PRINCIPLES OF HUMAN PHYSIOLOGY PRINCIPLES OF I MAN YSIOLOG\? T BY ERNEST H. STARLING M.D. (LOND.), F.R.C.P., F.R:§., HON. M.D. (BRESLAU) JODRELL PROFESSOR OF PHYSIOLOGY IN UNIVERSITY COLLEGE, LONDON LEA & PHILADELPHIA" NEW YORK 3P34 PREFACE 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 physio- logical 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 unexplpred 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 prin- ciples 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 trans- formed 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 " Ergebnisse der Physiologic," in NagePs "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 vi PREFACE 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 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 investigation 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 founda- tion for rational therapeutics is the proper understanding of the working of the healthy body. Until we know more about the physio- logy of nutrition, quacks will thrive and food faddists abound. Igno- rance 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 His- tology." I must also express my obligation 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 physio- logical 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 PAGE INTRODUCTION 1 BOOK I GENERAL PHYSIOLOGY CHAPTER II THE STRUCTURAL BASIS OP THE BODY 13 CHAPTER III THE MATERIAL BASIS OF THE BODY ECTION I. THE ELEMENTARY CONSTITUENTS OF LIVING CELLS 39 II. THE PROXIMATE CONSTITUENTS OF THE ANIMAL BODY 49 III. THE FATS 58 IV. THE CARBOHYDRATES 64 V. THE PROTEINS 78 VI. THE MECHANISM OF ORGANIC SYNTHESIS 121 CHAPTER IV THE ENERGETIC BASIS OF THE BODY I. THE ENERGY OF MOLECULES IN SOLUTION 136 II. THE PASSAGE OF WATER AND DISSOLVED SUBSTANCES ACROSS MEMBRANES 145 III. THE PROPERTIES OF COLLOIDS 154 IV. THE MECHANISM OF CHEMICAL CHANGES IN LIVING MATTER. FERMENTS 170 V. ELECTRICAL CHANGES IN LIVING TISSUES 189 BOOK II THE MECHANISMS OF MOVEMENT AND SENSATION CHAPTER V THE CONTRACTILE TISSUES I. THE STRUCTURE OF VOLUNTARY MUSCLE 197 II. THE EXCITATION OF MUSCLE 206 III. THE MECHANICAL CHANGES THAT A MUSCLE UNDERGOES WHEN IT CONTRACTS 215 vii viii CONTENTS CHAPTER V (continued) SECTION PAGE IV. THE CONDITIONS AFFECTING THE MECHANICAL RESPONSE OF A MUSCLE 230 V. THE CHEMICAL CHANGES IN MUSCLE 235 VI. THE PRODUCTION OF HEAT IN MUSCLE 246 VII. ELECTRICAL CHANGES IN MUSCLE 251 VIII. THE INTIMATE NATURE OF MUSCULAR CONTRACTION 263 IX. VOLUNTARY CONTRACTION 266 X. OTHER FORMS OF CONTRACTILE TISSUE 271 CHAPTER VI NERVE FIBRES (CONDUCTING TISSUES) I. THE STRUCTURE OF NERVE FIBRES 279 II. PROPAGATION ALONG NERVE FIBRES 283 III. EVENTS ACCOMPANYING THE PASSAGE OF A NERVOUS IMPULSE 287 IV. CONDITIONS AFFECTING THE PASSAGE OF A NERVOUS IMPULSE 289 V. THE EXCITATION OF NERVE FIBRES 294 VI. THE CONDITIONS WHICH DETERMINE ELECTRICAL STIMULATION 304 VII. THE NEURO-MUSCULAR JUNCTION 310 VIII. POLARISATION PHENOMENA IN NERVE 315 IX. THE NATURE OF THE EXCITATORY PROCESS 319 CHAPTER VII THE CENTRAL NERVOUS SYSTEM I. THE EVOLUTION AND SIGNIFICANCE OF THE NERVOUS SYSTEM 324 II. THE NERVOUS SYSTEM OF VERTEBRATES 334 III. GENERAL CHARACTERISTICS OF REFLEX ACTIONS 341 IV. NATURE OF THE CONNECTION BETWEEN NEURONS 346 V. FUNCTIONS OF THE NERVE -CELL 351 THE SPINAL CORD VI. STRUCTURE OF THE SPINAL CORD 355 VII. THE SPINAL CORD AS A REFLEX CENTRE 364 VIII. THE MECHANISM OF CO-ORDINATED MOVEMENTS 382 IX. TROPHIC FUNCTIONS OF THE CORD 394 X. THE SPINAL CORD AS A CONDUCTOR 396 THE BRAIN XI. THE STRUCTURE OF THE BRAIN STEM 407 XII. THE FUNCTIONS OF THE BRAIN STEM 441 XIII. THE FUNCTIONS OF THE CEREBELLUM 447 XIV. VISUAL REFLEXES 459 XV. SUMMARY OF THE CONNECTIONS AND FUNCTIONS OF THE CRANIAL NERVES 463 THE CEREBRAL HEMISPHERES XVL GENERAL STRUCTURAL ARRANGEMENTS OF THE CEREBRUM 470 XVII. THE FUNCTIONS OF THE CEREBRAL HEMISPHERES 491 XVIII. THE VISCERAL OR AUTONOMIC NERVOUS SYSTEM 520 CONTENTS ix CHAPTER VIII THE PHYSIOLOGY OP SENSATION SECTION PAGE I. ON THE RELATION OF SENSATION TO STIMULUS 533 II. CUTANEOUS SENSATIONS 542 III. SENSATIONS OP SMELL AND TASTE 555 IV. AUDITORY SENSATIONS 562 V. VOICE AND SPEECH 578 VISION VI. DIOPTRIC MECHANISMS OF THE EYEBALL 587 VII. THE RETINAL CHANGES INVOLVED IN VISION 622 VIII. VISUAL SENSATIONS 634 , IX. MOVEMENTS OF THE EYEBALLS 653 X. VISUAL JUDGMENTS 658 XI. THE NUTRITION OF THE EYEBALL 664 THE ORGANIC SENSATIONS XII. SENSATIONS OF MOVEMENT AND POSITION 669 XIII. THE LABYRINTHINE SENSATIONS 674 BOOK III THE MECHANISMS OF NUTRITION CHAPTER IX THE EXCHANGES OF MATTER AND ENERGY IN THE BODY (GENERAL METABOLISM) I. METHODS EMPLOYED IN DETERMINING THE TOTAL EXCHANGES OF THE BODY 685 II. THE METABOLISM DURING STARVATION 698 III. THE EFFECT OF FOOD ON THE METABOLISM OF THE BODY 706 IV. THE EFFECT OF MUSCULAR WORK ON METABOLISM 713 V. THE SIGNIFICANCE OF THE FOOD -STUFFS 718 VI. THE NORMAL DIET OF MAN 726 CHAPTER X THE PHYSIOLOGY OF DIGESTION CHANGES UNDERGONE BY THE FOOD -STUFFS IN THE ALIMENTARY CANAL 737 I. DIGESTION IN THE MOUTH 740 II. THE PASSAGE OF FOOD FROM THE MOUTH TO THE STOMACH 758 III. DIGESTION £N THE STOMACH 766 IV. THE MOVEMENTS OF THE STOMACH 782 x CONTENTS CHAPTER X (continued) INTESTINAL DIGESTION SECTION PAGE V. THE PANCREATIC JUICE 788 VI. THE BILE 803 VII. FUNCTIONS OF THE LARGE INTESTINE VIII. MOVEMENTS OF THE INTESTINES 817 IX. THE ABSORPTION OF THE FOOD -STUFFS . • X. THE FAECES 851 CHAPTER XI THE HISTORY OF THE FOOD-STUFFS I. PROTEIN METABOLISM 854 IL NUCLEEST OR PURINE METABOLISM 874 III. THE HISTORY OF FAT IN THE BODY 884 IV. THE METABOLISM OF CARBOHYDRATES 899 CHAPTER XII THE BLOOD GENERAL CHARACTERS OF THE BLOOD 915 I. THE WHITE BLOOD -CORPUSCLES 918 II. THE RED BLOOD -CORPUSCLES 924 III. THE BLOOD-PLATELETS 944 IV. THE COAGULATION OF THE BLOOD 947 V. THE QUANTITY AND COMPOSITION OF THE BLOOD IN MAN 964 CHAPTER XIII THE PHYSIOLOGY OF THE CIRCULATION I. GENERAL FEATURES OF THE CIRCULATION 979 II. THE BLOOD PRESSURE AT DIFFERENT PARTS OF THE VASCULAR CIRCUIT 986 III. THE VELOCITY OF THE BLOOD AT DIFFERENT PARTS OF THE VASCULAR SYSTEM 999 IV. THE MECHANISM OF THE HEART PUMP 1004 V. THE FLOW OF BLOOD THROUGH THE ARTERIES 1034 VI. THE FLOW OF BLOOD IN THE VEINS 1050 VII. THE PULMONARY CIRCULATION 1053 VIII. THE CAUSATION OF THE HEART BEAT 1057 IX. THE NERVOUS REGULATION OF THE HEART 1087 X. THE EFFECT OF MUSCULAR EXERCISE ON THE CIRCULATION 1099 XI. THE NERVOUS CONTROL OF THE BLOOD-VESSELS 1103 XII. THE INFLUENCE ON THE CIRCULATION OF VARIATIONS IN THE TOTAL QUANTITY OF BLOOD 1129 CONTENTS xi CHAPTER XIV • ACTION PAGE LYMPH AND TISSUE FLUIDS 1133 CHAPTER XV THE DEFENCE OF THE ORGANISM AGAINST INFECTION I. THE CELLULAR MECHANISMS OF DEFENCE 1143 II. THE CHEMICAL MECHANISMS OF DEFENCE 1152 CHAPTER XVI RESPIRATION I. THE MECHANICS OF THE RESPIRATORY MOVEMENTS 1162 II. THE CHEMISTRY OF RESPIRATION 1172 III. THE REGULATION OF THE RESPIRATORY MOVEMENTS 1200 THE CHEMICAL REGULATION OF THE RESPIRATORY MOVEMENTS 1204 THE REFLEX NERVOUS REGULATION OF RESPIRATION 1214 IV. THE EFFECTS ON RESPIRATION OF CHANGES IN THE AIR BREATHED 1225 V. THE MECHANISMS OF OXIDATION IN THE TISSUES 1233 CHAPTER XVII RENAL EXCRETION I. THE COMPOSITION AND CHARACTERS OF THE URINE 1240 II. THE SECRETION OF URINE 1264 III. THE PHYSIOLOGY OF MICTURITION 1289 CHAPTER XVIII THE SKIN AND THE SKIN GLANDS 1299 CHAPTER XIX THE TEMPERATURE OF THE BODY AND ITS REGULATION 1305 CHAPTER XX THE DUCTLESS GLANDS 1317 xii CONTENTS BOOK IV • REPRODUCTION CHAPTER XXI THE PHYSIOLOGY OF REPRODUCTION SECTION I. THE ESSENTIAL FEATURES OF THE SEXUAL PROCESS 1341 II. DEVELOPMENT AND HEREDITY 1356 III. REPRODUCTION IN MAN 1361 IV. PREGNANCY AND PARTURITION 1376 V. THE SECRETION AND PROPERTIES OF MILK 1384 INDEX 1395 CHAPTER I INTRODUCTION PHYSIOLOGY in its widest sense signifies the study of the phenomena presented by living organisms, the classification of these phenomena, and the recognition of their sequence and 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 com- prehensive 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 in 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 extir- pation 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 1 2 PHYSIOLOGY their simplest form in the most highly differentiated organisms. In the unicellular animal all the essential functions which we associate with living beings are carried out, often simultaneously, in one little speck of protoplasm. An analysis of these functions, the determina- tion of their conditions and mechanism, is obviously impossible under such 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 physiological 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 conception 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 respiration 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 environment. For the production of these movements, as for the maintenance of a constant body-tempera- ture, 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 food-stuffs 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. INTRODUCTION 3 Before we can make any accurate investigations of the conditions which determine these activities, We must know whether the two great laws of chemistry and physics, viz. the conservation 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 experi- ments 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 obtained, and we can be certain that any substance 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 the food- stuffs taken in to the carbon dioxide, water, &c., that are given out. We must then compare the figure so obtained with the actual outpi t 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 evaporation which is going on at the surface of the animal. The first accurate experiments of this nature were made by Rubner. This observer determined 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. Cal. Cal. Days. 1. Fasting < 259-3 261-0 5 2. 545-6 528-3 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 4 PHYSIOLOGY It will be seen that the average difference between the calculated and observed results amounts only to 1-01 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 food-stuffs 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 food -stuffs into kinetic energy, represented by the warmth and move- ments 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 food-stuffs after their entry into the body, we lose sight of 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 food-stuffs from this living matter, which therefore 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 integration, and the other, associated with activity, of destruction or disintegration. 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 food-stuffs, such as starch, with a high potential energy, is the necessary condition for the existence 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 indefi- nitely. 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 INTRODUCTION 5 allow the necessary conditions of life, viz. assimilation and disintegra- tion, to proceed. In all the higher forms, however, 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 environ- ment, until finally death of the organism takes place. All these phenomena, viz. assimilation, respiration, activity asso- ciated 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 definition 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 chemical system which tends to increase itself continuously under the average of the conditions to which it is subject, but under- goes 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 the 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 neutralisation, 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 com- pound 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 environ- ment. Out of the many such compounds which might have come into 6 PHYSIOLOGY being, only such would survive in which the process of exothermic disintegration tended towards a condition of greater stability, so that the process might come to an end, and the organism or compound be enabled to await the more favourable conditions necessary for the continuance of its growth. With the continued 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 pene- trating sun's rays and utilising them for the endothermic formation 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 there- fore 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 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 mankind against disease and death. The same law which determines the downward 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 defined 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 tempera- ture, 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 INTRODUCTION 7 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 tempera- ture of the body is maintained at a constant level, which represents the optimum for the discharge of the normal functions of the consti- tuent parts of the body. The presence of food material in the environ- ment 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 surrounding medium. In the higher forms however, the development of the complex digestive system has enabled the organism to utilise many different kinds of food, while the storage of any excess of food as reserve material in the organism, either in the form of fats or carbohydrates, provides for a constant supply of food to the constituent cells of the body, even when it is quite wanting in the environment. Since plants depend for their food in the first place on the carbohydrates produced within the chlorophyll corpuscles out of the atmospheric 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 the 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 constituent of all living matter, and takes part in all the changes which determine 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 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 their reaction to stains, are distinguished by the name of a nucleus, in the higher members this organisation becomes more and more marked. The 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, accepted by physiologists, that the structure is the deter- mining factor for the function. We might regard the histological differentiation as representing merely a continuation of the increasing molecular complexity, which We assumed must accompany and 8 PHYSIOLOGY 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 description of the chief reactions of the body to changes in its environ- ment, 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 deter- mine 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 disintegration or oxidation of the food-stuffs. Our next task must be, therefore, 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 the 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 gravita- tion ' 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 generalisations become wider and its laws summarise ever more extensive groups of phenomena. We have no reason for asserting that, in the course of research, 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 phenomena 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 * Karl Pearson, "Grammar of Science," p. 328 et seg. (2nd ed.) INTRODUCTION 9 deeply the physiologist endeavours to peer into the processes within the living cells, has led some, even at the present day, to the assump- tion 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 distinction 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 deter- mining 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 inter- ference, 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 food -stuffs. 