resembling in many respects chemical com- binations, but differing from these in the absence of definite quantitative relations between the reacting substances. This class of continuously varying chemical compounds has been designated by Van Bemmelen absorp- tion compounds. Since however the interaction must take place at the surface layer bounding the charged particles, it will be perhaps better, as Bayliss has done, to use the term adsorption. The huge molecules or aggre- gates of molecules which distinguish the colloidal state form a system with a considerable inertia, so that we have a tendency to the establishment of conditions of false equilibrium. Once a configuration is established, it is necessary, in consequence of the inertia, to overstep widely the conditions of its formation in order to destroy it. Thus a 10 per cent, gelatin solution sets at 21°C., but does not melt until warmed to 29-6°C. Solutions of agar in water set at about 35°C., but do not melt under 90°C. A gel of gelatin takes twenty-four hours after setting to attain a constant melting- point. The factors involved in the formation of adsorption or absorption com- binations are therefore : (1) Extent of surface. In a colloidal solution this must be enormous in proportion to the mass of substance in solution. Thus a 10 c.c. sphere 10 146 PHYSIOLOGY with a surface of 22 sq. cm., if reduced to a fine powder consisting of spherules of -(CCCCG25 cm. in diameter, will have a surface of 20,((0,((0 sq. cm., i.e. nearly half an acre. At the whole of this surface adsorption may take place, involving the concentration of dissolved electrolytes, ions, or gases. (2) Chemical nature of particle. (3) Electric charge on the surface. The sign of this may be determined by the chemical nature of the colloid and its relation to the electrolytes in the surrounding medium. Another factor which may determine the character of the charge on the particles has been pointed out by Coehn. This observer finds that, when various non-con- ducting bodies are immersed in 'fluids of different dielectric constants, they assume a positive or negative charge according as their own dielectric constants are higher or lower than the fluid with which they are in contact. For instance, glass (5 to 6) is negative in water (80) or alcohol (26), whereas in turpentine (2-2) it is positive. In water, as Quincke has found, nearly all non-conducting bodies take on a negative charge. Among these are cotton -wool and silk. Particles of these in water, exposed to an electric field, move towards the anode. The same is true, as Bayliss has shown, of paper. The conditions which determine the formation of these adsorption com- pounds can be studied in their simplest form on the adsorption of dyestuffs by substances such as paper. If we take a series of solutions of a dye, such as Congo-red, in progressively diminishing concentration, and place in each solution the same amount of filter-paper, we find that a part of the dye is taken up by the paper, and the proportion taken up is larger the more dilute the solution. This relation has been spoken of by Bayliss as the law of adsorption. This is illustrated by .the following Table of results of such an experiment: Concentration of Proportion of dye solution in solution Proportion of dye in paper Initial Final Per cent. Per cent. 0-014 0-0056 40 60 0-012 0-0024 20 80 0-010 0-0009 9-3 - 90-7 0-008 0-0003 4 96 0-006 0-00008 1-3 98-7 0-004 — trace practically all 0-002 — trace practically nil If put into the form of a curve, where the ordinates represent the per- centage of dye left in solution, and the abscissae the original concentration of the solution, the curve only approaches the ax^s (i.e. zero concentration) asymptotically. In other words, however dilute the original solution may be, there will always be a certain amount of the dye left unabsorbed by the paper. Similar relations are found to exist between proteins and electro- lytes. By continuously washing a protein or gelatin with distilled water, THE PROPERTIES OF COLLOIDS 147 the removal of electrolytes becomes slower and slower, but it is practically impossible within finite time to get rid in this way of the last traces of ash. Although the chemical behaviour of colloids is largely determined by surface phenomena, it presents at the same time analogies with more strictly chemical reactions, since it is conditioned by the chemical structure of the colloid molecule as well as by the charge carried by the latter. A good example of these adsorption combinations is presented by globulin, the behaviour of which has been studied by Hardy. This may be obtained from diluted blood-serum by precipitation with acetic acid. Four states can be recognised in both the solid condition and in solution, viz. globulin itself, compounds with acid or with alkali, and compounds with neutral salt. The amount of acid and alkali combining with the globulin is in- determinate, the effect of adding either acid or alkali to the neutral globulin being to cause a gradual conversion of an opaque, milky suspension into a limpid, transparent solution. On drying HC1 globulin, the dried solid is found to contain all the chlorine used to dissolve it. The acid may therefore be regarded as being in true combination. Both acid and alkali globulins act as electrolytes, the globulin being electrically charged and taking part in the transport of electricity. In order to produce the same extent of solution, the concentration of the alkali added must be double that of the acid. The relation of globulin to acids and alkalies is similar to that of the so-called amphoteric substances, such as the amino-acids. An ammo-acid, such as glycine, can react as a basic anhydride with other acids, thus : NH2 NH2HC1 CH2/ + HC1 - CH NC02H or as an acid anhydride with bases : CH2.NH2 CH2.NH2 | + NaHO = | +H2O COOH COONa Like these too, globulin forms soluble compounds with neutral salts. An amphoteric electrolyte thus acts as a base in the presence of a strong acid, and as an acid in the presence of a strong base. From true electrolytes, colloidal solutions differ in the fact that their particles are of varying size according to the conditions in which they exist and carry varying charges of electricity, whereas an ion such as Na or Cl has a mass which is constant for the ion in question, and always carries the same electric charge. The charged particles of an acid- or alkali-globulin may be distinguished therefore as pseudo-ions. In these adsorption combinations, although the chemical nature of the colloidal molecules is concerned, there is an absence of definite equilibrium points, such as we are accustomed to in most chemical reactions. The inertia of the system and the large size of the molecules determine the occurrence of false equilibria and of delayed reaction, so that the condition and behaviour of a colloidal system at any moment are determined, not entirely by the 148 PHYSIOLOGY quantitative relations of its components, but also by the past history of the system. COMBINATIONS BETWEEN COLLOIDS Besides the compounds between colloids and electrolytes, combination, or at least interaction, takes place between different colloids. Many colloids are precipitated by other colloidal solutions. This effect is always found to occur when the colloidal solutions carry different charges. Thus ferric hydrate in colloidal solution is precipitated by colloidal silica or colloidal gold, both colloids being thrown out of solution. On the other hand, certain colloids may exercise a protective influence on other colloidal solutions. Thus, as Faraday first showed, colloidal gold is much more stable in the presence of a little gelatin. The colloids of serum can dissolve a considerable amount of purified globulin. Although the latter in solution shows a drift in the electric field, the resulting solution is quite unaffected by the passage of a current through it. In these cases the protective colloids carry no charge, but the same protective effect may be observed if a large excess of, e.g. an electro-positive colloid be added to an electro-negative colloid. This interaction between different colloids probably plays an important part in many physiological phenomena. We have reason to believe that the reactions between toxin and antitoxin, and between ferment and sub- strate, which we shall study later, are of this character, and that the compounds formed belong to the class of adsorption combinations. THE COAGULATION OF COLLOIDS Most colloidal solutions are unstable, and the relations between the suspended particle or molecule and the surrounding fluid may be upset by slight changes of reaction or the presence of minute traces of salts. As a result the hydrosol is destroyed, the suspended particles aggregating to form larger complexes. These aggregations may settle to the bottom of the fluid as a precipitate, or may form a species of network, the result varying according to the nature of the colloid and its concentration. Thus gelatin changes from the condition of hydrosol to hydrogel with fall of temperature. The same is true of agar. On the other hand, by adding calcium chloride to an alkaline solution of casein, we obtain a mixture which sets to a jelly on warming, but becomes fluid again on cooling. Other agen- cies may lead to the production of changes which are irreversible. Thus a strong solution of colloidal silica sets to a solid jelly on the addition of a trace of neutral salt, and it is not possible to reform the hydrosol, however long the jelly is submitted to dialysis. Two methods of bringing about coagulation of hydrosols deserve special mention. The first of these is heat-coagulation. If a solution of egg- albumin or globulin be heated in neutral or slightly acid medium and in the presence of neutral salt, the whole of it is thrown down in an insoluble form. This coagulated protein is insoluble in dilute acids or alkalies. The same coagulative effect of heating is observed in the metallic sols. With con- THE PROPERTIES OF COLLOIDS 149 centrated solutions of protein, heat coagulation results in the formation of a .gel, i.e. a network of insoluble protein, containing water or a very dilute solution of protein in its meshes. In dilute solutions the result is the production of a flocculent precipitate. Another method is the so-called mechanical coagulation. If a solution of globulin or albumin be introduced into a bottle, which is then violently shaken, a shreddy precipitate makes its appearance in the solution, and this precipitate increases, so that by prolonged shaking it is possible to throw down 80 or 90 per cent, of the dissolved protein in the coagulated form. Ramsden has shown that this mechanical coagulation is a surface pheno- menon. It depends on the fact that a large number of substances in solution (viz. any which lower the surface tension of their solutions) undergo concen- tration at the free surface of the fluid. Such substances are proteins, bile- salts, quinine, saponin, &c. In the case of proteins the concentration reaches such an extent, and the molecules at the surface are so closely packed together, that they form an actual solid pellicle, which hinders the movement of any object, such as a compass needle, suspended in the surface. When the solution is violently shaken, new surfaces are constantly being formed, and as the older surfaces are withdrawn into the fluid, the solid pellicle on them is rolled up into a fine shred of coagulated protein, and this process will continue until there is no protein left to form a pellicle. We must conclude that colloidal solutions, although differing so widely from true solutions in many of their properties, are connected with these by all possible grades. In a solution of an ordinary crystalloid or electrolyte the molecules of the dissolved substance are distributed equally and homo- geneously among the molecules of the solvent. In the various grades of solution a colloid solution or hydrosol may be assumed to begin when the size of the molecule is increased out of all proportion to that of the molecules of the solvent, r* The * dissolved ' ^molecules now have the properties of matter in mass and to present surfaces with all their attendant attributes. The same sort of solution may be formed with smaller molecules, such as Si02, when these are aggregated together with adsorbed water into huge molecular complexes or, as in metallic sols, by the division of the solid metal into ultra-microscopic particles. The distinguishing features of a colloidal solution are due to this lack of homogeneity, and to the fact that in every solution there are two phases^— a fluid phase, and a second phase which is either solid or a concentrated or supersaturated solution of the colloid. The huge size of the molecules and the development of svttf ace not only determine the formation of adsorption combinations but, on account of the inertia of the system, cause a delay in changes of state, and tend to the formation of false equilibria dependent on the past history of the system. IMBIBITION All colloids, even those such as starch or gelatin, which are insoluble in cold water, exhibit a phenomenon, viz. * Quellung ' or imbibition, which many cases it is impossible to distinguish from the process of solution. 150 PHYSIOLOGY This phenomenon, which was long ago studied by Chevreul and has been the subject of a series of careful experiments by Overton, is exhibited by all animal tissues and all colloids. Thus elastic tissue dried in vacuo absorbs from a saturated solution of common salt 36-8 per cent, of water and salt. If dried colloids be suspended in a closed vessel over various solutions, they will take up water in the form of vapour from the solution, and the osmotic pressure of the solution in question will inform us as to the amount of work which would be necessary in order to separate the water again from the colloids. Thus it has been reckoned that to press out water from gelatin containing 284 parts of water to 100 parts of dried gelatin would require a pressure of over two hundred atmospheres. The imbibition pressure of colloids in- creases rapidly with the concentration of the colloid and at a greater rate than the latter. In this respect however imbibition pressure resembles osmotic, or indeed gaseous, pressure. At extreme pressures the pressure of hydrogen rises more rapidly than its volume diminishes. In solutions this effect is more marked the larger the size of the molecule. Thus a 6-7 per cent, solution of cane sugar has the same vapour-tension, and therefore the same osmotic pressure, as a -67 per cent. NaCl solution. A 67 per cent, cane-sugar solution has however the same osmotic pressure as an 18 \ per cent, solution of common salt. It is impossible to draw any hard line of distinction between imbibition pressure and osmotic pressure, or to say where a fluid ceases to be a solution and becomes a suspension. All grades are to be found between a solution such as that of common salt with a high osmotic pressure and optical homogeneity, and a solution such as that of starch, which scatters incident light and is therefore opalescent, and has no measurable osmotic pressure. The close connection between the processes of imbibition and of solution is shown also by the fact that the latter occurs only in certain media, the nature of the media being dependent on the chemical character of the sub- stances in question. Thus all the crystalline carbohydrates — such as grape sugar and cane sugar — are easily soluble in water, only slightly soluble in alcohol, and practically insoluble in ether and benzol. The amorphous carbohydrates which must be regarded as derived by a process of condensa- tion from the crystalline carbohydrates — e.g. starch, cellulose, gum arabic, &c. — have a strong power of imbibition for water. This power may be limited, as in the case of cellulose, or may be unlimited, as in the csfce of gum arabic, so that a so-called solution results. On the other hand, they swell up but slightly in alcohol, and are unaffected by ether and benzol. In the same way proteins all take up water and in many cases form a so-called solution, but are unaffected by ether and benzol — a behaviour which is repeated in the case of the amino-acids, out of which the proteins are built up, and which are easily soluble in water but are practically insoluble in ether and benzol. On the other hand, india-rubber and the various resins take up ether, benzol, and turpentine often to an indefinite extent, while they are un- touched by water. With this behaviour we may compare the easy solubility of the hydrocarbons, the aromatic acids, and esters in ether and benzol, and THE PROPERTIES OF COLLOIDS 151 their insolubility in water. As Over ton has pointed out, the power of amorphous carbohydrates to take up fluids is modified by alteration of their chemical structure in the same direction as the solubility of the corresponding crystalline carbohydrates. Thus, if the hydroxyl groups in the sugars be replaced by nitro, acetyl, or benzoyl groups, they become less soluble in water, while their solubility in alcohol, acetone, &c., is increased. In the same way the replacement of the hydroxyl groups in cellulose by N02 groups diminishes the power possessed by this substance of taking up water, but renders it capable of swelling up or dissolving in alcohol and acetone. SECTION IV THE MECHANISM OF CHEMICAL CHANGES IN LIVING MATTER. FERMENTS ALL the events which make up the life of plants and animals are accompanied and conditioned by chemical changes of the most varied character. In a previous chapter we have endeavoured to form an idea of the ways in which some of the synthetic processes that occur in the living body may be effected. We saw that, although it was possible to imitate in many respects the vital syntheses by ordinary laboratory methods, the imitation fell far short of the process as it actually occurs in the living cell, both in completeness of the reaction and in the ease with which it could be effected. We can, for instance, by passing carbon dioxide over red-hot charcoal, convert it into carbon monoxide, and this gas, acting on dry potassium hydrate, forms potassium formate. Formate of lime, on dry distillation, gives a small proportion of formaldehyde which, under the influence of dilute alkalies, will condense to the mixture of sugars known as acrose. The green leaf in sunlight absorbs the minimal quantities of carbon dioxide present in the atmosphere and converts it almost quantitatively into starch within a few minutes, and this change is effected in the absence of any concentrated reagents and at the ordinary temperature of the atmosphere. Many of the chemical transformations effected by living cells we have so far been quite unable to imitate. The problem of the synthesis of camphor, of the terpenes, of starch, of cellulose, is still unsolved ; and even in the case of those sub- stances which we can manufacture outside the living cell our methods involve the use of powerful reagents and of high temperatures, and result in most cases in the production of many side reactions, besides that reaction which it is our special object to imitate. The distinguishing characteristics of the chemical changes wrought by the living cell are : !-* (1) The rapidity with which they are effected at ordinary tempera- tures. (2) The specific direction of the process, which is therefore almost complete, with a surprising absence of the side reactions which interfere to such an extent with the yield of the methods employed in a chemical laboratory. This second characteristic may however be regarded as a consequence of the first, since an increase in the velocity of any given reaction will deter- 152 CHEMICAL CHANGES IN LIVING MATTER FERMENTS 153 mine a preponderance of this reaction over all other possible ones. A funda- mental question therefore in physiology must relate to the manner in which the cell is able to influence the velocity of some specific reaction. In spite of the enormous diversity of chemical reactions occurring in the body, they may be divided into a relatively small number of types. Nearly all the reactions are reversible. The chief types of chemical change are as follows : (1) HYDROLYSIS. In most cases this involves the taking up of water and a decomposition into smaller molecules. Thus the proteins are broken down in the intestine into their constituent amino-acids. The disaccharides, such as maltose or lactose, take up one molecule of water and give rise to two molecules of monosaccharide. The fats take up three molecules of water with the formation of fatty acid and glycerin. Hippuric acid is broken down into benzoic acid and glycine. The reverse change, that of dehydra- tion, is also effected apparently with equal facility by the living cell, the hexoses losing water and being converted into a complex starch or glycogen molecule, while the amino-acids are built up first into polypeptides, and these again into the complex proteins of the cell. Besides the reactions in which there is a difference in the amount of free water on the two sides of the equation, it seems probable that hydrolysis and simultaneous dehydrolysis at different parts of the molecule determine a number of chemical transforma- tions, which at first sight seem to involve a simple splitting of the molecule. An example of such a process is afforded by the conversion of glucose into lactic acid described on p. 113. (2) DEAMINATION. This process involves the splitting off of an NH2 group from an ammo-acid as ammonia, and its replacement by H or OH. Many tissues of the body appear to have this power. In most cases the nature of the change in the remaining fatty moiety of the molecule has not yet been ascertained. If, for instance, to a mass of liver cells some amino- acid, such as glycine, alanine, or leucine, be added, ammonia is set free in proportion to the amount of amino-acid which was added. This ammonia is therefore assumed to be derived from the amino-acid, and it has been sug- 3ted that here also the process of splitting off ammonia is a hydrolytic one id that the NH2 group is replaced by OH. Thus — CH3 CH3 ! I CH.NH2 + HaO = CH.OH + NH, I I COOH COOH (alanine) Recent work by Neubauer tends to show that deamination is accompanied the first place by oxidation, so that the first intermediate product formed not anaoxy-acid, but an a ketonic acid. A second atom of oxygen is bhen taken up, and carbon dioxide is split off, with the production of the lext lower acid of the series. 154 PHYSIOLOGY We might represent these changes as follows : (1) CH3 CH3 I I CHNH2 + 0 = CO + NH3 I I COOH COOH (2) CH3 I CH3 CO +0 = 1 + C02 I COOH COOH Is the reverse change ever effected in the animal body ? If it were possible to replace the OH group in an oxy-fatty acid by NH2 or the 0 in an a ketonic acid by HNH2, it ought also to be possible to nourish an animal from a mixture of carbohydrates and ammonia, or at any rate by supplying him with a mixture of the appropriate oxy-acids or ketonic acids and am- monia. Until recently there was no evidence that the animal body is able to utilise nitrogen, except in organic combination as amino-acids or the complex aggregate of amino-acids known as proteins. In the plant the process of synthesis of protein from ammonia and a carbohydrate such as hexose is continuously going on, and it is probable that the formation of amino-acid occurs by a process the reverse of that which we have just been studying. Knoop has shown that the same reversed change may occur even in a mammal, and that here again the intermediate substance is an a ketonic acid. On administering benzylpyruvic acid (C6H5.CH2.CH2.CO.COOH) to a dog, a certain amount of benzylalanine (CeHg.CHaCHgCHNHg.COOH) appeared in the urine. The first phase of the oxidative deamination of amino-acids is thus a reversible one and may be represented : R R R I I OH ^ il CHNH2 + 0 ^ > C< < ^ CO + NH3 I | NH2 | COOH COOH COOH (3) DECARBOXYLATION. Many amino-acids when subjected to the agency of bacteria lose a molecule of carbon dioxide and are converted into a corresponding amine. For instance, lysine, which is diamino-caproic acid, is converted into pentamethylene diamine or cadaverine. Thus : CH2.NH2 CH,.NHa I I CH2 CH, I I CH2 becomes CH2 I I CH2 CH2 I I CH.NH, CH2.NH2 I COOH CHEMICAL CHANGES IN LIVING MATTER. FERMENTS 155 In the same way ornithine derived from the breakdown of arginine is con- verted by putrefactive bacteria into tetra-methylene diamine or putrescine. Other examples of this process of decarboxylation are : Isoamylamine from leucine, (CH3)2.CH.CH2.CH2.NH2. /5 phenylethylamine from phenylalanine, C6H5.CH2JCH2.NH2. Para-oxyphenylethylamine from tyrosine, OH.C6H4.CH2.CH2.NH2. A similar process has been supposed to take place as a step in the suc- cessive oxidation of the carbon atoms in the long chain fatty acids or carbo- hydrates, but a thorough study of this process as it occurs in the higher animals is still wanting, and its very existence is indeed still hypothetical. In the case of the fats the oxidation takes place chiefly or entirely in the /? position. On the other hand, decarboxylation certainly takes place in substances such as the a amino-acids, where the first oxidation change occurs in the a group, and probably closely follows this oxidation change. The reverse reaction, namely, the insertion of the group CO.O at the end of the long carbon chain, is not known to take place, but would furnish a means ,by which the organism with apparent simplicity could build up long carbon chains and so imitate the process which in the laboratory is generally effected by attaching a CN group to the end of the molecule. In the case of the fats the building up, like the oxidative breakdown, appears to occur by two carbon atoms at a time ; hence all the fatty acids met with in the body have an even number of carbon atoms in their chain. It is worthy of note that all the changes which we have been considering — changes which not only account for the greater part of the chemical re- actions of the living body, but may lead to the production of the most complex substances known — are performed with little expenditure or evolu- tion of energy. This is evident if we examine the heat evolved by the total combustion of one gramme molecule of the initial and final substances in a number of typical reactions. In the following Table these are given for the substances involved in typical instances of the three classes of chemical change that we have just been considering: (1) HYDROLYSIS Initial Heat of com- 1 Heat substance bustion per Final of gram molecule Maltose . 1350 Glucose .... 677 Hippuric acid . . . .. 1013 substance combustion 2 Glucose . . . 1354 2 Lactic acid 659 /Glycine . . 235 \ \Benzoic acid . 773] 1008 (2) DEAMINATION Initial Heat of Final Heat of substance combustion substance combustion Alanine .... 389-2 Lactic acid . . 329-5 Leucine .... 855 Caproic acid . . 837 Aspartic acid . . .386 Succinic acid .. . 354 156 PHYSIOLOGY (3) DECABBOXYLATION Initial Heat of substance combustion Alanine .... 389 Leucine 855 Final Heat of substance combustion Ethylamine . . 409 Isoamylamine . . 867 (4) OXIDATION AND REDUCTION. The fourth class of chemical reactions differs from' those just described in being attended with a very considerable energy change. This class involves the processes of oxidation and reduction. In almost every living cell, by far the largest amount of the energy available for the discharge of the functions of the cell is derived from the oxidation of the food-stuffs, and even in the plant the energy is obtained from the oxidation of the food-stuffs, built up in the first instance at the cost of the energy of the sun's rays. If we take the final changes in the food-stuffs, the very large evolution of energy attending their oxida- tion is at once apparent. Thus in the conversion of glucose into C02 and water there is an evolution for each gramme "molecule of 677 calories. In the combustion of glycerin 397 calories are evolved. In the oxidation of a fat such as trimyristin there are 6650 calories evolved. The change does not in the living cell occur all at once, but the molecule is oxidised step by step. In each step the heat change will however be probably greater than the heat changes in the other types of chemical change which we have been considering. Since the mechanism of oxidation in the animal body will have to be discussed at length in a subsequent part of this work, we may at present confine our attention to the other types of chemical change. Of these, all which involve a splitting of a large molecule into smaller ones with the taking up of one or more molecules of water, as well as, in all probability, those in which the reverse change of dehydration and synthesis occur, are effected in the body by meajis of ferments. To the same agency are also ascribed the process of deamination which takes place in many organs of the body and, though with less certainty, the processes which involve decarboxylation. FERMENTS Under the name ferments we include a number of substances of indefinite composition whose existence is chiefly known to us by their action on other substances. A ferment has been defined as a body which on addition to a chemical system is able to effect changes in this system without supplying any energy to the reaction, without being used up, and without taking any part in the formation of the end products. It differs therefore from the reacting substances in the absence of any strict quantitative relation- ships between it and the substances included in the system in which its effects are produced. Minimal quantities of ferment can induce chemical changes involving almost indefinite quantities of other substances, the only result of increasing the quantity of ferment being to quicken the rate of the change. Since they are effective in minimal doses they occur in living tissues in minute quantities, and it is partly due to this fact that it has CHEMICAL CHANGES IN LIVING MATTER FERMENTS 157 hitherto proved impossible to obtain any preparation of a ferment which could be regarded as a pure substance. The difficulty in their isolation is increased by the fact that all of them are colloidal or semi-colloidal in character, and present therefore the tendency common to all colloids of adhering to other colloidal matter as well as to surfaces such as those pre- sented by a precipitate. A common method of isolating, or rather obtaining a concentrated preparation of a ferment, is to produce in its solution an inert precipitate such as cholesterin or calcium phosphate. The ferment is carried down on the precipitate and may be obtained in solution on washing the precipitate with water. A further difficulty in their preparation lies in the unstable character of many members of the group. Although they are not coagulated by alcohol, they are nevertheless gradually changed, so that every act of precipitation of a ferment tends to rob it of some of its powers, i.e. of the only characteristic by which we can establish its identity. Of these ferments a large number have already been described as taking part in the ordinary chemical processes of life. So wide is their dominion in cell chemistry that many physiologists have thought that the whole of life is really a continual series of ferment actions. The following list repre- sents some of the ferments whose existence has been definitely established in the animal body. The greater part of them are involved in the processes of digestion in the alimentary canal. The preponderance however of digestive ferments in the list is due to the fact that we know more about digestion than about the other chemical processes taking place within the cells of the body. LIST OF FERMENTS. Ferment Converting Into Amylase (of saliva, pancreatic Starch . Maltose and dextrin juice, liver, blood serum, &c.) . I Pepsin ..... Proteins . Proteoses and pep- tones Trypsin ..... Proteins . Peptones and ammo- acids Enterokinase .... Trypsinogen Trypsin Erepsin ..... Proteoses Amino -acids Lipase (of pancreatic juice, liver, Neutral fats Fatty acid and &c.) glycerin Maltase ..... Maltose . Glucose Laotase . . Milk sugar Glucose and galactose Invertase or sucrase . Cane sugar Glucose and laevulose Arginase ..... Arginin . Urea and ornithine Urease ..... Urea Ammonium carbonate Lactic acid ferment Glucose . Lactic acid Zymase ( ? present in the body) Glucose . Alcohol and CO2 Deaminating ferment (?), v. p. 153 Amino -acids Oxy -acids (?) 158 PHYSIOLOGY Many other ferments will probably be distinguished with increase in our knowledge of cellular metabolism. The long list which is here given suffices to show how great a part these bodies must play in the normal processes of life. - A study of the conditions of ferment actions is therefore essential if we would form a conception of the chemical mechanisms of the living cell. It is important to note that all the changes wrought by ferments can be effected by ordinary chemical means. Thus the disaccharides can be made to take up a molecule of water and undergo conversion into mono- saccharides. If a solution of maltose be taken and bacteria be excluded from the solution, it undergoes at ordinary temperatures practically no change. If the solution be warmed, a slow process of hydraticn takes place which is quickened by rise of temperature, so that if the solution be heated under pressure to, say, 110° C., hydrolysis occurs with considerable rapidity. If however a little maltase be added to the solution, the change of maltose into glucose takes place rapidly at a temperature of 30° C. In the same way a solution of protein may be kept almost indefinitely without undergoing hydrolysis, which however can be induced by heating the solution under pressure. The action of the ferments in these two cases is to quicken a process of hydrolysis which without their presence would take an infinity of time for its accomplishment. In this respect their action is similar to that of acids, and indeed of a whole class of bodies which are spoken of as catalysers or catalysts. A catalyser is a substance which will increase (or diminish) the velocity of a reaction without adding in any way to the energy changes involved in the reaction, or taking any part in the formation of the end-products. Since the catalyser is unchanged in the process, a very small quantity is able to influence reactions involving large quantities of other substances. By adding acids to a watery solution of the food-stuffs, the process of hydrolysis is quickened in proportion to the strength and concentration of the acid. The effective catalytic agents in this process appear to be the hydrogen ions of the free acid. There are many other bodies, besides the free acids, which may act as catalysers, and a study of the conditions under which catalysis takes place may throw some light on the essential nature of the action of ferments. The velocity of almost any reaction in chemistry can be altered by the addition of some catalytic agent, and there are few of the ordinary reactions in which catalysis does not play some part. Among such processes we may instance the action of spongy platinum on hydrogen peroxide. Hydrogen peroxide undergoes slow spontaneous decomposition into water and oxygen. If a little spongy platinum be added to it, it is at once seen to decompose rapidly with the evolution of bubbles of oxygen, and the action does not cease until the whole of the hydrogen peroxide has been destroyed. Spongy platinum is able in the same way to quicken a very large number of chemical reactions. Thus sulphur dioxide and oxygen when heated together will combine very slowly ; the combination becomes rapid if a mixture of the CHEMICAL CHANGES IN LIVING MATTER. FERMENTS 159 two gases be passed over heated platinum. The same reaction, namely, the combination of sulphur dioxide with oxygen, may be quickened by the addition of a small trace of nitric oxide, and this fact is made use of in the manufacture of sulphuric acid on a commercial scale by the ordinary lead- chamber process. Hydrogen peroxide and hydriodic acid slowly interact with the formation of water and iodine. This reaction may be quickened by the addition of many substances, among which we may mention molybdic acid. There is moreover a specificity in the action of catalysers, though not so well marked as with ferments. Whereas all the disaccharides are con- verted by acids into the corresponding monosaccharides, a ferment such as invertase acts only on cane sugar, and has no action on maltose or lactose, each of which requires a specific ferment (maltase, lactase) to effect their ' inversion.' But we find many examples of a restricted action even among inorganic catalysers. Thus potassium bichromate will act as the catalyser for the oxidation of hydriodic acid by bromic acid, but not for the oxidation of the same substance by iodic acid. Iron and copper salts in minute traces will quicken the oxidation of potassium iodide by potassium persulphate, but have no influence on the course of the oxidation of sulphur dioxide by potassium persulphate. Tungstic acid increases the velocity of oxida- tion of hydriodic acid by hydrogen peroxide, but has no effect on the velocity of oxidation of hydriodic acid by bromic acid, and these examples may be multiplied to any extent. One cannot therefore regard the limitation of action of the ferments as justifying any fundamental distinction being drawn between the action of this class of substances and catalysts. Whereas the influence of most catalysers on the velocity of a reaction increases rapidly with rise of temperature, in the case of ferments this in- crease occurs only up to a certain point. This point is spoken of as the optimum temperature of the ferment action. If the mixture be heated above this point the action of the ferment rapidly slows off and then ceases. This contrast again is only apparent. The ferments are unstable bodies easily altered by change in their physical conditions, and destroyed in all cases at a temperature considerably below that of boiling water. Thus ferment actions, like catalytic actions, are quickened by rise of temperature, but the effect of temperature is finally put a stop to by the destruction of the ferment. The same applies to those inorganic catalysers whose physical state is susceptible, like that of the ferments, to the action of heat. Thus the colloidal platinum ' sol ' exerts marked catalytic effects on various reactions, e.g. on the decomposition of hydrogen peroxide and on the combination of hydrogen and oxygen. The reaction presents an optimum temperature, owing to the fact that the colloidal platinum is altered, coagu- lated, and thrown out of solution when this is heated to near boiling-point. We may therefore employ either class of reactions in trying to form some conception of the processes which are actually involved. Very many theories have been put forward to account for this action of catalysers or of ferments. Many of them are merely transcriptions in 160 PHYSIOLOGY words of the processes which actually occur, and fail to throw any light on their real nature. The essential phenomena involved fall directly into two classes. In the first class we must place those which are determined by the influence of surface. In many cases the combination of gases can be hastened by increasing the surface to which they are exposed, as by passing them over broken porcelain or over powdered charcoal. This cata- lytic effect is certainly connected with the power of a solid to condense gases at its surface, and is therefore proportional to the extent of surface exposed. Thus the efficacy of platinum in hastening the combination of hydrogen and oxygen is in direct proportion to its fineness of subdivision, and is best marked when the metal is reduced to ultra-microscopic dimen- sions, as in the colloidal solution of platinum. Every colloidal solution must be regarded as presenting an enormous surface in proportion to the mass of substance in solution. There is therefore a direct proportionality between the power of a substance to condense a gas on its surface and its power to quicken the velocity of chemical changes in which the gas is in- volved. The same process of condensation may occur with dissolved sub- stances. In all cases where the presence of a substance in solution diminishes the surface tension of the solvent, there is a diffusion of dissolved substances into the surface, i.e. a concentration of dissolved substances at the surface of contact. It was suggested by Faraday that the catalytic property of surfaces was due to this condensation of molecules, and the consequent bringing of the two sets of molecules within each other's sphere of influence. Whether this is the sole factor involved is doubtful, since mere compression of gases or increased concentration of solutions does not in the majority of cases result in such a quickening of the velocity of reaction as is brought about by the effect of the surface. It is possible that this condensation effect or adsorption may be in every case combined with the second factor which we must now consider, namely, the formation of intermediate products If we boil an alkaline solution of indigo with some glucose, the indigo is reduced with oxidation of the glucose. The mixture therefore becomes colourless. On shaking up with air, the colourless reduction product of the indigo absorbs oxygen from the atmosphere, and is je-transformed into indigo. These two processes can be repeated until the whole of the glucose is oxidised, and the process can be made continuous if air or oxygen be bubbled through a heated solution of glucose containing a small trace of indigo. In this case the indigo does not add to the energy of the reaction. It appears unchanged among the final products and a small amount may be used to- effect the change of an infinite quantity of glucose. It therefore may be said to act as a ferment or catalytic agent. Instead of an alkaline solution of indigo, we may use an ammoniacal solution of cupric oxide for the purpose of carrying oxygen from the atmosphere to the glucose. This is reduced to cuprous hydrate on heating with the sugar, but cupric hydrate can be at once re-formed by shaking up the cuprous solution with air. It has been thought that many or all of the catalytic reactions occur in the same way by two stages, i.e. CHEMICAL CHANGES IN LIVING MATTER. FERMENTS 161 by the formation of an intermediate product. Thus, in the old lead chamber process for the manufacture of sulphuric acid, the nitric oxide may be sup- posed to combine with the oxygen of the air to form nitrogen peroxide. This interacts with sulphur dioxide, giving sulphur trioxide and nitric oxide once more. The nitric oxide, which we alluded to before as the catalyser, may in this way be regarded as the carrier of oxygen from air to sulphur dioxide. It has been suggested that the action of spongy platinum or colloidal platinum rests on the same process,, and that in the oxidation of hydrogen, for instance, PtO or Pt02 is formed and at once reduced by the hydrogen with the formation of water. There is a certain amount of experimental evidence in favour of this hypothesis. According to Engler and Wohler,* platinum black, which has been exposed to oxygen, in virtue of the gas which it has occluded, has the power of turning potassium iodide and starch blue. This power is not destroyed -by heating to 260° in an atmosphere of Co2, or by washing with hot water. On exposure of the platinum black to hydrochloric acid, a certain amount is dissolved, and the substance loses its effect on potassium iodide. The amount dissolved corresponds with the amount of iodine liberated from potassium iodide, and also with the amount of oxygen occluded, the (soluble) platinum and oxygen being in the proportions necessary to form the compound PtO. But why should a reaction take place more quickly if it occurs in two stages instead of one ? As Ostwald has pointed out, the formation of an intermediate compound can be regarded as a sufficient explanation of a catalytic process only when it can be demonstrated by actual experiment that the rapidity of formation of the intermediate compound and the rapidity of its decomposition into the end-products of the reaction are in sum greater than the velocity of the reaction without the formation of the intermediate body. In the case of one reaction this requirement has been fulfilled. The catalytic effect of molybdic acid on the interaction of hydriodic acid and hydrogen peroxide has been explained by assuming that the first action which takes place is the formation of permolybdic acid, and that this then interacts with the hydrogen iodide to form water and iodine. Now it has been actually shown — (1) that permolybdic acid is formed by the action of hydrogen peroxide on molybdic acid ; (2) that permolybdic acid with hydriodic acid produces water and iodine ; (3) that the velocity with which these two reactions occur is much greater than the velocity of the inter- §tion of hydrogen peroxide and hydriodic acid by themselves. Although we may find it difficult to explain why a reaction should occur more quickly in the presence of a catalyser by the formation of these intermediate bodies, certain simple analogies may help us to comprehend how a factor which introduces no energy can yet assist the process. Thus a man might stand to all eternity before a perpendicular wall twenty feet high. Since he cannot reach its top at one jump, he is unable to get there at all. The introduction of a ladder will not in any way alter the total energy he must expend on raising his body for twenty feet, but will enable him to attain the top. Or we might imagine a stone perched at the top of a high hill. The passive resistance of the system, the friction of the stone, and its inertia will tend to keep it at rest, even though it be on a sloping surface and therefore tending to slide or roll to the bottom. If however it be rolled to a point where there is a sudden increase in the rapidity of slope, it may roll over, and having once started its downward course, its * Quoted by Mellor, "Chemical Statics." 