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 changes occurring in their living substance. 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 as germane to the subject as if he were to call himself a Trinitarian or a Plymouth Brother. Throughout this chapter we have assumed no necessary dividing line between the different classes of phenomena in the conceptual universe, 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 objected that in taking up this attitude we had left out of account one supreme fact, viz. the existence of conscious- ness in ourselves. As a comparative and objective study, however, 10 PHYSIOLOGY 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 con- sciousness. 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 impossible to draw a sharp line between those animals which possess consciousness 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, anaesthetised, 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 hypothetical 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. Moreover, since this hypothetical quality does not claim to be a form of energy, it need not trouble us in our study of the energy-changes in the body and the conditions which determine them. BOOK I GENERAL PHYSIOLOGY CHAPTER II THE STRUCTURAL BASIS OF THE BODY THE CELL ALL the higher animals and plants, when submitted to microscopic examination, 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 uni- cellular condition represents the more primitive stage from which the higher organisms have been evolved in the course of ages is indi- cated by the fact that every one of these higher organisms in the course of its development passes through a unicellular stage, namely, the fertilised ovum. We may assume that the series of changes attending the development of the higher organism from the egg is a repetition in summary of the changes which have determined the evolution of the species from the primitive unicellular type.* The general characteristics of the cell present important simi- larities, whether we are considering a cell which forms the whole of an organism or a cell which is but an infinitesimal part of a highly developed animal. The name ' cell ' was first applied by botanists to the structural units found by them in plant tissues, and involved therefore the idea of certain qualities which do not enter into our present conception of the term. A section through the stem of a growing plant shows it to be made up of an aggregation of cells in the etymological sense of the * This assumption is often spoken of as the 'law of recapitulation.' 13 H PHYSIOLOGY word, i.e. small sacs bounded by a wall of cellulose and containing cell sap. Immediately inside the cellulose wall is a thin layer, the primordial utricle, which encloses at one point a spherical or oval structure known as the nucleus. If the section be 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 form the primordial utricle. This with a nucleus is 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 ; 6, nuclei preparing for division (spireme-stage) ; c, dividing cells showing mitotic figures ; e, pair of daughter-cells shortly after division. enclosed in a delicate cellulose wall. The wall is not an essential 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 of a unicellular animal such as an amoeba (Fig. 2). This is an organism frequenting stagnant pools, of varying size (from 0*1 to 0'3 mm. in diameter), 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 THE STRUCTURAL BASIS OF THE BODY 15 amoeba, the protoplasm or cytoplasm, often presents further differen- tiation 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 ""•••^'' •''•• ^ •?'*. •:?••.?•$ '-'^'^'''. v'"-'' ": v \ cv FIG. 2. Amcdba proteus, an animal consisting of a single naked cell, x 280. (From Sedgwick and Wilson's Biology.) n, the nucleus ; wv, water- vacuoles ; cv, contractile vacuole ; /w, food- vacuole. which we are acquainted, this twofold structure is also found. So that 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 Cyanophycese and Bacteria among the latter, no distinct nucleus can be demonstrated. In many of these forms the dimensions of the whole organism are too minute to allow of any definite statement being made as to the presence or absence of nuclear material. In the larger of them, however, the cytoplasm of the cell contains numerous scattered granules which stain with dyes in exactly 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. 16 PHYSIOLOGY We have defined a cell as a small mass of protoplasm containing a nucleus. Since we shall have to use the term ' protoplasm ' on many occasions in the course of this work, we must have a definite concep- tion of what we mean by it. The term is often used by histologists as implying a substance of certain definite chemical and staining characters. When employed by physiologists it generally implies any material which we can, on a study of its behaviour to changes in its environ- ment, regard as endowed with life. Huxley has defined it as " the physical basis of life." Though it may be convenient to have a word such as protoplasm signifying simply ' living material,' it is important to remember that there is no such thing as a single substance — proto- plasm. The reactions of every cell as well as its organisation are th resultant of the molecular structure of the matter of which it is bui' up. The gross methods of the chemist show him that the compositioi of the ' 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 peculiarities 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 reactions of its constituent cells. There is not one protoplasm therefore, but an infinity of proto- plasms, 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 cyto- plasm. Both are necessary for the life of the cell and both must be regarded, 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 proto- plasm ; 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 other parts are employed for the purpose of locomotion. Here again there must be chemical differences, and therefore different protoplasms. In this work, therefore, 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 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 THE STRUCTURAL BASIS OF THE BODY 17 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 Attraction-sphere enclosing two centrosomes Nucleus - ( Plasmosome or true nucleolus Chromatin- network Linin-network Karyosome, net-knot, or chromatin- micleolus Plastids lying in the cytoplasm Vacuole Passive bodies (meta- plasm or paraplasm) suspended in the cy- toplasmic meshwork FIG. 3. Diagram of a cell. Its basis consists of a meshwork containing numerous minute granules (microsomes) and traversing a transparent ground-substance. (WILSON.) often distinguished differentiated parts which may be regarded as organs of the cell. Thus in the amoeba we have the contractile vacuoles already mentioned. In the green parts of plants the cyto- plasm 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 ( 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- 2 18 PHYSIOLOGY cellular animals, for the service of the 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 corre- sponding uniform morphological organisation of the physical basis of these phenomena, namely, protoplasm. It is often impossible, even under the highest powers of the micro- scope, 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 demonstrated in almost all cells of the body (Fig. 4). These granules have been regarded by Altmann as the elementary particles of life, and he locates in them the various vital functions, the sum of which make up the life of the cell. According to Altmann these granules can only arise from the division of pre-existing granules, and he has formulated the phrase omne granulum e granulo, which is a further extension of Virchow's sentence omnis cellula e cellula. It is probable that a number of different kinds of structures of varying importance are included among Altmann's granules. In some cases they 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 plastids with the special metabolic functions assigned to all granules by Altmann. In many cases no treatment whatever will display the existence of granules. 2. THE FIBRILLAR 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 filling its meshes known as THE STRUCTURAL BASIS OF THE BODY 10 ' hyaloplasm.' A network is, however, one of the commonest pseudo- structures produced in the coagulation 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. Some- times a large portion of the protoplasm may take a fibrillar form which can be detected even in the unstained and unfixed cell, and there FIG. 4. Section of liver stained to show granules. (ALTMANN.) 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 will be deposited as spherules gradually increasing in size, so that the protoplasm 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 examined fresh or in the hardened and stained condition. Such a protoplasm would be practically an emulsion of one fluid in another, and according to Biitschli 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 20 PHYSIOLOGY 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 vacuoles ; c, double centrosome ; n, nu- cleus ; ri, nucleolus. 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 modifications 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 differen- tiation is still present, but is invisible owing .to the minute size of its constituent parts or an identity of refractive index between the alveolar walls and their con- tents. The fact that every chemical differ- entiation occurring within the colloidal mass will tend to cause differences of surface tension, and therefore formation of droplets, shows that an alveolar structure, i.e. one in which there is a large number of surfaces sepa- rating heterogeneous mixtures inside the cells, must be of very common occurrence, even in cases where it is not detect- able under the microscope. Such a structure must be present, at any rate, in those cases where, apart from the existence of a solid cell wall, the cell presents a certain degree of rigidity and resist- ance to deforming stress. ULTRAMICROSCOPIC STRUC- TURE 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) FIG. 6. A, protoplasm of an epidermal cell of the crayfish; B, foam-like appearance of an emulsion of olive oil. (BUTSCHLI.) THE STRUCTURAL BASIS OF THE BODY 21 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 differen- tiation 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 knowledge 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 advantage of this resistance to run freely over the surface of water, although their specific gravity may be greater than that of 22 PHYSIOLOGY water. The continued existence of protoplasm in a watery environ- ment 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 protoplasm 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 in- activity, 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 immiscible 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 concentration 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 practically solid and resists any turning of the needle. In consequence of the surface aggregation and solidification of the CDlloidal 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 produced gradually collect to form a solid mass of insoluble protein. Protoplasm may be regarded as essentially fluid in character, the form and rigidity which are acquired by most cells being due to chemical and physical differentiation 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 environ- THE STRUCTURAL BASIS OF THE BODY 23 ment, this layer acquires a prime importance for the life of the cell, and we may therefore consider here at greater length some of the properties of this layer, the Plasmahaut, as it has been called. The superficial layer of the protoplasm is not to be confounded with the cell 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 com- position 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 C^HjoOg. 