162 PHYSIOLOGY momentum will carry it to the bottom. The amount of energy set free by the stone in its fall will not vary whether the course be a uniform one, or whether it falls over a precipice at one time and rolls down a gentle slope at another. It is evident that by a mere alteration of the slope or, in the case of a chemical reaction, of the velocity of part of its course, a change in the system may be initiated and brought to a conclusion which without this alteration would never take place. Since the action of ferments, like that of catalysts, consists essentially in the quickening up of processes which would otherwise occur at an in- finitely slow velocity, it is possible that in their case also the formation of an intermediate compound may be involved in the reaction. Light may be thrown upon this question by a study of the velocity of the reaction induced by the action of a ferment. It is well known that the velocity of a reaction dep3nds on the number of molecules involved. As an illustration, we may take first the case of a reaction involving a change in one substance. If arseniuretted hydrogen be heated, it undergoes decom- position into hydrogen and arsenic. This decomposition is not immediate, but takes a certain time, and the velocity with which the change occurs depends on the tempera- ture. At any given temperature the amount of substance changed in the unit of time varies with the concentration of the substance. If, for instance, one-tenth of the gas be dissociated in the first minute, in the second minute a further tenth of the gas will also be dissociated. Thus, if we start with 1000 grammes of substance, at the end of the first minute 100 grammes will have been dissociated, and 900 of the original substance will be left. In the second minute one-tenth again of the remaining substance will be dissociated, i.e. 90 grammes, leaving 810 grammes. In the third minute 81 grammes will be dissociated, leaving 729 grammes. The amount changed in the unit of time will always bear the same ratio to the whole substance which is to be changed, and will therefore be a function of the concentration of this substance. Put in the form of an equation, we may say that 0, the amount changed in the unit of time, will be equal to KG, where K is a constant varying with the substance in question and with the temperature, and C represents the concentration of the substance. The equation <£ = KG applies to a monomolecular reaction. If two substances are involved, the equation will be rather different. In this case the amount of change in a unit of time will be a function of the concentration of each of the substances, and the form of the equation will be = K(CT -f- Cy). In the case of the unimolecular reaction, halving the concentration of the substance will halve the amount of substance changed in the unit of time. In the case of a bimolecular reaction, halving each of the substances will cause the amount of change in the unit of time to be reduced to one-quarter of its previous amount. If now either a unimolecular or a bimolecular reaction be quickened by the addition of a catalyser or ferment, and the ferment enter into combination with one of the substances at some stage of the reaction, it is evident that our equation must take account also of the concentration of the ferment or catalyser. In the case of the catalytic effect of molybdic acid on the inter- action between hydrogen peroxide and HI, there was definite evidence of a reaction taking place between the molybdic acid and the peroxide, resulting in the formation of an intermediate compound, namely, permolybdic acid. Erode has shown that the interaction of the molybdic acid is revealed in the equation representing the velocity of the reaction. Without the addition of molybdic acid the equation would be : 4>=K(CH202 XCHI) After the addition of molybdic acid, the equation becomes : <£ = K(CH Oj + 7 C molybdic acid)C,r], when y is another constant depending on the molybdic acid. If ferments act in a similar way by the formation of intermediate compounds, this fact should be revealed by a study of the velocity at which the ferment action takes place, CHEMICAL CHANGES IN LIVING MATTER. FERMENTS 163 Various methods may be adopted for the study of the velocity of ferment action. If, for instance, we are investigating the action of diastase upon starch, we should take solutions of starch and of diastase of known concen- trations, keep them in a water bath at 38°C., and at a certain point add, say, 20 c.c. of ferment solution to every 100 c.c. of the starch solution. At periods of five or ten minutes after the addition had been made, 5 c.c. of the mixture might be withdrawn by a pipette and at once run into boiling Fehling's solution. The precipitated cuprous oxide would be dried and weighed, and would give directly the amount of sugar formed by the action of the ferment. After obtaining a series of data in this way, a curve could be drawn, showing the amount of change of starch which had occurred in each unit of time. In the case of the action of invertase on cane sugar the investigation is still easier. Since the change from cane sugar to invert sugar is accompanied by a change in the rotatory power of the solution on polarised light, it is necessary only to put the mixture of ferment and cane sugar into a polarimeter tube, which is kept at a constant temperature by means of a water jacket, and read off at intervals of a few minutes the change in the rotatory power of the solution. From this change can be easily calculated the percentage of cane sugar still present, and therefore the total amount which has been converted into fructose and glucose. In investigating the action of proteolytic ferments, as, e.g. that of trypsin on caseinogen, samples are taken at five-minute intervals and run into some substance such as trichloracetic acid, which will precipitate all the unchanged protein, but will leave in solution the products of hydration of the protein. From the amount of nitrogen in the filtrate from the precipi- tate can be determined the total amount of protein which has undergone hydration in the sample under observation. Or the amount of albumoses and peptones present in each sample may be estimated by the intensity of the biuret reaction which can be obtained. This method however suffers from the drawback that the albumoses and peptones, at any rate in the action of trypsin, are formed merely as a stage in the process, and the in- tensity of the reaction will first rise to a maximum and then gradually dis- appear. A very convenient method is that employed by Henri and by Bayliss in the investigation of the kinetics of tryptic action, namely, the determination of the conductivity of the solution. In the disintegration of the molecule caused by the action of the ferment, there is a continuous increase in the conductivity of the solution,-and this increase can be regarded as an index to the rate of change in the substances undergoing disinte- gration. By such methods it has been found that, when the quantity of ferment iployed is very small in comparison with the substrate (the substance jted u£on), the amount of change in a given time is proportional to the lount of ferment present, and is (within limits) independent of the con- itration of the substrate. This is well shown by the two following Tables >resenting the action of lactase upon lactose (E. F- Armstrong) : 164 PHYSIOLOGY PROPORTIONS HYDROLYSED IN 100 c.c. OF A 5 PER CENT. SOLUTION OF LACTOSE Solutions containing — 1'5 hours 20 hours 45 hours 1 c.c. lactase . 0-15 2-2 3-9 10 c.c 1-6 23-3 38-6 20 c.c. „ 3-2 45-8 AMOUNT OF SUGAR (LACTOSE) HYDROLYSED 24 hours 46 hours Solutions containing — Proportion Weight Proportion Weight 10 per cent, lactose 14-2 1-42 22-2 2-22 20 „ 7-0 1-40 10-9 2-18 30 4-8 1-44 7-7 2-21 Moreover, if we take only the earlier stages of the ferment action, it is fpund that, with small proportions of ferment, equal amounts of substrate are changed in successive intervals of time until about 10 per cent, has been hydrolysed. This is shown in the following Table : 2 PER CENT. LACTOSE WITH Time £ hour * n 1 „ 2 hours 3 LACTASE Amount hydrolysed 3.2 6.4 . 9.6 . 16.4 20.8 These results can be interpreted only by assuming that the first stage in the reaction is a combination of ferment with substrate. It is only this compound which represents the active mass of the molecules, i.e. the mole- cules of substrate which are undergoing change. This compound, as soon as it is formed, takes up water and breaks down, setting free the hydrolysed substrate and the ferment, which is at once ready to combine with a further portion of the substrate. In such a case the velocity of reaction must be directly proportional to the amount of ferment, and the same absolute quantity of substance will continue tx> be changed in succeeding units of time. Supposing, for instance, we had a load of bricks at the bottom of a hill which had to be transferred to the top, and five men to effect the trans- ference. The rate of transference would be directly proportional to the number of men employed ; we could double the rate by doubling the men. CHEMICAL CHANGES IN LIVING MATTEB. FERMENTS 165 Moreover the number of bricks carried in each unit of time would be the same. Five men would carry as many bricks in the second ten minutes as they would in the first, and so on. On the other hand, the velocity with which the transference was effected would be independent of the number — that is, the concentration — of the bricks at the bottom of the hill. The active mass of bricks could be regarded as that number carried at any moment by the transferring factor, namely, the men. The equation of change would be 0 = KC, where C is the concentration of the ferment. This concen- tration is always being renewed, and kept constant by the breaking down of the intermediate product, so that the rate of change would be continuous throughout the experiment. On the other hand, when the amount of ferment is relatively large, the 'ate of change, though at first very rapid, tends continuously to diminish. This is shown by the following Table representing the rates of change, during succeeding intervals of ten minutes, in a caseinogen solution to which a strong solution of trypsin had been added (Bayliss) . VELOCITY OF TRYPSIN REACTION N 6 c.c. 8 per cent, caseinogen + 2 c.c.— AmHO + 2 c.c. 2 per cent. trypsin at 39°C. 1st 10 minutes K =0-0079 2nd 0-0046 3rd „ 0-0032 4th „ 0-0022 5th „ 0-0016 7bh „ 0-0009 &c. &c. The cause of this rapid diminution in the velocity of change is probably complex. One factor may be an auto-destruction of the ferment, which is known to occur in watery solution. ' That this is not the only, or even the chief, factor involved is shown by the fact that, when the action of trypsin on caseinogen has apparently come to an end, it may be renewed by further dilution of the mixture or by removal of the end-products of the action by dialysis. It is evident that, in this retardation of the later stages of ferment action, the end-products are concerned in some way or other, and the re- tardation can be augmented by adding to the digesting mixture the boiled end-products of a previous digestion. The retarding effect of the end- Eoducts resembles in many ways that observed in a whole series of reactions lich are known as reversible. As an example of such a reaction we may take the case of methyl acetate and water, hen methyl acetate is mixed with water, it undergoes decomposition with the forma- >n of methyl alcohol and acetic acid. On the other hand, if acetic acid be mixed with alcohol, an interaction takes place with the formation of methyl acetate and water. These changes are represented by the equation : [MeC2H302 + HOH = MeOH -f HC2H3O2. methylacetate water methylalcohol acetic acid Each of these changes has a certain velocity constant, and, since they are in opposite rections, there must be some equilibrium point where no change will occur, and ' 166 PHYSIOLOGY there will be a definite amount of all four substances present in the mixture, namely, water, alcohol, ester, and acid. This equilibrium point can be shifted by altering the amount of any of the four substances. Thus the interaction of methyl acetate and water can be diminished to any desired extent by adding to the mixture the products of the interaction, namely, methyl alcohol and acetic acid. There is evidence that some of the ferment actions are reversible. Thus maltase acts on maltose with the formation of two molecules of glucose. If the maltase be added to a concentrated solution of glucose, we get a reverse effect, with the production of a disaccharide which has been desig- nated as isomaltose or revertose. To this reverse action may be due a certain amount of the retardation observed in the action of trypsin on coagulable protein. A more important factor is probably the combination of the ferment itself with the end-products and the consequent removal of the ferment from the sphere of action. Several facts speak for such a mode of explanation. Thus the action of lactase on milk sugar is not retarded by both its end-products, namely, glucose and galactose, but only by galactose. In the same way the action of invertase on cane sugar is retarded by the end-product fructose, but not at all by the other end- product, glucose. So far therefore a study of the velocity of ferment actions would lead us to suspect that the ferment combines in the first place with the substrate, and that this combination is a necessary step in the alteration of the sub- strate. In the second place, the ferment is taken up to a certain extent by some or all of the end-products, and this combination acts in opposition to the first combination, tending to remove the ferment from the sphere of action, and therefore to retard the whole reaction. Other facts can be adduced in favour of these conclusions. Thus it has been shown that invertase ferment, which is destroyed when heated in watery solution at a temperature of 60 °C., can, if a large excess of its substrate, cane sugar, be present, be heated 25° higher without undergoing destruction. The same protective effect is observed in the case of trypsin. Trypsin in watery or weakly alkaline solutions undergoes rapid decomposition. At 37 °C. it may lose 50 per cent, of its proteolytic power within half an hour. If, on the other hand, trypsin be mixed with a protein such as egg albumin or caseinogen, or with the products of its own action, namely, albumoses and peptones, it can be kept many hours without undergoing any considerable loss of power. It has been found that, whereas maltase splits up all the a-glucosides, it has no power on the /?-glucosides ; that is to say, maltase will fit into a molecule of a certain configuration, but is powerless to affect a molecule which differs from the first only in its stereochemical structure. On the other hand, emulsin, which breaks up /S-gluco- sides, has no influence on a-glucosides. This specific affinity of the ferments for optically active groups of bodies suggests that the ferment itaelf may be optically active. We cannot of course isolate the ferment and determine its optical behaviour ; but that it is optically active is rendered probable both by these results and certain results obtained by Dakin on lipase, the fat-splitting ferment. Dakin carried out his experi- ments on the esters of mandelic acid. Mandelic acid is optically inactive, but this optically inactive modification consists of a mixture of equal parts of dextro-rotatory CHEMICAL CHANGES IN LIVING MATTER. FERMENTS 167 and lee vo -rotatory mandelic acid. The esters prepared from the optically inactive acids are themselves optically inactive. Dakin found that, when an optically inactive mandelic ester was acted upon by a lipase prepared from the liver, the final results of the action were also inactive ; but if the reaction were interrupted at the half-way point, the mandelic acid which had been liberated was dextro-rotatory, while the remainder of the ester was laevo -rotatory. Thus the rate of hydrolysis of the dextro- component of the ester is greater than that of the laevo -component, a result which can be best explained by the assumptions (a) that the enzyme or a substance closely associ- ated with it is a powerfully optically active substance ; (&) that actual combination takes place between the enzyme and the ester undergoing hydrolysis. Since the additive compounds thus formed in the case of the dextro- and laevo-components of the ester would not be optical opposites, they would be decomposed with unequal velocity, and thus account for the liberation of the optically active mandelic acid. We may conclude that in the action of ferments on the food substances, whether carbohydrate or protein, an essential factor is the combination of the ferment with the substrate. Only the part of the substrate, which is thus combined with the ferment, can be regarded as the active mass and as undergoing the hydrolytic change. What is the nature of this combination ? Ferments, which are all of a colloidal or semi-colloidal character, cannot be dealt with in the same way as the catalysts of definite chemical com- position, such as molybdic acid or nitric oxide. In many cases the substrate, e.g. starch or protein, is also colloidal, and the combination therefore falls into the class of combinations between colloids. In this we have an inter- action between two substances in which the adsorption by the surfaces of the molecules of one or both substances plays an important part, though this adsorption is itself determined or modified by the chemical configuration of the molecules. The combination of ferments with their substrates be- longs therefore to that special class of interactions, not entirely chemical and not entirely physical, but depending for their existence on a co-operation of both chemical and physical factors, which we have discussed earlier under the name of adsorption compounds. FERMENTS AS SYNTHETIC AGENTS If maltase, obtained from yeast, or from the so-called takadiastase (prepared from Aspergillus oryzce), be added to a solution of maltose, the latter is hydrolysed to glucose. The process of hydrolysis stops short of complete inversion at a point varying with the concentration of the sugar solution. Thus in a 10 per cent, solution of maltose, inversion proceeds until 98 per cent, of the maltose is converted into dextrose, whereas in a 40 per cent, solution the change stops short when 85 per cent, sugar has undergone inversion. Croft Hill showed that if the maltase were added to a 40 per cent, solution of dextrose, a change took place in the reverse direction, which proceeded until 85 per cent, of the glucose was left. The sugar formed, which is a disaccharide, was regarded by Croft Hill as maltose. According to Emmerling, however, it is the stereoisomeric sugar, iso-maltose, which is formed ; and Croft Hill in his later papers spoke of the sugar as revertose. In the same way it has been shown by Castle and Loewenhart that the 168 PHYSIOLOGY hydrolysis of esters by lipase is a reversible reaction, the action of lipase being simply to hasten the attainment of the equilibrium point between the four substances — ester (or neutral fat), water, fatty acid, and alcohol. Similar reversible effects have been described for other ferments. Thus the addition of pepsin to a strong solution of albumoses causes the appear- ance of an insoluble precipitate, which is called plastein, and has been regarded as produced by the resynthesis of the original protein molecule. If all ferment actions are in this way reversible, a possibility is opened of regarding the synthetic processes occurring in the living cell, as well as the processes of disintegration, as determined by the action of enzymes. It must be noted that these effects are obtained with distinctness only when dealing with concentrated solutions. The degree of synthesis which would be produced in the very dilute solutions of glucose &c. occurring in the animal cell would therefore be infinitesimal. But if a mechanism were provided for the immediate separation of the synthetical product from the sphere of reaction, either by removing it to a different part of the cell or by building it up into some more complex body which was not acted on by the ferment, the process of synthesis might go on indefinitely, and the infinitesimal quantities be summated to an appreciable amount. Some experiments by Bertrand on fat synthesis have been interpreted as showing that the process of synthesis by ferments is not the mere attainment of an equilibrium point in a reversible reaction. It has long been known that watery extracts of the fresh pancreas split neutral fats into the higher fatty acids and glycerine. This observer has shown that, if the pancreas be dried with alcohol and ether and powdered, addrtion of the dry powder to a mixture of the higher fatty acids and glycerine brings about a rapid synthesis of neutral fat. The process of synthesis is at once stopped by the addition of water. In this case either there are two ferments present, one a synthetising, the other a hydrolysing, ferment, differing in their conditions of activity, or there is one ferment which may act either as a fat-splitting or fat-forming agent according to the conditions under which it is placed. In the latter case the effect of the addition of water would be simply to alter the equilibrium point of the mixture. It has been shown that in all reversible reactions the equilibrium position is the same from which- ever side it be approached. The action of the ferment is to hasten the attainment of equilibrium, the position of the latter being determined by the relative concentration of the reacting molecules. SECTION V ELECTRICAL CHANGES IN LIVING TISSUES HE material composing living cells and tissues is permeated throughout with water containing electrolytes in solution. All salts, as we have seen, undergo ionic dissociation in watery solution — a dissociation which, in the concentrations occurring in the animal body, must be nearly complete. When an electric current passes through the living tissues it is carried by the charged ions formed by the dissociation of the salts. Thus, n /10 solution + of sodium chloride contains almost entirely Na and Cl ions. In addition to these charged inorganic ions, the cell protoplasm contains in solution or suspension various colloidal particles which in many cases are themselves charged. By the presence of these colloidal particles marked differences may be caused in the distribution of the inorganic ions owing to the power of adsorption possessed by the colloids for many inorganic salts. It is evident that any unequal distribution of the charged ions or colloidal particles in a tissue or on the two sides of a membrane may give rise to corresponding unequal distribution of electric charges, and therefore differences of potential between different parts of the tissue, which under suitable conditions may find their expression in an electric current. It is therefore not surprising that practically every functional change in a tissue has been shown to be associated with the production of differences of electrical potential. Thus all parts of an uninjured muscle are isopotential, and any two points may be led off to a galvanometer without any current being observed. If however one part of the muscle be strongly excited, as for instance by injury, so that it is brought into a state of lasting excitation, it will be found that, on leading off from this point and a point on the uninjured surface to a galvanometer, a current flows through the latter from the uninjured to the injured surface. Every beat of the heart, every twitch of a muscle, every state of secretion of a gland, is associated in the same way with electrical changes. In most cases the electrical changes associated with activity have the same general character, the excited part being found to be negative in reference to any other part of the tissue which is at rest. The uniform character of the electric response in different kinds of tissues suggests that an accurate know- ledge of the changes in the distribution of charged ions responsible for the response ought to throw important light on the intimate nature of excitation enerally. It may be therefore advisable to consider more closely the 169 170 PHYSIOLOGY conditions which determine differences of potential in a complex system of electrolytes. As a simple case we may take an ordinary concentration cell. Two vessels (Fig. 29), A and B, are united by a glass tube C. A contains a 10 per cent, solution of zinc sulphate and B a 1 per cent, solution of the same salt. A rod of pure zinc is immersed in each limb. On connecting the zinc by a zinc wire to a galvanometer a current is observed to flow from A to B through the galvanometer, and therefore from B to A through the cell. A - 7n ®Zn - ®2n Zn® - ©Z/7 7n © t l Zn Fio. 29. FIG. 30. solution of zinc sulphate contains partly undissociated ZnS04 and partly + dissociated Zn and S04 ions. If a rod of zinc be immersed in a watery fluid the zinc tends to dissolve. The Zn passing into the fluid is however directly ionised, and therefore carries a positive charge into the fluid, leaving the zinc negatively charged (Fig. 30). This process of solution will rapidly come to an end, since the positively charged ions in the fluid will repel back into the zinc any ions which may be escaping from the zinc. The amount of zinc actually dissolved in the fluid is infinitesimal, the process of solution ceasing when the pressure (osmotic pressure) of the Zn ions in the fluid equals what may be called the * electrolytic solution pressure ' of the zinc. The continued solution of the zinc is therefore possible only when means are supplied for the Zn ions in the fluid to get rid of their positive charges. In an ordinary Daniell cell the Zn ions which leave the zinc are dis- charged by combining with the S04 ions passing to the zinc from the copper sulphate in the outer cell. It is a well-known fact that pure zinc does not dissolve in acid until some other metal, such as copper, is brought into con- tact with it, so as to set up an electric couple, i.e. to provide means for the discharge of the Zn ions passing into the solution. When the zinc is immersed in the two solutions of zinc sulphate in the concentration battery, the same change will occur. The ZnS04 solution in the two limbs of the ELECTRICAL CHANGES IN LIVING TISSUES 171 concentration cell already contains Zn ions. Since their pressure in the 10 per cent, solution is greater than in the 1 per cent, solution, fewer Zn ions will leave the zinc in A than in B. The negative charge on the Zn in A will therefore be less than that on the rod in B, and positive electricity will there- fore flow from A to B. This will disturb the equilibrium at the surface both of B and A, so that Zn ions will be deposited from the fluid on the surface of the zinc in A and will continue to pass from zinc into solution in B. At the same- time there is a movement of S04 ions, set free at the surface of A towards B. The ultimate result therefore is that the zinc in B dissolves and the same amount of zinc is deposited on A. The solution of zinc sulphate on A becomes progressively weaker, while that in B becomes stronger, until finally the concentrations in the two limbs are identical and the current ceases. In this process no chemical energy is involved, the energy set free by the conversion of zinc into zinc sulphate in B being exactly balanced by the energy lost by the deposition of zinc from zinc sulphate in A. Yet the current which is produced has a certain amount of energy which can be utilised for heating a wire through which it is made to pass. Since this energy must be taken from the cell, the cell is cooled during the passage of the current. We have here a close analogy with the case of compressed gases. If the 10 per cent, and 1 per cent, solutions were mixed together in a calorimeter, no change of temperature would be produced, since no work is done in the process. In the same way no cooling effect is observed if compressed gas be allowed to expand into a vacuum. If however the compressed gas be allowed to expand from a narrow orifice against the pressure of the external air, so that it does work in the process, it is cooled, and this cooling effect is made use of in the working of refrigerat- ing machines or for the liquefaction of gases. We may therefore regard the concentration battery as a machine for making the substances in solution do work as they expand from a strong into a dilute solution. The differences of potential obtained from an ordinary concentration cell are very small and would not suffice to account for such a high electromotive force as is set up, e.g. in the contraction of a muscle. We have seen earlier however that even in isosmotic solutions differences of pressure may be brought about by differences in diffusibility of the sub- U V stances in solution, especially if the two solutions be separated by a mem- brane. Very large differences may be Fio. 31. produced if this membrane be prac- tically impermeable to one or other of the dissolved substances. In the same way a semipermeable membrane, i.e. a membrane with different permeabil- ities for the different ions of the two solutions, may suffice to bring the differences of potential of a concentration cell up to and beyond the extent m B UV 172 PHYSIOLOGY which is observed in living tissues. Supposing we have (Fig. 31) two solu- tions, A and B, each containing an electrolyte, UV, in different concentrations separated by a membrane m. If u represents the velocity of transmission of U through m, and v the velocity of V, then the electromotive force of the cell is given by the formula W~~Vo577.1og.10C2 Volt. U + V If v is taken as very small, the membrane may be regarded as semipermeable for the corresponding ion V. Supposing we take potassium chloride as the solution, we should have to make the concentration in B eight times that in A, in order to get a current of strength equal to that obtained from the olfactory nerve of the pike, for example. Macdonald has made such an assumption in order to explain the normal nerve current. He suggests that the axis cylinder contains an electrolyte which is equivalent to a 2-6 per cent, solution of potassium chloride. It is unnecessary however to assume such great differences of concentration if we regard the membrane as itself a S9lution of electrolytes, as has been suggested by Cremer, or* if we take different substances on the two sides of the membrane. In the case of two electrolytes, UjYj, U2V2 (U being the cation in each case), separated by a membrane with varying permeability for the different ions, the electro- motive force of the cell is given by the following formula : 0-0577 lo.10% + V* where uit DI, uz, v2, are the velocities of the corresponding ions. We assume that the concentrations of the two solutions are identical. Now it is evident that by making uz and vl very small, the expression log.10 — -2 may be U% "I" ^1 made to attain any quantity, and in the same way by making ul + vz infinitesimally small, the electromotive force of the combination will also become correspondingly small. The thickness of the membrane does not come into the formula, so that membranes of microscopic or even ultra- microscopic thickness, which we have seen reason to assume as present in and around cells and their parts, could perform all the functions required of the hypothetical membrane in the above example. This is also the case when Y! is the same as V2 — that is to say, there is a common anion or a common cation on the two sides of the membrane. It must be remembered that the passage of a current through a membrane impermeable to one or other ion in the surrounding fluid will cause an accu- mulation of the ion at the surface of the membrane, so that this will become polarised. Such an accumulation at any surface will naturally alter the properties of the surface, including its surface tension. The construction of the capillary electrometer depends on this fact. When mercury is in contact with dilute acid or mercuric sulphate solution it takes a positive charge from the fluid, and the state of stress at the surface of contact between the mercury and the negatively charged fluid diminishes the surface tension of ELECTRICAL CHANGES IN LIVING TISSUES 173 the mercury. If the mercury be in the form of a drop in a tube drawn out to a capillary, the mercury will run down the capillary and the drop will be deformed until the surface tension tending to pull the mercury into a spherical globule is just equal to the force of gravity tending to make the mercury run out through the end of the capillary (Fig. 32). If the mercury be immersed in sulphuric acid it will descend to a lower level in the capillary owing to the diminution of its surface tension, If now the acid and the mercury be con- nected with a source of current so as to charge the mercury negatively, the effect will be to diminish the charge previously taken up by the mercury. The state of tension at the contact with the acid is therefore diminished, the surface tension is increased, and the mercury withdraws itself from the point of the capillary. If however the mercury be connected with the positive pole, its charge will be increased and its surface tension correspondingly diminished, so that the meniscus will move towards the point of the capillary. The move- ment of the meniscus to or away from the point may thus be used, as in the capillary electrometer, to show the direction and amount of any moderate electric change occurring in a tissue, two points of which are connected with the mercury and the acid respectively. It is possible that this electrical alteration of surface tension may be a determining factor in many of the phenomena of movement observed in the animal body. We shall have occasion to discuss this question more fully when endeavouring to account for the ultimate nature of muscular contraction. FIG. 32. BOOK II THE MECHANISMS OF MOVEMENT AND SENSATION CHAPTER V THE CONTRACTILE TISSUES SECTION I THE STRUCTURE OF VOLUNTARY MUSCLE THE most striking features in the continual series of adaptations to the environment, which make up the life of an individual, are the movements carried out by contractions of the skeletal muscles. In fact, all the mechan- isms of nutrition can be regarded as directed to the maintenance of the neuro-muscular apparatus, i.e. of the mechanism for adapted movement. With the growth of the cerebral hemispheres, which determines the rise in the scale of animal life, the skeletal muscles become more and more the machinery of conscious reaction. Even the highest of the adaptations possessed by man, those involving the use of speech, are impossible without some kind of movement. A man's relation to his fellows, and his value in the community, are determined by these higher muscular adaptations. It is not therefore surprising that the organs of the body which present in the highest degree the reactivity characteristic of all living things should have early attracted the attention of physiologists and have been the object of numberless researches directed to determining the ultimate nature of the processes generally described as vital. The movements of the muscles are carried out in response to changes aroused in the central nervous system by events occurring in the environ- ment and acting on the surface of the body. Every movement of an animal is thus in its most primitive form a reflex action, and involves changes in a peripheral sense organ, in an afferent nerve fibre, in the central nervous system, and in an efferent nerve fibre, before the actual process of contrac- tion occurs in the muscle itself and gives rise to the resultant movement (Fig. 33). If we are to determine the nature of .the changes involved in this reflex action, we must be able to study them as they progress along the different elements which make up the reflex arc. This analysis is facilitated by the fact that we are able to arouse a condition of activity in the different parts of the arc, even when isolated from one another. Thus we can excite any given reflex movement by stimulation of the periphery of the body, Mof the afferent nerve passing from the surface to the central nervous 177 12 178 PHYSIOLOGY system. We can proceed further and cut the efferent nerve away from the central nervous system and still succeed in exciting a condition of activity in the efferent nerve or in its attached muscle. All parts of the reflex arc possess the property of excitability, and we are thus able to arouse the activity of eachx part in turn, to study its conditions, its time relations, and the physical and chemical changes concomitant with the state of activity. It will be convenient for our analysis to begin with the tissue whose II Sensory \\K Sensory nerve Surface\T r^ j 1 Centra/ Nervous System \\\\\\v FIG. 33. Diagram of a reflex arc. reaction forms an end link in the reflex chain, namely, the muscle, and to proceed from that to the consideration of the processes occurring in the conducting strand between central nervous system and muscle, namely, the nerve fibre, postponing to a future chapter the treatment of the more complex processes associated with the central nervous system. In the higher animals we may distinguish several varieties of muscle. All movements that require to be sharply and forcibly carried out are effected by means of striated muscular tissue and; as these movements are in nearly all cases under the control of the will, the muscles are generally spoken of as voluntary. Unstriated or involuntary muscles form sheets or closed tubes surrounding the hollow viscera. By their slow, prolonged contractions they serve to maintain and regulate the flow of the contents of these organs. Such fibres are found surrounding the blood-vessels, the alimentary canal, the bladder, &c. Intermediate in properties as well as structure between these two classes is the heart muscle. This, like voluntary muscle, is striated, but presents considerable variations both in structure and function from ordinary skeletal muscle. Many of its properties will be considered in treating of the physiology of the heart. The properties of contractile tissue's have been most fully investigated in the volun- tary muscles, almost exclusively on the muscles of cold-blooded animals, such as the frog. The choice of skeletal muscles for this purpose is justified by the fact that a function is most easily investigated in the organs in which it is most highly developed. The choice of cold-blooded animals is guided by the fact that it is possible to isolate the muscle from the rest of the body and to study its reactions during a considerable time without the research being interfered with by the death of the tissue. We may therefore deal at length with the properties of the skeletal muscles, pointing out inci- THE STRUCTURE OF VOLUNTARY MUSCLE 179 dentally in what respects the heart muscle and involuntary muscle differ from the skeletal muscle. • The voluntary or striated muscles form a large part of the body, and are known as the flesh or meat. Each muscle is embedded in a layer of connective tissue, and is made up of an aggregation of muscular fibres, which are united into bundles by means of areolar connective tissue. The individual fibres vary much in length, and may be as long as 4 or 5 cm. At each end of the muscle the fibres are firmly united to tough bundles of white fibres, which form the tendon of the muscle, and are attached as WTO FIG. 34. Muscular fibre of a mammal, examined iresh in serum, highly magnified. (SCHAFEE.) a rule to bones. Running in the connective tissue framework of the muscle we find a number of blood-vessels, capillaries and nerves. On examination of a living muscle, each fibre is seen to consist of a series of alternate light and dark striae, arranged at right angles to its long axis, and enclosed in a structureless sheath — the sarcolemma. Lying under the sarcolemma are a number of oval nuclei embedded in a small amount of granular protoplasm. In some animals these nuclei occupy a central position in the fibre. Each band may be considered to be made up of a number of prisms (sarcomeres) side by side, with interstitial substance (sarcoplasm) between them. The muscle prisms of adjacent discs are connected to form long columns (primitive fibrillse, or sarcostyles). Each muscle prism is more transparent at the two ends than in the middle, thus giving rise to the appearance of light and dark striae. In the middle of the light band is a line or row of dots (often appearing double), called Krause's membrane. The development of this regular cross and longitudinal stria tion is closely connected with the evolution and specialisation of the muscular function, i.e. contraction. Contractility is among others a function of all undifferentiated protoplasm. Undifferentiated cells, such as the amoeba, can effect only slow and weak contractions. Directly a specialisation of function is necessary and some cell or part of a, cell has to contract rapidly in response to some stimulus from within or without, we find a differentiation both of form and of internal structure. In many cases, as in the developing muscle of the embryo or the adult muscles of many invertebrates, this differentiation affects only part of the cell, so that while one part presents the ordinary granular appearance, the other half is finely and longitudinally 180 PHYSIOLOGY striated, the striation being apparently due to the development of special contractile fibrillse. In the slowly contracting unstriated muscle of the vertebrate intestine, the longitudinal striation is with difficulty made out, but as the muscle rises in the scale of efficiency, the longitudinal striation becomes more apparent, and in the striated muscle of vertebrates, and still more in the wonderful wing-muscles of insects, which can perform three hundred complete contractions in a second, the longitudinal is associated FIG. 36. FIG. 35. FIG. 35. Muscle fibre of an ascaris. a, the differentiated contractile portion of the cell. (After HERTWIG.) FIG. 36. Muscle fibres from the small intestine, showing the fine longitudinal stria- tion. (SCHAFER.) with and often apparently subordinated to a transverse striation, due to the regular segmentation of the contractile fibrillse or sarcostyles. Every muscular fibre, which presents any trace of histological differentiation, may be said to consist of contractile fibrillse (sarcostyles), each composed of a series of contractile elements (sarcous elements or sarcomeres), and embedded in a granular material known as sarcoplasm. The great divergence in the aspect of muscular fibres from different parts of the animal kingdom is THE STRUCTURE OF VOLUNTARY MUSCLE 181 largely conditioned by the varying relations, spatial and quantitative, of the sarcoplasm to the sacostyles. Thus in the higher vertebrates, two types of voluntary muscular fibre are distinguished, according to the • io. 37. Transverse sections of the pectoral muscles of a, the falcon, &, the goose, and c, the domestic fowl. It will be noticed that the relative amount of granular or red fibres present varies directly as the bird's power of sustained flight. (After KNOLL.) amount of sarcoplasm they contain : one rich in sarcoplasm, more granular in cross-section, and generally containing haemoglobin ; and the other poor in sarcoplasm, clear in cross-section, and containing no haemoglobin. From the fact that the granular fibres are B found chiefly in those muscles which have to carry out long-continued and powerful contractions, it seems reasonable to regard the interstitial sarcoplasm as the local food-supply of the active sarcostyles, although some authors have endowed the sarcoplasm with a contractile power of its own, differing only by its extremely prolonged character from the quick twitch of the sarcostyles. The connection between structure and activity of the muscle-fibres is well shown by Fig. 37. I In some animals, such as the rabbit, we find muscles consisting almost entirely of one or other of these varieties ; but in most animals (amongst which we may reckon frog and man) the two varieties occur together in one muscle, so that what we have to say about the properties of voluntary muscle, which rests nearly entirely on experiments with frog's muscle, really has reference to a mixed muscle, i.e. muscle containing both red and white fibres. Since the sarcous element represents the contractile unit of the muscle, a know- ledge of its intimate structure should be of great importance for the theory of muscular contraction. Unfortunately however we are here at the limits of the demons fcrably , 38. Fibrils of the wing- muscles of a wasp, prepared by Reliefs method. Highly magnified. (E. A. SCHAFER.) A, a contracted fibril. B, a stretched fibril, with its sarcous elements separated at the line oE Hensen. c, an uncon- tracted fibril, showing the porous struc- ture of the sarcous elements. 