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 interchanges 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 excre- tion. 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 exist- ence 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 24 PHYSIOLOGY water outwards so that the primordial utricle shrinks (Fig. 7). On immersing 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 to molecules of water, i.e. it behaves as a semi-permeable membrane. Similar experiments can be made on animal cells. The most convenient for this purpose are the red blood corpuscles. These also shrink when immersed in salt solutions with a greater molecular concentration than would 234 FIG. 7. Vegetable cells, showing varying degrees of plasmolysis. (DE VRIES.) 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 up and 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 in its effects on the cells to a decinormal solution of potassium nitrate or of potassium chloride. The imper- meability 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 amino-acids, it permits the easy passage of mon- atomic 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 mem- brane, 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 THE STRUCTURAL BASIS OF THE BODY 25 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 neces- sary 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. Overton 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 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 sub- stances 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 variable solubilities of the dissolved substance in the two menstrua. In the same way a mass of protoplasm will tend to absorb from the sur- rounding medium and to concentrate 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 26 PHYSIOLOGY 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 tension 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 surface 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 pro- trusion 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 food-stuff, may cause local variations in the surface tension of the plasma skin and thus 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 multicellular, can be regarded as compounded of two phases, assimilation and dissimilation. By assimilation we mean the building up of the living substance at the expense of material obtained 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 orga- nisms, 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 to 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 disintegration 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 THE STRUCTURAL BASIS OF THE BODY 27 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 amosba, or at a specialised portion, so-called ' mouth,' as in many of the infusoria. Digestion is apparently effected in most cases by the produc- tion and secretion around the ingested food particle of solutions con- taining ferments, i.e. agents which have the power of hydrolysing the different food-stuffs and rendering them soluble. In the vast majority of living organisms the energy for their activities is derived from the oxidation, ultimately of the food-stuffs, 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 surrounding 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 intramolecular oxygen) to be utilised for the formation of carbon dioxide when a discharge of energy is necessary, or whether it is only taken in 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, carbon dioxide and water. There are also many substances resulting from the oxidation of the nitro- genous 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 proto- plasm, 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 b. tetanus, and the bacillus of malignant oedema. In order to cultivate them it is necessary to displace 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 substances. 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 con- taining no oxygen, but rich in carbon dioxide. Here they are plentifully supplied with food-stuffs and can afford to adopt a wasteful method of nutrition, in which 28 PHYSIOLOGY 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 environmental 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 prevent 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 metabolism 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 appli- cation 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 disproportion between stimulus and reaction can be well illustrated on an excitatory tissue 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 gramme 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. 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 THE STRUCTURAL BASIS OF THE BODY 29 observed in the growing parts of plants, where the root always grows downwards and the stem upwards. This reaction to gravity is known as geotams, which is distinguished as ' negative ' or ' positive ' respectively, according as the plant grows in oppo- sition or in obedience 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 fertilisa- tion 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 sus- pended in a fluid will always swim towards any locality where there is a greater 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, (6) those which exercise negative chemiotactic influence on the leuco- cytes. Thus the introduction under the skin of an animal of a capillary tube containing a solution of substances 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 negative chemiotaxis. Tubes filled with these, after introduc- tion 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 30 PHYSIOLOGY simple proteins, but built up with other complex bodies to form con- jugated 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 B FIG. 8. Nucleated and non-nucleated fragments of Amaha. (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 contractile vacuole. chiefly by the 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, prota- mine. 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 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. THE STRUCTURAL BASIS OF THE BODY 31 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, found A FIG. 9. Regeneration in the unicellular animal Stentor. (From GRTJBER 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. 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). 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 regeneration. The wound quickly heals and the special organs — the mouth, with its surrounding cilia, and the contractile 32 PHYSIOLOGY vacuole — are regenerated, but all non- nucleated fragments quickly perish (Fig. 9). M Many similar observations have shown that the non-nucleated cytoplasm, though it may survive for some time and perform normal movements in response to stimuli, such as those of ingestion of food A ft C 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. <7. Root-hair of Marchantia ; all the fragments, connected by protoplasmic strands, have formed membranes. D. Leaf -hair of Cucurbita ; non-nucleated fragment, with membrane, connected with nucleated fragment of adjoining cell. particles, loses entirely the 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 rapidly comes to an end as the store of material in the cytoplasm is exhausted. In vegetable cells it is possible to break up the protoplasm by means of plasmolysis into nucleated and non-nucleated parts. The nucleated part quickly forms a new cell wall. The non-nucleated part is unable to effect this formation, and soon dies unless it is in connection with an adjacent THE STRUCTURAL BASIS OF THE BODY 33 cell containing a nucleus by means of fine threads of protoplasm which pass through pores in the intercellular septa (Fig. 10). In the higher animals 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 regeneration 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 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 next is effected by the entrance simply of the nuclear material of the male cell, the spermatozoon, into the ovum. In the words of Claude Bernard, " 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 apparatus for organic synthesis, an instrument of produc- tion, 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. 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 FIG. ll. Branched nucleus from 1 , , . , , . the spinning gland of butterfly the outgrowth always takes place in iarva (pieris). (KOESCHELT.) the immediate neighbourhood of the nucleus, which is carried forward and remains near the tip of the growing hair. The active growth of cytoplasm, 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 which interchanges can go on between 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 3 34 PHYSIOLOGY both morphologically 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 chromo- somes undergo characteristic changes during the entire growing period *8**t'J ^^i«X*wX FIG. 12. Chromosomes of the germinal vesicle 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. 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 meta- bolism or dissimilation, which determine the activity of the cell, have * Biickert, cited by Wilson, THE STRUCTURAL BASIS OF THE BODY 35 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 is shaped somewhat like a wine-glass, 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 sur- rounding fluid and so favour the passage of food particles towards the mouth. Food when ingested at this end passes only a 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 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 orga- nisms which are 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 me-ntioned the nucleus, with its chromosomes, arid the plastids, of which the 36 PHYSIOLOGY 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 centro- some, 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 ccelenterata 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 differen- tiation 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 transmission 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 therefore 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 ccelomata, the class to which all the higher animals belong. In these, by the forma- tion of a body cavity containing fluid, an internal medium is provided for all the working cells of the body. The composition 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 composition 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 constituents 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 coelom is later on formed a circulatory system which, by the circulation of the coelomic fluid or of blood throughout the whole THE STRUCTURAL BASIS OF THE BODY 37 body, can procure a still more perfect 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 independence of external con- ditions 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 environ- ment. 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 elabora- tion after absorption, and their preparation for utilisation by other cells of the body. Between these two surfaces are situated the support- ing 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 coelom or body cavity, represented in the higher animals by the pleural and peritoneal cavities. The alimentary canal projects for a considerable part of its course into this coelom, being attached to the body wall only by one side. From the ccelom is also developed the blood vascular system, surrounded by contractile and connective 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 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 functions and of the 38 PHYSIOLOGY 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. We have compared the higher animal in the foregoing lines 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 function 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 essentially composed of and determined by the recip- rocal 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 specialisa- tion. 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 con- venience. 