182 PHYSIOLOGY S.E, FIG. 39. Diagram of a sarcomere in a moderately extended condition, A, and in a contracted condition, B ; K, K, membranes of Krause : H, . line or plane of Hensen ; SE, poriferous sarcous elements. (SCHAFER.) visible. It becomes difficult to determine how far the appearances observed under the microscope are due to actual structural differences or are produced by the unequal diffraction of light by the various elements of the muscle fibre. All observers are agreed that the essential contractile element is the row A B of sarcous elements forming the muscle fibril or sarcostyle. Schafer, working on the highly differentiated wing-muscle of the wasp, concludes that each sarcostyle is divided by Krause's membranes (the lines in the middle of each light stripe) into sarcomeres. Each sarcomere contains a darker substance near the centre divided into two parts by Hensen's disc. At each end of the sarcomere the contents are clear and hyaline. In the act of contraction, the clear material flows, according to Schafer, into tubular pores in the central dark material. Most histologists agree in assigning to the middle part of the sarcous clement (the sarco- mere) a denser structure than to the two ends. According to Macdougall, however, the lighter appearance at each end of the sarco- mere is an optical illusion. He regards the sarcous element as a cylindrical bag with homogeneous contents, crossed only by one or three delicate transverse membranes. Krause's membrane would be rigid, while the lateral wall of the sarcous element is extensible, and is folded longi- tudinally, so that it can bulge out and produce a shortening and thickening of the whole sarcous element if by any means the pressure be raised in its interior. In favour of a differentiation within the sarcomere itself is the fact that under certain conditions it is possible to produce a precipitate, limited only to central part, i.e. to the sarcous element to which Schafer assigns a tubular structure. When a muscle fibre, killed by osmic acid or alcohol, is examined under the microscope by pol- arised light, it is seen to be made -tip of alternate bands *of singly and doubly refracting material. The doubly refracting (anisotrojwus) substance corresponds to the dark band, and the singly re- fracting (isotropous) to the light band. If the living fibre be examined in the same way, it is found that nearly the whole of it is doubly re- fracting, the singly refracting substance appearing only as a meshwork with long parallel meshes corresponding to the muscle prisms. In short, in a living fibre the muscle prisms are anisotropous, the sarcoplasm isotropous. When a muscle fibre contracts, there is an ap- parent reversal of the situations of the light and dark stripes, owing to the fact that the interstitial sarcoplasm is squeezed out from between the bulging sarcomeres, and accumulates on each side of the membranes of Krause. The accumulation of sarcoplasm in this situation makes the previously light striae appear dark, and the dark stri» by contrast lighter Fia. 40. ilr " ' Motor end -organ of a lizard, gold preparation. (KiJHNE.) n, nerve fibre dividing as it ap- proaches the end-organ ; r, ramifi- cation of axis cylinder upon 6, gran- ular bed or sole of the end-organ ; m, clear substance surrounding the ramifications of the_axis cylinder. THE STRUCTUKE OF VOLUNTARY MUSCLE 183 than they were before. That there is no true reversal of the striae is shown by exam- ining the muscle by polarised light, the two substances, isotropous and anisotropous, retaining their relative positions. Every skeletal muscle is connected with the central nervous system by nerve fibres, some conveying impressions from the muscle to the centre, the others acting as the path of the motor impulses from the centre to the muscle. These latter — the motor nerves — end in the muscular fibre itself, by means of a special end-organ — the motor end-plate. The neurilemma of the nerve fibre, becomes continuous with the sarcolemma, the medullary sheath ends suddenly, while the axis cylinder ramifies in a mass of un- Eerentiated protoplasm, containing nuclei, and lying in contact with the Crural is Add. magn.' Tib ant. long. Sartorius Add. magn. Gracilis Tendo AchilHs FIG. 41. Muscles of hinder extremity of frog. (After ECKEB.) mtractile substance of the muscle immediately under the sarcolemma (Fig. 40). This mass of protoplasm is known as the * sole plate.' It is not marked in all animals. Thus in the frog the axis cylinder ends in a series of branches at right angles to one another, distributed over a considerable length of the muscle fibre. The sole plate in this case seems to be limited to scat- tered nuclei lying in close contact with the terminal branches of the nerve fibre. So far as we can tell at present, the ultimate ramifications of the axis cylinder end freely and do not enter into organic connection with the contractile substance itself. 184 PHYSIOLOGY Most of our knowledge on the subject of muscle has been derived from the study of the gastrocnemius and sartorius muscles of the frog. The position of these muscles is shown in the accompanyirtg diagram (Fig. 41). The gastrocnemius which, with the attached sciatic nerve, is most frequently employed as a nerve-muscle preparation, forms a thick belly immediately under the skin at the back of the leg, and arises by two tendons from the lower end of the femur and the outer side of the knee-joint. The two tendons converge towards the centre of the muscle, uniting about its middle, and from them a number of short muscular fibres arise, passing backwards and dorsally to be inserted into a flat aponeurosis covering the lower half of the muscle, which ends in the tendo Achillis. On account of this irregular arrangement of the muscular fibres, the gastrocnemius can be employed only«when the contraction of the muscle as a whole is the object of investigation. The effective cross-area of the fibres is much greater than the actual cross-section of the muscle, so that, while the actual shortening of the gastrocnemius is but small, its strength of contraction is considerable. The sar'torius muscle consists of a thin band of muscle fibres running parallel from one end of the muscle to the other. It lies on the ventral surface of the thigh, arising from the symphysis publis by a thin flat tendon, and is inserted by a narrow tendon into the inner side of the head of the tibia. On account of the regularity with which its fibres are disposed, this muscle is of especial value in experiments on the local con- ditions of a muscle fibre accompanying its activity. When a greater mass of approxi- mately parallel fibres is necessary, recourse may be had to a preparation consisting of the gracilis and semi-membranosus muscles together. This latter muscle lies dorsally to the gracilis muscle which is shown in the illustration. Other muscles in the frog used for particular purposes are the mylohyoid and the dorsocutaneous muscles. The mylohyoid muscle of the frog, which lies on the ventral surface of the tongue, has the advantage that its fibres lie in close contact with a lymph-, space occupying the centre of the tongue. If any drug be injected into this lymph - space it acts with extreme rapidity on the muscle fibres, so that the tongue-prepanit ion of the frog is a useful one for the study of the action of different substances on muscle- fibres. SECTION II EXCITATION OF MUSCLE MUSCLE may be caused to contract in various ways. Normally it con- tracts only in response to impulses starting in the central nervous system and transmitted down the nerves. But contraction may be artificially excited in various ways in a muscle removed from the body. If we make a muscle-nerve preparation (i.e. a .muscle with as long a piece of its nerve as possible attached to it), such as the gastrocnemius of the frog with the sciatic nerve, we find we can cause contraction by various forms of stimuli — mechanical, thermal, or electrical — applied to the muscle or the nerve (direct and indirect stimulation). Thus the muscle responds with a twitch if we pass an induction shock through it or its nerve, or pinch either with a pair of forceps. Or we may use chemical stimuli, and cause contraction by the application of strong glycerin or salt solution to the nerve. These experiments do not prove conclusively that muscle itself is irritable. It might be urged that, when we pinched or burnt the muscle we stimu- lated, not the muscle substance itself, but the terminal ramifications of the nerve in the muscle, and that these in their turn incited the muscle to contract. But the independent excitability of muscle is shown clearly by the following experiment by Claude Bernard. A frog, whose brain has been previously destroyed, is pinned on a board, and the sciatic nerves on each side exposed. A ligature is then passed round the right thigh underneath the nerve, and tied tightly so as effectually to close all the blood-vessels supplying the limbs, without interfering with the blood-supply to the nerve. Two drops of a 1 per cent, solution of curare are then injected into the dorsal lymph-sac. After the lapse of a quarter of an hour it is found that the strongest stimuli may be applied to the left sciatic nerve without causing any contraction of the muscles it supplies. On the right side, stimulation of the nerve is as efficacious as before. Both gastrocnemii respond readily to direct stimulation, showing that the muscles are not affected by the drug. Since both sciatic nerves have been exposed to the influence of the curare, it is evident that the difference on the two sides cannot be due to any deleterious effect on them by the curare. We have also excluded the muscles themselves ; so we must conclude that the curare paralyses the muscles by affecting the terminations the nerve within the muscle, and probably the end-plates themselves. H 186 PHYSIOLOGY This experiment teaches us that muscle can be excited to contract by direct stimulation, even when the terminal ramifications of the nerve within it are paralysed, so that stimulation of them would be without effect. The same fact may be demonstrated in a different way by means of, chemical stimuli. It is found that whereas strong glycerin excites nerve fibres, it is without effect on muscle fibres, while on the other hand weak ammonia is a strong excitant for muscle, but is without effect on nerve. If the frog's sartorius be dissected out and the lower end dipped in glycerin, no twitch is produced. On snipping off the lower third of the muscle and then immersing the cut end in glycerin, a twitch at once occurs. The lower end contains no nerve fibres (Fig. 42), and it is only when a section containing nerve fibres is ex- posed to the action of glycerin that contraction takes place. On the other hand, mere exposure of muscle to the vapour of dilute ammonia causes contraction (and subsequent death), although the nerve to the muscle can be immersed in the solution without any excitation being produced. Of all the different stimuli capable of exciting muscular contraction, the electrical is that most frequently employed. It is easy, using this form, to graduate accurately the intensity and duration of the stimulus. At the same time the stimulus PIG. 42. The-ramifioation T be aWlied "^ timeS tO "* P0^ °n ^ oi the nerve fibres within muscle or nerve without killing the part stimulated, the sartorius muscle of whereas with other forms of stimulus it is difficult the frog, showing the free- . . . . dom of the lower portion to obtain excitatory effects without injuring to a of the muscle from nerve greater or less extent the part stimulated. fibres. (KUHNE.) METHODS EMPLOYED FOR THE STIMULATION OF MUSCLE AND NERVE The two commonest forms of electrical stimuli employed are (1) the make and break of a constant current, (2) the induction currents of high intensity and short duration obtained from an induction coil. (1) CONSTANT CURRENT. As a source of constant current a Daniell's cell is generally employed. This consists of an outer pot containing a saturated solution of copper sulphate, in which is immersed a copper cylinder. To the cylinder at the top a binding screw is attached, by which the connection of the copper with a wire terminal is effected. Within the copper cylinder is a second pot of porous clay, filled with dilute sulphuric acid, in which is immersed a rod of amalgamated zinc. In this cell the zinc is the positive and the copper the negative dement. Hence the current flows (in the cell) from zinc to copper, and if the binding screws of the two elements are connected by a wire, the current flows in the wire (outer circuit) from copper to zinc, thus completing the circuit. Since in the outer circuit the current flows from copper to zinc, the terminal attached to the copper is called the positive pole, and that to the zinc the negative pole. When the current is required to be very constant, the zinc may be immersed in a saturated solution of zinc sulphate instead of dilute sulphuric acid. A Daniell's cell, though very constant, gives only a small current, owing to its small electromotive force and high internal resistance. When a stronger current is required it is best to use a storage battery. In this, EXCITATION OF MUSCLE 187 when charged, the two elements are lead and lead oxide, Pb02. It has the advantage that it may be used over and over again, being recharged through a resistance from the electrical mains when it has run down. Another useful type of cell is the Leclanch^ cell. This consists of a glass jar con- taining a solution of sal ammoniac. Into this dips an amalgamated rod of zinc, which is the positive plate. A piece of gas carbon forms the negative plate. This is sur- rounded by peroxide of manganese (Mn02) which is kept in contact with the surface of the carbon by being placed in a porous pot. In some forms of Leclanche the manganese and carbon are ground up together and pressed into a cylinder which surrounds the zinc rod. When the cell is on open circuit — that is, when the terminals are not con- nected and no current is passing — very little action takes place ; but when the circuit is closed and the current passes, the zinc dissolves in the sal ammoniac, forming a double chloride of zinc and ammonia, while ammonia gas and hydrogen are liberated at the carbon pole. The nascent hydrogen reduces the peroxide of manganese and so polarisa- tion is prevented. On account of its great solubility in water the ammonia has no polarising action. The Leclanche is a convenient form of cell, as when once set up it requires a minimum of attention. If it is worked through a considerable resistance, it will keep in order for some time, particularly if the work is intermittent ; but if it is used with a small resistance in circuit it polarises very rapidly. The E.M.F. of one Leclanche cell is 1'4 volt in the external circuit. The positive current is conventionally said to run from the zinc to the carbon in the cell, and from the carbon to the zinc in the circuit outside. The wire attached to the carbon is the positive pole, that to the zinc the negative pole. Dry cells are usually Leclanche cells, in which the solution of sal ammoniac is prevented from spilling by absorption with sawdust or plaster of Paris. The E.M.F. is the same as the Leclanche, but they polarise much more readily. If the poles of a DanielFs cell be connected by wires with a nerve or muscle of a nerve-muscle preparation (as in Fig. 43), the current will flow from copper to the nerve at A, and along the nerve from A to K. At K the current will leave the nerve to flow to the zinc of the battery, so com- pleting the circuit. The point at which the current enters the nerve (i.e. the point of the nerve connected with the positive pole of the battery) is called the g^g""" \-'''''':\\^ K\ -*~ [A jy anode, and the point at which the current ^J^^^^^' Ifathode. Anode. leaves the nerve is called the cathode. The F 43 wires by which the current is conducted to and from the nerve are called 'the electrodes. As electrodes we generaDy employ two platinum wires mounted together on a piece of vulcanite. For the purpose of making or breaking the current at will, various forms of keys are employed. The ordinary make and break key consists of a hinged wire dipping into a mercury cup. When the wire is depressed so that it dips into the mercury, the circuit is complete. On raising the wire by means of the handle, the circuit is broken. Du Bois Reymond's key consists of two pieces of brass, each of which has two bind- ing screws for the attachment of wires. These are connected by a third piece, or bridge, which is jointed to one of the two side bits, so that it may be raised or lowered at pleasure (v. Fig. 44). It may be used either as a simple make-and-break key, or, as is more usual, as a short-circuiting key. In the first case one brass bank is attached to one terminal, the other to the other terminal. If the bridge be now lowered, the connection is made and the current passes. If the bridge be raised, the current is broken. Fig. 44 A and B shows the way in which the key is arranged for short-circuit- ing. It will be seen that four wires are attached to the key ; two going to the battery, and two we may suppose going to a nerve. When the bridge is down, as in Fig. 44 A, the current from the cell on coming to the key has a choice of two routes. It may either go through the brass bridge, or through the other wires and nerve. The resistance of 188 PHYSIOLOGY the nerve however is about 100,000 ohms, whereas that of the bridge is not the thou- sandth part of an ohm. When a current divides, the amount of current that goes along any branch is inversely proportional to the resistance. Here the resistance in the nerve- circuit is practically infinite compared with that in the brass bridge, and so all the A B Fia. 44. Du Bois key. closed. Du Bois key. open. current goes through the bridge and none through the nerve. We say then that the current is short-circuited. It is often necessary to reverse the direction of a current through a nerve -muscle preparation or a galvanometer in the course of an experiment. For this purpose Pohl's reverser may be used. It consists of a slab of ebonite or paraffin or other in- sulating material, in which are six small holes filled with mercury. A binding screw is in connection with the mercury in each of these holes. Two cross-wires (not in contact with one another) join two sets of pools together, as shown in Fig. 45. A cradle con- sisting of two wires joined by an insulating handle carries two arcs of wire by which the pools at a and 6 may be put into connection with either x and y, or the corresponding pools on the opposite side. It will be seen that with the cradle tipped to one side, as in Fig. 45 A, the current from the battery enters thie reverser at a ; this proceeds up the wire of the cradle, down towards the right, then along the cross -wire to the pool at x. x is therefore the anode, and y the cathode. In Fig. 45 B the cradle has been swung over to the other side. Here the cross -wires are not used at all by the current, which passes from a up the sides and down the curved wire to y. In this case y is now the anode and x the cathode, and the direction of the current through the circuit connected with x and y is reversed. By taking out the cross-wires, Pohl's reverser may be used as a simple switch, by which the current may be led into two different circuits in turn. With this form of reverser difficulty is often experienced owing to dirt accumulating on the mercury and forming an insulating layer be- tween it and the binding screw or copper wire. Several improved forms of reverser are now made where the mercury poles are replaced by brass banks, and these are generally to J?e preferred in practice. (2) INDUCED CURRENTS. In using these the muscle or nerve is stimulated by the current of momentary duration produced in the secondary circuit of an induction-coil by the make or break of a constant current in the primary. The construction of the induction-coil or inductorium is founded on the fact that if a coil of wire in connection with a galvanometer be placed close to (but insulated from) FIG. 45. Diagram of Pohl's reverser. EXCITATION OF MUSCLE 189 another coil through which a current may be led from a battery, it is found that on make and break of the current of the second coil a momentary current is induced in the first. The induced current on make is in the reverse direction, that on break in the same direction as the primary current. The electromotive force of the induced current is proportional to the number of turns of wire in the coils. The induction-coil consists of two coils, each containing many turns of wire. The smaller coil (R15 Fig. 46), consisting of a few turns of comparatively thick wire, is the pri- mary coil, and is put into connec- tion with a battery. It has within it a core of soft iron wires, which has the effect of attracting the lines of force, concentrating them, and so increasing its power of in- ducing secondary currents. The secondary coil, R2» °* a large num- ber of turns of very thin wire, is arranged so as to slide over the primary coil. It is provided with two terminals, which may be con- nected with the nerve or other FlG< 46 Diagram of inductorium. Rt, primary: tissue that we wish to stimulate. R2? secondary coil, m, electro-magnet of Wagner's Since the electromotive force of the hammer, w, Helmholtz's side wire, induced current is proportional to the number of turns of wire, it is evident that the electromotive force of the current delivered by the induction coil may be many thousand times that of the battery cur- rent flowing through the primary coil. The induced currents increase rapidly in strength as the coils are approached to one another ; the strength of these therefore may be regulated by shoving the secondary up to or away from the primary coil. A short-circuiting key is always placed between the secondary coil and the nerve to be stimulated. If only single induction shocks are to be used, a make-and-break key is put in the primary battery circuit, and the two wires from the battery and key are attached to the two top screws of the primary coil (c and d, Fig. 46). It is then found that the shock given by the induced current on break of the primary current is much stronger than that on make. In endeavouring to explain this difference in the intensity of the make-and-break induction shocks, it must be remembered that the intensity of the momentary current induced in the secondary coil at make or break of the primary current is proportional (1) to the number of turns of wire in each coil ; (2) inversely to the mean distance between the coils (i.e. the nearer the coils, the stronger the induced current) ; (3) to the rate of change in strength of the primary current. Now, when a current is made through the primary coil, induction takes place, not only between primary and secondary coils, but also between the individual turns of the primary coil itself. This current of self- induction, being opposed in direction to the battery current, hinders and delays the attainment by the latter of its full strength, and so slows the rate of change of current in the primary coil. Hence the intensity of the momentary current induced in the secondary coil is less than it would have been without the retarding effect of self-induc- tion. At break of the current, an extra current is also produced in the primary coil in the same direction as the battery current, and therefore tending to reduce the rate of change of the current from full strength to nothing. In this case however the primary circuit being broken, the current of self-induction cannot pass without jumping the great resistance offered by the air, so that its retarding effect on the rate of dis- appearance of the primary current may be practically disregarded. In Fig. 47 the line, a, b, c, d, will represent the changes occurring in the primary current at make and break, a & corresponding to the make and c d to the break. The lower line represents 190 PHYSIOLOGY the momentary currents induced in the secondary circuit, m being the current of low intensity and long duration produced by the make, and £ the shock of high intensity and short duration caused by the sharp break of the primary current. When we desire to use faradic stimulation — that is, secondary induced shocks rapidly repeated 50 to 100 times a second — we make use of the apparatus attached to the coil, known as Wagner's hammer (Figs. 48A and 48fi). In this case the wires from the battery are connected to the two lower screws (a and b, Fig. 46). Fig. 48A shows the direc- tion of the current when Wag- ner's hammer is used. The cur- rent enters at a, runs up the pillar and along the spring to the screw x. Here it passes up through the screw, and through the primary coil EJ. From the primary coil it passes up the small coil m, and from this to the terminal 6 and back to the battery. But in this course FIG. 47. the coil m is converted into an electro -magnet. The hammer h attached to the spring ia attracted down, and so the spring is drawn away from the screw x, and the current is therefore broken. The break of the current destroys the magnetic power of the coil, the spring jumps up again and once more makes circuit with the screw x, only to be drawn down again directly this occurs. In this way the spring is kept vibrating, and the primary circuit is continually made and broken, with the production at each make-and-break of an induced current in the secondary coil. It is evident that, when the primary current is made and broke;i fifty times in the second, there will be a hundred momentary currents produced during the same period in the secondary coil. Every alternate one of these produced by the break of current A. T T * W=^ El * Fia. 48A. Diagram showing course of current in inductorium when Wagner's hammer is used. T *y m m FIG. 48E. Diagram showing course of current when the Helmholtz side wire is used. in the primary will be much stronger than the intervening currents produced by the make. In order to equalise make and break induction-shocks, so that a regular series of momentary currents of nearly equal intensity may be produced, the arrangement known as Helmholtz's is used. In this arrangement the side wire w, shown in Fig. 46, and diagrammatically in Fig. 48s, is used to connect the binding screw o with the binding screw c at the top of the coil. The screw x is raised, so as not to touch the spring, and the lower screw y is moved up till it comes nearly in contact with the under EXCITATION OF MUSCLE 191 surface of the spring. If we consider the direction of the current now, we see that it enters as before at the terminal, travels up the Helmholtz wire w to the screw c, thence through the primary coil B1? then through the coil m of the Wagner's hammer, and so back to the battery. The coil m, thus becoming an electro -magnet, draws down the hammer h. In this act the under surface of the spring comes in contact with the screw y. The current then has a choice of two ways. It may either go through the coil as before, or take a short cut from the terminal a, up the pillar, along the spring, through the screw y, and down to the terminal b back to the battery. As the resistance of this latter route is very small compared with the resistance of the primary coil, &c., the greater part of the current takes this way. The infinitesimal current which now passes through the coil of Wagner's arrangement is insufficient to magnetise this, and the hammer springs up again ; thup the process is restarted, and the spring vibrates rhythmically. With this arrangement the primary current is never broken, but only short-circuited, and so diminished very largely. Hence the retarding influence of self-induction is as potent with break as with make of the current, and the effects on the secondary coil in the two cases are approximately equal. In Fig. 47 ce represents the change hi the primary current when the current is short-circuited instead of being broken, and b represents the effect produced in the secondary coil. It will be seen that the currents m and b are practically identical in intensity and duration. When the induction-coil is used for stimulating, it is usual to graduate the strength the shock administered to the excitable tissue by moving the secondary coil nearer to or further away from the primary coil. It must be remembered that the strength of the induced current does not vary in numerical proportion with the distance of the two coils from one another. If one coil is some distance, say, 20 cms. from the primary coil, the induced current produced by make or break of the primary current is very smallj and on moving the secondary from 20 up to 10 cms. the increase in strength of the current will not bo very rapid. The increase will however become more and more rapid as the two coils are brought closer together. Using the same strength of current in the primary coil and the same resistance in the secondary coil, we can say that the make or break current will be uniform so long as the distance of the coils remains constant. We are not able however to say by how much the current will increase as the secondary coil is moved, say, from 11 to 10 cms. distant from the primary coil. If it is required to know the exact increment in the exciting current which is used, it is necessary to graduate the induction-coil by sending the induction shocks, obtained at different distances of secondary from primary coil, through a ballistic galvanometer. Another method which may be adopted for the excitation of muscle or nerve is the discharge of a condenser. The advantage of this method is that we can determine not only the amount of electricity discharged through the preparation, but the actual energy employed. If two plates of metal separated from one another by a thin insulating layer of dielectric such as air, glass, mica, or j paraffined paper, be connected with the two poles of a battery, each plate acquires the potential of the pole of the battery with which it is connected, and receives therefrom a charge of electricity (positive or negative). If the connections be broken the two plates retain their charge. If now they be connected by a wire they discharge through the wire, and if a nerve be inserted in the course of the wire, it may be excited by the discharge. The amount of electricity that may be stored up in this way will depend on the extent of the plates and their proximity to one another, as well as on the E.M.F. FIG. of the charging battery. In order to get great extent of surface, a condenser is built up, as in the diagram (Fig. 49), of a very large number of plates of tinfoil, separated by discs of mica or paraffined paper. Alternate discs are connected together : 49. Diagram to show the mode of construction of a condenser. 192 PHYSIOLOGY thus, 1, 3, 5 are connected to one pole, while 2, 4, 6 are connected to the other. The rheocord is used to modify the amount or strength of current flowing through a preparation. One form of it is represented in Fig. 50. A constant source of current at B causes a flow of electricity from a to b through a straight wire. As the resistance of this wire is the same throughout its length, the fall of potential from a to 6 must be constant. The nerve, or whatever preparation is used, is connected with the straight wire at two points, at a and at c, by means of a sliding contact or rider. Supposing that there is an electromotive difference of one volt between a and b, it is evident that if c is pushed close to 6, the B.M.F. acting on the nerve will be also one volt. The E.M.F, however may be made as small as we like by sliding c nearer to a. Thus if ab is one metre, and there is a difference of one volt between the two ends, then if c be one centimetre from a, the E.M.F. acting on the nerve will be T^- volt. Thus we alter the current passing through the nerve by altering the E.M.F. which drives the current. If a weak current from a Daniell's cell (or any other form of battery; be passed through a muscle or any part of its nerve, at the make of the current the muscle gives a single sharp contraction — a muscle twitch. In this contraction the whole of the muscle fibres may be involved. During the passage of the current no effect is apparently produced and the muscle seems to be quiescent, though on careful observation we may see that there is a state of continued contraction limited to the immediate neighbourhood of the cathode, which lasts as long as the current is passing through the muscle, and is not propagated to the rest of the muscle. If the current be now broken, the muscle may remain quiescent. If however the current is above a certain strength, the muscle responds to the break of the current with another single rapid contraction. With a current of moderate strength we may get a contraction both at make and break of the current, but the make- contraction may be stronger than the break contraction. Thus stimulation is caused by the make and break of a constant current, the make stimulus being more effective than" the break stimulus. If the duration of the passage of the current is sufficiently short, no contraction is produced at the break of the current, however strong this may be. The same phenomenon of a single twitch may be evoked by the passage of an induction shock. This is the current of momentary duration produced in the second circuit of an induction-coil by the make or break of a constant current in the primary. Using this mode of stimulus, it is found that the contraction on break of the primary current is much stronger than that on make. It must not be imagined however that there is any contradiction between this and the fact that the make of a constant current is a stronger stimulus than the break. When we put a muscle in the secondary circuit and make a current in the primary, there is a current of momentary duration induced in the secondary, so that there is a current made and broken through the muscle ; and the same thing takes place again when the primary circuit is broken. It has been shown that, when we use currents of such short EXCITATION OF MUSCLE 193 duration, the break stimulus is ineffective ; so in both cases, whether we make or break the current in the primary circuit, we are dealing with a make stimulus in the muscle. The difference in the efficacy of make and break induction shocks is purely physical, and depends on the fact that the current induced in the secondary coil on make is of slower rise and smaller potential than that produced at break. In using either of these modes of stimulation we find that there is a certain intensity which the stimulating current must possess in order that any effect shall be produced. Any strength of stimulus below this is known as a subminimal stimulus. A minimal stimulus (sometimes known as liminal or threshold stimulus) is the weakest stimulus that will produce any result, i.e. in muscle a contraction. A maximal stimulus is one that produces the strongest contraction a muscle is capable of under the effects of a single stimulus. A submaximal stimulus is any strength of stimulus between these two extremes. SECTION III THE MECHANICAL CHANGES THAT A MUSCLE UNDERGOES WHEN IT CONTRACTS IF a skeletal muscle, such as the gastrocnemius, be stimulated either directly or by the intermediation of its nerve by any of the means mentioned in the foregoing chapter, it responds by a single short sharp contraction, followed immediately by a relaxation. The volume of the muscle does not alter in the slightest degree, but each muscle-fibre and the whole muscle become shorter and thicker. At the same time, if a weight be tied on to the tendon of the muscle, the muscle during contraction may raise the weight and thus perform mechanical work. In order to determine the time relations of the simple muscle contraction or the muscle-twitch, and to studj its conditions, it is necessary to employ the graphic method, so as to obtain a record of the changes in shape of the muscle during contraction. We may uss the graphic method either for recording the changes in shape or for registering changes in tension of a muscle which is prevented from contracting. In order to record the contraction of the frog's gastrocnemius, the muscle is excised together with a portion of the femur to which it is attached, and the whole length of the sciatic nerve from its origin in the spinal canal to its insertion into the muscle. The femur, to which the gastrocnemius is attached, is clamped firmly, and the tendo Achillis attached by a thread to a light lever, free to move round an axis at one end. The point of this lever is armed with a bristle (anything that is stiff and pointed will do), which just touches the blackened surface of a piece of glazed paper. This paper is stretched round a cylinder (drum) which can be made to rotate at any constant speed required. If the drum is moving, the point of the bristle draws a horizontal white line on the smoked paper. If a single induction shock be sent through the nerve of the preparation the lever is jerked up, falling again almost directly, and a curve is drawn like that shown in Fig. 52. A similar curve is obtained if the muscle be stimulated directly. In all such graphic records we should have also — (1) A time record. This is furnished by means of a small electro-magnet, armed with a pointed lever writing on the smoked surface. This electro-magnet (time marker or signal) is made to vibrate 100 times a second (more or less as may be required) by putting it in a circuit which is made and broken 100 times a second by means of a tuning-fork vibrating at that rate. The tuning-fork is maintained in vibration in the same way as the Wagner's hammer of an induction-coil. (2) A record of the exact point at which the nerve or muscle is stimulated. This may be obtained in two ways : (a) When using the pendulum or trigger myograph, in both of which the recording 'surface is a smoked flat surface on a glass plate, this latter is so arranged that it knocks 194 THE MECHANICAL CHANGES OF MUSCLE 195 over a key as it shoots across, and so breaks the primary circuit and excites the nerve or muscle of the preparation. As we know the exact point that the plate reaches when it knocks over the key, we can mark on the contraction curve the exact moment at which stimulation took place. (6) If we wish to make and break the primary circuit at will by means of a key, a small electro -magnetic signal, interposed in the circuit, is arranged to write on the revolving drum, and so mark the point of stimulation. Kia. 51. Arrangement of apparatus for recording simple muscle-twitch. the figure (Fig. 52) the upper line is the curve drawn by the lever of the muscle t contracts ; the small upright line shows the point at which the muscle was stimu- lated ; and the second line is the tracing of the chronograph, every vibration repre- senting ¥^4 of a second. In the pendulum myograph (Fig. 53) a smoked glass plate is carried on a heavy ron pendulum. At each side the pendulum is armed with a catch, which fits on to FIG. 52. Curve of single muscle-twitch taken on a rapidly moving surface (pendulum myograph). (YEO.) other catches at the side of the triangular box, from the apex of which the pendulum i? suspended. At its lower part the pendulum carries a projecting piece which can knock over the * kick-over ' key K, thus breaking a circuit in which is included the primary coil of an induction-coil. The lever attached to the muscle is arranged so as to write lightly on the glass plate. Everything being ready, and the key K closed, the pendulum is raised to A, the catch A is then released, and the pendulum falls at an ever-accelerating rate and then rises again, gradually slowing off until it is caught again at B. As it passes by the key it breaks the circuit. A break induction shock i;} sent into the muscle or nerve, which contracts, and a curve is obtained similar to that shown in Fig. 52. Since the rate of the pendulum is constantly varying throughout its course, it is necessary to have a tuning-fork, or time-marker actuated electrically by a tuning-fork, writing just below the muscle-lever. In the spring myograph, otherwise known as .the trigger or shooter myograph (Fig. 54), a smoked glass plate is also used. "The frame supporting the glass plate sides on two horizontal steel wires. .To make the instrument ready for use, the frame is moved to one side, which compresses a short spring. When the catch holding it in this position is released by the trigger, the spring, which only acts for a short space, 196 PHYSIOLOGY gives the frame and the glass plate a rapid horizontal motion ; and the momentum carries the glass plate through the rest of the distance, till stopped by the buffers. Th(» FIG. 53. Simple form of pendulum myograph. velocity during this time is nearly constant, as the friction of the guides is small. Two keys are knocked over by pins on the frame and break electric circuits. The rr'ative positions at which the circuits are broken can be altered by a convenient adjustment. FIG. 54. Diagram of spring myograph. or ' shooter.' A tuning-fork vibrating about 100 per second fixed to the base of the instrument marks the time ; its prongs are sprung apart by a block between their ends, and the same action which releases the glass plate also frees the fork by removing the block and allows it to vibrate ; a writing style then draws a sinuous line on the smoked surface of the moving glass plate. A muscle lever with a scale-pan attached also forms part of the instrument." The record obtained in either of these ways may, in consequence of instrumental THE MECHANICAL CHANGES OF MUSCLE 197 inertia, be a very inaccurate reproduction of the true events occurring in the muscle itself. When the muscle begins to contract it imparts a very rapid movement to the lever, which therefore tends to overshoot the mark and deform the curve. This source of error may be almost avoided by making the lever as light as possible, and hanging the extending weight in close proximity to the axle of the lever, as shown in Fig. 55. Since the energy of a moving mass is proportional to the square of the velocity ( = %mv2,) and the tension due to the weight as well as the velocity on contraction is directly proportional to the distance of the weight from the axis, it follows that it is better to Ct_ r FIG. 55. Blix apparatus for recording isometric and isotonic curves synchronically. (Miss BUCHANAN.) p, the steel cylindrical support with jointed steel arm to bear the isotonic lever I, which consists of a strip of bamboo with an aluminium tip. t, the isometric lever, also of bamboo, except for a short metal part t', in which are holes for fixing the muscle. The two wires from an induction coil are brought, one to X , which is in con- nection with the support and hence with the metal bar t', the other to y, which is insulated from the support but connected by a copper wire with a thin piece of copper surrounding the isotonic lever at the point where the muscle is attached to it. Cl, clamp for fixing the lower end of the muscle when an isometric curve is to be taken. The axis of the isotonic lever is at x, close to 'which is hung the weight of 50 grm. oad the muscle with 40 grams 1 millimetre from the axis than with 1 gram 40 milli- etres from the axis, though the tension put on the muscle will be the same in both ases. In the first case the energy of the moving tmass, will be proportional to 0}- L = 800, and it is this energy which deter- ) X (I)2 1 = 20, and in the second to — nines the overshooting of the lever and the deformation of the curve. Since throughout le contraction in the latter arrangement the lever follows the muscle in its movement, le tension on the muscle remains the same throughout, and the method is therefore •:nown as the isotonic method. It is of importance to be able to record the development of the energy (i.e. the ten- on) of the active muscle apart from any changes in its length. For this purpose the luscle is allowed to contract against a strong spring, the movements of which are lagnified by means of a very long lever. Thus the shortening of the muscle is almost ntirely prevented, but the increase in its tension causes a minute but proportionate lovement of the spring, which is recorded by means of the lever. Since in this case ae length or measurement of the muscle remains approximately constant, while the 3nsion is continually varying throughout the contraction, it is known as the isometric lethod. The great magnification necessary in this method introduces serious sources of rror ; but it seems that if all due precautions be taken to avoid these errors, the isometric 198 PHYSIOLOGY curve differs very little in form from the isotonic, displaying only a somewhat quicker development of energy at the beginning of contraction. It is better to eliminate the lever altogether and magnify the minute movements of the spring by attaching to it a small hinged mirror by which a ray of light is reflected through a slit on to a travelling photographic plate. Since the ray of light has no inertia, magnification of the movements may be carried to any extent without increasing the instrumental deformation of the curve (Fig. 56). A simple muscular contraction or twitch, such as that in Fig. 52, produced by a momentary stimulus, consists of three main phases : (1) A phase during which no apparent change takes place in the muscle, or at any rate none which gives rise to any movement of the lever. This is called the latent period. FIG. 50. Myograph for optical registration of muscular contraction. (K. LUCAS.) (2) A phase of shortening, or contraction. (3) A phase of relaxation, or return to the original length. The small curves seen after the main curve are due to elastic vibrations of the lever, and do not indicate any changes occurring in the muscle itself. From the time-marking below the tracing we see that the latent period occupies about T -(J () second, the phase of shortening T * (T, and the relaxation yg-^ second. Thus a single muscle-twitch is completed in about r\T second. It must be remembered however that this number is only approximate, and varies with the temperature of the muscle and its condition, being much longer in a fatigued muscle. Moreover it is almost impossible to avoid some deforma- tion of the curve due to defects of the recording instruments used. Thus the relative period during which no mechanical changes are taking place in the muscle must always be shorter than is apparent from a curve obtained THE MECHANICAL CHANGES OF MUSCLE 199 by the foregoing method. The elasticity and extensibility of the muscle must prolong the apparent latent period, since the first effect of contraction of any part of the muscle will be to stretch the adjacent part, and only later to Fia. 57. Burdon Sanderson's method for photographic record of muscle-twitch. The exciting shock is sent into the muscle by the wires d and d'. ove the tendon to which the lever is attached. Thus if we have a weight pported by a rigid wire, and suddenly pull the upper end of the wire so as to ise the weight, the latter will rise instantaneously. If however the eight be suspended by a piece of elastic, it will not follow the pull exactly, >ut will lag behind, the first part of the pull being occupied with stretching india-rubber, and only when this is stretched to a certain degree will the' weight begin to rise. The same re- tardation of the pull would be observed if, instead of india- rubber, we used a piece of living muscle. It is possible to obviate this instrumental inertia by employing solely photographic methods for the record and magnification of the muscle- twitch. In the experiments of Sanderson and Burch the thickening of the muscle at the point stimulated was recorded graphically by photo- graphing the movement on a FIG. 58. Photographic record of muscle-twitch . slit (Fio- 57^ behind which (B' SANDERS°N.) The upper curve is the move- rig. 01), I wnicn ment of the muscle> the middle curve the signal Was a moving sensitive plate. showing the moment of excitation, and the lower • _ . i 1 _ t f t • l» 1 '1 _ J • __ *>/\f\ A • Thus avoiding all instrumental inertia, and diminishing the inertia of the muscle to a minimum, the mechanical latent period was found to be only 0*0025 second (Fig. 58). This figure we can take as the average latent period for the skeletal muscle of the frog at the ordinary mperature of the laboratory (about 16°C.). We shall have occasion later * that of a t™ing-fork vibrating 500 times 200 PHYSIOLOGY on to consider the changes which occur in the muscle between the application of the stimulus and the moment at which the first mechanical change makes its appearance. The relaxation of muscle is helped by a moderate load, and in a normal condition is complete. It is not active — that'is to say, is not due to a con- FIG. 59. V. Kries1 apparatus for taking ' after- loading ' and 'arrested con- traction curves.' traction in the transverse direction — but is a passive effect of extension and elastic rebound. This may be shown by allowing a muscle to contract while floating on mercury. The subsequent lengthening on relaxation is very incomplete. Even with the most careful arrangements for securing isotonicity in the record of the contraction there is probably a certain amount of over-shoot of the lever whenever, as at high temperatures, the contraction is sufficiently rapid. The effect of this is that one cannot assume the existence of an act ual pull on the lever during the whole time of the ascent of the latter. We can therefore speak of a period during which there is contractile stress — that is to say, when the muscle is actu- ally pulling on the lever, which l will occupy only a part of the AAAAAAAAAAAAAAAAAAAAA ascent of the curve. The dura- _ . , , . . , , FIG. 60. Curves of isotonio and arrested contractions lion Ot this period Ot COntraC- Of an unloaded ...nsclr (KAISER.) tile stress may be shown by recording what is known as • anvMt-d ' c -ontrac-.tions. One mechanism for this purpose is shown in the figure (Fig. 59). The stop Su is THE MECHANICAL CHANGES OF MUSCLE 201 used simply for after- loading the muscle so that the weight shall not act upon the muscle until it begins to contract. The stop. So may be regu- lated so that it suddenly checks the movement of the lever at any desired height above the base line. We may thus get a series of contractions such as those shown in Fig. 60. It will be seen that at the points x', x", and x"' the muscle was still pulling on the lever, and therefore held it up against the stop. At the point A the arrested twitch returns rapidly to the base line, showing that the movement of the lever in the unarrested curve above this point was due to the inertia of the moving parts and not to the actual pull of the muscle. In this case the period of contractile stress was about 0'02 seconds. THE ENERGY OF CONTRACTION. When a muscle contracts we may nceive of it as converted into a body with elastic properties other than hose which it possesses during rest. Directly after it has been excited t possesses potential energy which can be measured by the isometric method tension and which will degenerate in a few hundredths of a second into eat, or can be turned into work by allowing the muscle to shorten and to raise a weight, as in the isotonic method of recording muscular contractions. Under the conditions of an ordinary physiological experiment, a contracted muscle loaded only by a light lever is shorter than the non-contracted, but can be stretched to the length of the latter by a certain weight, when it will be in a condition of tension. In their natural position in the body muscles may possess any length between extreme shortening and extreme elongation whether they are in a resting or in an excited condition. Since the relaxed muscle requires only a minimal force to extend it to the maximal length possible in its natural relationships in the body, it is" usual to speak of the different lengths of an excited and unexcited muscle, the lengths being in this case those which are impressed on the muscle by a minimal load. When we measure by means of the isometric method the maximum energy set free in a muscle as the result of excitation, we find, as Blix first pointed out, that this energy depends on the length of the muscle fibres during the period of contractile stress set up by the excitation. With increase in the length of the muscle the tension developed on excitation increases until the length of the muscle is somewhat greater than that which it possesses in its normal relationships in the body. To lengthen the muscle beyond this point a certain stretching force must be applied to it which rapidly increases. The tension developed on excitation however soon begins to iminish. These relationships are shown by the diagram (Fig. 61), where the ordinates repre- snt the length of the muscle and the abscissae the tension on the muscle. The left- id thick line represents the muscle in a state of rest, the right-hand curved line the muscle in a state of excitation. The horizontal distance between the two lines gives the increase of tension (as measured by the isometric method) produced when the muscle passes from the resting into the excited state as the result of stimulation by a single induction shock. t Since the tension set free on excitation depends on the length of the 202 PHYSIOLOGY muscle fibres during the production of the condition of tension, the tension developed will be diminished if the muscle be allowed to shorten before its maximum tension has been reached. This is the case with all isotonic records of muscular contraction, so that it becomes difficult to give any exact expression for the total energy changes in a muscle which is allowed to shorten. On the other hand, in the body the bony levers are so arranged that the muscles at their greatest length work at a maximum mechanical disadvantage which lessens continuously as the muscles shorten and approxi- mate their points of attachment. The load on a muscle is thus lessened Tension FIG. 61. Diagram to show the relation between the initial length of a muscle and the tension developed in it during excitation (as measured by the isometric method). The tension developed at each initial length is measured by the horizontal distance between the two thick lines, the left line representing the resting muscle, and the curved thick line on the right the contracted muscle. (From BLIX.) continuously as the muscle contracts. A muscle is a machine primarily for developing tension, and the potential energy thus set up may be used for the production of work to any degree the conditions of loading allow. The work done by a muscle when it contracts is measured by multiplying the weight lifted by the height through which it is lifted, w X h. Since however the result will vary according to the conditions of loading of the muscle, a much more useful quantity is obtained by measuring the tension produced in a muscle which is stimulated but not allowed to shorten. The potential energy available due to the new elastic conditions of the fibres is found to be approximately ^ 17, where T is the maximum tension developed in the twitch and I is the length of the muscle (A. V. Hill). THE MECHANICAL CHANGES OF MUSCLE 203 THE EXTENSIBILITY OF MUSCLE Living muscle in a perfectly normal condition is distinguished by its slight but ^ perfect elasticity ; that is to say, it is con- siderably stretched by a slight force (in the longitudinal direction), but returns to its original length when the extending weight is removed. The length to which muscle is stretched is not proportional to the weight used, but any given increment of weight gives rise to less elongation the more the FIG. 62. Extensibility of india-rubber (a) mugcle ig alread stretched> The accom_ compared with that of a irog s gastroc- ... • nemius muscle (6). panying curves show diagrammatically the elongation of muscle as compared with a piece of india-rubber when the weight on it is uniformly increased. Dead muscle is less extensible and its elasticity is less perfect. A given weight applied to a dead muscle will not stretch it so much as when the muscle was alive, but the dead muscle does not return to its original length when the weight is removed. A contracted muscle, on the other hand, is more extensible than a muscle at rest. A gramme applied to a contracted gastrocnemius will cause greater lengthening than if it were applied to the same muscle at rest. The relation between the exci- tability of a muscle under the two conditions of contraction and rest are shown in the diagram in Fig. 63. At the point y the muscle is unable to shor- ten at all against a weight. It is evident from this diagram that FIG. 63. Curve showing the length of a muscle under various the height of contraction loads in the contracted condition by, and uncontracted of a iscle dim condition cy. The double lines a, 6, &c., represent the con- tracted muscle, while the long single lines a c, &c., show the the load is increased, very length of the inactive muscle. rapidly if the muscle is after-loaded, less rapidly if the weight applied to the muscle be allowed to extend it at rest. It is evident however that in either case the diminution in height is not in proportion to the load and that the work done by the muscle, w X h, as the weight is increased, rises at first quickly, then more slowly to a maximum to sink finally to zero. By inspection of diagram (Fig. 63) it will be seen that O.h <20.h2< 30.h3 > 40.h4 > 50.h5 so that in this case the maximal mechanical work is obtained when the muscle is loaded with about 30 gms. PROPAGATION OF CONTRACTION. THE CONTRACTION WAVE The whole muscle does not as a rule contract simultaneously. When excited from its nerve the contraction begins at the end-plates and spreads in both directions through the muscle. The rate of propagation of the con- traction wave can only be measured by employing a curarised muscle, so as to avoid the wide spreading of the excitatory change by means of the intra- muscular nerve-endings. For this purpose a curarised saitorius muscle 204 PHYSIOLOGY is taken, stimulated at one end, and the thickening of the muscle recorded by means of two levers placed one near the exciting electrodes and the second at the other end of the muscle, as shown in the diagram (Fig. 64). The difference between the latent periods of the two curves represents th'e time taken by the contraction wave in travelling from a to b. By measurements carried out in this way it is found that the rate of propagation of the con- traction in frog's muscle is 3 to 4 metres per second ; in the muscle of warm-blooded animals it may amount to 6 metres. FIG. 04. Diagram of arrangement for recording the contraction wave in a curarised sartorius. The actual duration of the shortening at any given point is necessarily smaller than that of the whole muscle, and amounts in frog's muscle to only 0-05-0-09 sec., about half the duration of the contraction of a whole muscle of moderate length. The length of the wave is obtained by multiplying the rate of transmission by the duration of the wave at any one point. It varies therefore in frog's muscle between 3COO x -05 ( = 150) and 4CCO x -09 ( = 360) millimetres. Thus the muscle fibres in the frog are much too short to accommodate the whole length of the wave, and the contraction of the whole muscle must be made up of the summated effects of the contraction wave as it passes from point to point. Hence the longer the muscle, the more must the contraction be lengthened by the time taken up in propagation from one end to another. SECTION IV - THE CONDITIONS AFFECTING THE MECHANICAL RESPONSE OF A MUSCLE STRENGTH OF STIMULUS. If a series of single break-shocks be applied to a muscle or nerve at intervals of not less than five seconds, it will be found that beyond a certain distance of the secondary from the primary coil no :ffect at all is produced. The shocks are said to be subminimal. On pushing the secondary coil nearer the primary a point will be reached at which a small contraction will be observed. On then pushing in the coil a millimetre at a time the contrac- tion will become greater for the next couple of centimetres (e.g. as the coil is moved from 12 to 10 cm. distance). Further increase of current by approximation of the coils is without effect, although the current actually used may be increased a hundred times in moving the coil from 10 to 0. It was formerly thought that this limited gradation of the muscular response according to strength of stimulus was due to a similar gradation in the response of each individual muscle fibre of which the muscle is composed. It seems more probable how- ever that, when a minimal or subminimal response is obtained, not all the fibres making up the muscle are contracting. A min- imal contraction is in fact a contraction in which some fibres of the whole muscle are stimulated. A maximal contraction is one in which all the fibres are stimulated. So far as concerns each individual muscle fibre every contraction is a maximal contraction. The fibre either contracts to its utmost or it does not contract at all. The rule of ' all or none ' which was first nunciated for heart-muscle is probably true for every con- actile element. ' jThe difference between skeletal and heart muscle lies in the fact that in the former the excitatory process does not spread from one fibre to its neighbours. If for instance we take a curarised sartorius and split its lower end, as in Fig. 65, the stimulus applied to A causes a contraction only of the left side of the muscle, while a stimu- lus applied to B is in the same way limited to the right side. If a piece of ventricular or auricular muscle of the frog or tortoise were treated in the same way, a stimulus applied at A would cause a contraction which would travel across the bridge at the upper end and' extend to B. 205 FIG. 65. 206 PHYSIOLOGY It was shown by Gotcti that, if each of the three roots which make up the sciatic nerve and send fibres to the gastrocnemius be stimulated in turn, it is often impossible to evoke a maximal contraction of the gastrocnemius, however strongly each root be stimulated. Keith Lucas has shown that if stimuli in gradually increasing strength be applied to the motor nerve (containing only seven to nine fibres), which supplies the dorso-cutaneous muscle of the frog, the contraction of the muscle increases, not gradually, but by a series of steps. This can be explained only by assuming that the smallest effective stimulus excites perhaps four out of the seven nerve fibres, those immediately in contact with the electrodes. With increasing strength of current the stimulus becomes effective for the three fibres lying next to these, and finally still further increase of current may excite all the fibres making up the nerve (Fig. 66). SO 100 150 200 FIG. 66. Curve showing relation of height of contraction of dorso-cutaneous muscle to strength of stimulus. - Ordinates = height of contraction ; abscissa = strength of stimulus. (K. LUCAS.) THE REPETITION OF STIMULUS SUMMATION. The response of a muscle fibre to a single shock, whether measured by the isotonic or the isometric method, i.e. as shortening or as tension, is independent of the strength of stimulus and varies only with the length of the fibre during the rise of the excitatory condition. If however a second shock is sent in during this period a further evolution of energy is possible, and the effect is still further increased by putting a series of stimuli into the muscle or its attached nerve before the development of the contractile stress due to the first stimulus has feached its maximum. If two shocks at intervals of one hundredth of a second be sent into a muscle, the response, whether shortening or rise of tension, will be greater than that produced by one shock. If a series of shocks be sent in, the excitatory condition is main- tained, so that instead of a simple muscle twitch rising to a maximum and then falling, the muscle lever rises to a given point, which in the muscle con- tracting isometrically may be double that due to a single stimulus, and then remains at this height during the continuation of the repeated excitations. If the muscle be allowed to contract isotonically, the continued contraction produced by a series of stimuli may with a heavy load be three or four times as considerable as that produced by a single stimulus. This condition of apparently continued stimulation brought about by continued application of stimuli is said to be surnmated. REFRACTORY PERIOD. If the interval between two stimuli sent into a muscle be successively shortened in a series of observations, we finally arrive at a point at which summation is no longer apparent, i.e. the effect of THE MECHANICAL RESPONSE OF MUSCLE 207 the two stimuli is no greater than the effect of a single stimulus. This means that the second stimulus has become ineffective, and this ineffective- ness we must ascribe to the condition set up in the muscle as the result of the first stimulus. For a very short period of time after stimulation a muscle is inexcitable to a second stimulus. The period during which it is inexcitable is known as the refractory period and amounts in skeletal muscle to about -0015 second. The same phenomenon is better marked in certain other excitable tissues, such as the heart muscle, but it seems to be a common property of excitable tissues generally. When a loaded muscle is made to record its contractions isotonically we may get summation of effects, though the interval between the stimuli is greater than that which corresponds to the duration of the rise of contractile stress. Thus if the interval is just so long that the second becomes effective just as the contraction due to the first has com- menced to die away, the second con- i 7 traction seems to start from the point _ . , FIG. 67. Muscle curves showing summation of to which the muscle has been raised by the first (Fig. 67). By repeating these stimuli in a heavily loaded muscle, the contraction may be made three or four times as extensive as a single twitch. With slow stimuli the summation is however rather mechan- ical than physiological. The period of contractile stress, which lasts only about •03 second, is so short that it has no time to raise the weight to the maximum height before it has passed away. 'This is shown by the fact that if the muscle be after- loaded, so that the lever is raised to the top of the curve of a single twitch, application of the stimulus will make it shorten still more, and by repeated after-loading in this fashion, it is possible to make the muscle raise a weight in response to a single stimulus to the same height that it would if excited by a series of stimuli. This mechanical factor in sum- mation is shown in Fig. 68. It will be noted however that the tetanus is not a steady one and is probably due to stimuli FIG. 68. Contractions of a frog's muscle. Two single twitches repeated at intervals of are followed by a tetanus, which is almost twice as high as a about * of a second. stimuli, r and r'. the points at which the stimuli were sent into the nerve. From the first stimulus alone the curve abc would be obtained. From r' the curve def is obtained. These two curves are summaled to form the curve aghik when both stimuli are sent in at the interval r r'. single contraction. After two more single twitches, the drum was made to rotate more slowly, and single shocks employed, at the same time as the ' after-loading ' was continually were increased to 50 or the rate of If stimulation increased. It can be seen that the curve obtained in this way is as high as the original tetanus. (V. FREY.) 100 per second, a tetanus .e — „ i&^w, „««.„«,. v,.A-»Jai.y would be produced and the curve would be prob- ably twice as high as that represented in the figure. We thus see that for the over- coming of a resistance a single twitch is not economical. It is doubtful whether any contractions of muscles which occur in the body are other than tetani of varying duration. TEMPERATURE. Speaking generally, the effect of warming a muscle 208 PHYSIOLOGY is to quicken all its processes. The latent period becomes shorter and the muscle curve steeper and shorter. It is very often observed that the height of contraction of the warmed muscle is greater than that obtained at ordinary temperatures. It seems that this apparent increase in height is really instrumental in origin, the quicker-moving muscle jerking the lever beyond the real extent of the contraction. If proper means are taken to eliminate this overshooting of the lever, it is found that the height of contraction is unaltered between 5° and 20°C., the only change being in the time-relations of the curves. This is especially well shown in the so-called ' arrest ' curves (Fig. 69). FIG. 69. Isotonic and 'arrest ' curves of muscle-twitch : (1) unloaded at 14°C. ; (2) at 25°C. ; (3) at 0°C. ; (4) loaded at 14°C. Note that the arrest curves attain the same height throughout. (KAISER.) If a muscle be heated gradually (without stimulation) up to about 45°C., it begins to contract slowly at about 34°C., and this contraction reaches its maximum at 45°C., at which point the muscle has entered into pronounced rigor mortis. Cold has the reverse effect. The intra-molecular processes which lie at the root of the muscular activity are slowed, so that the latent period and 1 he contraction period are prolonged. The action of cold on the r./r //////// of muscle is to increase it, so. that any form of stimulus is more effective at "> ( . than at 25°C. Moreover, when maximal stimuli are being used, and the muscle is heavily loaded, the first effect of the application of cold may be to increase the height as well as the duration of contraction, for the same reason that a gentle push is more efficacious in closing a door than would be a heavy blow with a hammer. If however a muscle be cooled for a short time to zero or a little below, it loses its irritability, which returns if the muscle be gradually warmed again. Prolonged exposure to severe cold irrevocably destroys its irritability. Warming the muscle will now simply bring about rigor mortis. FATIGUE. A muscle will not go on contracting indefinitely. If it be repeatedly stimulated, changes soon become apparent in the curve of contraction. The latent period is prolonged, as well as the length of the contractions ; the absolute height and work done are diminished. At the same time the muscle does not return to its original length ; the shorten- THE MECHANICAL RESPONSE 'OF MUSCLE 209 ing which remains is spoken of as ' contraction remainder' After an initial rise during the first few contractions, these diminish uniformly in height till they are no longer apparent, so that the muscle is now said to have lost its irritability. At the same time there is a great prolongation of the curve, occasioned almost entirely by a retardation of the relaxation, so that after forty or fifty contractions several seconds may elapse before the lever returns to the base line (Fig. 70). FIG. 70. Muscle curves showing fatigue in consequence of repeated stimulation. The first six contractions are numbered, and show the initial increase of the first three contractions. (BRODIE.) The fact that the relaxation part of the muscle curve is affected by various conditions. jcially fatigue, apparently independently of the contraction part, led Fick to put forward a theory that two distinct processes were concerned in the response of a muscle to excitation, one process causing the active shortening and the other the relaxa- tion. (It must be noted that this is not the same as saying that the lengthening is an active process, a statement negatived by the behaviour of a muscle when caused to contract on mercury.) He suggested that the disintegration associated with activity might be conceived as occurring in two stages : the first resulting in the production of sarcolactic acid and the active shortening of the muscle ; the second in the further conversion of the acid into CO2, with a consequent relaxation. A retardation of this second phase would cause the prolonged curve with ' contraction remainder ' observed in a fatigued muscle. We shall return to this point when discussing the chemical and heat chan'ges which accompany contraction. If left to itself, the muscle which has been exhausted by repeated stimula- tion will recover. The recovery is hastened by passing a stream of blood, or even of salt solution, through the blood-vessels of the muscle. Recovery in a muscle outside the body is never complete. »The phenomena of fatigue probably depend on two factors : (1) The consumption of the contractile material or the substances avail- e for the supply of potential energy to this material. (2) A more important factor is the accumulation of waste products of contraction. Among these waste products the lactic acid is probably of great mportance. Fatigue may be artificially induced in a muscle by ' feeding ' t with a dilute solution of lactic acid, and again removed by washing out ;he muscle with normal saline solution containing a small percentage of alkali. 14 210 PHYSIOLOGY THE ACTION OF SALTS The action of sodium salts on muscle is of considerable interest. We are accustomed to use a 0-6 per cent, solution of NaCl as a ' normal fluid ' to keep muscle preparations moist. If however the solution be made with distilled water, it has a distinctly excitatory effect upon the muscle, B FIG. 71. A. Tracing of the contraction of a frog's sartorius, poisoned with veratrin, in response to a momentary stimulus. The time-marking indicates seconds. B. Tetanic contraction of normal Sartorius in response to rapidly interrupted stimuli. (The duration of the stimulus is indicated by the words ' on ' and 'off.') It will be noticed that the two curves are practically identical. BUCHANAN.) (Miss so that single induction shocks may cause tetaniform contractions. The same excitatory effect is still better marked with solutions of Na2C08. If a thin muscle, such as a frog's sartorius, be immersed in a solution con- taining 0-5 per cent. NaCl, 0-2 per cent. Na2HP04, and 0-04 per cent. Na2C03 (Biedermann's fluid), the muscle enters into a series of frequent contractions, so that it may wriggle from side to side, or may even ' beat ' for a time with the regularity of heart-muscle, though at a much greater rate. This excitatory action of sodium salts is neutralised by the addition of traces of calcium salts. Hence the normal saline used in the laboratory should always be made with tap water, containing calcium salts. FIG. 72. Tracing of the contraction of a Potassium Salts, although form- muscle poisoned by the injection of a strong innr go important a constituent of __l__i; _« i_i_ _1 : j.U~ J~.-l-l_ the ash of muscle, act as muscle poisons, quickly and permanently destroying its irritability. If a muscle be transfused with normal fluids containing minute traces of potas- sium salts, it at once shows all the signs of fatigue, signs which may be removed by washing out the potassium salts by means of 0-6 percent. NaCl solution. It is possible that the setting free of potassium salts may be one of the factors involved in the development of the normal fatigue of muscle. solution of veratrin, showing the double contraction due to unequal poisoning of different fibres. (BIEDERMANN.) THE MECHANICAL RESPONSE OF MUSCLE 211 THE ACTION OF DRUGS Of the drugs that have a direct action on muscle, the most remarkable is veratrin, which causes an excessive prolongation of a muscular contraction (produced by a single stimulus). Thus the ' twitch ' of a muscle poisoned with veratrin may last fifty or sixty seconds, instead of the normal one-tenth of a second (Fig. 71). Barium salts have a similar, though less marked effect. In order to carry out the poisoning with veratrin, very weak solutions (1 in 100,000 or 1 in 1,000,000 of normal saline) should be used and the muscle exposed to its action for some time. We get then on a single stimulus a response lasting many seconds and exactly similar in height and form to a tetanus obtained by discontinuous stimu- lation. If stronger solutions be used, the action of the drug is apt to affect the fibres unequally, so that we may have a sharp normal twitch preceding the prolonged con- traction (Fig. 72). If the muscle be excited several times immediately after the pro- longed contraction has passed away, it responds with twitches like those of a normal muscle, but if allowed to rest a few minutes, stimulation is again followed by the peculiar long-drawn-out contraction. SECTION V CHEMICAL CHANGES IN MUSCLE CHEMICAL COMPOSITION OF VOLUNTARY MUSCLE VOLUNTARY muscle consists of elongated cells, the muscle fibres being embedded in a connective tissue framework ; and, as in all cellular tissues, proteins form its chief chemical constituents. The contents of the fibres are semi-fluid and can be expressed from the finely divided muscle as a viscous fluid known as muscle-plasma. Muscle- plasma is obtained in the following way. The living muscle of frogs is frozen, minced with ice-cold knives and pounded in a mortar with four times its weight of sand containing -6 of common salt. The mixture is then thrown on to a filter kept at 0° C. when an opalescent fluid filters through. The filters soon become clogged and therefore must be frequently changed, and their temperature must not be allowed to rise above 2° to 3°C. If the temperature of the muscle-plasma be allowed to rise, clotting takes place, the clot later on contracting and squeezing out a serum, as is the case with blood-plasma. The muscle-plasma is neutral or slightly alkaline. When coagulation takes place however, it becomes distinctly acid, and this acidity is due to the formation of sarcolactic acid in the process. Arguing chiefly from analogy with the blood-plasma, the muscle-plasma has been said to contain a body, myosinogen, which is converted when clotting takes place into myosin. The exact nature of the prot3ins in muscle-plasma, as well as of the protein con- stituent of the clot, which we have called myosin, is still a subject of debate. Kiihne, to whom we owe our first acquaintance with muscle-plasma, described the clot as consisting of myosin, a globulin, soluble in 5 per cent, solutions of neutral salts, such as NaCl or MgS04, precipitated by complete saturation with MgS04, and coagulated on heating to 56° C. In the muscle-serum, obtained after separation of the clot, he found three proteins, one coagulating at 45°C., one he called an albumate (i.e. a derived albumen or metaprotein), and the third coagulating about 75°C., and apparently identical with serum albumen. Halliburton extended these researches to the muscles of warm-blooded animals. He described four proteins as existing in muscle-plasma. of which two, paramyosinogen and myosinogen, gave rise to the clot of myosin. In no case however is it possible entirely to dissolve up the clot when once formed, and it seems that the so-called solution in dilute salt solutions Was merely an extraction of still soluble protein in the meshes of the clot. Von Fiirth has shown that if the muscles of a mammal are washed free of adherent lymph and blood, the plasma obtained by extraction with normal salt solution contains only two proteins. Those proteins a iv extremely unstable, and are gradually transformed on standing into insoluble 212 CHEMICAL CHANGES IN MUSCLE 213 protein, giving rise to a precipitate in dilute solutions, or forming a jelly-like clot in strong solutions. The properties of these proteins may be summarised as follows : (1) Myosin (paramyosinogen of Halliburton). A globulin, coagulating at about 47°- 50°C., precipitated by half saturation with ammonium sulphate or oh dialysis. Trans- formed slowly in solution, rapidly on precipitation, into an insoluble protein, myosin fibrin. (2) Myogen (myosinogen of Halliburton). A protein allied to the albumens in that it is not precipitated by dialysis. Coagulates on heating at 55°-60°C. It changes slowly into an insoluble protein, myogen fibrin, but passes through an intermediate soluble stage called soluble myogen fibrin. This latter body coagulates on heating to 40°C., being instantly converted at this temperature into insoluble myogen fibrin. It does not seem that any ferment action is associated with these changes, which we may represent by the following schema : Muscle-plasma. I I 1 myosin or paramyosinogen. i myogen (myosinogen of Halliburton, albumate of Kiihne). Soluble myogen fibrin. I Myosin fibrin. Insoluble myogen fibrin. Muscle clot. Soluble myogen fibrin, which in mammalian muscle-plasma forms only on standing, exists apparently preformed in frog's muscle. Hence the instantaneous clotting of 's muscle-plasma on warming to 40°C. The residue left after the expression of the muscle-plasma consists defly of connective tissue, sarcolemma, and nuclei, and as such contains ilatin (or rather collagen), mucin, nuclein, and adherent traces of the proteins of the muscle-plasma itself. The muscle-serum contains the greater part of the soluble constituents of muscle. OTHER CONSTITUENTS OF MUSCLE. A number of other sub- stances are found in muscle in small quantities, those which are soluble being contained to a great part in the muscle- serum. It will suffice here to enumerate the chief of these. t(a) Colour ing -matter. All red muscles contain a considerable amount of haemo- bin. A special muscle pigment allied to haemoglobin has been described by MacMunn as myohaematin. The only evidence for its existence is spectroscopic. (b) Nitrogenous extractives. ' Of these, the most important is creatine (C4H9N302) of which 0*2 to 0*3 per cent, may be found in muscle. Its significance will be the subject of consideration later. Other nitrogenous bodies occurring in smaller quantities are >xanthine, xanthine, and traces of urea and amino-acids. (c) Non-nitrogenous constituents. Fats, in variable amount. Olycogen. This substance is invariably found in healthy muscle. Fresh skeletal le contains about 1 per cent. In the embryo the muscles may contain many this quantity of glycogen. Glucose is present in fresh muscle in minimal quantities, about -01 per cent. When muscle is allowed to stand, especially in a warm place, the glycogen under- goes partial conversion into glucose, so that the latter increases at the expense of the 214 PHYSIOLOGY Inosit (C6H1206 2H20) or ' muscle sugar ' occurs in minute traces in muscle. It does not belong to the group of carbohydrates at all, being a hexahydrobenzene. It is nonfermentable and does not rotate polarised light nor does it reduce Fehling's solution. Its significance is quite unknown. (d) Inorganic constituents. Muscle contains about 75 per cent, of water. A.e not allowed to shorten, the state of tension passes off and the whole lergy which has been set free must appear as heat. The potential energy ieveloped in a muscle twitch is approximately equal to ^ Tl, where T is the 222 PHYSIOLOGY tension developed and I the length of the muscle, and it is this amount which must be compared with the heat production measured in the muscle by one of the methods described. A. V. Hill has shown that the heat production in a contracting skeletal muscle occurs in two phases, a rapid production of heat which apparently is synchronous with the contraction itself, and a slow production of heat which continues for some time after fehe muscle has relaxed. The second phase of h$at production depends on the presence of oxygen, and is observed at its best when the muscle is kept in pure oxygen. If the muscle be allowed to contract in nitrogen only the initial heat production is observed. The heat production of the second phase is stated by Hill to be approximately equal to that in the first phase. These results have been interpreted as showing that the initial change in muscular contraction is the development of lactic acid. The appearance of this lactic acid in some way changes the muscle and sets up potential energy at the surface of its ultimate fibrils, which will result in a shortening of the muscle if any movement of its ends be allowed. A comparison of the energy of the tension set up with the actual heat evolved in the initial stage when a muscle is not allowed to contract shows that the two quantities are approximately equal. In a series of experiments Hill found that the ratio J- T/ : H in the sartorius muscle under low initial tensions and in comparatively Weak contractions approximated to the value 1, the mean value being -91. Under high initial tensions and in strong contractions of the sartorius muscle, it is lower, being roughly from 04 to 0-6. He concludes from this that under certain conditions the initial process of con- traction consists largely, if not entirely, of the liberation of free potential energy manifested as tension in the muscle. This potential energy may be used for the accomplishment of work or for the production of heat. The efficiency of the initial stage of contraction is therefore almost 100 per cent. If however a muscle is to go on contracting without rapidly showing signs of fatigue, it must be kept in oxygen, so that the processes of replace- ment or of removal of the lactic acid may take place. Under these circum- stances there is a further evolution of heat after the contraction, equal to that set free during the initial stage. So that the total efficiency of a muscle kept in oxygen would not be more than 50 per cent. This is assuming that the process of oxidation of the lactic acid and its repl;u •<• ment in whole or in" part in the muscle molecule is completely carried out during the time of the observation. It is improbable that such is the case, and it seems possible that the evolution of heat during the so-called recovery stage of the muscle has been under-estimated. If a series of observations of the heat production and tension developed during isometric contractions be made with varying initial length of the muscle, it is found that while the ratio of tension developed to heat produced is approximately constant, both these quantities first increase and then finally diminish. The optimum of the heat production in some experiments seems to fall later than the optimum of the tension developed. Thus the longer the muscle fibre, within limits, when it is excited, the greater the ten- PRODUCTION OF HEAT IN MUSCLE 223 sion and the greater the heat production developed, i.e. we may assume that increased length of muscle fibre increases the chemical changes, ensuing on excitation, which are responsible both for the development of mechanical energy and the production of heat. The significance of these results for the essential nature of muscular contraction we shall discuss in a later chapter. SECTION VII Fia. 75. Diagram of non- ELECTRICAL CHANGES IN MUSCLE IF a current from a battery be passed between two plates of platinum immersed in acidulated water or salt solution, electrolysis of the water takes place, bubbles of oxygen appearing on the positive plate (anode), and bubbles of hydrogen on the negative plate (cathode). If now we remove the battery, and connect the two plates (electrodes) by wires with a galvanometer, a current passes through the galvanometer and water in the reverse direction to the previous battery current. This current is called the polarisation current, and is due to the electrolysis of the water that has taken place. The vessel in which the electrodes are immersed has in fact become a galvanic cell, the platinum covered with oxygen bubbles being the positive element, and that covered with hydrogen