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 proto- plasmic 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 forma- tion of living matter. Every living organism without exception contains the following elements : carbon, hydrogen, oxygen, nitrogen, sulphur, phosphorus, chlorine, potassium, sodium, calcium, magne- sium, 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 orga- nisms, 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, i.e. those which are 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 sur- rounding 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 which is 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, 39 40 PHYSIOLOGY which was originally the chemistry of substances produced by the agency of living organisms, has come to be synonymous with the chemistry of carbon compounds. The carbon compounds which make up the living cell are combustible, 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 provided 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 (NH8) into the molecules of fatty acids, amino-acids may be formed, from which the complex proteins are built up to form the chief constituents of the living protoplasm. THE ELEMENTARY CONSTITUENTS OF PROTOPLASM 41 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, undergoing a gradual oxidation, and finally leave the body in the form of carbon dioxide, water, ammonia or some related compound, and sulphates. A sharp line of demarcation has therefore often been drawn between the metabolism of plants and animals, plants being regarded as essentially assimilatory in character while animals are dissimilatory, utilising the stores of energy which have been accumulated by the plant. There is, however, no sharp 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 respec- tively 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 evolution 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 42 PHYSIOLOGY silica. The action of water charged with carbon dioxide on a silicate is to cause its gradual decomposition 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 carbo- nate 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 carbonate 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 vol- canoes must get less and less, so that one can conceive a time when the whole of the carbon dioxide will be bound up 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 food-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 respiration. Like the three elements we have already considered, nitrogen is also derived directly or indirectly from the surrounding atmosphere. In consequence 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 the only source of nitrogen to this class 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 combined nitrogen which is available. In view of 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 THE ELEMENTARY CONSTITUENTS OF PROTOPLASM 43 factors which result in the production 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 quantities of ammo- nium nitrite, which will be washed down with the rain and serve as a source of combined nitrogen to the soil. Every decaying vege- table 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 car- bonate 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 con- verted into this highly oxidised form. This 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 nitromonas) 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 A, the fluid will be found to contain, not nitrates, but nitrites. In this conversion the two kinds of microbes mentioned above are concerned. At the top of the cylinder the nitrous bacterium is present, in the bottom of the FIG. 13. Arrangement for studying the nitrifica- tion of sewage. (Miss H. CHICK.) 44 PHYSIOLOGY 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 pre- paring 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 quantities produced by atmospheric discharges ? Of late years definite evidence has been brought forward that such is not the case and that organisms exist whicih can utilise and bring into com- bination 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 he isolated by growing on gelatinous silica free from any trace of combined nitrogen, so that the organism had to procure its entire nitrogen from the atmosphere. Under such con- ditions the numerous other micro-organisms of the soil died of nitrogen starvation, and only the microbe survived which was 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 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 dis- sociation either of ammonium nitrite or of nitrous acid into nitrogen and water, as is seen from the following equation : HN02Aq. + 308 Cal. = H -f N + 02 -f- 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 increased 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 sainfoin. 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 combined 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, THE ELEMENTARY CONSTITUENTS OF PROTOPLASM 45 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 therefore 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 nitrogen at all, the beans developed nodules on their roots 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 bacteria 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 soil free from combined nitrogen, e.g. FlG- }*- .^)ot °f & . . vetch with nod- conifers, but it is in the legummosae that their ules. 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 dis- charges in the 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 deoxidation 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 amino- acids such as cystine, and these, together with other amino-acids, are synthetised to form proteins. Practically the whole of the sulphur taken in by animals is 46 PHYSIOLOGY in the form of proteins. It shares the oxidation of the protein molecule in the animal body which it leaves in the form of sulphates. The output of sulphates by an animal can therefore 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. Iron, although forming but a minute proportion of the material basis of living organisms (the whole body of man contains only six FIG. 15. Section of a root nodule of Dorychnium. (VUILLEMIN.) a, cortical tissue ; b, cells containing bacteria. 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 chloro- phyll molecule, plants grown in the absence of this 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 con- tinually 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 THE ELEMENTARY CONSTITUENTS OF PROTOPLASM 47 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, as well as the latter, is excreted almost entirely with the faeces in the form of sul- phide. 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 protoplasm it is built up with fatty acids and other organic radicals to form complex compounds such as lecithin, a phosphorised fat, and nuclein, a combination of phosphorus with nitrogenous bases of great variety. Both lecithin 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 pro- cesses of dissociation and oxidation, with the production, as a final result, of phosphates, which are excreted with the urine or fa3ces 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 proto- plasm. They are therefore taken up also by animals in the form of salts, and as such are again excreted with the urine. 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 48 PHYSIOLOGY 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 hsemo- cyanine, 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 oxida- tion are volatile, namely, 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 bases 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 IN spite 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 con- stituents includes the ttudy 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 relation- ships of these groups to one another and to the hydrocarbons is given here. THE HYDROCARBONS (FATTY SERIES). These form a continuous homo- logous 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 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 un- changed. In order to render them, accessible to the action of the living cell they must first undergo oxidation. 49 4 50 PHYSIOLOGY The unsaturated hydrocarbons have the general formulae CnH2n, C H2n .2, CH2n_4, &c. Examples of the first two groups are ethylene CH2 II CH2 and acetylene CH III 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 C2H5OH ethyl » C3H7OH propyl » C4H9OH butyl V C6HUOH amyl n C6H13OH capryl >» and so on, the general formula for the group being CnH2n + 1OH. 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 an 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 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 is formed together with phosphorus oxychloride and hydrochloric acid. Thus : Et.OH + PC15 - POC13 + HC1 + 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 sulphate and water. Thus : Et.OH + H2S04 *= Et.HS04 + 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 + HC2H3O2 = 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. PROXIMATE CONSTITUENTS OF THE ANIMAL BODY 51 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 = NaC2H3O2 + 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 ' 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 C3H6 (OH3), which is known as glycerin, or glycerol. Other alcohols of physiological importance are cholesterol and cetyl alcohol. Cholesterol 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 constituent 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 Another alcohol — cetyl alcohol — C^H^O — (CH2)14 occurs in the feather I ' CH2OH glands of 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 we obtain another group of compounds — the aldehydes. From ethyl alcohol, for instance, by warming with potassium bichromate and dilute sulphuric acid, ethyl aldehyde /H is produced and given off. In these aldehydes the group C\-H is converted into H IX°H the group C *= O, and it is the 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) 52 PHYSIOLOGY 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, hydrazones 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 With sodium sulphite the following reaction takes place : CH3 CH3 + NaHS03 = /OH CHO CH<( XS03Na These compounds of aldehydes with sodium sulphite can be readily obtained in a crystalline form and furnish a convenient means of separating the alde- hydes from their solutions. (4) All the aldehydes possess a strong tendency towards polymerisation. Ethyl or acetic aldehyde treated with strong sulphuric acid gives the com- pound 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, CH2O, 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 : 6CH20 = 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. CH3 CH3 I +0- | CHO COOH. Since these acids are derived from the paraffins a whole series of them exists PROXIMATE CONSTITUENTS OF THE ANIMAL BODY 53 corresponding 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 COOH CH2 CH2 I OOOH 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 : CH2.NH2 CH3 I CH2 or CH.NH2 I I COOH COOH. The second form, the o-amino acid, is the only one which occurs in the body. OXYACIDS are formed by the replacement of one H atom by the group OH. Thus: CH3 I CHOH is oxypropionic acid or lactic acid. I OOOH 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 oxida- tion of a ketone. Thus : CO CO CO 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 . CH8 from CO.NH2 COOH, (acetamide) (acetic acid) 54 PHYSIOLOGY AMINES. These may be regarded as formed from ammonia NH8 by replacing one or more of the H atoms by an organic radical. Thus we may have: /CH3 /CH3 /CH3 N(-H Ne-CH3 N^CH3 \H \H 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 - CO2 - 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 com- pound is benzene C6H6. It behaves as a saturated compound. It is represented as a hexagon with a hydrogen atom at each angle. H I ) H 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 : C6H5.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 \^J H H ixy X H k^y H H H X ortho- meta- para- The following are some of the most important monosubstitution derivatives of benzene : Nitrobenzene C6H5.NO2. Aniline C6H$.NH2. Benzene sulphonic acid C6H6 .SO3H. Phenol C6H5.OH. Toluene C6H6 . CH8. PROXIMATE CONSTITUENTS OF THE ANIMAL BODY 55 Benzyl alcohol Benzylaldehyde Benzole acid C6H5.CH2OH. C6H5.CHO. C6H5.COOH. Of the disubstitution compounds, we need only mention the following : The dihydroxybenzenes : Pyrocatechin or catechol Resorcinol Hydroquinone OH OH OH ortho- meta- Salicylic acid (o-hydroxybenzoic acid) C6H5< Tyrosin (parahydroxyphenyl a-alanine) : OH COOH. CH2.CH(NH2)COOH. Examples of trisubstitution derivatives of benzene are : OH XX OH Pyrogallol OH Homogentisic acid CH2.COOH Adrenaline Picric acid OH CH.OH CH2.NH(CHS) OH N09 f7^ N02 N0a 56 PHYSIOLOGY 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) FIG. 16. Diagram of polarimeter. B, polariser; D, analyser; o, tube containing solution under examination. can be rotated round the axis of the beam of light passing through the first. When both prisms are parallel light passes through the analyser. On inter- posing 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 I I COOH COOH is asymmetric, i.e. it is unequally loaded on the four sides. We can imagine such a carbon atom as occupying the interior of a tetra- hedron. A B Fio. 17. PROXIMATE CONSTITUENTS OF THE ANIMAL BODY 57 In this tetrahedron, if we represent the four groups combining with the carbon by B!, 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 tetra- hedron B, but that, if we hold A before a mirror, its image in the mirror will be represented by B. One arrangement 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 will 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 laevo- rotatory, 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 i 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 2n 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, Z, racemic or i, and mesotartaric, also inactive, in which internal compensa- tion occurs. These four varieties may be represented as follows : COOH COOH COOH HCOH HOCH HCOH HOCH HCOH HCOH COOH COOH COOH rf-tartaric acid I -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. SECTION III THE FATS THESE substances are widely distributed throughout the animal and vegetable kingdoms. In the higher animals they form 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 CHOH CH— 0— OC.CH, 0-U2O — OO . O.H.3 Cxi2OJti a-monacetin /3-monacetin monoglycerides (3) (4) (5) CH2— 0— OC.CH3 CH2OH CH2— 0— OC.CHj CHOH CH— 0— OC.CH3 CH— 0— OC.CH3 CH2— 0— OC.CH3 CH2— 0— OC.CH3 CH2— 0— OC.CH3 a, a diacetin a, /3 diacetin triacetin triglyceride 58 THE FATS 59 In these compounds the phenomenon of isomerism occurs owing to the presence of primary and secondary alcohol groups in glycerol. In the case of the diglycerides and the triglycerides mixed esters, in which the fatty acid radical varies, are possible : (6) (7) CH2— 0— OC . CH3 CH2OH CHOH CH— 0— OC.CIL, CH2 — 0— 00 . CH2 . CH3 CH2- 0— OC . CH2CH3 (8) CH2— 0— OC.CH3 CH— 0— OC.CH2.CH3 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 composi- tion 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 tri- glycerides 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 Behenicacid, CH3(CH2)20.COOH Lignoceric acid, CH3(CH2)22 . COOH 60 PHYSIOLOGY 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 series, e.g. linolenic acid (CnH2n_602) Of the long list of fatty acids given above only a few occur to any extent 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 derived from the fatty acids palmitic, stearic, and oleic, i.e. tripalmitin, tristearin, 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., tristearin 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 tem- perature, or a fat containing 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 devoid of smell. They are insoluble in water, in which they float. They are soluble in warm absolute alcohol, but separate out into crystal- line form on cooling. They are easily soluble in ether. If they are strongly heated with potassium bisulphate they give off 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., C3H6(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 thus 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 saponifica- THE FATS 61 tion; giving the alkaline salt of a fatty acid and glycerin. The former campound 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 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 deter - N mined 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) The 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 secre- 62 PHYSIOLOGY tion of the sebaceous glands in man and the higher animals, which furnishes the natural oil of hair, wool, and feathers, consists, with small traces of glycerides, of cholesterol esters. 