bubbles the negative element. Exactly the same process of electrolysis or polarisation takes place when we pass currents through the tissues of the body by means of metallic electrodes. Hence before we can study accurately the delicate electrical changes that may occur normally in living polarisable electrode. tissues, it is necessary to have some form of electrodes in a,covered wire; b, amal- which this polarisation will not occur. The ' non-polari- gamated zinc rod ; c, sable ' electrodes which are most generally used for this glass tube ; d, saturated purpose are made in the following way. A glass tube ZnSO4 solution ; e,plug of f,-,. _„.. , , , ., * , , °,. zinc sulphate clay ; /,plug 1S closed at one end Wlth a PluS of kaolm made of normal saline clay. into a paste with a saturated solution of zinc sulphate. The rest of the tube is filled with a similar solution. Dip- ping into the zinc sulphate solution is a rod of pure zinc, amalgamated. Just before use, a plug of china clay made with normal saline solution is put on the end of the tubs, so as to effect a connection between the zinc sulphate clay and the nerve or muscle which it is desired to stimulate or lead off. In these electrodes there is no contact of metals with fluids that can produce dissimilar ions (e.g. hy- drogen or oxygen) at the surface of contact, and hence they may be regarded as practically non- polarisable. A more convenient form is that employed by Burdon Sanderson, in which the glass tube is bent into a U (Fig. 76). The mouth of the tube is closed by a smaller glass tube plugged with clay, and bearing a plug of normal saline clay. • In such electrodes the conduction of the cur- rent through the nerve or muscle to the metal I i< part of the circuit may be represented as shown on the opposite page (see Fig. 77). If a muscle such as the sartorius be removed from the body, and two imn- polarisable electrodes connected with a delicate galvanometer be applied to two points of its surface, there will be a deflection of the mirror attached to the galvanometer, showing the presence of a current in the muscle from 224 FIG. 76. U-shaped non-polarisftbie electrodes. ELECTEICAL CHANGES IN MUSCLE 225 the ends to the middle, and in the external circuit from the middle (or equator) to the ends. It was formerly thought that this current was always present in all normal muscles, and it was spoken of as the ' natural muscle current ' ; the muscle was said to be made up of a series of electromotive molecules, the equator of each molecule being positive to the two poles (du Bois Raymond). It has been conclusively shown however (by Hermann and others) that this Zn so* a a so* Zn + FIG. 77. FIG. 78. Current of rest. current of resting muscle is not a natural current at all, but is due to the effects of injury in making the preparation. The less the preparation is injured, the smaller is the current to be obtained from it, and in some con- tractile tissues, such as the heart, there may be absolutely no current during quiescence. Hermann describes the fact of the existence of currents of rest thus ; " In partially injured muscles every point of the injured part is negative towards the points of the uninjured surface." Fig. 78 shows the direction of the current in a muscle with two cut ends. , ^ When the whole muscle is quite dead, this cur- fj'~J J J J '* \ 1 rent of rest, or ' demarcation current ' (Her- — mann), disappears. -The current is due to the electrical differences at the junction of living and dying (not dead] tissue. If the sartorius of the frog be cut out and immersed for twenty- four hours in 0-6 per cent. NaCl solution made with tap water (i.e. con- taining lime), all the injured fibres die, and the uninjured fibres are then found to be iso-electric and therefore currentless. The existence of this current may be demonstrated without using a galva- nometer. If the nerve of a sensitive muscle-nerve preparation a, (Fig. 79) be allowed to fall on an excised muscle 6, so that two points of the nerve are in contact with the cut end and with the surface of the second muscle b, the muscle a will contract each time the nerve touches b so as to complete the circuit. Whatever be the explanation of this current of resting muscle, there is no doubt that a very definite IRheoscopic frog electrical change occurs in a muscle when it contracts. To show this change, we may lead off two points, one the cut end and one on the surface of the muscle of a muscle-nerve pre- paration,to a galvanometer. We shall then obtain a deflection of the mirror of 225 16 226 PHYSIOLOGY the magnet, due to the current of rest or demarcation current. If now the nerve be stimulated with an interrupted current so as to throw the muscle into a tetanus, the ray of light from the galvanometer mirror swings back towards the zero of the scale, showing that the current which was present before is diminished. When the excitation of the nerve is discontinued, the galvanometer indicates once more the original current of rest. This diminution of the current of rest during activity of a muscle is spoken of as the ' negative variation.' In carrying out this experiment it is usual to compensate the demarcation current by sending in a small fraction of the current from a constant cell. The arrangement of the apparatus is represented in the accompanying diagram. Two non-polarisable D 'h e.c. FIG. 80. electrodes np are applied to the surface and cross-section of a muscle m. These are connected with the shunt of the galvanometer, one of the wires however being con- nected with a Pohl's reverser p, and this in its turn with the shunts. The two end- terminals of the reverser are connected with a rheochord, through the wire of which ab a constant current is passing from the Daniell cell D. By means of the rider c the fraction of current passing through the reverser can be modified to any extent. The key k being open, the muscle is connected with the shunt and galvanometer, and the direction and -extent of the swing noticed. The key k is then closed, and by means of the reverser the current is sent through the galvanometer in the opposite direction to the demarcation current, and the rider c shifted until the two currents exactly balance one another, and the needle of the galvanometer returns to zero of the scale. This adjustment is first made, using only ,,,',,,, of the total current, and then by means of th<- shunt, ,,',„, ,',,, and finally the whole current is thrown into the galvanometer. If this precaution be not taken, much too large a current may in the first case be sent through the galvanometer, to the detriment of the instrument. If we know the difference of potential between the two ends of the wire, the proportion— will give us the E.M.F. of the demarcation current. The galvanometer needle having by compensation been brought to zero, stimulation of the nerve at e by interrupted currents causes the needle to swing at once in the opposite direction to the first variation. This swing is the measure of the negative variation or current of action. In order to study the electrical changes accompanying a single muscle twitch, it is necessary to employ some instrument which can react much more rapidly than the ELECTRICAL CHANGES IN MUSCLE 227 ordinary galvanometer. For this purpose we may employ either the capillary electro- meter or the string galvanometer of Einthoven. The capillary electrometer is an instrument for recording and measuring difference of potential. That is to say, if connected with two points, it measures the force which would make a current flow between these two points if they were connected by a wire. Its structure is very simple. It consists of a glass tube drawn out to a fine capillary point. This tube with the capillary is filled with mercury. The point dips into a wide tube containing dilute sulphuric acid, at the bottom of which is a little mercury. Two platinum wires fused into the glass and dipping into the mercury serve as terminals. When the instrument is used, the meniscus of the mercury in the capillary at its junction with the acid is observed under the microscope, or a magnified image of it is thrown on a screen with the aid of the electric light. If now the capillary and acid be con- nected with two points, it will be observed that any difference in the potential of these two points causes a movement of the meniscus. If the point connected to acid be negative as compared with the point connected to mercury in capillary, the meniscus moves towards the point of the capillary. If the acid be positive as compared with the capillary, the meniscus moves away from the point. The extent of the excursion is proportional to the difference of potential. Since the capillary electrometer appears to have no latent period, and is free from instru- mental vibrations, it is extremely useful in recording the quick changes in potential occurring in the diphasic rloctrical changes that accompany every contraction-wave in the body. The excursions lend themselves well to photography, so that we may obtain a graphic record of every electrical variation, and thus determine its extent and its time-relations. It must be remembered that this instrument is an electrometer (measurer of differ- FIG. 81. Capillary electrometer. (BUBCH.) FIG. R 82. FIG. 83. ence of potential), and not a galvanometer (current measurer). When the electrometer is connected with two points at different potential, current passeb into it for a fraction 228 PHYSIOLOGY of a second, and polarises the surface of the mercury, so that it takes up a new position in the capillary. This polarisation causes an electromotive force which exactly balances the E.M.F., setting up the polarisation so that no current passes the surface. Hence the use of non-polarisable electrodes is not so essential in experiments with this instru- ment as when we make use of the galvanometer. In the D'Arsonval galvanometer (Fig. 82) the current is sent through a coil of fine wire hung between the poles of a permanent magnet. The same principle is made use of in the string galvanometer of Einthoven (Fig. 83). In this a very delicate thread of silvered quartz or of platinum is stretched between the poles of a strong magnet. The poles of the magnet are pierced by holes so that the thread may be illumined by an electric light from one side, and from the other may be observed by means of a microscope ; or a magnified image of the thread may be thrown upon a screen. When- ever a current passes through the thread it moves laterally, and the lateral movement may be photographed on a moving photographic screen. Owing to the minute dimen- sions of the thread the instrument is one of extreme delicacy. It will detect very minute currents and will respond accurately to very rapid changes in potential. If a perfectly uninjured regular muscle (Fig. 84), such as the sartorius be stimulated with a single in- duction shock at one end, x, and two points, a and 6, be led off to a capillary electro- meter, each stimulus applied at x gives rise to an excursion of the meniscus of the electro- meter, known as a' spike/ and FIG. 84. Diagram showing diphasic variation of uninjured muscle. shown in Fig. 85-. Knowing the constants of the instrument used, we can analyse this spike, and we find that it represents a diphasic change. Our study of the mechanical changes in muscle has shown that, when the muscle is stimu- lated at x, a contraction wave commences which travels down the muscle through a and b. The electrical . investigation of the muscle shows that excita- tion of x arouses an electrical change which also passes down the muscle at the same rate as the mechanical change which it precedes. If we are leading off from x and a, the electrical change ensues immediately upon stimulus, i.e. there is no latent period to the electrical change. On leading off from a FIG. 85. A typical electrometer record from a sar- nnrl 7> fhprp iq a Intpnt rjpriod toriu8 muscle excited by a single induction shock. Time-marking=200 D.V. (KEITH LUCAS.) between the stimulus and the first change, representing the time taken for the change to travel ELECTRICAL CHANGES IK MUSCLE 229 from x to a. When the change reaches a this becomes the seat of an electromotive force of such a direction that the current would pass in the outer circuit from b to a. We may say therefore that a is negative to b. A fraction of a second later the excitatory change has passed on to b and has died away at a. Now b is negative to a,* and the current therefore passes in the opposite direction. Between a and b therefore, there is a diphasic current, the first phase representing negativity of a to b, and the second phase A B FIG. 86. Diphasi9 response of uninjured sartorius (obtained by analysis of curves such as Fig. 85). A, .at 8°C. ; B, at 18°C. (KEITH LUCAS.) representing negativity of b to a. A diphasic change is thus also a sign of a propagated change. Every excitation of a normal muscle gives rise to a * The statement that the excited portion of the muscle becomes ' negative,' though sanctioned by long usage, is not very exact and may give rise to misconception. When we lead off the terminals of a copper- zinc couple or cell to a galvanometer, a current flows outside the cell from copper to zinc and inside the cell from zinc to copper. In this case the zinc is said to be electropositive to the copper, and in the same way we must assume that the excited portion of a muscle is electropositive to the unexcited portions. When therefore we speak of any part of a tissue being negative, we are using a conventional expression to indicate the direction of the current in the outer circuit, and not the electrical condition of the tissue itself. In order to avoid the con- fusion which might result from an attempt to replace the loose expression ' negative ' by the more correct expression ' electropositive,' Waller has suggested the employ- ment of the term * zincative ' to indicate the electrical condition accompanying excita- tion. This term would also serve to emphasise the fact that the excited portion, like the zinc in a zinc-copper cell, is the chief seat of chemical change. 230 PHYSIOLOGY diphasic variation of such a direction that the point stimulated first becomes negative to all other points of the muscle, and this ' negativity,' to use a loose but convenient expression, passes as a wave down the muscle, preceding the wave of contraction and travelling at the same rate. If one leading-off point be injured, e.g. at b, the change accompanying excitation is absent at that point. A single stimulus applied at x will in this case give only a monophasic variation in which a is relatively negative to b. When we study the time relation of the electrical variation ensuing on a single stimulus, we find that the electrical change under the electrodes begins at the moment that the stimulus is applied. It takes about -CG25 sec. to attain its culminating point. At this point the mechanical .change or contraction of the muscle begins. These time-relations vary with the temperature of the muscle. We have alread}^ seen that the effect of lowering the temperature is to increase that latent period of the contraction. In the same way it slows the rise of the electrical change and the rate of propagation of the wave of electrical change. This is shown in Fig. 86, in which are given the diphasic response of the sartorius first at 8°C. and secondly at 18°C. ' We are therefore justified in regarding the electrical change as an index to the chemical changes evoked in the muscle as the direct result of the stimulus. The flow of material, which is responsible for the change in form of each contracting unit, is secondary to these changes. As the result of stimulation, a chemical change is. aroused at the point of excitation and travels thence along the muscle fibres at a rate of about three metres per second, i.e. the same rate as that of the following wave of mechanical change and, like this, varying with the temperature. Under certain conditions an excitatory condition may be propagated without the presence of a visible contraction. Thus, if the middle third of the sartorius be soaked for a time in water, it passes into a condition known as ' water rigor,' in which it is incapable of contracting, although capable of transmitting an excitation from one end of the muscle to the other. The connection of a diphasic current of action with an excited condition of the tissues passing as a wave from one end to the other is shown still more clearly on a slowly contracting tissue, such as the ventricle of the frog or tortoise. Fig. 87, A, is a photographic record of the variation obtained from the tortoise ventricle, which is led off to a capillary electrometer, one (acid) terminal being connected with the base of the ventricle, the other (mercury) with the apex. Each part of the ventricle remains contracted for a period of 1 j to 2 seconds, and then the contraction passes off, first at the base and later at the apex. The electrical events are an exact replica of the mechanical. Directly after the stimulus has been applied, the base becomes negative and the column of mercury moves up. A moment later the excitatory condition extends to the apex. There is thus a sudden equalisation of potential between the two terminals, and the mercury comes back quickly to the base line. Here it stays for 1 J to 2 seconds. During this time the whole heart is in an excited condition. Both base and apex are equally excited, and there ELECTRICAL CHANGES IN MUSCLE 231 can be no difference of potential between them. The excitatory condition then passes off, first at the base and then at the apex. There is thus a small period of time in which the apex is still contracted or excited while the base is relaxed, and the apex is therefore negative to the base. This terminal negativity of the apex is shown on the photograph by the excursion of the column of mercury away from the point of the capillary. If one terminal, e.g. the apex, be injured, we obtain quite a different variation, which is shown in Fig. 87, B. It is evident from this figure that the electrical sign lasts practi- Fia. 87. Electrometer records of the electrical variations in a tortoise ventricle, excited to beat rhythmically by single shocks. A, Ventricle uninjured. B, One leading off spot injured. (B. SANDERSON.) cally as long as the mechanical sign of the excited state, and that we are not justified in regarding the first spike of the diphasic variation as indicative of an excitatory wave attended by an electrical change which is independent of the succeeding mechanical change. The only difference between the electrical changes in this case and in that of voluntary muscle is that in the latter all processes are very much quicker, so that as a rule the point a (Fig. 84) has ceased to be negative before the negativity of b has attained its full height, and there is thus no prolonged fquipotential stage. Although in the case of the slowly contracting ventricle of the tortoise, the record obtained of the electrical changes accompanying its contraction by means of the capillary electrometer shows with great clearness the diphasic nature of the variation, and there- fore the wave character of the electrical change, considerable difficulty is experienced sometimes in recognising that the ' spike ' record of the electrical change in voluntary 232 PHYSIOLOGY muscle or in nerve is also due to a diphasic variation. In this case the electrical change at any spot lasts only about ^ -^ second, and there is not a prolonged equipotential period, as in the case of the heart. The nature of the variation is however obvious, if we compare the electrometer record of an intact and therefore currentless muscle with that of a muscle in which one of the leading-off points has been injured, so as to give rise to a demarcation current. The two curves are given in Fig. 88, the uppel shadowy tracing being that obtained from the injured muscle. It will be seen that the dis- tinguishing character of an electrometer record of a diphasic variation in the rapidly contracting striated muscle consists in the fact that the down- stroke of the image of the meniscus is as rapid as the upstroke, whereas FIG. 88. Superimposed photographs of the electrical varia- the monophasic variation tion of the sartorius in response to a single stimulus, of the injured muscle (BUEDON SANDERSON.) presents a slow fall pro- duced by the gradual leakage of the charge imparted to the instrument back through the electrodes and muscle. When such a record is analysed, we obtain a curve similar to those in Fig. 89, which repre- sent monophasic variations of a sartorius injured at one end, under different conditions of temperature. A similar curve to the diphasic variation can be obtained by putting in a current of similar E.M.F. from a battery, first in one direction for ^^ second, and then in a reverse direction for another 2^ second. It must be remembered that a diphasic variation does not mean that one part of a muscle changes from normal in one direction, and then swings back past the normal in another direction, but that a change in one direc- tion at one electrode dies away and is succeeded by a similar change in the same direction, which also dies away, at the second electrode: that is to say, a diphasic variation implies the pro- gression of a wave of electri- cal change between the lead- ing-off points. Using a string galvanometer, which reacts much more rapidly, the diphasic nature of the varia- tion is immediately apparent SEC. .oos .„, from the photographic record Flo 89j Monophasic variations of an injured sartorius. even with voluntary muscle, A, at 18°C. ; B, at 8°C. (KEITH LUCAS.) or nerve. The electrical variation obtained by leading off a heart beating normally is a much more complex affair. The question will be discussed more fully in chapter xiii. ELECTRICAL CHANGES IN MUSCLE 233 THE DEMARCATION CURRENT OR CURRENT OF INJURY Muscle or nerve may become negative under two conditions : (1) During activity ; (2) when dying as the result of injury. It is doubtful however whether these two conditions are really distinct. Section or injury of a muscle causes a prolonged stimulation of the adjacent parts of the muscle fibres. These parts therefore being excited, must be negative to the unexcited parts which are further away from the seat of injury so that a demarcation current is really an excitatory current. We thus come to the conclusion, para- doxical only in terms, that the so-called currents of rest are really currents of action and are due to excitation around the injured spot.* SECONDARY CONTRACTION. RHEOSCOPIC FROG The negative variation of one muscle may be used to make another contract. If the nerve of the preparation a (in Fig. 90) be laid so as to touch at two points the cut end and surface of the i tiiscle 6, and the nerve of b then stimulated with single induction shocks, every contraction of b will be attended by a contraction of a, excited by the negative variation of the current passing through its nerve from the point touching the cut end to that in contact with the equator of 6. If the nerve of b is tetanised, a as well as b enters mfc *frog. into a continued contraction. This 'secondary tetanus ' is of interest as showing that, although the contractions of b are fused, the excitatory process and negative variations are still quite distinct. * If the demarcation current is really due only to excitation, we should expect to find it weaker than the action current obtained by exciting the whole muscle to contract. And this is the case. The E.M.F. of the demarcation current of a sar- torius equals about 0'05 of a Daniell cell. The action current of the same muscle may attain to an E.M.F. = O08 of a DanieU cell (Gotch). SECTION VIII THE INTIMATE NATURE OF MUSCULAR CONTRACTION EXPERIMENTS on the metabolism of the body as a whole show that the energy of muscular work is derived from the oxidation of the food-stuffs. In man the performance of work involves an increase of the oxidative processes of the body with a corresponding evolution of energy, of which four-fifths will appear as heat while one-fifth may be transformed into mechanical work. In this respect the physiological mechanisms for the production of mechanical energy resemble the greater number of the machines employed by man for the same purpose. In nearly all these .the prime source of energy is the oxidation of carbon and hydrogen in the form of coal or oil. In the steam-engine and internal-combustion engine the whole energy set free by the process of oxidation appears first as heat, and then a certain portion of the heat is converted into mechanical work. There is a limit to the efficiency of such heat engines, depending on the maximum differences of temperature available between the two sides of the working part of the machine. The efficiency of any heat engine is expressed by ^ fl the formula E = - — — -, where T is the highest temperature (in absolute measurement) obtained by the working substance and T1 is the lowest temperature of the same substance. Ordinary engines rarely attain more than half this ideal efficiency, but it is evident that the greater the difference of temperature available the greater will be the efficiency of the machine. Internal-combustion engines, such as the gas engine or the oil-engine, therefore give a greater percentage of the total energy of the fuel out as mechanical energy than is the case with the steam-engine. Engelmann has maintained that in muscle there is a similar transforma- tion of heat into mechanical energy. He has found that non-living sub- stances, which contain doubly refractive particles and possess the property of imbibition (e.g. catgut) when soaked with water, will contract on heating and relax again on cooling. He has constructed a model in which a thread of catgut in water, surrounded by a platinum coil, can be made to simulate muscular contractions and relaxations by passing a heating current through the platinum coil. He imagined that the chemical changes in the muscle liberate heat and that the effect of this heat upon the doubly refractive particles is to make them iml>il>r t lie surrounding water so that they change 234 THE INTIMATE NATUEE OF MUSCULAR CONTRACTION 235 from an oval to a spherical shape. It would be impossible however for any large changes of temperature to take place in the muscle without entirely destroying its chemical character, and with small differences of tempera- ture it would be impossible to attain the efficiency of 50 to 100 per cent, which characterises muscle. Under certain conditions we may obtain by a machine almost the entire energy of a chemical change. The condition is that the chemical change shall be susceptible of taking place in a galvanic battery. We may use, for instance, a series of Daniell cells to drive an electric motor and allow the motor to perform mechanical work. Under these circumstances we could theoretically obtain ICO per cent, of the total chemical energy avail- able, and in conditions of practice the efficiency of the machine may attain to 70 or 80 per cent. A similar arrangement might be present in the ultimate contracting elements of the muscle fibre. The mechanism in the fibre must be one which will provide for a more or less direct transformation of chemical energy into mechanical energy without a previous conversion of the chemical into heat energy. In the living body, where everything is in solution, all the energies may be reduced to one of two kinds, osmotic energy and surface energy. The contractile machine must therefore be one which employs one or other, or both, of these forms of energy. We might with Macdougall, regard the contractile element as a cylindrical structure differing in its contents from the surrounding sarcoplasm. When the muscle is at rest the contents of the muscle prism will be in equilibrium with the surrounding sarcoplasm. We might imagine the excitatory process to consist in a sudden chemical change occurring in the contents of the muscle prism. The production of a number of new molecules within the muscle prism (e.g. of lactic acid) would raise the osmotic pressure within the prism and occasion a rapid flow of water from the sarcoplasm. As a result the pressure in the muscle prism would rise and cause a bulging of its lateral wall and a shorten- ing of the whole element. The subsequent phase of relaxation may be due either to a secondary change, e.g. oxidation, leading to the formation of a substance to which the walls of the prism are freely permeable, or to the gradual leak of the primary products of oxidation or disintegration into the sarcoplasm. The substance or substances giving rise to the osmotic differ- ences which determine contraction may be either products such as lactic acid and carbon dioxide, which are formed during contraction, or may possibly be of the nature of neutral salts set free from some condition of combination with the proteins of the sarcous element. Macdonald has brought forward micro-chemical evidence of the appearance of potassium salts in the sarcous element during the state of activity of the muscle. On the other hand, Bernstein has suggested that the changes during muscular contraction are determined by alterations in surface tension. If a little mercury be spilt on a plate the particles form globules. They are kept from spreading themselves out in a thin film under the influence of gravity in consequence of the surface tension of the mercury. Any modifica- tion of the surface will alter the tension, and therefore state of expansion, of 236 PHYSIOLOGY the globule. Thus, if the globule be in sulphuric acid it undergoes a certain amount of polarisation, and becomes positively charged. By altering the charge of such a globule we can change its shape, as is shown diagram- matically in Fig. 91. If B represents the shape of the globule lying on the plate in some weak sulphuric acid, A will represent the shape of the globule when it is connected with the negative pole of a battery, while c will repre- sent its shape when it is connected A o C with the positive pole of a battery, the other pole in each case being connected with the acid. If we FlG 91 consider muscle as made up of a series of chains of oval particles, a chemical change in the surface of these particles, causing an increase of surface tension, will tend to make them assume the globular shape, and will therefore cause a shortening and thickening of the whole fibre. According to Schafer, contraction is associated with a flow of the outer hyaline contents of the sarcous element into the tubular structure forming the middle portion. Such a flow may be determined either by osmotic differences between the centre/ and periphery of the sarcous element, or by a change in the surface tension obtaining between the isotropic fluid at the ends and the anisotropic structures in the centre of the muscle prism. The tendency of recent investigation is all in favour of the second hypo- thesis, namely, that the essential factor in the processes of excitation and contraction is an alteration of surface. In the first place the electrical changes accompanying the excitatory process denote a polarisation or accumulation of ions on the surfaces situated in the excited area. The chemical change which is responsible for the current of action, or the negative charge at the excited spot, takes place almost instantaneously and disappears somewhat more slowly. It would seem that the excitatory process consists essentially in the setting free of certain ions on the surface or surfaces in the contractile tissue, and that the passing away of the excitatory state is due to the disappearance of these ions, either by diffusion away into tin- surrounding fluid or by further chemical changes, such as oxidation. A study of the development of tension and of heat production in a muscle on excitation has shown that in both cases the yield of energy on excitation is increased by lengthening and diminished by shortening the muscle. Now alteration in length of the muscle will not alter its volume, but will alter the extent of its longitudinal surfaces, and it appears therefore that the production of heat as well as of mechanical energy is not a volume, but a surface effect. Finally the work of A. V. Hill on the heat production in muscle seems to show that the rise of tension in a muscle on excitation is due to the liberation of chemical bodies, of which lactic acid is certainly one, in the neighbourhood of certain longitudinal surfaces or membranes, and that the presence of these bodies changes the tension at such surfaces and thereby the longitudinal tension of the fibre. The extent and intensity of the production of these bodies must depend on the area of the chemically THE INTIMATE NATURE OF MUSCULAR CONTRACTION 237 active surfaces and therefore on the length of the muscle fibres. The muscle reacts at the end of the excitatory stage, not by any active process of lengthening, but by neutralisation, or simply physical diffusion of the active chemical bodies away from the interfaces or membranes. Later on, lactic acid is removed or replaced by its previously unstable precursor under the influence of oxygen with the production of some carbon dioxide and a certain amount of heat. We have seen already that the efficiency" of the initial chemical change in which lactic acid is set free may approximate ICO per cent. It must be noted that, although the oxidative processes are responsible ultimately for all the energies of the higher animal, no oxidative change is involved in the production of lactic acid from e.g. glucose, nor is the presence of oxygen necessary for the contraction of muscle to take place. On the other hand, if we wish to obtain the maximum amount of work from a muscle, we must supply it richly with oxygen, the presence of which seems essential not to the contractile process but to the stage of recovery. In this stage a certain amount of heat is evolved, set free by the oxidation of the lactic acid, and we must assume that part of the energy so available is utilised for building up the precursor from which the lactic acid is derived. It is as if the process of oxidation furnished the energy for winding up a spring, whereas excitation removed a catch and allowed the spring to run down, setting free this energy for the performance of work or for conversion into heat.* For many years it was imagined, as a result of experiments by Hermann, Pfliiger, and others, that the oxygen supplied to a muscle was built up with its other constituents, especially carbohydrates, into a complex ' inogen ' molecule. On stimulation this mole- cule underwent an explosive rearrangement, the carbohydrate and oxygen parts of the molecule combining to form carbonic acid, another product of the decomposition being lactic acid. The careful experiments of Fletcher have shown however that in the absence of oxygen there is no evidence of the formation of carbonic acid during contraction, and therefore no reason to assume the presence of oxygen in the muscle in an intramolecular form. Everything points to oxygen being taken in and applied forthwith to the purposes of oxidation, so that the output of carbon dioxide and water keeps pace with the intake of oxygen. It is at present quite impossible to come to any conclusion as to the nature of the - * Peters has shown that, if a muscle be stimulated to exhaustion under anaerobic conditions, about 0-2 per cent, lactic acid is formed with the evolution of '9 calories per gramme of muscle substance. The production of 1 gm. of lactic acid is therefore accompanied by the evolution of 450 calories. According to A. V. Hill the ' recovery heat production ' in oxygen is of about the same order as the initial heat production, so that in the oxidative removal of 1 gm. of lactic acid there would also be an evolution of about 450 calories. The oxidation of 1 gm. of lactic acid produces 3700 calories, about eight times as much as the quantity observed. Hill considers this amount far too large to have escaped detection in his experiments, and therefore concludes that the lactic acid is not oxidised but replaced in its previous position under the influence and with the energy of the oxidation either : (a) of a small part of the lactic acid itself, or (&) some other body. He regards the latter alternative as the more probable, and concludes therefore that the lactic acid is part of the machine and not part of the fuel of the muscle. 238 PHYSIOLOGY precursor from which the lactic acid is derived. The immediate precursor cannot be glucose or glycogen since the heat evolved in the initial stage of contraction is two or three times as great as could be derived from the mere conversion of either of these substances into lactic acid. We must therefore conclude that the oxidation of lactic acid which goes on during the process of recovery is used to yield the energy necessary for building up the active molecules, which are the precursors of lactic acid and which have a higher potential energy than glucose itself, so that when it rapidly decomposes sufficient energy is set free to account for the observed heat production. Some such utilisation of the energy of oxidation of the lactic acid is indicated by the results of Parnas, who found that the heat evolved during this recovery process corresponded to only about one half the heat which would be evolved by the formation of the carbon dioxide output of the muscle during the same time as a result of the oxidation of lactic acid. SECTION IX VOLUNTARY CONTRACTION THE whole of our analysis of the processes accompanying the contraction of a skeletal muscle has so far had reference merely to the contractions evoked by artificial stimuli, mainly electric. These contractions have either been the simple twitch, with a duration of about one- tenth of a second, evoked by a momentary stimulus, or the tetanus, a continued contraction composed of a number of single twitches, summated and fused together. Under normal circumstances the contraction of skeletal muscles is brought about either reflexly, or in response to some stimulus descending from the cerebral cortex, the so-called ' voluntary contraction/ These contractions may have a duration of almost any extent. The quickest contractions carried out by man have a duration of about 0-1 sec: Considerable effort and training are required to reduce a muscular movement to this degree, and nearly all contractions, even the rapid ones, last considerably over 0-1 sec. Since we have no certain means of producing contractions of any given length, except by means of repeated stimuli, it is natural that physiolo- gists have regarded voluntary contractions as similar to the artificial tetanus, and as like this composed of fused single contractions, and have endeavoured to determine the number of contractions per second, i.e. the natural rhythm of the tetanus. If however every muscular contraction in the body is to be regarded as of the nature of a tetanus, effected by rapidly repeating stimuli sent down the motor nerve from the central nervous system, we must assume a similar discontinuity for the process underlying the normal tone of muscles, and for the continued contraction of unstriated muscles, e.g. of the arteries. Is this discontinuity of muscles really essential for the production of a prolonged contraction ? So far as our present knowledge of the intimate nature of muscular contraction goes, it would seem quite possible that the continuous state of "contraction is dependent on a1 continuous evolution of energy in the muscle. We have seen reason to regard the chemical processes in a contracting muscle as presenting two phases, namely, (1) the production of a substance which increases the osmotic pressure within the sarcous elements, or raises the surface tension of the ultimate contractile elements of the muscle, thus causing a shortening and thickening of those elements ; and (2) the further change of this substance into one which can escape by diffusion, or into a substance with a low surface tension, so that now the muscle relaxes and can be stretched by any extending 239 240 PHYSIOLOGY force. If these two phases went on continuously, but the first phase kept ahead of the second one, a continuous state of contraction would be produced in the muscle. Since the contraction of the muscle occurs only in response to impulses from the central nervous system, we should have to imagine also a continuous stream, e.g. of negatively charged ions, descending the nerve and evoking an excitatory change in the muscle fibres as they impinge on the neuro-muscular junction. We have evidence that a state of excita- tion of a nerve, which is apparently continuous, may excite a correspondingly continuous state of excitation in the muscle attached. During the passage of a constant current through muscle there is a continuous contraction in FIG. 92. Continued contraction followed by rhythmic contractions of a muscle in response to a constant stimulus. (BIEDERMANN.) The muscle was excited by the passage of a constant current, the cathoda end having been moistened with a weak solution of NaC03. the neighbourhood of the cathode. If the irritability of the muscle at this point be increased by the application of a solution of sodium carbonate, Biedermann has shown that this excitation is propagated to the rest of the muscle, and on closure of the current we obtain a prolonged contraction followed by rhythmic contractions (Fig. 92). Moreover in frogs, the ex- citability of which has been heightened by keeping them at 2° to 3° C. for some days, the closure of a descending current through the sciatic nerve causes a prolonged contraction of the gastrocnemius ; and in the same way there may be a prolonged contraction produced by the opening of an ascend- ing current through the nerve. The question however can only be decided by experiment. If a volun- tary or reflex contraction is of the nature of a tetanus, we should be able, by a study of the mechanical and electrical phenomena combining the contraction, to obtain distinct evidence of this causation. It was shown by Wollaston that, on listening to a contracting muscle, a low sound was heard, which, according to him, corresponded to a- vibration frequency of 36 to 40 per second. The same observation was made by Helmholtz, and can be repeated by any one who will place the end of a stethoscope on a muscle, e.g. the biceps, and listen to the sound produced when it contracts. Helmholtz pointed out however that the tone heard corresponded to the resonance tone of the external ear, and was the same as that noted when listening to any irregular sound of low intensity. Thus the roar of London that we hear in the middle of Hyde Park has the same pitch as the muscle sound of the contracting biceps. The muscle sound therefore teaches us nothing as to the pitch or number of contractions per second making up t he VOLUNTARY CONTRACTION 241 voluntary tetanus. It merely points to an irregularity or discontinuity in this contraction. By bringing vibrating reeds of different frequency in contact with the contracting muscles of the frog, Helmholtz came to the conclusion that the chief element in the muscle sound was the first over-tone of a sound with a vibration frequency of 18 to 20 per second, which, according to him, was to be taken as representing the number of single contractions in every voluntary muscular contraction. Nearly all voluntary contractions present a certain degree of irregularity, and the same irregularities are observed when a tetanic spasm in the muscle of the body is caused by strong excitation of the cerebral cortex, as in epilepsy. On taking a record of such contractions, Schafer and Horsley showed that in nearly all cases the tracing presents superposed undulations repeated at the rate of eight to twelve per second. These observers concluded that this was the normal rate at which the impulses descend the nerve to arouse a voluntary contraction. One difficulty in this conclusion is that when human muscle is excited by eight to twelve stimuli per second, we obtain, not a tetanic contraction with a few irregularities superposed on it, but a series of single contractions, the so-called clonus. In order to produce a nearly continuous contraction we must employ a vibration frequency of about 30 per second. It has been suggested to get over this difficulty that under normal circumstances the discharge does not travel along all the nerve fibres at the same time, so that the different muscle fibres composing the muscle will be in different phases of contraction, and there will be never any large degree of relaxation between the individual contractions of the whole muscle. Von Kries has found that the duration of a muscle twitch may be lengthened by increasing the duration of the electrical change used to excite the nerve, and has suggested that the normal excitatory process may resemble the prolonged electrical change which can be produced electro-magnetically, rather than the short sudden shock represented by the induced current of an induction-coil. Attempts have been made to decide the question by recording the electrical changes accompanying the natural contractions of a muscle, i.e. those excited reflexly from the central nervous system. It was long ago shown by Loven that a certain discontinuity could be seen in records of the electrical changes obtained from a frog's muscle in the tetanic spasms produced by an injection of strychnine, but according to Burdon Sanderson this discontinuity represents a series of spasms discharged from the central nervous system, fiach discharge produces, not a twitch, but a continued contraction of short duration. On photographing the electrical changes of strychnine spasm as obtained by a capillary electrometer, he found that each individual spasm could be compared only to a short tetanus. The most recent investigations of the question we owe to Piper, who made use of the string galvanometer, an instrument much more delicate in the reproduction of rapid changes than is. the capillary electrometer. Piper led off two points in the fore-arm, one electrode being placed about two inches below the bend of the elbow, and the other about four inches above the wrist. A single stimulus of the median nerve was found by him to give a typical diphasic variation in the muscles. When the muscles were contracted voluntarily, well-marked oscillations of the galvanometer wire were obtained, indicating the existence in the muscle of forty- eight to fifty complete diphasic variations in the second (Fig. 93). Piper obtained similar records on leading off other muscles of the body when these were placed voluntarily in a state of contraction, and he concludes therefore that each voluntary contraction, short or long, is a tetanus composed of about fifty fused twitches per second. These results would indicate that 16 I 242 PHYSIOLOGY the impulse, which normally travels down the motor nerve from the anterior cornual cell to the muscle, is discontinuous, and therefore that on leading off a motor nerve to a galvanometer we ought to obtain electrical oscillations of fifty distinct stimuli per second. Dittler has investigated by means of the string galvanometer the electrical changes accompanying the ordinary contractions of the diaphragm, and also those occurring in the phrenic FIG. 93. Electrical variations produced by voluntary contractions of human muscle. (PiPER.) nerve. He finds that both in the muscle and in the nerve there is evidence that each contraction is a fused series of single contractions, evoked by the discharge along the nerve of between fifty and seventy excitations per second. So far therefore the evidence is in favour of the view that volun- tary contraction and, one must add, the tonic contractions of all skeletal muscles, are discontinuous in nature and analogous to the tetanus which we may evoke artificially by rapid stimulation either of muscle or of its motor nerve. SECTION X OTHER FORMS OF CONTRACTILE TISSUE SMOOTH OR UNSTRIATED MUSCLE THE little we know about the physiology of unstriated muscle is derived chiefly from experiments on the intestine, ureter, bladder, and retractor penis.