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 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 PHOSPHATIDES 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 Thudichum, who isolated various compounds of this nature from brain, suggested the term phospha- tides 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 compound 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 compounds, 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 * According to Gardner, cholesterol may be absorbed from the intestine. THE FATS 63 different fatty acid radicals, as oleyl-lecithin, stearyl-lecithin. The following formula represents distearyl-lecithin : CH2-0-OC.(CH2)16CH3 CH— 0-OC.(CH2)16CH3 CH2— 0 HO/ \).CH2.CH2.N(CH3)3 OH On warming with baryta water lecithin is broken down into fatty acid, glycerophosphoric acid, and choline. The latter base, which is trimethyl-oxethyl-ammonium hydrate, N - (CH3)3 must be distin- OH guished from neurine, N -j (CH3)3 which is trimethyl- vinyl- ammonium [OH hydrate, and is 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 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 inter? preted 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. 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 pro- portions 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 contain- ing 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 glycerol we may obtain glyceryl aldehyde COH CH2OH I CHOH and dioxy acetone CO. Both these substances behave as CH2OH CH2OH sugars and belong to the group of trioses. They are generally obtained together and are called glycerose. From the hexatomic CH2OH COH alcohol (CHOH)4 we may obtain either the aldehyde (CHOH)4 or I I CH2OH CH2OH THE CARBOHYDRATES 65 CH2OH CO the ketone (CHOH)3. These two oxidation products of the polyatomic CH2OH alcohols are known as aldoses and ketoses respectively. All these compounds are distinguished by the termination ' ose.' It is con- venient 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 atoms contained in the sugar molecule, e.g. the aldose CH2OH (CHOH)4, four are asymmetric, i.e. their four combining affinities are COH saturated with groups of different kinds, viz. several carbon atoms, one H atom, and one OH group : C H- C— OH C They must therefore present many stereoisomeric forms. If n repre- sent 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, in 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 twelve have been found or have been artificially prepared. Only a small number are, however, of any physiological importance. These in- clude the aldoses, glucose, mannose, and galactose, and the ketrse, 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 repre- sent them as d-, 1-, and i- varieties respectively, i.e. dextro-rotatory, laevo-rotatory, and inactive. On Fischer's suggestion, however, this mode of nomenclature has been altered in favour of representing, by the 5 66 PHYSIOLOGY etter 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 corresponding to the dextro- rotatory glucose, d-fructose itself being laevo-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 particular form which are assimilable, and therefore of physio- logical importance. The small differences in the configuration -of the four d-sugars can be readily seen if their graphic formulae be com- pared : CHO CHO CH,OH CHO H.C.OH HO.C.H H.C.OH H.C.OH CH2OH d-glucose HO.C.H HO.C.H H.C.OH H.C.OH CH2OH d-mannose CO HO.C.H H.C.OH H.C.OH CH2OH d-fructose H.C.OH HO.C.H HO.C.H H.C.OH CH2OH d-galactose THE PENTOSES. C5H1005 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, 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, however, 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 absorption 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 CARBOHYDRATES 67 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 C6H1206, examples of which are glucose, fructose, &c. (2) Disaccharides, which are derived from two molecules of a mono- saccharide with the elimination of a molecule of water, as follows : 2C6H1206 — H20 = C12H22On. (Examples, maltose and cane sugar.) (3) Polysaccharides, composed of three or more molecules of a mono- saccharide. The number of molecules which are associated in the compounds of this group may be very large. We can regard their general formation as represented by the following equation : . - 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 formalde- hyde polymerises with the formation of a mixture of hexoses known as acrose. From this mixture a-acrose can be isolated by the forma- tion 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 1-fructose behind. For the preparation of d-fructose 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 substances, and, like aldehydes, reduce ammoniacal solution of silver to metallic silver, and many of the higher oxides of metals 68 . PHYSIOLOGY to lower oxides. On this behaviour is founded the commonest of all the tests for the presence of reducing sugar — Trommer's test. (b) On oxydising a monosaccharide the COH group becomes con- verted 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, and 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 C6H1406. (d) Another important general reaction of the monosaccharides depending on the COH or the CO group is the reaction with phenyl hydrazine. On warming a solution 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 + H2N.NH.C6H5 == CH2OH(CHOH)3CHOH.CH : N : NH.C6H5 + H20. The hydrazone then reacts with another molecule of phenyl hydrazine with the production of an osazone : CH2(OH)(CHOH)3CHOH.CH : N.NH.C6H5 -f 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 carbohydrates. 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 con- stituent 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 CARBOHYDRATES 69 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 propor- tions 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 Allilm'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 C02, and form alcohol with small traces of amyl alcohol, glycerin, and succinic acid. With phenyl hydrazihe 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 insoluble 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. In order to identify glucose in a normal fluid, the following tests may be applied, after removing any protein which may be present : (1) Reduction of cupric hydrate or Fehling's solution. (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 mono- saccharide 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 LEVULOSE, occurs mixed with dextrose in honey and in fruit sugar. It is also, with glucose, formed by the 70 PHYSIOLOGY digestion or inversion of cane sugar. It is difficultly crystallisable. Its watery solution is laevo-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 con- stituent 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 extremely slowly. One species of yeast is known, namely, saccharomyces apiculatus, which, while fermenting d-fructose and glucose, has no effect on galactose. 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 occur- rence 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, C6H13N05, has the structural formula : CH2OH (CH.OH)3 CH.NH2 CHO It is obtained from chitin, which forms the exoskeleton of large numbers of the invertebrata, by boiling this with concentrated hydro- chloric acid. It is stated to have been obtained as a decomposi- tion 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- THE CARBOHYDRATES 71 rotatory, reduces Fehling's solution, and gives an osazone resembling that derived from glucose. GLYCURONIC ACID, C6H1007, 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 (CH.OH)4 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 Isevo- 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 distinguish 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 formula given on p. 66 do not explain all the possible modes of arrangement 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 solution, 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 + 53° D. This change in rotatory power seems to be associated with a change in the arrangement of the groups, the aldose, for example, assuming, by the shifting of a mobile oxygen atom, what is known as a lactone arrangement. Thus glucose COH(CHOH)2CHOH.CHOH.CH2OH be omes CHOH . (CHOH)2 . CH . CHOH . CH2OH 0 This change in the arrangement of the molecule renders a further 72 PHYSIOLOGY stereoisomerism possible, owing to the fact that now the end group which was formerly COH becomes H 0— C— OH C so that now there are five instead of four asymmetric carbon atoms. The two isomers of glucose, which are thus rendered possible, are represented by the following structural formulae : H— O- OH OH— a— H or HCOH HCOH CH2OH CH.OH In these molecules the OH attached to the 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 /3 methyl glucosides, the formulae of which would be represented as follows : H— 0— OCH CH,0— C— H CH2OH 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 THE CARBOHYDRATES 73 a glucoside of glucose, lactose as the ft galactoside of glucose. The mode of combination of the two hexose groups to form these disac- charides may be represented as follows : H H OH H CH2OH — C — C — C — C OH X H OH H C glucose rest HO H HO HO OHO — C — C— C — C— CH2 glucose rest H OH H H maltose. H CH2OH — C — C — C - - C — C galactose rest ) OH H H OH 0 lactose. HO H HO HO OHC — C — C — C — C — CH2 glucose rest H OH H H 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 monosaccharides with the elimination of one molecule of water, and can be regarded, 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 monosaccharides. Thus, cane sugar gives equal parts of glucose ani 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 dis- tributed throughout the vegetable kingdom, and forms an important article of diet. It has no reducing power on Fehling's solution. It is strongly dextro-rotatory and has a specific rotatory power of -f 66-5°. On hydrolysis it is converted into equal molecules of glucose and 74 * PHYSIOLOGY 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 Igevo-rotatory. On this account the change from free cane sugar to the mixture of mono- saccharides 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 monosac- charides. With yeast, 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 gtarch 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 260° 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 fermentation can occur the lactose must be split by the agency of acids or by a ferment, lactase, which occurs in the animal body and in certain moulds, into the monosaccharides glucose and galactose. Lactose reduces Fehling's solution and gives with phenyl hydrazine lactosazone, which is easily soluble in 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 protec- tive substances, and many of their reserve materials consist of members of this group. In the animal body, where the supporting tissues are composed chiefly of derivatives 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 (C6H1005) 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 THE CARBOHYDRATES 75 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 microscopic grains, each of which presents the characteristic concentric striation. It is in- soluble 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 intermediate 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 decomposi- tions 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 reduce 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, 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 hydrolysed to form malto-dextrin : *2T vlj\ \ one molecule of water being taken up. The malto-dextrin 76 PHYSIOLOGY 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 achroo- dextrin. 