* This tissue differs from voluntary muscle in containing numerous plexuses of nerve fibres (non-medullated) and ganglion cells, so that in all our researches it is difficult to be certain whether the results are due to the muscle fibres themselves, or to the nerves and nerve cells which are so inti- mately connected with them ; especially as we have as yet no convenient drug like curare, by aid of which we might discriminate between action on muscle and action on nerve. The differences between unstriated and voluntary muscle, although at first sight very pronounced, on further investigation prove to be in most cases differences of degree only, qualities and reactions which are marked in involuntary muscle being also present in a minor degree in the more highly differentiated tissue. The contraction of smooth muscle is so sluggish that the various stages of latent period, shortening, and relaxation can be easily followed with the eye. The latent period may be from 0-2 to 0-8 second, and the contraction may last from three seconds to three minutes. Smooth muscle preserves many of the properties of undifferentiated protoplasm, especially an automatic power of contraction, which is regulated by the condition of the muscle. Thus whereas the voluntary muscle is intimately dependent on its connection with the central nervous system, and in the absence of this is reduced to a flabby inert tissue, the smooth muscle, isolated from all its nervous connections, presents in many cases rhythmic contractions, and can carry out a peripheral adaptation to its environment. These rhythmic contractions are almost invariably observed if the muscular tissue be subjected to a certain amount of tension, after * The retractor penis, which is found in the dog, cat, horse, hedgehog (but not in rabbit or man), is a thin band of longitudinally arranged unstriated muscle, which is inserted at the attachment of the prepuce, and is continued backwards in a sheath of Connective tissue to the bulb, where it divides into two slips, which pass on either side of the anus. It is innervated from two sources, the motor fibres being derived from the lumbar sympathetic and running to the muscle in the internal pudic nerve, while the inhibitory fibres run in the pelvic visceral nerves (nervi erigentes) and are derived from the second and third sacral nerve-roots. . 243 244 PHYSIOLOGY separation from the central nervous system. The rhythm of the contrac- tions may vary from one (spleen) to twelve (small intestine) contractions in the minute. The stimuli for smooth muscle are essentially the same as for striated. As we should expect however from the sluggish response of this kind of contractile tissue, the optimum rate of change of current which excites is very much slower than in the case of striated muscle. Thus in many in- stances a single induction shock, even if very strong, is powerless to excite contraction, and the make-induction shock of long duration and low intensity is always more efficacious than the short sharp break-induction current. A still better stimulus is the make or break of a constant current. When the latter form of stimulation is used, response occurs at the make sooner than at the break, and, just as in the voluntary muscle, the make excitation starts from the cathode and the break excitation from the anode. An apparent exception to this statement is afforded by the behaviour of certain forms of involuntary muscle. In the intestine, in the skin of worms, and in many other muscular tubes the smooth muscle-fibres are arranged in two different sheets, one consisting of longi- tudinal, the other of circular fibres. If non-polarisable electrodes, connected with a constant source of current, be applied to the surface of the small intestine, when the current is made there will be apparently a strong con- FIG. 94. At the cathode K there is a small line traction of the circular coat at the of constriction, surrounded by an area of relaxation. At the anode itself the muscle is relaxed, but is strongly contracted on each _, side of the anode, so that on rough observation the longitudinal coat at the cathode, it would be thought that contraction occurred The same result is observed in the at the anode itself. earthworm and leech. But careful observation shows in each case that the irregularity is really only apparent, and that in the immediate neighbourhood of the anode there is relaxation of both coats, with a contraction of the circular coat on each side, and that at the cathode there is a contraction of both coats. The accompanying diagram (Fig. 94) will serve to show the condition of the circular coat at each electrode. As a matter of fact, in con- sequence of the arrangement of the fibres, we have in the neigh- bourhood of the anode a num- ber of places (virtual cathodes) where the current is leaving the muscle-cells to enter inert con- ducting tissues, and in the same way there will be in the neigh- bourhood of the cathode a num- ber of virtual anodes (Fig. 95). anode, which spreads up and down the intestine, and a linear contraction of FIG. 95. Diagram to show the spread of current which occurs when a current is led through a tube such as the ureter by means of two electrodes applied to its surface. It will be noticed that while + E is the anode, there are immediately below and around it a number of cathodes, E,, E/y, E,,,, E///y due to the current leaving the muscle to flow tlirough indifferent tissues. (BIEDERMANN.) Thus if we take the ureter and lead a current through it while it is slung up in thread loops serving as electrodes, there is contraction of both coats at the cathode and relaxation of both at the anode. If however the ureter be packed in a pulp of blotting-paper OTHER FORMS OF CONTRACTILE TISSUE 245 moistened with normal saline, thus allowing the current to leave the contractile tissues anywhere along the ureter, we get the same aberrant results of stimulation as are obtained with the intestine. In .voluntary muscle, if one stimulus follows another at an interval which, is not too large, a summated contraction is produced which is greater in amplitude than that due to a single stimulus. This summation may be mechanical or physiological, the former being observed when the stimulus is repeated during the decline of the excitatory process and being due simply to the after-loading of a muscle by the first contraction. It is best marked when the muscle is heavily loaded. If however the stimuli be sent in at sufficiently short intervals so that two stimuli fall within the period of rise of contractile stress, an increased height of contraction is obtained under all conditions, and under isometric conditions the tension developed is greater than that with a single stimulus. If the interval between two stimuli be so short that the second falls within what we have called the refractory period due to the first stimulus, no summation is obtained, the second stimulus being ineffective. In the slow contraction of involuntary muscle we could hardly expect mechanical summation to come into play. Most types of this tissue show however the true summation, i.e. the increased liberation of energy due to repetition of the stimulus during the rise of the excitatory condition. As might be expected the refractory period is also longer in involuntary muscle, since all the processes of this muscle are slowed in comparison with those of voluntary muscle. In certain types of tissue, and especially in heart muscle, the refractory period lasts during the whole of the period of contraction. During this time therefore a second shock will be ineffective. As the contraction dies away the muscle fibre gradually recovers its sus- ceptibility to stimulation, but it does not recover its full irritability until it has entirely relaxed. On this account it is impossible to obtain summa- tion in or to tetanise heart muscle, the application of interrupted currents to this tissue producing only a series of rhythmic contractions. In all involuntary muscle we may observe summation of the effects of stimuli even when the individual stimuli are insufficient to produce any excitation. Thus in a muscle such as the retractor penis, we may find a strength of induction shock which, applied singly, is just insufficient to evoke any response. If however the shocks are repeated at intervals of a second, it will be found that the first three or four stimuli are ineffective and then the muscle enters into a contraction which increases with each succeeding stimulus until it has attained its maximum. There is thus summation before any contraction has occurred, a summation of stimuli. Each stimulus, in fact, alters the state of the contractile tissue and makes it more ready to respond to the next stimulus, so that the stimuli become more and more effective. If time is allowed for the muscle to relax between successive stimuli, this summation is evidenced by a continually increasing height of contraction, the so-called ' staircase.' The same initial increase of 246 PHYSIOLOGY effect is observed when voluntary muscle is excited by continually recurring stimuli (v. Fig. 70, p. 209). We shall meet with other examples of this summation of stimuli when dealing with the physiology of the central nervous system. It is indeed a fundamental phenomenon in the physiology of excitation. CHEMICAL STIMULATION. Strong salt solution excites contractions just as in the case of skeletal muscle. Many drugs, such as physostigmine, ergot, salts of lead and barium, digitalis, may act directly on smooth muscle and cause contraction. As one would expect however from the greater independence of the smooth muscle, the action of these drugs varies from organ to organ, muscle-fibres, which apparently are histologically identical, reacting diversely according to their origin. MECHANICAL STIMULATION. Smooth muscle may react to a local pinch or blow with a local or a general (propagated) contraction. The most important form of mechanical stimulation is that produced by tension. The effect of increasing the tension on smooth muscle may be twofold : causing in the first place relaxation and in the second excitation with in- creased contraction. These two effects may be illustrated by taking the case of the bladder. If this viscus (which is surrounded by a complete coat of smooth muscle) has all its connections with the central nervous system severed, it is when empty in a state of tonic contraction. If fluid be injected into it rapidly there is a great rise of pressure in its cavity, due to the forcible distension. If however the fluid be injected slowly, the bladder muscle relaxes to make room for it, so that a considerable amount of fluid may be accommodated in the bladder without any great rise of pressure. This process of relaxation has its limit. If the injection of fluid be continued, the walls begin to be stretched passively, and this increased tension acts as a stimulus causing marked rhythmic contractions of the whole bladder. In the same way the response of a smooth muscle to an electrical stimulus is much increased by previous increase of the tension on the muscle fibres. PROPAGATION OF THE EXCITATORY STATE, OR WAVE OF CONTRACTION. On stimulating any part of a voluntary muscle film-. a wave of contraction is started which travels to each end of the fibre, but no further. There is no propagation from muscle fibre to muscle fibre, the synchronous contraction of the whole muscle being brought about l»v simultaneous excitation of all its fibres. It is doubtful whether this isolation of the excitatory state is found in smooth muscle. As ;i rule a stimulus applied to any part of a sheet of smooth fibres may travel all over the sheet just as if it were a single fibre. It seems probable indeed that there is protoplasmic continuity by means of fine bridge-like processes between adjacent muscle cells. And even in the absence of such bridges the propaga- tion of the contraction could be easily accounted for. Although in the case of voluntary muscle the rule is isolated contraction, yet a very small change in the muscle, such as that produced by partial drying or by pressure, is sufficient to cause the contraction to spread from one fibre to another, OTHER FORMS OF CONTRACTILE TISSUE 247 Indeed by clamping two curarised sartorius muscles together, as in the diagram (Fig. 96), it is found that stimulation of the muscle A causes con- traction of the muscle B. The current of action of A in this case has served to excite a contraction in B. It must be remembered that in all unstriated muscle the fibres are sur- rounded by a network of non-medullated nerve fibres. Some physiologists are inclined to ascribe to these fibres an important part in the propagation of the contraction wave. In the case of the heart muscle however, it can be shown almost conclusively that the propagation takes place independently of nerve FIG. 96. fibres, and probably the same is true for many kinds of involuntary muscle. INFLUENCE OF TEMPERATURE. Smooth muscle is extremely sus- ceptible to changes of temperature ; as a rule warming causes relaxation, while 'application of cold causes a tonic contraction. The condition of the muscle at any given time depends not only on its actual temperature, but also on the rapidity with which this temperature has been reached. Thus a rapid cooling of the retractor penis muscle of a dog from 35° to 25° may cause a contraction as extensive as would be produced by a slow cooling to 5°C. On warming a muscle from 30° to 50°C. it lengthens gradually up to about 40°, and it may then undergo a marked heat contraction (varying in degree in different muscles) at about 50°C., which may pass off at a somewhat higher temperature. It is killed somewhere between 40° and 50°C. It seems very doubtful whether any true rigor mortis occurs in smooth muscle. The hard contracted appearance of the smooth muscle in a recently dead animal is chiefly conditioned by the fall of temperature. On excising the muscle and warming it up to body temperature it may again relax and show signs of irritability two or three days after the death of the animal. Different smooth muscles however vary very much in their tenacity of life. DOUBLE INNERVATION. Voluntary muscle is absolutely dependent for its activity on the central nervous system. Cut off from^this it is flabby and motion- less. Its sole function is to con- tract efficiently and smartly on re- ceipt of impulses arriving along its nerve. It is only necessary therefore FIG. 97. Tracing from the retractor penis muscle that these impulses should be of one of the dog, showing lengthening (inhibition) i -, , on stimulation of the nSrvus erigens, and a character — motor,andweknowthat smart contraction on stimulating the pudic each fibre of a muscle, such as the (motor) nerve. (Movements of muscle re- «. duced £. ) sartorius, receives one efferent nerve fibre terminating in an end-plate. In the case of smooth muscle we have a tissue which has an activity and reactive power of its own, and apart from its innervation may be at one time in a state of relaxation, at another in a state of tonic contraction, 248 PHYSIOLOGY In order that the central nervous system should have efficient control over such a tissue, it must be able to influence it in two directions : it must be able to induce a contraction or increase a contraction already present, and it must also be able to put an end to a spontaneous contraction, i.e. to induce relaxation. In order to carry out these two effects, smooth muscle receives nerve fibres of two kinds from the central nervous system, one kind motor, analogous to the motor nerves of skeletal muscle, the other kind inhibitory, causing relaxation or cessation of a previous contraction. All these fibres belong to the visceral or ' autonomic ' system. They are connected with ganglion-cells in their course outside the central nervous system, and their ultimate ramifications in the muscle are always non-medullated. A typical tracing of the opposite effects of these two sets of nerves is given in Fig. 97. In the invertebrata many ' voluntary ' striated muscles probably possess a double innervation. Thus in the crayfish the adductor muscle of the claw consists of striated muscular fibres, every fibre of which is supplied with two kinds of nerve fibres. By exciting these fibres one may get, according to the conditions of the experiment, either contraction of a relaxed muscle or relaxation of a tonically con- j f J t t j 4 _ t tracted muscle (Fig. 98). FIG 98 Tracing of contraction AMCEBOID MOVEMENT of adductor muscle of claw of crayfish, showing inhibition re- Amoeboid movement is seen in the uni- sulting from stimulation of its ,, , . , ,, -, nerve (at 6) by means of a con- cellular organisms such as the amoeba and stant current. The break of the m the white blood corpuscles. It Can OCCUr current^ causes a second smaller , .., . , . ,. ., ,. . inhibition. (BIEDERMANN.) only within certain limits of temperature (about 0°C. to 40°) ; within these limits it is the more active the higher the temperature. At about 45° the cell goes into a condition resembling heat rigor. The fluid in which the corpuscles are suspended is of great importance. Distilled water, almost all salts, acids and alkalies, if strong enough, stop the action and kill the cell. The movements are also stopped by C02 or by absence of oxygen. Artificial excitation, whether electrical, chemical, or thermal, causes universal contraction of the corpuscle, which therefore assumes the spherical form. CILIARY MOVEMENT Cilia are met with in man in nearly the whole of the respiratory passages and the cavities opening into them, in the generative organs, in the uterus and Fallopian tubes of the female, and the epididymis of the male, and on the ependyma of the central canal of the spinal cord and its continuation into the cerebral ventricles. The cilia (Fig. 99) are delicate tapering filaments which project from the hyaline border of the epithelial cells. There are about twenty or thirty to each cell. The hyaline border is really made up of the enlarged basal portions of the cilia. OTHER FORMS OF CONTRACTILE TISSUE 249 in be su: In action the cilia bend suddenly down into a hook or sickle form, and then return slowly to the erect position. This movement is repeated many (twelve to twenty) times a second, and thus serves to move forward mucus, dust, or an ovum, as the case may be. The movement seems to be entirely automatic, and it is quite unaffected by nerves, at any rate in all the higher animals. There seems to be a functional connection tween all the cells of a ciliated epithelial urf ace, so that movement of the cilia, started in one cell, spreads forward as a wave, just as, when the wind blows, waves of bending pass over a field of corn. The conditions of ciliary action are the same FIG. 99. Ciliated columnar epithelium from the trachea of a rabbit ; m\ m2, cells. as those for amo3boid movement of naked cells. The minuteness of the object has up to now prevented us from deciding whether the cilium is itself actively contractile, or whether it is simply passively moved by the action of the basal part situated in the hyaline border of the cell. m3, mucus-secreting (SCHAFER.) CHAPTER VI NERVE FIBRES (CONDUCTING TISSUES) SECTION I THE STRUCTURE OF NERVE FIBRES ON stimulating the nerve of a nerve-muscle preparation at any part by electrical, thermal, or mechanical means, the stimulus is followed, after a very short interval, by a contraction of the muscle. This observation illus- trates the two functions of nerve fibres, irritability and conductivity — that is to say, a suitable stimulus can set up changes in any part of the nerve, which are trans- mitted down the nerve without any visible effects occurring in it, and it is not until this nervous change has reached the muscle that a visible effect takes place in the shape of a contraction. In the animal body a direct excitation of the nerve fibre in its course never takes place under normal circumstances. The only function the nerve fibre has to perform is that of conducting impulses from the sense organs at - the periphery to the central nervous system, and efferent impulses from this to the muscles and other of its servants. Hence it is absolutely es- sential that there should be vital continuity along the whole length of the fibre. Dam- age to any part, such as by crushing, heat, or any other injurious condition, infallibly causes a block to the passage of JIM impulse. FIG. 100. Diagram of a motor nerve- cell with its nerve-fibre. (After hillock ; rf, dendrites ; a.x. axis cylinder; m, medullary sheath ; n.R. node of Ranvirr. A nerve fibre is essentially a long process or arm of a nerve-cell (Fig. 100). The cell may either be situated on the surface of the body or, ag in mogfc cagcg in tjie m-gher animals, may be *ithd»WH ^om the surface into a special collection of cells such as the posterior root ganglion, or may be one of the mass of cells and 250 THE STRUCTURE OF NERVE FIBRES 251 interlacing processes making up a central nervous system. All nerves are alike in possess- ing as their conducting part the continuous strand of protoplasm produced from the nerve-cell and known as the axon or axis cylinder. By special methods the axon may be shown to be made up of fibrillse or neuro -fibrils, embedded in a more fluid material (Fig. 101 ). These neuro-fibrils are supposed to be continuous throughout the cell and the axis FIG. 101. Medullated nerve fibres, showing continuity of the neuro-fibrils across the node of Ranvier. (BETHE.) a, longitudinal ; &, transverse section. cylinder and to represent the essential conducting constituents of the nerve. In the course of growth the nerves develop certain histological differences, which appear to bear some relation to the nature of the processes they conduct or to the character of their parent cell. Thus all the fibres which are given off from and which enter the central nervous system, i.e. the brain and spinal cord, belong to the class known as medullated. In this type the conducting core or axis cylinder is surrounded with a layer of apparently insulating material known as myelin, forming the medullary sheath, or the sheath of Schwann. This sheath consists of a fatty material composed largely of lecithin, and staining black with osmic acid, supported in the interstices of a network formed of a horny substance known as neurokeratin. The medullary sheath is sur- rounded by a structureless membrane, the primitive sheath or neurilemma. At regular intervals a break occurs in the medullary sheath, the neurilemma coming in close contact with the axis cylinder. This break is the node of Ranvier, the intervening portions .of medullated nerve being the internodes. In each internode, lying closely under the neurilemma, is an oval nucleus embedded in a little granular protoplasm. The medullated nerve fibres vary considerably in diameter, the largest fibres being distributed to the muscles and skin, the smallest carrying impulses from the central nervous system to the viscera. The latter all come to an end in some collection of ganglion-cells of the sympathetic chain or peripheral ganglia, the impulses being carried on to their destination by a fresh relay of non-medullated nerve fibres. 252 PHYSIOLOGY The non-medullated fibres (Fig. 102) differ from the medullated simply in the absence of a medullary sheath. They possess, in many cases at any rate, a primitive sheath, under which we find nuclei lying closely on the side of the fibre and bulging out the sheath. In their ultimate ramifications they tend to form close networks or plexuses and appear to lose the last traces of a sheath. The medullated nerves are bound together by connective tissue (endoneurium) into small bundles, which ?re again united by tougher connective tissue into larger nerve-trunks. These fibres as a rule branch only when in close proximity to their destination, and then the branching always occurs at a node of Ranvier. Fro. 102. Non-medullated nerve fibres. (ScnlFEB.) As to the functions of the myelin sheath in the medullated nerve fibre very little is known. It does not make its appearance until the axis cylinder is formed, and is apparently derived from a series of cells which grow out from the spongioblasts of the central nervous system and form a chain surrounding the out-growing axons. In the regeneration of a nerve fibre after section the myelin sheath appears later than the axon in the peripheral part of the nerve. It has been supposed by some to act as a sort of insulator ensuring isolated conduction within any given nerve fibre. We have how- ever no proof that equally isolated conduction is not possible in the non-medullated fibres of the visceral system, although it is certainly true that a finer ordering of move- ments is required in the skeletal muscles than in the visceral unstriated muscles. More- over in the central nervous system the main tracts cannot be shown to be functional before the date a't which they acquire their medullary sheaths, suggesting that pre- viously any impulse making its way along the tract underwent dissipation before arriving at its destination. It is possible too that the myelin sheath may serve as a source of nutrition to the enclosed axis cylinder which, in the greater part of its course, is far removed from its trophic centre, namely the cell of which it is an outgrowth. This trophic function of the myelin sheath has a certain basis of fact in that the myelin sheath is as a rule larger in those fibres which take the longer course. SECTION II PROPAGATION ALONG NERVE FIBRES * r THE velocity of propagation along a nerve fibre may be measured, although in early times it was thought to be as instantaneous as the lightning flash. To measure the velocity of propagation in a motor nerve, a frog's .gastroc- nemius is prepared, with a long piece of sciatic nerve attached. The muscle is arranged (Fig. 103) so that its contraction may be recorded on a rapidly moving surface, on which are also recorded, by means of electro-magnetic = 103. Diagram of arrangement of experiment for the determination of the velocity of transmission of a motor impulse down a nerve. The battery current passes through the primary coil of the inductorium c, and a ' kick over ' key k. By means of the switch #, the break shock in the secondary circuit can be sent through the nerve n, either at b or at a. The muscle m is arranged to write on the blackened surface of a trigger or pendulum myograph, and is excited during the passage of the recording surf ace by the automatic opening of the key k. (The time-marker is not shown.) ignals, the moment at which the stimulus is sent into the nerve, and also a time-marking showing -%±-Q sec. Tracings are now taken of the contraction of the muscle : first, when the nerve is stimulated at its extreme upper end ; secondly, as close as possible to the muscle. It will be found that the latent period, which elapses between the point at which the stimulus is sent into the nerve and the point at which the lever begins to rise, is rather longer in the t case than in the second. The difference in the two latent periods gives the time that the nervous impulse has taken to travel down the length of nerve between the two stimulated points. Calculated in this way, the elocity of propagation in frog's nerve is about 28 metres per second. In man and in warm-blooded animals the velocity has been variously timated at from 60 to 120 metres per second. The higher of these figures probably nearer the truth. 253 254 PHYSIOLOGY On the other hand, in in vertebra ta the velocity of propagation along nerve fibres may be quite slow. The following Table represents the velocity of transmission along a number of different fibres, as determined by Carlson, compared with the duration of a single muscle twitch in the same animal. Muscle Nerve Species Contrac- Rate of Muscle tion Nerve the impulse i i mo in in metres seconds per second Frog Gastrocnemius 0-10 Sciatic 27-00 • (medullated) Snake . Hyoglossus 0-15 Hyoglossal 14-004 (medullated) Lobster . Adductor of 0-25 Ambulacral 12-00 (Homarus) forceps (non -medullated ) Hagfish . Retractor of jaw . 0-18 Mandibular 4-50 (non-medullated) Limulus . Adductor of 1-00 Ambulacral 3-25 forceps (non-medullated) Octopus . Mantle 0-50 Pallial 2-00 (non-medullated) Slug (Limax) . Foot 4-00 Pedal 1-25 (non-medullated) Limulus . Heart 2.25 Nerve plexus in heart 0.40 (non -medullated ) The velocity of propagation in sensory nerves is more difficult to deter- mine owing to the fact that a sensory impulse, on arrival at the receiving organ — i.e. some part of the central nervous system — does not at once give rise to some definite recordable mechanical change, such as a muscular con- traction. There is another method of determining the velocity of conduction, which may be used also with sensory fibres. The passage of a nerve- impulse down a nerve, just as the passage of a wave of contraction along a muscle-fibre, is immediately preceded or accompanied by an electrical change, which also travels along the nerve as a wave of ' negativity.' The velocity of propagation of this wave may be measured, and is found to give the sain** numbers as the velocity determined by the preceding method. The existence of this electrical change enables us to show that a nerve- impulse, excited at any point in .the course of a nerve fibre, travels in both directions along the fibre. The power of nerves to transmit impulses in either direction is shown further by the experiment known as Kiihne's gnu -ills experiment. The gracilis muscle of the frog is separated into two portions by a tendinous intersection, so that there is no muscular continuity between the two halves. The nerve to the muscle divides into two branches, one to each half, and at the point of junction there is division of the axis cylinder* themselves. If the section a in the diagram (Fig. 104), which is quite isolated from the rest of the muscle, be stimulated, as by snipping it with scissors, PKOPAGATION ALONG NERVE FIBRES 255 the whole muscle contracts. If the portion of the muscle which is free from nerve fibres be stimulated in the same way, the contraction is limited to the fibres directly stimulated, showing that in the first case the stimulus excited nerve fibres which transmitted the impulse up the nerve to the point of division and then doivn again to the other half of the muscle. Since nerves have this power of conduction in both directions, it might be thought that a single set of nerve fibres might very well subserve both afferent and efferent functions, at one time conducting sensory impulses from periphery to cord, at another time motor impulses from cord to muscles. But this is not the case. As a matter of fact we find in the body a marked differentiation of function between various nerve fibres. Thus Bell and Majendie showed that the spinal roots might be divided into afferent and efferent, the anterior roots carrying only impulses from spinal cord to periphery, while the posterior roots carried impulses from periphery to central nervous system. The law known by the name of these observers states indeed that a nerve fibre cannot be both motor and sensory. We may find both kinds of fibres joined together into a single nerve- trunk, but the fibres in each case are isolated and conduct impulses only in one or other direction. Under normal conditions the afferent fibres are excited only at their endings on the surface of the body, while the efferent fibres are excited only at their origin from the spinal cord. The difference in the function of different nerve fibres depends there- fore not so much on the structure of the nerve fibre itself as on the connections of the fibre. We can show this experimentally by graft- ing one set of nerve fibres on to another. If the cervical sympathetic be united to the lingual nerve, stimulation of the sympathetic, instead of causing, as usual, constriction of the* vessels of the head and neck, will cause dilatation of the vessels of the tongue and secretion of watery saliva. In the same way the finer functional differences between the various forms of sensory nerves seem to be determined by their connections within the central nervous system. Stimulation of the optic nerve by any means whatsoever evokes a sensation of light. One and the same stimulus applied to different nerves will evoke different sensations, e.g. a tuning-fork applied to the skin will give a sensation of vibration, to the ear a sensation of sound. We shall have occasion to return to this question of the restricted function of nerve fibres when we deal with Miiller's ' law of specific irritability ' in the chapter on Sensations. FIG. 104. Kiihne's gracilis experiment. SECTION III EVENTS ACCOMPANYING THE PASSAGE OF A NERVOUS IMPULSE IN muscle we saw that the passage of an excitatory wave was accompanied or followed by electrical changes, production of heat, and mechanical change, all pointing to an evolution of energy from the explosive breaking-down of contractile material. In nerve however which serves merely as a conducting medium, we should not expect so much expenditure of energy, or in fact any expenditure at all. All that is necessary is that each section of the nerve should transmit to the next section just so much kinetic energy as it has received from the section above it. And experiment bears out this conclusion. The most refined methods have failed to detect the slightest development of heat in a nerve during the passage of an excitatory process, and we know already that there is no mechanical change in the nerve. The only physical change in a nerve under these circumstances is the development of a current of action. A nerve becomes, when excited at any point, negative at this point to all other parts of the nerve and, just as in muscle, this ' negativity ' is propagated in the form of a wave in both directions along the nerve. That the excitatory process in nerves is probably accompanied by certain small chemical changes is indicated by the facts that, in the complete absence of oxygen, the nerve fibres lose their irritability, and that this loss of irritability is hastened by repeated stimulation of the nerve. When the irritability has been abolished by stimulation in the absence of oxygen, it may be restored within a few minutes by readmission of oxygen to the nerve. If we connect a galvanometer to two points of an uninjured nerve, no current is observed, all points of a living nerve at rest being isoelectric. On making a cross-section of the nerve at one leading-of? point, a current is at once set up, which passes from the surface through the galvanometer to the cross-section. This is a demarcation current, set up at the junction between living and dying nerve. This current rapidly diminishes in strength and finally disappears, owing partly to the fact that the dying process started in the nerve by the section extends only as far as the next node of Ranvier and there ceases, so that after a short time the electrode applied to the cross- section is simply leading off an intact living axis cylinder through the dead portion of the nerve, which acts as an ordinary moist conductor. On making a fresh section just above the previous one, the process of dying is again set 256 EVENTS ACCOMPANYING A NERVOUS IMPULSE 257 up, and the demarcation current is restored to its original strength. If, while the demarcation current is at its height, we stimulate the other end of the nerve with an interrupted current, the needle of the galvanometer swings back towards zero, i.e. there is a negative variation of the resting current. In order to demonstrate the wave-like progression of the electrical change from the excited spot along the nerve, it is necessary, as in the case of muscle, to make use of a very sensitive capillary electrometer or a string galvano- meter. It is then found that the change progresses along the nerve at the same rate as the nervous impulse, i.e. 28 to 33 metres per second in the frog. But it lasts only an extremely short interval of time at each spot, viz. six to eight ten- thousandths of a second. Thus the length of the excitatory wave in nerve is about 18 mm. 17 SECTION IV CONDITIONS AFFECTING THE PASSAGE OF A NERVOUS IMPULSE TEMPERATURE. Below a certain temperature the propagation of the excitatory process in the nerve is absolutely abolished. The exact tempera- ture at which this occurs varies according as we use a warm- or a cold-blooded i • animal. In the frog it is necessary to cool the nerve below 0°C. before conduction is abolished, whereas in the mammal it is sufficient to cool the nerve to somewhere between 0° and 5°C. Since cooling the Q nerve does not excite it, this procedure forms a convenient method for blocking the passage of impulses along a nerve without using the irritating procedure of section. On warming the nerve again the conductivity returns. The rapidity with which the excita- tory process is propagated along either a nerve or a muscle fibre depends on the temperature. Thus the mean rate of conduction in the frog's nerve at 8° to 9°G. is about 16 metres per second. The temperature coefficient of the velocity of nerve propagation, i.e. velocity at Tn + 10 , — — -, — — has been round by Lucas velocity at Tn to be about 1-79. The same value WMS found by Maxwell for conduction in molluscan nerve, and in frog's striated muscle Wool Icy found the temperature coefficient for con- duction of the excitatory process to vary between 1-8 and 2. An ingenious method (Fig. 105) has been used by Keith Lucas for the determination of the conduction rates in nerve at different temperatures. The glass vessel repre- sented in the figure is filled with Ringer's solution, in which the whole nerve-muscle preparation is immersed. The muscle used was the flexor longus digitorum, so that the whole length of the sciatic, tibial, and sural nerves could be used. The nerve is passed up through the constrictions in the inner glass vessels at c and D, and is attached to the thread E. F, I, and a are three non-polarisable electrodes composed of porous 258 CONDITIONS AFFECTING A NERVOUS IMPULSE 259 FIG. 106. clay, containing saturated zinc sulphate, in which a zinc rod is immersed. If the current is passed in at G and out at r the effective cathode is at the lower end of the constriction c, and similarly if the current is passed in at I and out at G, the effective cathode is at D. The tendon of the muscle A is attached by a thin glass rod H to a very light recording lever, the movement of which is magnified by placing it in the focal plane of a projecting eye-piece and recording its image on a moving sensitive plate. The whole apparatus, with the exception of the glass rod at H, can be immersed in a water bath at any given tempera- ture. Two records are taken with the whole apparatus, first stimulating at c, and secondly stimulating at D. The difference between the latent periods in these two cases is the time taken for the excitatory wave to travel from D to c. The rate of propagation is similarly recorded when the water bath is raised to 18°C. or to any desired tempera- ture. Since we are only dealing with differences in latent periods the effect of the rise of temperature on the latent period of the muscle itself does not affect the determinations. THE INFLUENCE OF FATIGUE. In the description of the phenomena of mus- cular fatigue* given in the last chapter, it was assumed that the muscle was being excited directly. The same phenomena are observed when the muscle is excited through its nerve, though in this case fatigue comes on much more quickly. If, after the muscle has been excited in this way until exhausted, it be excited directly, it will respond with a contraction nearly as high as at the beginning of the experiment. We see therefore that the nervous structures are more susceptible to the influences causing fatigue than the muscle itself, and it can be shown that the weak point in the nerve- muscle preparation is not the nerve, but the end-plates. In fact it is not possible to demonstrate any phenomena of fatigue in the nerve-trunk.* This fact can be shown in mammals by poisoning the animal with curare, and then stimulating a motor nerve continuously while the animal is kept alive by means of artificial respiration. As the effect of the curare on the end- plates begins to wear off in consequence of its excretion, the muscles supplied by the stimulated nerve enter into tetanus. The action of the curare may be cut short at any time by the injection of salicylate of physostigmine, when the muscles will at once begin to react to the excitation. The same fact may be shown on the excised nerve-muscle preparation of the frog. The gastrocnemii of the two sides with the sciatic nerves are dissected out, and an exciting circuit is so arranged that the interrupted * Unless it be asphyxiated by total deprivation of oxygen. Curve of muscle-twitch obtained by foregoing method. (KEITH LUCAS.) A = moment of excitation. B = movement of muscle, c = time-marker. 260 PHYSIOLOGY secondary currents pass through the upper ends of both nerves in series (Fig. 107). At the same time a constant cell is connected with two non-polarisable electrodes (np, np) placed on the nerve of B, so that a current runs in the nerve in an ascending direction. The effect of passing a constant current through a nerve is to block the passage of impulses through the part traversed by the current. When the con- stant polarising current is made, the muscle may give a single twitch, and then remains quies- cent. The exciting current is then sent through both nerves by the electrodes e^ and e2. The muscle A enters into tetanus, which gradually subsides owing to ' fatigue.' When A no longer responds to the stimulation, the constant current through the nerve of B is broken. B at once enters into tetanus, which lasts as long as the contraction did in the case of A, and gradually sub- Fio. 107. Arrangement of experiment fordemon- • -i r,- RITIPP strating the absence of fatigue in medullated SldeS aS latl8U( nerve fibres. both nerves have been excited EC, exciting circuit ; CP, polarising circuit. throughout, it is evident that the fatigue does not affect the nerve-trunk. We have already seen that a muscle will respond to direct stimulation when stimulation of its nerve is without effect, and must therefore conclude that the first seat of fatigue is the junction of nerve and muscle, i.e. the end-plates. In the normal intact animal the break in the neuro-muscular chain which is the expression of fatigue occurs still higher up, i.e. in the central nervous system, and is probably due to some reflex inhibition of the central motor apparatus from the muscle itself. Thus after complete fatigue has been produced in a muscle so far as regards voluntary efforts, direct stimulation of the muscle itself or its nerve will produce a contrac- tion as great as would have been the case at the beginning of the experiment. THE INFLUENCE OF DRUGS. The most important drugs with an influence on nerve fibres are those belonging to the class of anaesthetics. Of these we may mention carbon dioxide, ether, chloroform, and alcohol. The action of any of these substances on the excitability and conductivity of a nerve be studied by means of the simple apparatus, represented, in Fig, 108, The nerve CONDITIONS AFFECTING A NERVOUS IMPULSE 26l of a nerve-muscle preparation is passed through a glass tube which is made air-tight by plugs of normal saline clay surrounding the nerve at the two ends of the tube. By means of two lateral tubulures a current of CO2, or air charged with vapour of ether or other narcotic, can be passed through the tube. The nerve is armed with two pairs of elec- trodes which are stimulated alternately, the pair within the tube serving to test the action of the drug on the excitability, while the pair outside the tube show the presence or absence of any effect on the conducting power of the nerve below it. Of the gases and vapours mentioned above, C02 and ether both diminish and finally abolish the excitability and conductivity of the nerve fibres. The conductivity however persists after all trace of excitability has dis- appeared, before in its turn being also abolished. On removing the gas FIG. 109. Tracing to show the effect of ether on excitability and conductivity of nerve. Nerve excited by single induction shocks alternately within and above ether chamber. The vertical lines indicate contractions of the muscle (gastrocnemius. ) The lower line indicates the periods during which the nerve was exposed to the action of ether. A, disappearance of excitability, B, reappearance of excitability, c, disappearance of conductivity ; D, reappearance of conductivity. (From a tracing kindly lent by PROF. GOTCH.) or vapour by blowing air over the nerve, the conductivity and excitability gradually return in the reverse order to their disappearance (Fig. 109). Alcohol is said to increase the excitability or leave it unaffected, while diminishing the conductivity of the nerve. Chloroform rapidly abolishes both excitability and conductivity. It is a much more severe poison than the drugs just mentioned, so that in many cases its effects are permanent, and no, or only a very partial, recovery of the nerve is obtained on removal of the chloroform vapour from the apparatus. SECTION V THE EXCITATION OF NERVE FIBRES MANY different forms of stimuli may be used to arouse the activity of an excitable tissue such as muscle or nerve. Thus we may use thermal, mechani- cal, or chemical stimuli. If the temperature of a motor nerve be gradually raised, no effect is noticed till about 40°C. is reached, when the muscle may enter into weak quivering contractions. Sudden warming of the nerve always gives rise to excitation. At about 45°G. the nerve loses its irritability and dies. On the other hand, a nerve may be rapidly cooled without any excitation taking place. A nerve may be excited mechanically by crushing or cutting. These methods destroy the nerve. It is possible to excite a nerve mechanically, without any serious injury to it, by carefully graduated taps, and this method has been used in investigating the phenomena of electrotonus. All chemical stimuli applied to the nerve have a speedy effect in destroying its irritability. The chemical stimuli most used are strong salt solutions, glycerin, or weak acids. If any one of these be applied to a motor nerve, the muscle enters into an irregular tetanus, which lasts till the irritability of the nerve is destroyed at the part stimulated. None of these forms of stimuli can be adequately controlled either as to strength or duration. Moreover, owing to their destructive effects, any repetition of the stimulus will fall on a nerve or muscle more or less altered by the first stimulus. We are therefore justified in the use of electrical stimuli not only for arousing the activity of excitable tissues, but also for determining the conditions of excitation of muscle and nerve. For this pur- pose we may use either the make and break of a constant current, the ind IK cl short duration produced in a secondary coil of an inductorium by the make or break of the primary circuit, or the discharge of a condenser. Tin- last-named method of stimulation is especially useful when we desire to deter- mine, tin- total amount of energy involved in the electrical stimulation of a nerve or muscle. The arrangement of such an experiment is shown in Fig. 110. By means of tin- switch S the condenser can be put into connection either with the battery from "•ni<-h it r. charge or with tin- nerve through which it can discharge. By lsn..uiiiL' th«- capacity of the condenser and the electromotive force by which it is ''• we -.in estimate tin- energy «>f tin* charge sent through the nerve. K (energy in er^s)* fiKV- (F capacity in ini.-rofarads ; V electromotive force in volts). * An erg is the amount of \\ork produced or energy expended by the action of one ,/////, thiuiiLrh on,- centimetre. A dyne is the force which will give to a mass of one gram an acceleration of one centimetre per second. 262 THE EXCITATION OF NERVE FIBRES 263 In this way it has been found that the energy of a minimal effective stimulus for frog's nerve is about 1TrVo of an erg- The amount of energy necessary to excite the nerve will vary with the rate at which the condenser is allowed to discharge through the nerve. Its rate can be modified by altering the resistance in the discharging circuit or by altering the electromotive force of the charge. This method has been adopted by Waller in determining the rate of change at which excitation is obtained with a minimal ex- penditure of energy, which he calls the "characteristic" of the tissue in question. To this point we shall have occasion to refer later. 8 FIG. 110. Arrangement of apparatus for the excitation of a nerve by means of condenser discharges. B, battery ; R, rheochord ; c, rider of rheochord ; s, switch (Pohl's re- verser without cross wires) ; c, con- denser ; n, nerve ; m, muscle ; e, non- polarisable electrodes. When using the make and break of a constant current as a stimulus, the first fact of importance is the relation of the seat of excitation to the poles by which the current is led into or out of the ex- citable tissue. We have already seen that when a current is passed through a muscle or nerve the muscle contracts only at make or at break of the current, no propagated excitatory effect being produced during the passage of the current. The excitation at make is obtained with a smaller current than the excitation at break. Besides this difference in intensity, there is a difference in the point from which excitation starts. A make contraction starts from the cathode, a break contraction from the anode. This is well shown by the two following experiments : (a) A curarised sartorius muscle of the frog (Fig. Ill), with its bony insertions still attached, is fastened at the two ends to two electrodes, which are able to swing when the muscle contracts, and are attached by threads to levers which serve to record the contraction. The middle of the muscle is then fixed by clamping it lightly. A circuit is arranged so that a con- stant current can be sent through the electrodes and the whole length of the muscle. It is found, on making the .current, that the lever attached to the cathode — that is, to the elec- trode by which the current leaves the muscle — rises before the other lever. On the other hand, on breaking the current, the lever at the anode rises first, showing that the anodic half of the muscle contracts before the cathodic half. (b) The irritability of a muscle, i.e. its power of responding to a stimulus by contracting, is intimately dependent on the life of the muscle. If the muscle be injured or killed at any spot, its irritability at this spot will be therefore diminished or destroyed. Hence, if we stimulate a muscle at the injured spot, no contraction will ensue. This fact may be used to demon- FIG. 111. Sartorius clamped in middle and attached to levers at either end. 264 PHYSIOLOGY strate the production of excitation at cathode on make, and at anode on break of a constant current. A muscle with parallel fibres, such as the sartorius, is injured at one end, and a constant current passed, first from the injured to the uninjured end, and then in the reverse direction (Fig. 112). It is found in the former case, when the anode is on the injured part (which is therefore less excitable), that break of the current is ineffec- tive, and in the latter, when the contraction at make. cathode is on tne injured surface, that the make stimulus is ineffec- tive, showing that the part excited kath°d8 ^lnJured^ corresponds to the cathode at make T"= J no contraction at make. and to the anode at break *Xv^|^-v'V With a current of very short b duration no excitation is produced Fio. 112. Diagram to show the effect of local, f IT EVPTV induction shork injury on the excitability of a muscle. &, at break' -KjV6r3r battery ; m. muscle. The arrows indicate can be therefore regarded as a the direction of the current. make stimulus, and when such a shock is led through a muscle the contraction in each case will start at the cathode, i.e. the point at which the induction shock leaves the muscle. The results of stimulating nerve fibres are similar to those obtained by stimulating muscle fibres directly. Under normal circumstances, if a constant current be passed through the nerve of a nerve-muscle preparation for a short time, the muscle responds only at the make and the break of the current, remaining perfectly quiescent all the time the current is passing. If the nerve be in a very excitable condition, it is possible that the muscle may be thrown into a tetanus or continued contraction during the whole time that the current is passing (' closing tetanus '). On the other hand, if a strong ascending current be passed through the nerve for a considerable time, the muscle when the current is broken may go into continued contraction, which may last some time. Normally the muscle simply responds with a single twitch at the make and break of the current, although, on investigating the condition of the nerve during the passage of the current, we find that it is considerably modified. This modification in the condition of the nerve is spoken of as electrotonus^nd includes change* in its irritability and its electrical condition. To investigate these changes the following apparatus is necessary : two constant batteries, induction-coil, a reverser and keys, a pair of non-polarisable electrodes, and a pair of ordinary platinum electrodes. Fig. 113 represents roughly the arrange- ment of the experiment. A constant current from the battery is led through a part of the nerve by means of non-polarisable electrodes, which are about one inch apart. In this circuit we put a reverser, by means of which the direction of the current of the nerve may be changed at will, and a key to make or break the current. This is the polarising circuit. The other battery is arranged in the primary circuit of the coil, a key being interposed, so that we may use make or break induction shocks, which are applied to the nerve by means of the small platinum electrodes. The tendon of the muscle is connected by a thread with a lever, which is arranged to write on a smoked surface, so that the height of the contraction can be recorded. THE EXCITATION OF NERVE FIBRES 265 We first find the position of the secondary coilj at which the break induction shock is a submaximal stimulus, and we employ this strength of stimulus throughout the experiment. The make induction shock is prevented from acting on the nerve by closing a shortcircuiting key in the circuit of the secondary coil. The nerve is now stimulated at various points with a single break induction shock, and the contractions recorded. The heights of these contractions serve to indicate the irritability of the nerve at the point stimulated. We now throw the polarising current into the nerve. At the make of this current the muscle will prob- ably respond with a twitch which is not recorded. We then test once more the irritability of different points of the nerve, and we find that, when the stimulus is applied near a, the point where the current enters the nerve (anode), the stimulus, which before gave a moderately large con- traction of the muscle, now has either no effect or else produces a very weak contraction. On the other hand, in the region of the cathode the stimulus, which before was submaximal, has now become maximal, as is shown by the increase in the height of the con- FlQ- 113« Arrangement of apparatus for showing traction evoked by the induction electronic changes in irritability, shock. e, exciting current ; p, polarising current ; We now reverse the direction of the *, Pohl's reverser. polarising current, so that the current of the nerve runs from k to a. With this reversal of current there is also a reversal of the changes in the nerve ; that is to say, the normally submaximal stimulus is maximal when applied near a, and minimal when applied near k. On break of the polarising current the condition of the nerve returns to normal, and the submaximal stimulus is once more submaximal throughout. This return to normal conditions, is not immediate, since the first effect of breaking the current is a swing-back, so to speak, past the normal, the diminished irritability at the anode giving place to an increased irritability, which only gradually subsides. In the same way, immediately after the polarising current has ceased to flow, the neighbourhood of the cathode acquires a condition of diminished irritability, and this only gradually gives place to a normal condition. This experiment teaches us that, when a constant current is passed through a nerve, there is increase in the irritability in the nerve near the cathode, and a diminution in irritability near the anode. These conditions of increased and diminished irritability are spoken of as catelectrotonus and anelectrotonus respectively. In muscle we have seen that a make contraction always starts from the cathode, and a break contraction from the anode. Now the event that takes place at the cathode on make and at the anode on break of a constant current is, as the last experiment shows us, a rise in irritability, in the former case from normal to above normal, in the latter from subnormal to normal. Hence we may say that the excitation is caused by a sudden rise of irritability, which may be due either to a sudden appear- ance of catelectrotonus or a sudden disappearance of anelectrotonus. I have said sudden because the steepness of the rise of irritability is a necessary factor in causing excitation. If the polarising current passing through PHYSIOLOGY a nerve be slowly and gradually increased to considerable strength, it will give rise to no contraction. The degree of suddenness of the rise, which is most beneficial in causing contraction, varies with the nature of the tissue stimulated. Thus it is more rapid in nerve than in muscle, ajid in pale muscle than in red muscle, and in voluntary muscle than in unstriated muscle. It is evident that there must be, somewhere between the anode and cathode, an indifferent point — that is to say, a region where the irritability I i.:. 111. Diagram to show the variations of irritability in a nerve during the passage of polarising currents of different strengths. The degree of change is represented by the distance of the curves from the base line ; the part of the curve below the line signifying decrease, that above the line increase of irritability. A, anode ; B, cathode ; y{ , effect of weak current ; y2, medium current ; ?/3. strong current. It will be noticed that the indifferent point, x, where the curve crosses the horizontal line, approaches nearer and nearer the cathode as the current is increased in strength. (From FOSTER, after PFLUGKR.) is neither increased nor diminished. We find experimentally that this in- different point is nearer the anode when the polarising current is weak, and approaches the cathode as the current is strengthened, so that with very strong currents nearly the whole intrapolar length is in a condition of anelectrotonus (Fig. 114). When a strong polarising current is used, the depression of irritability at,the anode is so marked that no impulse can pass ascendinq current make excitation blocked kath! anT^"^^" at anode. break excitation at anode blocked at kathode. an. kathT"-^'^ Fio. 115. Diagram to show the blocking effect of a strong constant current passed through the nerve of a nerve-muscle preparation. this region. Thus if we send a very strong ascending current through tho nerve, there is no contraction at make. This is owing to the fact that the impulse started at the cathode on make of the current cannot reach the fa pa-suu,. ,|,,WM th<> nrm- boinjr blocked in the region of tin* anode H.\ A). Tin- result- of stiimilatiiiL' nmlor nerves h\ means of constant currents were studied I iy IMliiL'.-r ami. .•ml.o.li.-d in a TaUe, make up uliat is knmui as PlIuL'.-r's law. The i. .nit ni' iv> with a certain number of nerve cells at the nodes of the netxvork. From this network fibres pass more deeply to end in a finer net- work situated among a layer ot muscle fibres formed, like the sensory cells, by a differentiation of the primitive epithelium or epiblast (Fig. 134). Besides Fio. 133. Diagrammatic view of a jelly-fish. (HEBTWIO.) U, umbrella ; M, manubrium ; TL, T2, tentacles ; v, velum ; N, nerve ring ; R. ' marginal body.' EVOLUTION OF THE NERVOUS SYSTEM 291 this diffuse nervous system, there is a continuous ring of nerve fibres round the margin of the umbrella, thickened at intervals by the accumulation of nerve cells, which are in close relation to special collections of sensory cells in the ' marginal bodies.' These sensory cells present a differentiation among themselves, some being apparently determined for the reception of mechani- FIG. 134. Diagram of su be pit-he lial plexus of nerve fibres and nerve cells, communicat- ing on the one side with the sensory epithelium, and on the other side with the sub- umbrellar sheet of muscle fibres. (After BETHE.) cal stimuli, others for the reception of light stimuli, while others again are found in close relation with little masses of calcium carbonate crystals, by the direction of the weight of which the cells are able to react to changes in the position of the animal in space. In the jelly-fish therefore the nervous or reactive system has already acquired a considerable degree of differentiation. FIG. 135. Figure of a jelly-fish in which all the marginal bodies except one have been removed, and which has been incised in various diiecticns so as to divide the nerve ring and all the ' long paths,' so that only the diffuse nerve network remains functional. (ROMANES.) • We may study the behaviour of a more primitive system if we remove the special sense-organs of the medusa by cutting off the whole of the marginal ring with its contained marginal bodies (Fig. 135). We have then a layer 292 PHYSIOLOGY of contractile tissue, innervated by a nerve network, and covered by a layer of epithelium containing sense cells. To this network is attached the manubrium, which represents the mouth and stomach of the animal. In such a mutilated jelly-fish it is easy to show that a stimulus applied to one spot on the surface travels outwards from the excited spot to all parts of the bell. The stimulus is propagated also to the manubrium, which in some species bends in the direction of the excited spot — that is to say, in the direc- tion which represents the shortest possible path from the excited spot to the manubrium. This preparation rarely presents any automatic activity. It may react to a constant stimulus by a rhythmic series of contractions, but remains perfectly motionless in the absence of stimulus. The unmutilated Fio. 136. Schema showing the utility of the multiplication of neurons and their grouping in central ganglia. (CAJAL.) A. an ideal invertebrate with only cutaneous * sensory ' neurons. B, invertebrate, such as a medusa, with sensory and motor neurons, but no central nervous system. c, invertebrate (e.g. Annelid) in which the motor neurons are concentrated in central ganglia. «, sensory neuron ; 6, muscle ; c, motor neuron. jelly-fish presents rhythmic contractions of its sub-umbrella tissue which are inaugurated in any or all of the marginal bodies and serve to drive the animal onwards through the water in which it is immersed. The rhythmic contractions may be initiated, augmented, or diminished, in response to stimuli of light, mechanical irritation, or changes in the position of the whole animal acting on the marginal bodies. In the reaction of an animal to ex- ternal stimuli it must be an advantage if the energies of the whole can be concentrated in defence of any one part and be evoked by a stimulus applied at one point. Such a co-operation of the whole for the benefit of the part involves the existence of direct paths from the stimulated point to all parts of the animal if the reaction is to take place with any promptitude. In the medusa we find a beginning of such ' long paths.' The general direction of the fibres of the network is radial, and there is a concentration of such fibres in the neighbourhood of the marginal bodies, so that an excitation can pass more readily from a sense organ to the manubrium than it can laterally along the circumference of the animal. Moreover a stimulus which is too slight to excite a reflex contraction of the muscular* tissue may travel along the nerve tissue to each of the marginal ganglia and arouse these to a discharge of motor impulses. We have therefore in the medusa sensory cells of different sensibilities ; central cells specially adapted to reacting to and reinforcing a EVOLUTION OF THE NERVOUS SYSTEM 293 nerve stimulus started by a small change in the environment ; a general nerve network propagating the excitatory changes in all directions, but with special ease in certain directions ; and a reactive muscular tissue which carries out movements at the end of the chain of excitation, all the elements forming the chain being derived from epiblastic cells. A further differentiation of a nervous system, such as that just described, must in the first place involve the laying down of more ' long paths ' and the collection of the special ' central ' cells into closely connected masses (ganglia), so as to concentrate the control of the reactions of the body, and to permit of the ready subordination of every part to the needs of the whole. A special direction is given to this development by the evolution of animals, such as worms and crustaceans, which are segmented and capable of locomotion. The fact that these animals are segmented determines the collection of the central cells into a chain of ganglia, one ganglion or pair of ganglia being provided for each segment. In the act of locomotion it is of advantage to the animal that those sense-organs or sensory cells which are proficient, i.e. which are stimulated by changes in the environment originating at a dis- tance from the animal, should be collected together near that part which goes first, namely, the head end. Thus the projected sensations of sight, those which are excited by chemical changes in the surrounding medium and represent the sense of smell, and those which are specially aroused by vibrations in the surrounding medium and correspond to those which we call the sense of sound, are in the majority of these animals sub- served by organs situated near the head end. The wisdom of a man is measured by his fore- sight. The chances of an animal in the struggle for existence are determined by the degree to which the reactions of the animal to its immediate en- vironment are held in check in response to stimuli arising from approaching events. An animal with- out power to see, smell, or hear its enemy will re- ceive no impulse to flee until it is already within its enemy's jaws. It must therefore be of ad- vantage to a segmented animal that the activities of the whole chain of segmented ganglia should be subservient to those central nerve-cells which are in direct connection with the projicient sense-organs at the head. The influence exerted by the head ganglia will be in the first place inhibitory of FIG. 137. View of central nervous system of cray- fish. (After YUNG and VOGT.) a, cerebral ganglion. b, commissure. e,subcesophageal ganglion. g. first abdominal ganglion 7, oesophagus. m, optic nerve. p, antennary nerve. ,s, stomato-gastric nerve. 294 PHYSIOLOGY the direct reaction excited in each segment by stimulation of its surface, and, for this influence to be propagated, long tracks must be laid down, joining up ganglion to ganglion and propagating impulses from the head ganglia to the most distant part of the chain. As a type of such a system wo may refer to the crayfish. In this animal the central nervous system (Fig. 137) consists of a chain of thirti •• -n ganglia, namely, six abdominal ganglia, six thoracic ganglia, and one supracesophageal or cerebral ganglion. In the abdomen and thorax the ganglia form a longitudinal series situated in the middle line of the ventral aspect of the body close to the integu- OAN&UON - CHAIN FIG. 138. Diagram of nervous system of a segmented invertebrate (earthworm or crayfish). (From SCHAFER, after RETZIUS.) Ofl >r\ cells ; s, afferent nerve fibres; m, motor neuron ; i, central or intermediate cell. ment. All rive origin to a variable number of nerves, which are distributed partly ID tin IMIIX -li -s. partly to tin- skin and sense-organs. They are connected by longitu- dinal bands of nerve fibres or eommissures, which arc double, each ganglion being bilobed. The moM anterior of tin- thoracic ganglia, which is the largest, is marked at the side by notches, as if it \\ere made up of several pairs of ganglia fused together. From this <_ra Million two commissures pass forward round the gullet to unite in front of this tube, behind tin- eyes, \\ith the transversely elongated mass of ganglion cells and fibres .•ailed the MI j HM . . ->oj .ha u'ejil ganglion. This ganglion consists of three fused pairs of LMiiL'lia. \\hii-h have been termed the /i/'nfnct , •, hroti, the dcuterocerebron, and the trito- cerebrou. The most anterior gives origin to the optic nerves, which run by the optic -t ilk- t.i the • yes. From the middle ganglion on each side a temimentary nerve passes to ramify in the internment and from the inferior surface the antennulary nerves pass to the internal antenna-. Krom these small Itranches are distributed to the organ of hearing. The posterior protuberance of the brain gives origin to the antennary \\hieh pa-s to the large external antenna' of the animal. The first thoracic, EVOLUTION OF THE NERVOUS SYSTEM 295 or subcesophageal, ganglion gives origin to ten pairs of nerves which are distributed to the mandibles, to the jaws or maxillae, to the maxillipedes, and to the branchial appendages of the latter. When we investigate the structural basis of such a nervous system we find that, as in medusa, the starting-point of the reflex arc is in certain neuro-epiihelial cells (Fig. 138) lying on the surface of the body. These cells are spindle-shaped, and have one short process passing to the surface, and a long process which runs in a nerve fibre or collection of fibres towards the ganglion of the segment. Arrived at the ganglion it divides into two branches, which pass towards the two ends of the body and become lost in Nerve Cell. NerveCell FIG. 139. Diagram of a reflex arc in a (neuro-fibrillar) invertebrate nervous system. (BETHE.) The efferent paths are coloured red, the afferent black. the granular material forming the inner part of each ganglion. The ganglia themselves consist internally of this ' punctuated substance ' or granular material, and externally of a capsule of ganglion- cells. Each of the ganglion- cells sends one thick process towards the centre, which rapidly divides, some of the branches passing into the granular material, while one branch passes outwards in a nerve to end in a network of fine fibrils within the muscles on the surface of the body. The nervous impulse excited in the sensory cell on the periphery travels therefore up a nerve fibre into the granu- lar substance of the ganglion. From this granular substance it is collected by the fine branches of the ganglion-cells and is transmitted by them along the motor nerve fibre to the muscles. The central granular material consists entirely of a close felt- work of fibres, which may be regarded as processes either of the sensory nerve fibres or of the nerve cells. The typical reflex arc in this case therefore is formed by two nerve cells with their processes. Such a nerve cell with its processes is spoken of as a neuron. The first neuron, the recipient neuron, or receptor, is represented by the sensory cell with its 296 PHYSIOLOGY two processes in the granular material. The second neuron is formed by the ganglion-cell with its finely branched dendritic processes in the granular matter and its motor axon. which passes into the muscle fibres. As to the manner in which the impulse passes from the branches of one cell into those of the other, opinions are still divided. The question will have to be more fully considered when we come to deal with the vertebrate nervous system. Many believe that there is no anatomical continuity between the two neurons, and that the excitatory change is transmitted by a mere contiguity, a change in one set of nerve-endings exciting a corresponding change in another set of nerve-endings in immediate contact with them. By certain methods however it is possible to show the existence of an anato- mical continuum throughout the whole nervous system in these inverte- brate animals. Apathy and Bethe have demonstrated the presence of a continuous system of neurofibrils (much smaller than an individual nerve fibre), which, starting in a sensory cell, pass into a network of fibrils forming the greater part of the central granular matter. From this network neuro- fibrils run along the dendrites into the ganglion cells, forming there a small network through the centre of which a neurofibril is continued down the nerve processes again, and passes out along the motor nerve to end in a network of fibrils among the muscle fibres. In a system so constituted it is evident that, although an excitatory process passing along a given fibril may find certain paths easier than others, and so maintain a constant prescribed path through the nerve system, yet it will be possible, by sufficiently increas- ing the strength of the excitatory process, to cause it to travel in all direc- tions in the central nervous system and to evoke in this way a general activity of all parts of the body, a condition in fact found to obtain in the normal animal. It is significant that, although a great number of fibrils pass into the bodies of the ganglion cells, yet in many cases, especially in crustaceans, fibrils are to be found sweeping from the neuropilem or nerve network of the granular substance into a nerve process, and thence into its motor axon without at any time entering the body of the cell (Fig. 1 :»(.n. SECTION II THE NERVOUS SYSTEM OF VERTEBRATES IN these, as in the invertebrata, the central nervous system is developed by an involution of the epiblast, revealing thereby its primitive relations to the surface of the body. At an early period in foetal life, shortly after the formation of the two layers of epiblast and hypoblast, a thickening is ob- served in the epiblast. This thickening soon gives place to a groove, the neurab groove (Fig. 140), and the walls of the groove folding over form a FIG. ]40. Transverse section of human embryo of 2 '4 mm. to show developing neural canal. (T. H. BRYCE.) nc, neural canal ; me, muscleplate ; my, outer wall of somite ; sc, sclerotome. canal, the neural canal, which is dilated at the head end of the embryo to form three enlargements known as the cerebral vesicles. When first formed the canal is oval in cross-section, its wall being made up of a layer of columnar cells between the outer extremities of which are seen smaller rounded cells. The internal layer of columnar cells sends a process peripherally which branches at the end so as to form a close meshwork of fibres. These fibres branch more and more as development progresses, and eventually form the supporting tissue of the adult central nervous tissue, known as the neuroglia. As the wall of the canal grows in thickness, some of the cells may wander outwards and form neuroglia-cells with numerous radiating branches. In the adult nervous system little is left of these cells except their nuclei, so that the neuroglia appears as a close felt- work of fibres, to which here and there nuclei are attached. These cells 297 298 PHYSIOLOGY ff. are formed from the most superficial layer of the invaginated epiblast, and are spoken of as spongioblasts. The deeper layer of cells, which are to give rise to the permanent nerve-cells, and are therefore known as neuroblasts, rapidly divide and form a thick layer surrounding the internal layer of spongioblasts, through which pass the peripheral processes of the latter. When first formed these cells have no processes. Later on each neuroblast acquires a pear shape, the stalk of the pear having a somewhat bulbous extremity (Fig. 142). The stalk continually elongates, and the elongated process may leave the spinal cord altogether and growr outwards to any part of the body of the embryo, or may pass to other parts of the central nervous sys- tem. This long process of the developing nerve cell is known as the axon. Some time after its formation other processes grow out from the cell, which soon branch and end in the immediate neighbourhood of the cell. The axons of the cells near the ventral part of the neural tube grow out to the different muscles of the body, where they end in close connection with the muscular fibres by an arborisation i which forms the end-plate. They provide an efferent path for impulses running from the central nervous system to the musculature of the body. The afferent channel is formed in a somewhat different manner. Even before the neural groove has closed in, a thickening of the epiblast is seen immediately external to the groove on each side. This thickening becomes divided into a series of collections of cells lying immediately under the epiblast on the lateral and dorsal surface of the neural canal. The cells, which are at first round or oval, send off two pro- cesses in opposite directions so that they become bi-polar (Fig. 142). One process passes into the central nervous system, where it divides, some "I 11- branches being distributed in the nervous system at the same level, win!.' others run a considerable distance towards the head immediately out- side the tube of nerve cells. The other process grows downwards, along with tin- processes from the ventral cells of the tube, towards the periphery of the body, where it ends in close connection with the surface in the various sense urL'aii* of t lie skin and muscles. These collections of bi-polar cells form the posterior root ganglia. In fishes they retain their primitive character throughout life, but in mammals the bi-polar cell is to be found only in the spiral and vestibular ganglia which give origin to the fibres of the eighth nerve. In all the other ganglia the shape of the cell becomes modified by an approximation of the points of attachment of the two processes until FIG. 141. Neuroblasts from the spinal cord of a chick embryo. (CAJAL.) A, three neuroblasts stained to show neurofibrils ; a. a bi-polar cell. B. a neuroblast showing the ' incre- mental cone ' r. THE NERVOUS SYSTEM OF VERTEBRATES 299 finally the cell becomes uni-polar, giving off one process which divides by a T-shaped junction into two, one of which runs towards the spinal cord, while the other takes a peripheral course as the afferent nerve fibre. The central nervous system thus becomes provided with a ' way in ' and a ' way out ' for the chain of impulses concerned in a nervous reaction or reflex action. The further development of the spinal cord is mainly determined by the exten- FIG. 142. Section through developing spinal cord and nerve roots from chick embryo of fifth day. (CAJAL.) •A, ventral root ; B, dorsal root ; c, motor nerve cells ; D, sympathetic ganglion cells ; E, spinal ganglion cells still bi-polar ; F, mixed nerve ; b, c, d, motor iierv,e fibres to J, developing spinal muscles ; e, a sensory nerve-trunk. , sion of the axons of the cells outside the tube of cells themselves, and by the provision of the ' long paths ' which are a necessary condition of increased efficiency of the reacting organ. Some time after the outgrowth of the axon a medullary sheath is formed, apparently by the agency of the axon itself, so that each group of axons leaving or entering the cord form& a bundle of medullated nerve fibres. The long branches of the posterior or dorsal roots running up towards the head form a mass of fibres behind the tube of cells known as the posterior columns. Fibres starting in the spinal cord itself run upwards and downwards to end in other parts of the cord, or in the more anterior divisions of the central nervous system forming the brain, and surround the neural tube on its ventral and lateral aspects with a sheath of white matter. To these white fibres are added others, which take origin in the brain and pass all the way down the cord. Meanwhile the cells 300 PHYSIOLOGY themselves become separated by the ramifications between them of the branches of axons entering the cord, as well as of the dendrites of the cells themselves. Thus, in its adult form, the spinal cord consists of a central mass of nerve cells and fibres, known as the grey matter, which is encased in a sheath of white matter formed of medullated nerve fibres. The cord itself is cylindrical in shape, and is divided into two symmetrical halves by the anterior and posterior fissures. In each half of the cord the grey matter on cross-section is crescentic in shape, presenting an anterior or ventral horn and a posterior or dorsal horn, and is connected with the corresponding mass in the other half of the cord by grey matter known as the anterior and posterior grey commissures. Between the two grey commissures is the central canal, relatively very minute when compared with the condition in the foetus and lined by a single layer of columnar ciliated epithelium, the cells of which are directly descended from the neural epithelium lining the medullary canal. THE STRUCTURE OF NERVE CELLS In the adult animal a typical nerve cell, such as those forming a prominent feature in the anterior horn of the spinal cord, is a large cell with many branches. It has a large vesicular nucleus with very little chromatin, Fio. 143. Nerve cell from the spinal cord, stained by Nissl's method, a, axis-cylinder process or axon ; &, proto- plasm of cell, consisting of c, fibrillated L'n. Mini substance, and e, the granules of Nissl; d, nucleus. (LENHOSSEK.) FIG. 144. The point of oriiriti of the axon, the 'nrru-- hillock, highly magnified, to show absence of Nissl's granules from the origin of the process. (HELD.) which may be collected into one or two nucleoli. The body of the cell presents different appearances according to the manner in which it has been treated for histological examination. When separated from the sur- rounding tissues by means of dissociating fluids it may present traces of striation. the individual striae running from one process to another of the cell. When treated fresh with mrthylene blue, or hardened by alcohol THE NERVOUS SYSTEM OF VERTEBRATES 301 or corrosive sublimate and stained with methylene blue or toluidine blue, the protoplasm is seen to contain angular masses which are deeply coloured with the dye (Fig. 143). These masses are known as the Nissl granules or bodies. By other methods it is possible to demonstrate that the whole protoplasm of the cell between the Nissl bodies is pervaded by fine fibrils, which enter the cell from the processes and may run out of the cell by the axon or may run into some of the other shorter processes (Fig. 146). The processes of the cell, as is evident from their development, are of two kinds. The axon which becomes continuous with the axis cvlinder of the medullated FIGS. 145 and 146. Nerve cells from spinal cord. (BETHE.) Fig. 145, showing Golgi network, and neurofibrils : d, e, f, junctions of axons with Golgi network. Fig. 146, showing neurofibrils and Nissl bodies. nerve fibre arises from a part of the cell body known as the axon hillock, which is the only part of the cell free from Nissl bodies (Fig. 144). The other processes, which may be very numerous, are known as the dendrites. They are generally thicker than the axon at their origin from the cell, but rapidly diminish in size as they give off branches, the branches apparently terminating freely in the grey matter in the immediate neighbourhood of the cell. In specimens stained by the Golgi method the dendrites may sometimes present a somewhat serrated outline. The Nissl bodies of the cell extend some way into the dendrites. A nerve cell with all its processes, axon, and dendrites is spoken of as a neuron. From the development of the central nervous system in vertebrates, it is evident that the nervous path of every reaction must be made up of two or more neurons. If we take, for example, the simplest possible reaction which might be effected through a single segment of the spinal cord, we see that the afferent impulse might be started by some stimulus applied to the ramifications in the skin of the distal processes of the posterior root ganglion cell (c/. Fig. 132). The nerve impulse so started is carried by the nerve fibre 302 PHYSIOLOGY past the T-shaped junction in the posterior root ganglion into the cord, and along a branch of the entering nerve fibre which runs right across the cord to terminate in the neighbourhood of the anterior horn cells. Here the impulse must be transmitted in some way to the dendrites or body of one of the large motor nerve cells in the anterior horn, whence it is carried along the axon of the cell, leaving the cord by the anterior root and passing down a peripheral nerve to the end-plate on- a muscle fibre. Here again by some means the arrival of the impulse excites the muscle to contract. This reaction never takes place in the contrary sense, i.e. no impulse started in the motor nerve can travel back through the spinal cord and along the sensory nerve. Although an impulse excited in the nerve passes easily to the muscle, an excitatory process started in the muscle itself is confined to this tissue and never extends to the nerve fibre. Apparently the same rule holds good within the grey matter of the central nervous system, where two neurons come into relation with one another. An impulse passes easily from the axon of one into the dendrites and cell of the other neuron, but, so far