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 : ° °\ C^B^iOjo^ the sign -' being used to denote the open terminal CHO group. They further found that the stable dextrin remaining at the end of the diastatic hydrolysis of starch probably had the formula of 40C6H1005H2O, and might be regarded as a condensation of forty glucose molecules with the elimi- nation of thirty -nine molecules of water. The starch molecule cannot be less than five times that of the stable achroodextrin. Since the latter has a molecular weight of 6498, the molecular weight of starch cannot be less than 32,400, and its empirical formula can be represented by : o, or (80d2H20010.40C6H1005). INULIN. Another kind of starch, known as inulin, occurs in dahlia tubers. It is easily hydrolysed by weak acids, and is entirely con- verted into d-fructose, or levulose. GLYCOGEN, or animal starch, is found in the liver, muscles, and other tissues of the body, and occurs in large quantities in all foetal tissues. It is a white powder, soluble in water, forming an opalescent solution. It is precipitated from its solution on the addition of alcohol to 60 per cent., or by saturation with solid ammonium sul- phate. On boiling with acids, it is entirely converted into glucose. It is affected by the ferments diastase and amylase, in the same way as vegetable starch, giving first dextrins and finally a mixture of maltose and dextrin. With iodine it gives a mahogany-red colour, which, like the blue colour produced in starch, is destroyed by boiling, to return again on cooling. We shall have occasion to consider its properties more fully when we are dealing with the functions of the liver. THE CELLULOSES. Cellulose (C6H1005)X is a colourless, in- soluble material, or mixture of materials, which compose the cell walls of the younger parts of plants, and therefore forms a constituent of most of our vegetable foods. It is insoluble in water or dilute acids or alkalies, its only solvent being an ammoniacal cupric oxide solution. On boiling with strong acids, it gradually undergoes hydrolysis and yields sugar, the nature of which varies according to the source of the cellulose. In herbivorous animals cellulose undergoes digestive THE CARBOHYDRATES 77 changes and forms an important constituent of their food. The solu- tion of the cellulose in this case is effected by the agency, not of fer- ments secreted by the wall of the gut, but of micro-organisms which swarm in the paunch of ruminants and in the coecum of other herbi- vora. In some cases the effective agent is a cytase present in the vegetable cells themselves. Since this ferment is destroyed by boiling, cooked hay is much less digestible than in the raw condition. In certain invertebrata it seems probable that a true cellulose-digesting ferment, or cytase, is secreted by the walls of the alimentary canal. In man cellulose undergoes practically no change in digestion, and serves merely by its bulk to promote peristalsis and the normal evacuation of the bowels. A further consideration of its chemical properties, as well as of the closely allied vegetable materials, gums, pectins, mucilages, derived for the most part from the condensation of pentose molecules, may be dispensed with here. SECTION V THE PROTEINS As sources of energy to the organism all three classes of food -stuffs are valuable in proportion to their heat equivalents, and it is often a matter of indifference whether the main bulk of the energy required is supplied at the expense of fat or at the expense of carbohydrate. The proteins, however, form the most important constituent of living protoplasm. On this account protein must always be present in the food to supply the material necessary for building up new protoplasm in the growing animal and for replacing the waste of living material which is taking place in the discharge of its normal functions. Regard- ing the complexity of reaction presented by living protoplasm as deter- mined in the first instance by the chemical and physical complexity of this material itself, we should expect to find that the proteins, forming its main constituents, would themselves partake of some of this quality. The carbohydrates and fats, although ir. many cases made up of huge molecules, are nevertheless built up on a very simple type. Starch, for instance, with a molecular weight of over 30,000, is formed simply by the polymerisation of glucose molecules. The ordinary fats, stearin and palmitin, consist of fatty acids with long- straight chains of CH2 groups, combined with the glyceryl radical. Their molecular weight is very large, but their molecules are simple in structure. When, however, we break up a protein molecule we meet with a great number of subsidiary groups, the presence of which is essential to the making of a nutritive protein. Owing to this complexity of structure it is not easy to give a simple definition in chemical terms of what we mean by the term ' protein.' It is necessary rather to describe certain of the qualities presented by this group, the possession of which we regard as essential to the conception of a protein. Elementary Composition. All proteins contain oxygen, hydrogen, nitrogen; carbon, and sulphur. The proportion of these elements in the various proteins may be represented as follows : C 50-6-54-5 per cent. H 6-5-7-3 „ „ N 15-0-17-6 ;, „ S 0-3- 2-2 „ „ 0 21-5-23-5 „ „ 78 THE PROTEINS 79 Nearly all the proteins contain a small trace of phosphorus varying from 04 to 0-8 per cent. It is doubtful, however, how far this phos- phorus forms an integral part of the protein molecule. Physical Characters. The proteins are amorphous indiffusible substances belonging to the class of bodies known as colloids. Most of them are soluble either in water, weak salt solutions, or in dilute acids or alkalies. They are inert bodies and tasteless. Although they form compounds with various metallic salts, acids, or alkalies, these compounds are but ill defined, and the relative proportions of the ingredients vary according to the conditions under which the com- pound was formed. As is the case with most colloids when in solu- tion or pseudo -solution, they can be brought into an insoluble form by various simple agencies, such as shaking, change of temperature, alteration of reaction, or addition of neutral salts. Coagulation by heat forms a distinguishing feature of a number of members of this class, which are therefore spoken of as ' coagulable proteins.' For instance, white of egg is a solution of different proteins. On diluting it with weak salt solution no precipitation takes place. If, however, the solution be heated to about 80° C. a precipitate of coagu- lated protein is formed. If a strong solution be boiled the whole fluid sets to a solid white mass (hydrogel). This change is irrever- sible, i.e. it is not possible by lowering the temperature to bring the white of egg again into solution, and many properties of the protein have been changed in the act of coagulation. With certain proteins and their allies the coagulation on change of temperature is a reversible process. Thus an alkaline solution of caseinogen, the chief protein of milk, if treated with a little calcium chloride and heated, undergoes coagulation and sets into a jelly, but on cooling the mixture the coagulum once more enters into solution. Ordinary gelatin, which is closely allied to the proteins, with water forms a solid jelly below 20° C., and a fluid solution above this temperature. If a protein be heated in a current of air or oxygen it undergoes combustion. In all cases a certain amount of incombustible material is left, consisting of inorganic salts which were closely attached to the protein molecule. If a solution of protein be subjected to long- continued dialysis, the proportion of ash may be diminished very largely, but in no case has any experimenter succeeded in obtaining a prepara- tion of protein absolutely ash-free. On this account it has been thought that the salts of the ash must be in chemical combination with the protein ; but having regard to the physical character of colloidal solutions, which we shall study in the next chapter, and the power of adsorption of substances possessed by such solutions, there is no need to regard these salts as essential constituents of the protein. Crystallisation of Proteins. Although the indiffusibility of protein 80 PHYSIOLOGY solutions differentiates them from the crystalloid substances such as sugar or sodium chloride, under certain conditions it is possible to obtain crystals consisting, largely at any rate, of proteins. Thus in the seeds of certain plants, e.g. hemp seeds, Brazil nut, pumpkin, and castor-oil seeds, the so-called aleurone crystals may be seen under the microscope enclosed in the protoplasm of the cells. These crystals consist of proteins belonging to the class of globulins. By chemical means they can be separated from the surrounding tissues and, after washing, dissolved in a solution of magnesia. Drechsel showed that on dialysing such a solution against alcohol, the fluid undergoes gradual concentration, and crystalline granules of the magnesia compound of the protein separate out. These crystals contain T4 p.c. MgO. A better method of obtaining such crystals has been devised by Osborne. The ground seeds are extracted with 10 per cent, sodium chloride solution, and filtered. The filtrate is diluted with water heated to 50° or 60° 0. until a slight turbidity forms. After warming the diluted solution until this turbidity disappears, and then allowing it to cool slowly, the protein separates in well-developed crystals. It is possible also to obtain crystals of animal proteins. Hemoglobin, the oxygen- carrying protein of the red blood corpuscles, can be made to crystallise with extreme ease. With some animals, such as the rat, it is only necessary to bring the hemoglobin into solution, by the addition of a little distilled water and ether to the blood, to cause the crystallisation of the liberated haemoglobin. Egg albumin and serum albumin may also be crystallised with ease by a method demised by Hofmeister and improved by Hopkins. If, for instance, we wish to crystallise egg albumin, white of eggs is treated with an equal bulk of saturated solution of ammonium sul- phate in order to precipitate the globulin. It is then filtered, and the filtrate is treated with saturated ammonium solution until a slight permanent precipitate is produced. This precipitate is then just redissolved by the cautious addition of water, and dilute acetic acid (10 per cent.) is added drop by drop until a slight precipitate is produced. The flask is now corked and allowed to stand for twenty- four hours, when the precipitate, which will have increased in quantity, will be found to consist entirely of acicular crystals. A similar method may be used for serum albumin. In each case the crystals contain a considerable proportion of ammonium sulphate. This may be replaced by sodium chloride by washing the crystals with a saturated solution of this salt. By absolute alcohol the crystals may be coagulated and may be then washed practically free from salt, but it is not possible to obtain crystals of coagulable protein free from the presence of some salt. Although by repeated crystallisation of egg albumin a product THE PROTEINS 81 may be obtained which is absolutely constant in both its physical and chemical characters, we cannot ascribe to crystallisation the same importance in securing purity and homogeneity of the substance that we can when we are dealing with inorganic salts. This is due to the fact that these crystals take up other colloids with great ease. When haemoglobin, for instance, is crystallised from blood, the first crop of crystals, although thoroughly washed from their mother liquor, always contain a considerable proportion of serum albumin. Indeed, the presence of colloidal material seems to render the production of the so-called mixed crystals much more easy. Thus Schultz has shown that in urine mixed inorganic crystals can be obtained. Human urine is allowed to stand twenty-four to forty- eight hours -with di- calcium phosphate and then filtered. On allowing the filtrate to evaporate slowly, a crystalline precipitate is produced consisting of whetstone-shaped crystals which are doubly refracting. On treating these crystals with dilute acetic acid this acid extracts calcium phosphate from the crystals. The original shape of the crystals is, however, retained. The only difference under the microscope consists in the fact that they have now lost their doubly refracting power on polarised light. They consisted of a mixture of calcium sulphate and calcium phosphate, from which, on treatment with acid, only the calcium phosphate was dis- solved out. The Molecular Weight of Proteins. We may arrive at an approxi- mate idea of the minimum size of the protein molecule in various ways, though in all cases our calculations are apt to be vitiated by the diffi- culty of obtaining a preparation which is homogeneous, i.e. chemically pure, and by the ease with which molecules of the size which we must assume for proteins form adsorption combinations in varying propor- tions with other substances. If we assume that each molecule of the respective protein contains only one atom of sulphur, we can calculate its molecular weight. It is evident that the protein which contains 1 per cent, of sulphur will have a molecular weight of 3200. In this way the following molecular weights have been arrived at (Abderhalden) : Sulphur per cent. Molecular weight Edestin . . . 0-87 .. 3680 Oxyhaimoglobiii . 043 . . 7440 (horse) Serum albumin . . 1-89 . . 1700 (horse) Egg albumin . . 1-30 .. 2460 Globulin . . . 1-38 .. 2320 The greater part at any rate of the sulphur in the protein molecule occurs as a constituent of a substance, cystine, each molecule of which contains two atoms of sulphur. Each molecule of protein must also 6 82 PHYSIOLOGY contain two atoms of sulphur, and we must regard double the molecular weight given in this table as the minimum molecular weights of these various proteins. Some idea of the molecular complexity repre- sented by these weights may be gained by writing out the empirical formulse of the various proteins, e.g., ' Egg albumin . . . C204 H322 N52066S2 Protein in hemoglobin (from horse) . . C680H1098N2100241S2 Protein in haemoglobin (from dog) . . . C725H1171N1940214S2 Crystallised globulin (from pumpkin seeds) C292H481N20083S2 With some proteins we may make use of other elements to arrive at an idea of the approximate molecular weight. Thus, oxyhsemo- globin contains between 04 and 0-5 per cent iron. If we assume that each molecule of oxyhaemoglobin contains one atom of iron, its molecular weight must be from 11,200 to 14,000. Attempts have been made to solve the same question by studying the compounds of proteins with inorganic salts or oxides. Thus, the crystals of globulin from pumpkin seeds prepared with magnesia contain 14 per cent. MgO. Assuming that one molecule of protein has combined with one molecule MgO, the molecular weight of the protein must be about 2800. (If x be the molecular weight x 100-14 40 14 .'. x -2817) Harnack has shown that many proteins are precipitated from their solutions as a copper compound by the addition of copper sulphate. Harnack found that this precipitate of copper contained either 1-34-1-37 Cu. or 2-48-2-73 per cent, Cu. The smaller percentage would correspond to a molecular weight of 4700, while the second number might be accounted for on the hypothesis that each molecule of protein was combined with two atoms of copper. Similar attempts have been made by determining the amount of acid or alkali necessary to keep certain types of protein in solution. We shall see later on, however, that the amounts vary largely with the physical condition and previous history of the colloidal substance. We are dealing here not with compounds in the strict chemical sense of the term, but with adsorption compounds, where the quantities taken up are determined not only by the chemical nature of the protein itself, but by the state of aggregation of its molecules. It is therefore impossible to lay any great stress on the determinations of the mole- cular weight which have been effected in this way. THE PROTEINS 83 Some clue to the size of the protein molecule is afforded by deter- minations of the osmotic pressure or molecular concentration of their solutions by physical methods. When we determine the freez- ing-point or boiling-point of protein solutions, the depression of freezing-point, or elevation of boiling-point, is so small that it falls within the limit of experimental error or is no greater than can be accounted for by the inorganic salts present in the solution. Since, however, colloidal membranes, such as films of gelatin or vegetable parchment, are impervious to proteins, we can directly determine the osmotic pressure of their solutions. In many cases no osmotic pressure whatever is found. In other cases, e.g. egg albumin, or serum, the colloidal constituents of these solutions are found to give an osmotic pressure of such a height that 1 per cent, protein corre- sponds to about 4 mm. Hg. pressure. Such an osmotic pressure would indicate a molecular weight for the serum proteins of about 30,000. A determination of the osmotic pressure of haemoglobin by Hiifner gave a molecular Weight about 16,000. These results, however, must be received with caution, since we are not justified in applying to these gigantic molecules data derived from a study of smaller mole- cules such as salt or sugar. Even if we accept these, determinations of osmotic pressure as indicating the molecular weights I have just quoted, it is evident that a very slight degree of aggregation of the molecules into larger complexes will bring the osmotic pressure below the point at which it is measurable, and would transform the solution into a suspension of particles in which one could not expect to find any osmotic pressure whatsoever. THE STRUCTURE OF THE PROTEIN MOLECULE We can arrive at some idea of the manner in which the protein molecule is built up only by breaking it down bit by bit, employing methods which, while resolving the large molecule into its proximate constituents, will not act too forcibly in changing the whole arrange- ments of these constituents. The relation of the starches or poly- saccharides to the sugars was found by studying the hydrolysis of the former, and it is by the hydrolysis of the proteins that we have arrived at most of our present knowledge of their constitution. Contributory evidence may also be gained by the use of oxidising agents or by employing the refined methods of analysis possessed by certain living organisms — bacteria, by which means we can effect limited oxidations or reductions or can replace an NH2 group by H, or a COOH group by H. ACID HYDROLYSIS OF PROTEINS. For this purpose rather stronger acids are used than for the hydrolysis of starch. The pro- tein is heated for twenty-four hours in a flask fitted with a reflux 84 PHYSIOLOGY condenser either with concentrated hydrochloric acid or with a 25 per cent, sulphuric acid. Hydrochloric acid was first made use of by Hlasiwetz and Habermann, who added a certain amount of stannous chloride to the mixture in order to prevent any oxidation taking place. We obtain in this way an acid fluid containing an extremely complex mixture of various substances, all of which belong to the class of amino-acids, and must be regarded as the proximate constituents of the protein molecule. A similar hydrolytic change may be effected by the use of digestive ferments obtained either from the alimentary canal of higher verte- brates or from certain plants. Thus we may use pepsin, the active constituent of the gastric juice, trypsin, -the proteolytic ferment secreted by the pancreas, papaine or other vegetable ferments obtained from papaya, from pineapple juice, and so on. These ferments are all milder in their action than the strong acids. Pepsin, for instance, only effects a partial decomposition of the protein molecule. Its action results in the formation of substances which still present all the protein reactions and are classified as hydrated proteins or as proteoses and peptones. Trypsin carries the protein a stage further and gives a mixture of amino-acids. Certain groups, however, of the protein molecule present a considerable resistance to the action of trypsin, so that when its action is complete these groups are still found not yet broken down into their constituent amino- acids. The putrefactive processes determined by the presence of bacteria in solutions of proteins are somewhat too complicated in their results to throw much illumination on the structure of the protein molecule itself. This method is, however, of extreme value when it is applied to isolated constituents of the proteins. Under the action of these bacteria we may have a process of deamination which may be accompanied by simple hydrolysis, or by reduction. In the former case an amino-acid may be converted into an oxyacid, in the latter case into a fatty acid. Thus tyrosine under the action of bacteria of putrefaction may split up into ammonia and oxyphenyl propionic acid. OH.C6H4.CH2.CHNH2.COOH + H2 = HO . C6H4 . CH2 . CH2 . COOH + NH3 Under the action of yeasts an amine may become an alcohol. C5Hn.NH2 + H20 = C5Hn.OH + NH3 (amylamine) (amylalcohol) .On the other hand, the effect of the bacteria may be to split THE PROTEINS . 85 off carbon dioxide from the amino-acids. Thus, the diamino-acid, lysine, CH2NH2 CH2NH2 CH2 CH2 I I CH2 becomes CH2 pentamethylene diamine. CH2 CH2 CH.NH2 CH2NH2 COOH Tyrosine becomes p. oxyphenylethylamine, a substance having marked physiological effects, and an important constituent of ergot. Phenylalanine C6H5 . CH2 . CH . NH2 . COOH, becomes phenylethyla- mine C6H5.CH2.CH2.NH2. These reactions are therefore of value in determining the exact grouping of the atoms in the more complex of the proximate constituents of the proteins. Since all the known disintegration products of the proteins belong to the class of amino-acids, it may be of value to point out some of the distinguishing features of this class of bodies. PROPERTIES OF AMINO-ACIDS. An amino-acid is derived from an organic acid by the replacing of one atom of hydrogen by the amino group NH2. Thus from the acids, acetic acid propionic acid CH3 CH3 COOH CH2 COOH we may obtain the mono-amino-acids, amino-acetic acid alanine or a-amino-propionic acid CH2NH2 CH3 COOH CH.NH2 COOH It will be noticed that in the fatty acids with more than two atoms of carbon the position of the NH2 group may be varied. Thus, 86 PHYSIOLOGY instead of alanine we may have another amino-propionic acid, namely : CH2NH2 CH2 COOH This acid would be spoken of as /3-amino-propionic acid, alanine being a-amino-propionic acid. This nomenclature is always used to distinguish the position of the NH2 group, so that we may have mono- amino- acids a, ft, y-> S, e • • • and so on. Practically all the amino- acids which occur as constituents of the protoplasmic molecule belong to the a group. On inspection of the formula of glycine it is evident that only one isomer of this body is possible. In alanine, however, the carbon atom to which NH2 is attached is asymmetric, since its four combining affinities are each attached to different groups. Thus : C H— C— NH2 C In this case, therefore, there is a possibility of stereoisomerism, and alanine must have an influence on polarised light. If the compound CH3 HCNH2 COOH is dextro-rotatory, then its stereoisomer CH3 I H2NCH COOH will be laevo-rotatory, and it will be possible to obtain a racemic modification without any influence on polarised light by mixing equal molecules of these two isomeric forms. All the amino- acids derived from proteins are optically active, whereas those obtained THE PROTEINS 87 by synthesis are inactive, and special means have to be devised in order to obtain from the artificially formed racemic amino-acid either the d- or Z-amino-acid. If more than one hydrogen atom in an organic acid be replaced by NH2 we obtain diamino- and triamino-acids. Thus, ornithine, obtained by the splitting up of arginine, one of the commonest dis- integration products of protein, is a- §- diamino- valerianic acid. CH2NH2 CH2 CH2 CH.NH2 COOH The presence in the amino- acids of the basic radical NH2 and of the acid group COOH lends to these bodies a double character. In themselves devoid of strong chemical qualities, possessing neither acid nor alkaline reaction, they are able in the presence of strong acids or bases to act either as base or acid. When in solution by themselves it is possible that there is an actual closing of the ring by a soluble union between the NH2 group and the COOH group, so that, e.g. the formula of glycine may be : CH2-NH3 I I CO — 0 When such a neutral compound is treated with acid this bond is loosed and we have the salt of the amino-acid. Thus, with hydrochloric acid, glycine forms glycine hydrochlorate : CH2NH2HC1 COOH a salt which still possesses an acid group and which is therefore capable of combining with ethyl to form the hydrochlorate of the ester of the amino-acid. Thus : CH2.NH2HC1 COOC2H5 88 PHYSIOLOGY With bases the amino-acids form salt- like compounds such as potassium ami no- acetate : CH2NH2 COOK With neutral salts crystalline compounds may be also formed. With sodium chloride glycine will form the double salt C2H5N02.NaCl, which may perhaps be represented : CH2NH3C1 COONa Not only do the amino-acids form compounds with salts, but they also combine with one another. This power of combination much increases the difficulty of separating the constituents from a mixture of amino-acids. Amino-acids, which singly are extremely insoluble, are readily soluble when in the presence of other amino-acids. On account of the dual nature of the amino-acid molecule, these substances act as feeble conductors of the electric current, i.e. as electrolytes. The charge carried by an amino-acid and its ionisation depends upon the conditions in which it is placed. Since it may act either as the cation or the anion, it is spoken of as an amphoteric electrolyte. One reaction of the amino-acids is of special interest in connection with the respiratory functions of the body, namely, the formation of carbamino-acids. If a stream of carbon dioxide be passed into a mixture of an amino-acid, e.g. glycine, with lime, the carbon dioxide is taken up. On filtering the mixture a clear liquid passes through which gradually in course of time deposits a precipitate of calcium carbonate. The filtrate first obtained contains a compound of cal- cium, calcium glycine carbonate. The formula is as follows : CH9.NH COO Ca METHODS OF SEPARATING AMINO-ACIDS. By the hydrolysis of protein by means of acid or of trypsin, we obtain a complex mixture of amino-acids. From this mixture certain amino-acids are separated with ease. Thus, tyrosine, which is extremely insoluble, crystallises out on concentrating the fluid, and further concentration leads to the separation of leucine. The other acids, which keep each other mutually in solution, are however very difficult to isolate. We owe to Fischer THE PROTEINS 89 the first general method for their separation. We may take one experiment as an example. Five hundred grammes of casein are heated for some hours under a reflux condenser with 1| litres of strong hydrochloric acid. The liquid is then saturated with gaseous hydrochloric acid and allowed to stand for three