as we are aware, it is impossible by exciting an axon to cause a retrograde wave of excitation to pass through its corresponding cell and into the terminations of the axons in immediate contact with the cell. This statement has been called by Sherrington the ' Law of Forward Direc- tion.' It might be also spoken of as the irreciprocal conduction of the nerve arc. The character of a reaction to any stimulus, applied to the surface of the body, is determined by the course which the impulse, excited in the afferent nerves, takes on entrance into the central nervous system. This course is laid down by the connections of the neurons through which the nerve impulse passes. In the central nervous system therefore, more than in any other part of the body, function is directly dependent on struc- ture. Theoretically if we had a perfect knowledge of the connections of the neurons in the central nervous system and knew the nerve fibres affected by any given stimulus, we should be able to prophesy exactly the result of such stimulus. In the case of the simpler reactions this is already possible, but in the higher parts of the nervous system the enormous complexity of the systems of neurons excludes any possibility of our forming more than a general idea as to the nerve paths traversed in any given reaction ; and the variations which exist from individual to individual must always prevent in the intact animal an absolute prediction of the results of any stimulus. SECTION III GENERAL CHARACTERISTICS OF REFLEX ACTIONS THERE are certain features common to all reactions, carried out through the intervention of the central nervous system, which must be regarded as determined by the properties of the neurons, i.e. the conducting links in the chain of excitatory tissues intervening between the stimulated spot on the exterior and the reacting tissue, muscle or gland. These charac- teristics may be roughly classified as follows : (1) LOCALISATION. In a simple system of neurons a given stimulus will nearly always produce the same reaction. In a frog possessing only a spinal cord, the upper parts of the central nervous system having been destroyed, any harmful stimulus applied to a toe will cause a lifting of the leg. If the motor nerve to the gastrocnemius be excited, the whole muscle con-tracts. If one of the nerve roots entering into the formation of the sciatic nerve be excited, only certain fibres of the gastrocnemius contract, the locality of the reacting fibres being determined by their connection with the excited nerve fibres. In the same way the contraction of certain muscles of the leg, in response to a stimulus applied to the skin of the foot, is deter- mined by the fact that the nerve fibres, which carry the impulses from the toe into the spinal cord, divide there and make connections with the motor neurons, whose axons are distributed to the several muscles involved in the reaction. The connection of the sensory with the motor neuron may be direct, but in most cases the impulse has to pass through intermediate neurons before arriving at the motor neurons. The path of the impulse however, in spite of its enormous extension, is as definite as is the path from an excited motor nerve root to a muscle fibre. (2) DELAY. Instead of one nerve-ending intervening between the stimulated nerve and the reacting tissue, there will be, in the case of the reflex action, two, three, or more nerve-endings interpolated in the path «f the impulse. These nerve-endings are the fields of conjunction, the synapses, between the axon of one neuron and the dendrites and cell body of the neuron next in the chain. We have seen that there is a distinct difference between the latent period of a muscle excited through its nerve as compared with the latent period when excited directly, and we ascribed this latent period to a delay in the motor end-plate. We should expect therefore to find that the delay or 303 304 PHYSIOLOGY latent period in the case of a reflex action, i.e. the lost time in the conversion of an afferent into an efferent impulse in the central nervous system, would be appreciable and would increase with the complexity of the response — that is, with the number of neurons involved in the reaction. Such is indeed the case. In determining the actual ' lost time ' in the central nervous system for any given reflex, it is necessary to subtract from the total delay, inter- posed between the application of the stimulus and the resultant movement, the time taken by the impulse in travelling to and from the central nervous system, as well as the latent period of the muscles themselves. The re- mainder is known as the ' reduced reflex time.' Wundt found in the frog, when a reflex contraction of the gastrocnemius was excited by a stimulation of a posterior root of the same side, that the reduced reflex time was -C08 sec. For a crossed reflex the delay was increased by -C04 sec. If we assume that one additional neuron is involved in the crossed »reflex, the lost time at a synapse would be -C04 sec. ; if two cells are intercalated, the synapse delay would be only -002 sec. Since the uncrossed reflex has a delay of *C08 sec., at least two, and possibly four, synapses are involved in the path of this simple reflex. The blinking excited by stimulation of the eyelid has a reduced reflex time of -047 sec. (3) SUMMATION. When contractile tissues, such as striated or un- striated muscle, are excited by single shocks, a certain minimal strength of stimulus is necessary in order to produce a contraction. Weaker stimuli are spoken of as sub-minimal, and when applied singly have apparently no effect on the muscle. In dealing with the properties of involuntary muscle we saw that a sub-minimal stimulus is not necessarily devoid of effect because it fails to evoke a contraction, since, if repeated at sufficiently frequent intervals, a summation of stimulus occurs, so that at the fifth or sixth application a stimulus, which was previously ineffective, becomes effective and a contraction results. The muscle will now continue to respond to each stimulus, but, if the excitations be discontinued for a time, reapplica- tion of a stimulus of the same strength becomes once more ineffective. This summation of stimulus is a prominent feature in all reflex actions, so much so that it may be often impossible to evoke a reaction to a very strong single induction shock, whereas the application of a tetanising current too weak to be felt on the tongue may produce a marked reaction. We shall have occasion later on to deal with special examples of this summation of stimulus. • (4) FATIGUE. In the muscle-nerve preparation the weakest point and that which soonest suffers from fatigue is the end-plate, or rather the field of conjunction of nerve fibre -and muscle fibre. In the central nervous system the synapses of the different neurons are equally susceptible, and since several of such synapses are involved in every reflex action, we should expect to find that the central nervous system would show signs of fatigue before the peripheral structures. If a given reaction be repeatedly elicited by applying a stimulus to a certain area of the surface, the reaction becomes CHARACTERISTICS OF REFLEX ACTIONS 305 feebler and finally disappears altogether long before any signs of fatigue in the motor apparatus can be detected by stimulation of the motor nerve itself. The fatigue is produced equally well if the reaction be excited by stimulating a sensory nerve directly, and since we know that it is practically impossible to fatigue nerve fibres, we must conclude that the seat of fatigue is in the grey matter of the spinal cord itself. (5) ' BLOCK ' OR RESISTANCE. In the central nervous system there is an absolute block to the passage of an impulse backwards through a synapse, i.e. from a nerve-cell or its dendrites into the end ramifications of an axon. The phenomena of fatigue show that there is a certain degree of resistance at the synapse to the passage of an impulse in the normal direction, and that this resistance is rapidly increased under the conditions which produce fatigue. When we study the structure of the central nervous system more fully, we find that although there are certain shortest possible paths, i.e. ones involving few neurons, for every impulse arriving at the central nervous system, yet so extensive is the branching of the entering nerve fibres and so complex are the neuron systems with which they come in connection that an impulse entering along one given fibre could spread to practically every neuron in the spinal cord and brain. Such a result is indeed observed in animals poisoned by strychnine. In such animals the slightest stimulus applied to any part of the skin excites strong tonic spasms in the whole musculature of the body. Every single nerve fibre, that is to say, can discharge into every motor neuron of the cord. That this result does not ensue on localised stimulation in a normal animal is dependent on the varying resistance to the passage of an impulse into the several neurons with which the entrant fibre comes in relation. A small stimulus will dis- charge only along the few neurons where the resistance is lowest. Increase of the stimulus, either by increase of its strength or by summation of weak stimuli, will enable the impulse to spread along more neurons and therefore will elicit a more widespread response. Only when the * blocks ' are entirely removed- by the administration of strychnine, or when the stimuli are abnormally powerful and long continued, will the impulse spread to all regions of the central nervous system, so that response becomes general and inco-ordinate instead of local and adapted to the stimulus. (6) FACILITATION OR < BAHNUNG.' The passage of a nervous impulse across a synapse or series of synapses in the central nervous system has a twofold effect. If the passage be too often repeated, phenomena of fatigue are produced and there is an increase of the block at each synapse. If however the stimulus be not excessive and the reaction not too frequently evoked, the effect of passage of an impulse once is to diminish the resistance, so that a second application of the stimulus evokes the reaction more easily. The process of summation in fact is chiefly in the direction of removal of block. We have a close analogy to this process of facilitation in the ' stair- case phenomenon ' observed in cardiac and unstriated muscle. In these tissues the repetition of a sub-minimal stimulus renders it in time effective, and then repetition of the now effective stimulus causes a gradually 20 PHYSIOLOGY increasing height of contraction, which depends on the state of the contracting tissue itself and cannot be evoked by changes in the strength of the stimulus. This process of facilitation or ' Bahnung ' is of great interest in connection with the development of ' long paths ' in the central nervous system, and more especially with the acquirement of new reactions by the higher animals. The Law of Facilitation is really the Law of Habit. When an impulse has passed once through a certain set of neurons to the exclusion of others it will tend, other things being equal, to take the same course on a future occasion, and each time that it traverses this path the resistances in the path will be smaller. Education is the laying down of nerve channels in the central nervous system, while still plastic, by this process of 'facilitation' along fit paths, combined with inhibition (by pain) in the other unfit paths. Memory itself has the process of facilitation as its neural basis. (7) INHIBITION. The constant occurrence of a reaction in response to a giv spinal'nMitres, produced by the strong stimulation of the injury to thr brain and medulla, plays at any rate an important part. We may say that the passage of an impulse through a chain of neurons diminishes the block for subsequent impulses at each synapse that it traverses, but increases during its passage the block in all the adjacent synapses. In dealing with the special reactions of the spinal cord we shall have, occasion to refer more fully and in greater detail to many of these pro- perties which are characteristic of all reflexes. Before treating of the functions of the separate parts of the central nervous system in the higher mammals, it may be of interest to consider the exact nature of the structure intervening between neuron and neuron at each field of conjunction or synapse, as well as the significance of. the two chief elements of the central nervous system, nerve cell and nerve fibre, in the production of co-ordinated purposive reactions. SECTION IV NATURE OF THE CONNECTION BETWEEN NEURONS THE study of the development of the central nervous system in higher animals has shown that this system is made up of neurons, the connections of which determine the possible paths of impulses in the adult cord. The first stage in the development of the neuron is a single cell without processes, and it is only by the growth of these processes out from the cell that the spinal cord becomes capable of serving as an aggregate of conducting paths. Moreover the deferred acquisition of an influence of one neuron on the next neuron in the line of impulse, or at any rate on the peripheral tissue which receives the end arborisation of its axon, is shown by the fact that entire destruction of the spinal cord in the embryo at an early stage in its develop- ment does not prevent in any way the development of the voluntary muscles (Harrison) ; although, after birth, a severance of the connection between spinal cord and skeletal muscle leads to a rapid degeneration and atrophy of the latter. In the muscle-nerve preparation there is an apparent break of structure at the, termination of the nerve in the muscle fibre, any con- tinuity between' nerve-ending and contractile substance being subserved by undifferentiated protoplasm. There is therefore no difficulty in con- ceiving a propagation across a similar nerve-ending or synapse, between the axon of one neuron and the cell body or dendrites of another neuron. If however the conception we have formed above of the evolution of a nervous system from a continuous conducting protoplasmic network, by a process of ' facilitation ' attended by histological differentiation, be correct, we should expect to find in the fully developed brain and spinal cord some traces at any rate of continuity throughout the whole system of neurons. The question as to the existence of anatomical continuity from neuron to neuron has been hotly discussed both for vertebrates and in- vertebrates. In the case of the latter, evidence in favour of the continuity of neuro-fibrillae from sensory surface to reacting tissue is very strong. Many observers, especially Apathy, Bethe, and Held, have described a similar continuity in the nervous system of mammals. The last-named observer regards this continuity as a product of later development and as due to a process of concrescence occurring between the axon terminations and the bodies of the nerve cells with which they come in contact. It is. easy to show the existence of a fibrillar structure both in the nerve cell and in the 307 308 PHYSIOLOGY nerve fibre (Fig. 147). The axis cylinder of the nerve fibre can be regarded as made up of fine fibriUse embedded in an interfibrillar substance. At the nodes of Ranvier the interfibrillar substance is interrupted, the fibrillse alone extending into the next internode and representing the continuous structure which determines the conducting power of the nerve fibre. In the nerve cell the fibrillae occupy all the space between the Nissl bodies, passing from dendrite to dendrite, and many of them from all the dendrites and all parts of the cell sweeping through the axon hillock to form the fibrillse of the nerve- fibre. The existence of these fibrillar structures in nerve cell and nerve Fio. 147. Part of an anterior cornual cell from the calf's spinal cord, stained to show neurofibrils. (BETHE.) Ax, axon ; a, b, r. dendrites. 148. Arborisation of collaterals from the pos- terior root-fibres round the cells of the posterior horn. (RAM6N Y CAJAL.) fibre is accepted by most histologists. The question however of the con- nection between the fibrill® of one axon and those of the next neuron, i.e. the histology of the synapse, presents much greater difficulties and has excited much difference of opinion. If we examine a nerve cell such as a cell of Purkinje of the cerebellum, or a cell of Clarke's column in the cord, we find that it is surrounded by a thick basket-work of fibres whicli are the arborisations or end terminations of the axons which pass to the cell to enter into functional relationships with it (Figs. 148 and 149). This peri- cellular network is of great extent and may equal in total diameter the diameter of the cell itself. Whether the basket-work is really a network, NATURE OF CONNECTION BETWEEN NEURONS so§ or merely a felt- work in which the fine fibres intertwine among each other without becoming actually continuous at any points, is difficult to make out. On the periphery of the cell itself another network has been described and is known as the Golgi network (Fig. 150). This has been displayed both by the process of impregnation with silver chromate (Golgi method), as well as by staining with methylene blue. Some authors have regarded this network as an artefact due to precipitation of albuminous fluids on the surface of the cell. According to Bethe however, the Golgi network on the one hand receives fibres from the encircling pericellular basket-work of axons, FIG. 149. Basket-work of fibres around two cells of Purkinje. (CAJAL.) a, axis-cylinder or nerve-fibre process of one of the corpuscles of Purkinje ; b, fibres prolonged over the beginning of the axis- cylinder process ; c, branches of the nerve-fibre processes of cells of the molecular layer, felted together around the bodies of the corpuscles of Purkinje. FIG. 150. Superficial network of Golgi surrounding two cells from the cerebral cortex of the cat ; Erlich's method. (CAJAL. ) A, large cell ; B, small cell ; a, a, folds in the network ; b. a ring-like condensation of the network at the poles of the larger cell ; c, spinous projections from the surface. and on the other hand gives off towards the interior of the cell fine fibrils, which are continuous with the neurofibrillae of the cell and pass out in its axon. The diagrammatic course of a nerve impulse according to Bethe is represented in the accompanying diagram (Fig. 151). An impulse starting from the periphery of the body travels up the distal process of the posterior root ganglion-cell, passes either through the cell or directly to the central process, and travels along this to the terminations of the posterior root fibres round a posterior horn-cell. Here it passes into the peri-cellular basket-work or axon network, thence into the Golgi network and along the fibrillae of the cell out by the fibrillse of the axon and so to a fresh synapse with a cell of the anterior horn. There are certain physiological difficulties in the acceptance of this 310 PHYSIOLOGY doctrine of continuity through the central nervous system. Even if it be true, it would not in any way upset the importance of the neuron theory. Every plant or animal individual must be regarded as a protoplasmic con- tinuum. With growth of the living matter, its metabolic functions demand the dispersion of nuclear material through the protoplasm, and this is effected by division of the nucleus. Considerations of strength and rigidity demand the division of the protoplasm into compartments or cells which, at first at any rate, remain in protoplasmic continuity. This division has probably a further advantage in that lesions of parts of the individual entail merely the death of the cells immediately affected and do not necessarily Fio. 151. Schema of the neurofibrillar continuum, involvediin an ordinary reflex act, in a vertebrate nervous system. (BKTHE.) spread to the whole organism. Thus in the central nervous system injury to one axon causes degeneration of the axon below the point of section, but the degeneration stops short at the end arborisation and does not spread into the next neuron. If we assume that, in consequence of the straitness of the path, the propagation through the fibrillae is especially difficult in the synapse, most of the phenomena described above as characteristic of the reactions which take place in the central nervous system can be easily ex- plained on the theory of continuity of the fibrillae. The serious difficulty in the acceptance of this theory is however the * Law of Forward Direction,' i.e. the fact that an impulse will pass from an axon to the next neunm. but will not pass backwards across the synapse from the cell body to the OOntigUOUJ axon. llcthe surest s thai this rule of Forward Direction, which is possibly present only in tin- HIM iv hi-rbly developed n.-rvous systems, may be due to a Bpedea of ' pol.-.rif \'' • •I Hi-- nerve fibril, of such ;i nature that an impulse is si relict liened and so assisted on its passage in the normal din •« I ion. but is diminished and finally abolished uheii it passes in tin- opposite direction. Such an explanation is unsatisfactory, since then- is absolutely noexperii.ici,tale\ideiice of the existence of such polarity in a nerve libre: all the evidence I hat \s .- have at present points to a nerve fibre Lmnj.' the power of NATURE OF CONNECTION BETWEEN NEURONS 311 propagating equally well in either direction. It is certainly more useful to regard a synapse as of the nature of a motor nerve-ending, in which an impulse arriving along the branches of an axon excites a fresh impulse in the excitable tissue, i.e. the nerve- cell, with which the branches of the axon come in contact. Moreover the neurons are formed without any structural connection with the future destination of their axons. These grow out as processes with thickened amoeboid extremities. Harrison has shown that the growth of the axon from the cell may be observed under the microscope in a neuroblast separated altogether from the body, and kept on a warm stage in a thin layer of coagulated lymph. It is possible that we may have to distinguish two types of nervous systems, viz. : (a) A neurofibrillar type, peculiar to invertebrata, with conduction in all directions. (6) A synaptic type, in which the Law of Forward Direction holds, of later evolution, and forming the greater part of the nervous system of vertebrata. SECTION V FUNCTIONS OF THE NERVE CELL WHEN a unicellular organism, containing a single nucleus, is cut into two parts, both continue to live for some time, each performing active move- ments and evincing all the phenomena which we associate with activity and therefore with destructive katabolism. For the continued existence of a cell the processes of constructive metabolism, or anabolism, must take place pari passu with those of disintegration, and for this the presence of the nucleus is necessary. Hence, in a few days, the half cell with the nucleus has repaired its loss and become once again a normal individual, whereas the half without a nucleus undergoes degeneration and death. The axon of a nerve cell can be regarded as analogous to a long pseudopodium of an amoeba. Like this, if cut away from that part of the cell containing the nucleus, though capable for a time of discharging its active function of propagation of excitatory impulses, yet it finally dies, death of the nerve fibre occurring in the mammal within three to five days after separation of the axon from the cell. Every nerve cell therefore may be looked upon as a trophic centre of the nerve fibre proceeding from it as well as of the medullary sheath, which is practically a product or secretion of the axis cylinder. But has the nerve cell any more important functions to dis- charge ? It has long been customary to endow the nerve cell with all the properties which are Distinctive of a nervous system, and to ascribe to it the active part in the origination of automatic actions, in the reflection of afferent impulses, and in the supply of energy to all nervous processes. That the passage of impulses through the nerve centres requires the ex- penditure of energy by these centres can be proved in various ways. In the first place, we have the fact that in all nervous systems, at any rate of the higher animals, arrangements are made for their free supply with oxygen. Very short deprivation of oxygen causes a complete block throughout the system, in many cases preceded by a short period of increased excitability or ease of transmission. If, in the rabbit, the thoracic aorta be clamped for a few minutes, the hind limbs become paralysed, and if the obstruction be continued for half an hour, there is widespread degeneration and death of the cells with their fibres in the grey matter of the lumbar and sacral cord. In the second place, the ready production of fatigue of the nervous system points to a considerable using up of material as a condition of the passage of nerve impulses. In many instances moreover, an infinitesimal stimulus travelling up a few nerve fibres may excite widespread activity of the whole central nervous system with the discharge of impulses along 312 FUNCTIONS OF THE NERVE CELL 313 practically every nerve of the body. Thus the presence of a crumb on the larynx will excite impulses travelling up the superior laryngeal nerve, which in themselves can involve but little expenditure of energy. The result however of their arrival at the central nervous system is the discharge of impulses along the motor nerves causing spasmodic contractions of almost every muscle in the body. It seems beyond doubt then that energy is evolved in the central nervous system as a result of metabolic changes, and that energy may be added to impulses passing through the central nervous FIG. 152. Diagrammatic representation of the brain of Carcinus to show the parts involved in Bethe's experiment. The dotted line x shows the incision employed to isolate the neuropilem of the ganglion of the second tentacle. system, which therefore acts as a relay of force. But this activity does not necessarily require the presence or co-operation of nucleated cells. In dealing with the nature of a nerve impulse we had reason to conclude that there may be an actual, though minimal, liberation of energy in the axis cylinder with the passage of each nerve impulse. The non-nucleated parts of a cell, whether the axon or the cell body, are equally capable of this evolution of energy, and we might conceive therefore of a nervous system which, existing for a few days, might act as a normal reflex centre in the entire absence of the nucleated cell bodies. This conception has been realised by Bethe in an experiment in the crab (Carcinus menas). In this animal the reflex movements of the tentacle are carried out by a gang- lion situated at its base. As in the other Crustacea, the cell bodies in this ganglion lie outside the mass of neurofibrils in the centre, forming a sort of capsule (Fig. 152). Bethe was able, under the dissecting microscope, to remove the cell bodies without interfering with the nerves entering or leaving the central mass of fibrils. All the nerve processes with their connections were therefore left intact. In animals, operated in this way, Bethe found that for two or three days the tentacle reacted normally to stimuli applied to its surface. The reflex functions of the ganglion were not in any way affected by the removal of the nucleated bodies of the cells. A similar experi- 314 PHYSIOLOGY ment would be impossible in the central nervous system of vertebrates, since impukes must of necessity pass through the cell body on their way from the termination of one axon to the beginning of the next. In the spinal root ganglion however, most of the cells lie on the surface. In the rabbit Steinach exposed a posterior root ganglion, separating it from all its vascular supply, but leaving its nervous attachments intact. The wound was opened every day for the next few days and an instrument passed under the ganglion so as to divide any newly forming vessels. As a result of the deprivation of blood-supply the ganglion- cells died. But Steinach found that nerve impulses were still conducted perfectly well through the ganglion at a time when microscopic examination showed a complete atrophy of all cells. It is therefore only in virtue of the fact that the nerve-cell is the seat of the nucleus, and therefore of the assimilative functions of the neuron, that any pre-eminent importance can be ascribed to it in the building up of a reactive nervous system. Prominent among the functions with which the nerve cell has been endowed is that of automaticity of action in the absence of stimulus other than that supplied by its own metabolism or by the fluids which bathe it. A priori there is no reason to deny to the neuron a property which is pos- sessed by other cells of the body, such as the muscular cells of the heart, and is a fundamental quality of undifferentiated protoplasm. The purpose however for which these cells have been evolved and differentiated is that of reaction, of adapting the organism to changes in its environment, and it is doubtful whether, in this differentiation, it has retained any auto- matic properties whatsoever. In the absence of any afferent impulse the whole central nervous system would probably be inert. In a frog retaining only the spinal cord Hering divided all the posterior roots. The frog remained flaccid and motionless. Injection of strychnine was powerless to evoke the usual tetanic spasms. In such a strychninised frog however, it was necessary only to open the wound and touch one of the divided posterior roots to throw the whole body into convulsions. As shown by Sherrington and Mott, division of all the afferent nerves coming from the upper limb in monkey or man entirely abolishes all such contractions of the limb, as are usually affected through the intermediation of the cerebral cortex. Cutting off the major portion of the afferent impulses to the respira- tory centre does not, it is true, abolish all respiratory discharges, but converts the rhythmic respirations into a series of inspiratory spasms which are repeated at long intervals and are entirely inadequate for the proper aeration of the blood. According to Sherrington a repetition on the mammal of Hering' s experiment does not lead to the same results, since a spasmodic discharge is produced from the isolated spinal cord as a result of asphyxia. But it is doubtful whether in this case there was not some continuous excita- tion of the cord going on, as a result of the closure of the demarcation current in the cut ends of the posterior roots by the body fluids. It is possible that the neurons possess some automatic power, i.e. some power of initiating nervous piOftMMg, as a result of changes in the fluids surrounding them. Tliis automat ieity however is not a prominent feature of the nervous system, which has l,een evoked as a purely reactive mechanism to the afferent impulses resulting from the material changes continually taking place in the '•n\ iroimieiit of the animal. SECTION VI STRUCTURE OF THE SPINAL CORD IN the higher representatives of the invertebrate class, the central nervous system consists, as we have seen, of a chain of ganglia, each ganglion or pair of ganglia presiding over the reactions of its own segment, but connected by long paths with the other ganglia and with the head ganglia. The latter, being especially developed in connection with the organs of special sense which are projicient in function, acquire a control over the rest of the ganglia (Fig. 153). The vertebrate spinal cord may be looked upon as a chain of ganglia which Neuj-eatcric canal Spinal Cord. * Segmealal Nerves In/undifculum VENTRAL Altui.i DORSAL Aa VENTRAL Fiu. 153. Vertebrate central nervous system compared with that of the arthropod. (GASKELL.) (Note that according to Gaskell the ventricles of the brain and the primitive neural canal correspond to the invertebrate stomach and intestine.) have become fused concurrently with a diminution in the importance of the local segmental reactions and with a growth in the solidarity of the whole system ; so that in the higher vertebrates, at any rate, little trace of the primitive segmental arrangement is evident in the internal structure of the cord. Some remains of this arrangement still persist however in the origin from the cord of nerve roots, which are distributed roughly within the area of tin1, corresponding segment of the body. In man the spinal cord is an elongated cylindrical structure slightly flattened from before backwards and about eighteen inches long. It gives oft' a series of nerve roots, which are arranged in .thirty-one pairs :MI.| juv distributed symmetrically to the two sides of the body. Each 315 _ . . ICNVW- 316 PHYSIOLOGY nerve arises by t\vo roots, an anterior and a posterior, the anterior being composed of a series of rootlets spread over a considerable area of the cord, while the posterior roots arise as a compact bundle from a groove on the postero-lateral aspect of the cord. The posterior nerve-roots pass through a ganglion and join the anterior roots in the intervertebral foramina to form the mixed nerve. < )i>. section the cord is seen to consist of a, core of grey matter surrounded on all sides by white matter. The white matter is made up of medullated nerve fibres which are devoid of a neurilemma, and run within tunnels or tubes in the supporting neuroglia. The grey matter has roughly the form of a letter H, and consists, in cross-section, of a comma-shaped mass on each side of the cord, joined across the middle line by a band of grey matter. On the anterior aspect of the cord is a furrow, the anterior fissure, which contains a process of the enveloping membrane of the cord, the pia mater, and is limited at its bottom by a band of white matter, the anterior white commissure, which unites the anterior columns of white matter. On the hinder aspect of the cord is another fissure, the posterior fissure, which is very narrow and is built up chiefly by neuroglia. A third fissure at the point of origin of the posterior nerve-roots serves to divide the white matter of the cord into an antero-lateral column and a posterior column, and the former is imperfectly separated by the spread-out anterior rootlets into anterior and lateral columns. The cord in cross-section (Fig. 154) is circular in the dorsal region and oval in the cervical and lumbar regions. It presents two marked enlargements, namely, the cervical enlargement, corresponding to the outflow of the nerves going to the upper limb, and the lum bo-sacral enlargement, which gives off the nerves to the lower limb. In the sacral region it rapidly tapers off to a blunt point. In the centre of the band of grey matter, connecting the two masses on each side of the middle line, is the central canal of the cord, the remains of the primitive neural canal of the embryo. The grey matter in front of it is called the anterior grey commissure, that behind the posterior grey commissure. The comma- shaped mass of grey matter on each side of the cord presents in front the broad f anterior cornu, and behind the narrower posterior cornu, which extends up to the postero-lateral groove in the line of emergence of the posterior roots. In the dorsal region of the cord the grey matter projects into the lateral column of white matter to form the lateral horn. The grey matter consists of a supporting tissue of neuroglia in which are embedded nerve cells and their processes and the endings of nerve fibres. The neuroglia is formed of a thick felt-work of fibres with here and there nuclei applied to the fibres. Occasionally we may meet cells provided with a very large number of branches and representing the cells from which all the fibres of the neuroglia have been derived. The neuroglia is present in specially large amount in two situations, namely, immediately around the central canal and as a capsule to the enlargement of the posterior cornu, known as the head or caput cornu posterioris. In this latter situation the neuroglia con- tains a large number of small richly-branched nerve cells and is spoken of as the substantia gelatinosa of Rolando. The nerve cells are arranged in STRUCTURE OF THE SPINAL CORD 317 distinct groups. In the anterior horn we may distinguish three groups, a median group of cells near the middle line, majiy of which send their processes across to the other side in the anterior white commissure, and an external CERVICAL. DORSAL. LUMBAR. ANTERIOR ROOT-BUNDLES FIG. 154. Sections of human spinal cord from the lower cervical, mid-dorsal, and mid-lumbar regions, showing the principal groups of nerve cells, and on the right side of each section the conducting tracts as they occur in the several regions (magnified about 7 diameters). (E. A. SCHAFER.) a, b, c, groups of cells of the anterior horn ; d, cells of the lateral horn ; e, middle group of cells ; /, cells of Clarke's column ; g, cells of posterior horn ; cc, central canal ; ac, anterior commissure. IMS IMIYSIOMHJY group often subdivided into a postero-external and an antero-external. The latter group is especially developed in the regions of the cervical and lumbar enlargements and consists of very large multipolar cells with many dendrites which send their axons into the anterior roots and by these to the muscles of the limbs. Another group of rather smaller cells is found in the lateral horn, in that region of the cord where this is marked. A very definite group of cells may be seen in the dorsal region of the cord in the inner aspect of the root of the posterior horn. This, which is known as Clarke's column, is formed by large cells elongated in the longitudinal direction of the cord. Besides these definite columns a number of nerve cells are distributed irregu- larly through the grey matter, especially of the posterior horn. Atit. lot — j aac. tract. Direct Cerebellar Asferua Roots, witii collaterals. FIG. 155. Spinal cord. (After LENHOSSEK-.) On left side of figure are shown the nerve cells with their axis-cylinder processes. On the right side the dis- tribution of the chief collaterals. 1, motor cells ; 2, cells of the columns ; -2n, cells of Clarke's column, sending processes across into direct cerebellar tract ; 3, 4, and 5, commissural cells. According to the destiny of their axons these nerve cells may be divided into four (Fig. IT).-)). (1) THE MOTOR CELLS, the largeel of all, which eend their axons info i he anterior roots, when, t hey run to supply skeletal muscle fibres. As a sub-group of these cells we may class the somewhat smaller cells of the lateral horn, which in all probability send ixons by the anterior roots to supply visceral muscles. Their axons. can he distinguished from the motor axons by the smaller diameter of the nerve fibres they form. They pass later from the mixed nerve along a white ramus com in un leans into the sympathetic system, in the ganglia of which they end. (2) CELLS OF THE COLUMNS. As typical of these cells we may take those which form Clarke's column. Their axons do not leave the central nervous system, but pass out into the white matter to some other part of the central nervous system, contributing thus to form the white columns of the cord. (3) COMMISSURAL CELLS. These cells send their axon across the middle line to the opposite side of the cord, making up a great part of the anterior white commissure. (4) CELLS OF GOLGI. These cells are found chiefly in the posterior horn. They are multipolar and are distinguished from all the other cells by the fact that their STRUCTURE OF THE SPINAL CORD 319 axon does not pass far from the cell, but rapidly breaks up into a number of branches which terminate in the near neighbourhood of the cell giving off the axon. They may be regarded as association cells, i.e. as serving to establish a functional connection between many different cells at any given level of the grey matter. The, white matter of the cord is divided by the fissures already described into anterior, lateral, and posterior columris. The nerve fibres of which it is composed are all of them axons of nerve cells situated at different levels of the central nervous system or outside the cord. Since the whole object of the study of the anatomy of the cord is the tracing out of the systems of neurons of which it is made up, and therefore of the possible paths of any reflexes or nerve impulses through the cord, a mere, anatomical differentiation of different columns is quite useless unless we can determine in each column the origin and destination of the fibres of which it is composed. For tracing out the course of the different axon systems in the central nervous system several methods arc available. (a) HISTOLOGICAL. Two methods may be employed for staining a nerve cell with all its processes, namely, the intravitum staining with methylene blue and the impregnation method invented by Golgi. In the latter method, of which there are many modifications, the nervous tissue is hardened in some chromate or bichromate, and is then soaked in a solution of silver nitrate or mercuric chloride. In this way a precipitate of silver or mercuric chromate is formed within the nerve cells and their processes ; but for some unexplained reason the impregnation is not general, and is confined to a small percentage of the neurons. If the precipitate were diffuse, even a thin section would be absolutely opaque ; since it is partial, thick sections may be cut and, after clearing, allow the tracing of the processes of the few impregnated nerve cells through the whole thickness of the section. We may in this way get sections 0-1 mm. thick at the point of entrance of a posterior nerve root, and trace out the course and ending of a large number of the fibres composing the nerve root, or we may in a r/fc— - section involving the anterior nerve root trace the course of an axon of an anterior cornual cell out of the cord into the root. This method is of no use in tracing any given nerve fibre through the whole length of the cord. For this purpose however several methods are available. (6) MYELINATION METHOD OF FLECHSIG. Nerve fibres at their first formation as axons of a T , „ , , jll FIG. 156. Section throusrh the cer- nerve cell are non-medullated, the medullary sheath vical 8pinal cord of a new.born being formed later with the beginning of function of child, stained by -Weigert's the nerve. It has been shown by Flechsig that the myelination does not occur simultaneously through all parts of the central nervous system, but that it is later in proportion as the nerve fibre is more recent in the phylogenetic history of the animal. root zone ; The cord in its most primitive form can be regarded fibres. as a series of ganglia presiding over the different segments of the body. The most primitive fibres therefore would be those which run from the periphery of the body to each segment and from each segment out to the muscles, and so a medullary sheath is first formed in a number of the fibres entering and leaving the cord in the nerve-roots. Next in order of myelination are those method, to show absence of medullation in pyramidal tract. ca> anterior commissure ; Fp, rp', posterior (BECHTEREW.) root 320 PHYSIOLOGY fibres which connect different segments of the cord, the internuncial or intra-spinal fibres. Next come those fibres which connect the spinal cord with the cerebellum. Last of all to receive a medullary sheath are the fibres which take a direct course from the cerebral cortex to the spinal cord. These are called the pyramidal tracts, and in man are not medullated until the first month after birth (Fig. 156). (c) THE WALLERIAN METHOD. A nerve fibre, when cut off from t>he nerve cell of which it is a process, degenerates. This degeneration is marked by a breaking up of the medullary sheath and a conversion of the phosphorised fat, myelin, of which it is composed, into ordinary fat. Later on the fat is absorbed and the nerve becomes replaced by a strand of fibrous tissue in the case of peripheral nerves, of neuroglia in the central nervous system. If the white matter of one half of the spinal cord be divided in the dorsal region, and the animal be killed about three weeks after the opera- tion, sections of the cord both above and below the lesion will show the presence of degenerated fibres. In order to display these fibres pieces of the cord are hardened in a solution containing bichromates and are then immersed in a mixture of osmic FIG. 157. Cells from the oculo-motor nuclei thirteen days after section of the nerve on one side. ". coll from healthy side ; b, cell from side on which nerve was divided. (FLATATJ.) ;tri