days in the ice- chest. Crystals of hydrochlorate of glutamic acid separate out. The filtrate from these crystals is evaporated at 40° C. under diminished pressure to a syrupy consistence, and is then dissolved in 1J litres of absolute alcohol. Hydrochloric acid is then passed into the solution to complete saturation, the mixture being warmed for a short time on the water bath, and the mixture is once more evapo- rated to a syrupy consistence. By this treatment all the amino -acids have been converted into the hydrochlorates of their esters, e.g. : CH2NH2HC1 G,H4NH2HC1 i r COOC2H5 COOC2H5 &c. From the hydrochlorates the esters are set free by the addition of potassium carbonate, the mixture being cooled in a freezing mixture. By this means the esters of aspartic and glutamic acids are separated and are extracted by shaking with ether. The remaining esters are then liberated by the addition of 33 per cent, caustic soda together with potassium carbonate, and are again extracted by ether. The combined ethereal solutions are dried by standing over fused sulphate of soda and then evaporated, when a residue containing the free esters is obtained. These esters are then separated by fractional distillation under a very low pressure obtained by means of the Fleuss pump, the second receiver of the apparatus being cooled in liquid air. The various fractions of amino- esters obtained in this way are hydrolysed — the lower fractions by boiling for some hours with water, the higher fractions by boiling with baryta. The acids obtained by the hydrolysis can then be further purified by means of fractional crystallisation. T;HE DISINTEGRATION PRODUCTS OF THE PROTEINS By the methods just described the following substances have been isolated from proteins : A. FATTY SERIES (1) Mono-amino-aeids (Monobasic) GLYCINE or GLYCOCOLL. This, the simplest member of the group, is amino-acetic acid : CH2NH2 COOH It occurs in considerable quantities among the disintegration products of gelatin and to a slight extent among those derived from certain of the proteins. Like the other a-amino-acids, it has a sweetish taste, whence its name was derived (y\vKoCO (urea) and NH(CH3)CH2COOH (methyl glycine). H2N This latter substance is known as sarcosine and is derived from glycine by the replacement of one atom of hydrogen by a methyl group CH3. Arginine has a similar formula. On the left-hand side of the dotted line the formula would be identical with that of creatine. On the right- hand side the sarcosine group is replaced by a diamino-acid of the fatty series, diamino-valerianic acid or ornithine. DIAMINO-TRIOXYDODECOIC acid is, as its formula implies, a derivative of a twelve-carbon acid. Its constitutional formula has not yet been made out. B. AMINO- ACIDS CONTAINING AN AROMATIC NUCLEUS The best known of these is TYROSINE, which has the formula OH CH2CH.NH2COOH It is paraoxyphenyl «-alanine. It is one of the first of the amino- acids to be split off from the protein molecule under the influence of hydrolytic agents. Owing to its insolubility it rapidly separates out as bundles of fine needle- shaped crystals at the sides and bottom of the vessel. When tyrosine is treated with an acid solu- tion of mercuric nitrate containing a little nitrous acid, a precipitate is produced, and on boiling, the precipitate and the supernatant fluid assume a deep red colour. This reaction is given by all FIG. 18. Tyrosine crystals, (PUMMER.) 94 PHYSIOLOGY benzene derivatives in which one atom of hydrogen in the ring is replaced by one OH group. This is known as Hoffmann's test, but is identical with Millon's reaction, which is given by all proteins con- taining tyrosine in their molecules. Closely allied to the foregoing compound is another aromatic ammo-acid, namely, phenyl a-alanine : |C6H5| \/ CH2CH.NH2COOH It is an almost constant constituent of proteins. TRYPTOPHANE was known long before it had been isolated, owing to the fact that with bromine water it gives a rose-red colour. It had long been observed that this substance was to be obtained at a certain stage in the digestion of proteins by pancreatic juice, but nothing was known about its constitution until Hopkins succeeded in isolating it by precipitation with mercuric sulphate dissolved in 5 per cent, sulphuric acid. Cystine is also precipitated by this reagent, but comes down with a less concentration of the salt than tryptophane, so that it is possible to separate the two substances by a species of fractional precipitation. Tryptophane can be isolated by decomposing the mercury salt with sulphuretted hydrogen, and is obtained in a crystallised form. On distillation it gives an abundant yield of indol and skatol, bodies also obtained during the putrefaction of proteins. Tryptophane itself is indol amino-propionic acid : .CH2CHNH2.COOH CH NH C. AMINO-ACIDS OF HETEROCYCLIC COMPOUNDS Three of the disintegration products of proteins can be grouped in this class. Two of them contain the pyrrol ring, namely, proline and oxyproline. PROLINE, which was first isolated by Fischer, is a-pyrrolidin carboxylic acid and has the formula CH2— CH2 CH2 CH.COOH \/ NH THE PROTEINS 95 OXYPROLINE is the oxy- derivative of this body and has the formula C5H9N03, the exact position of the oxy- group having not yet been determined. Doubts have been expressed whether the pyrrol group is present as such in the protein molecule, or whether proline, for example, is not formed by the closing of an open chain of a compound belonging to the amino- acids in the fatty series. Thus from an oxy-amino- valerianic acid CH2OH . CH2 . CH2 . CH . NH2 . COOH we can by dehy- dration make the compound CH2CH2 . C.H2 . CH . COOH, which will be Ntf seen to be identical with that given for proline. The third member of this group contains the iminazol ring : CH— NH II >H C —W and is known as HISTIDINE. Its structural formula is as follows : CH— NH II /CH C -- W CH2.CH.NH2.COOH i.e. it is iminazol a-amino-propionic acid or iminazol alanine. Since it occurs in the phosphotungstic precipitate from the products of acid disintegration of proteins and contains six carbon atoms, it was formerly classified with lysine and arginine as a hexone base. D. SULPHUR-CONTAINING AMINO-ACIDS Sulphur forms an integral part of the molecule of all classes of proteins except protamines. In some substances allied to proteins, such as keratin, it may occur to the extent of 3 per cent. On boil- ing proteins with caustic potash or soda, a portion of the sulphur is split off to form a sulphide, which gives a black precipitate on addition of copper salts. On this account it was formerly thought that the sulphur must be present in two forms, the oxidised and the unoxidised, in the protein molecule. Recent investigation has shown, however, that practically the whole of the sulphur is present in the form of CYSTINE, and that this body on boiling with alkaline solutions gives up only a little more than half its content in sulphur. This substance, which has been known for many years as the chief constituent of a rare form of urinary calculus and as occurring in the urine in certain cases of disordered metabolism, is again a deriva- 96 PHYSIOLOGY tive of the three-carbon propionic acid. On reduction it gives a body known as cysteine, which is a-amino-thiopropionic acid. CH2SH CH.NH2 COOH Cystine itself is compounded of two cysteine molecules joined together by their sulphur atoms and has the formula CH2— S— S— CH2 CH.NH2 CH.NH2 COOH COOH E. OTHER CONSTITUENTS OF THE PROTEIN MOLECULE When we* add together the total amino- acids obtainable by the acid disintegration of any given protein, a considerable proportion of the original protein remains unaccounted for. This remainder must have a greater content in hydrogen and oxygen than the amino- acids enumerated above, and it has been suggested that among the missing unascertained constituents of proteins may be oxyamino-acids, of which serine would form one of the lowest members. The isolation of such substances would present considerable interest, in that it would supply the intermediate stages between the constituent groups of the protein molecule and the carbohydrates, the first product of assimilation by living organisms. Only one such intermediate body has so far been isolated, namely, glucosamine. an amino- derivative of glucose. It was first shown by Pavy that from the products of disintegration of a protein such as egg-white it was possible to obtain a reducing sub- stance 'and to isolate an osazone resembling in its characters those derived from the sugars. Since then various observers have shown that this reducing substance is most probably glucosamine : CH2OH (CHOH)3 CH.NH2 CHO Although this substance may be obtained from crystallised egg albumin or crystallised serum albumin, authorities are not yet THE PROTEINS 97 convinced that^it forms an integral part of these proteins. Both egg- white and serum contain proteins belonging to the class of mucins, ovomucoid and serum mucoid, each of which yields on acid hydrolysis from 16 to 30 per cent, glucosamine. Since various observers have obtained results varying from 1 to 16 per cent, glucosamine for crystal- lised egg albumin, it seems possible that in every case the crystals carried down with them some of the carbohydrate-rich mucoid, and that the varying results were due to the different amounts of mucoid present in the crystals. By our ordinary methods it is impossible to prepare a specimen of either egg albumin or serum albumin which is entirely free from this amino -derivative of carbohydrate. Connected with this group of proteins may be reckoned the diamino- trioxydodecoic acid already mentioned as occurring among the dis- integration products of proteins. THE BUILDING UP OF THE PROTEIN MOLECULE By simple hydrolysis the protein molecule may be broken down into a large number of amino-acids. Analyses of various proteins show that these amino-acids are present in different proportions in the individual proteins, so that in many cases a large number of identical amino-acid groups must be present in the protein molecule with smaller numbers of other groups. In endeavouring to form an idea of the manner in which the amino-acids can be linked together into one gigantic molecule, Hofmeister first put forward the idea that the linkage follows the general formula : -CH2— NH— CO— or _NH— CH2— CO— NH— This theory of the constitution of proteins was based on the fact that a similar grouping was known to occur in leucinimide, obtained by the condensation of two molecules of leucine, /CH\ NH CO I I CO NH C4H9 and also by the fact that only a small proportion of the NH2 groups present in the separated amino-acids exist free in the protein molecule. 98 PHYSIOLOGY By the action of nitrous acid the terminal NH2 groups are split off and replaced by OH. When proteins are treated with nitrous acid only a small proportion of the total nitrogen is split off in this way. The linking of the amino groups must therefore take place by means of the nitrogen, i.e. by NH groups. Synthetic experiments have fully con- firmed this hypothesis. In 1883 Curt i us obtained a substance giving the biuret reaction, the so-called ' biuret base,' by the spontaneous polymerisation of glycocoll ester. This base has been shown by recent- researches to consist of four glycine molecules arranged together in an open chain. The clue to the structure of this base was given by Fischer, who has devised a number of ingenious methods for combining together amino-acids of any character and in any number. Thus from two molecules of glycine we may obtain the compound glycyl glycine, as follows : NH2 . CH2 . COOH + HNH . CH2 . COOH — H20 - NH2 . CH2.CO.NH.CH2 . COOH This may be prepared in various ways. In one method glycine is converted into its ester CH2.NH2.CO.OCH3. In a watery solution this undergoes spon- taneous conversion into glycine anhydride, which belongs to the class of bodies known as diketopiperazins, as follows : /CH2— C(\ 2NH2.CH2CO.OCH3 = 2CH3OH + NH<^ / >NH methyl alcohol N:CO— CH/ On treating this with dilute alkali it takes up water, splitting in the situation of the dotted line and forming glycyl glycine, NH2CH2CO . NH . CH2COOH. More general methods have been devised by Fischer for the same purpose, depending on the use of the halogen acyl chlorides. Thus chloracetylchloride and alanihe yield chloracetalanine : C1.CH2.COC1 + NH2.CH(CH3).COOH = C1.CH2.CO - NH.CH(CH3)COOH + HC1. By the subsequent action of ammonia, the halogen group is replaced by the amino group, and a dipeptide results : C1.CH2.CO - NH.CH(CH3)COOH + 2NH3 = NH2.CH2.CO - NH.CH(CH3)COOH + NH4C1. Different halogen acyl chlorides are used for introducing the various ammo- acid radicals, e.g. chloracetylchloride for glycyl, a -bromopropionyl chloride for alanyl, &c. By various such methods Fischer has succeeded in combining compounds containing as many as eighteen amino-acids, e.g. alanyl leucine, glycyl tyrosine, dialanyl cystine, dileucyl cystine, leucyl pentaglycyl glycine, and so on. The last named would be built up out of one molecule of leucine and six molecules of glycine. These compounds have been designated by Fischer as polypeptides, to sig- nify their close connection with the peptones produced by the agency THE PROTEINS 99 of digestive ferments on the proteins. He distinguishes di-, tri-, tetra-, &c., peptides according to the number of individual amino-acids taking part in the formation of the compound. The polypeptides resemble in all respects the peptones. Most of them, even if derived from relatively insoluble amino-acids, are soluble in water, insoluble in absolute alcohol. They dissolve in mineral acids and in alkalies with the formation of salts, thus resembling in their behaviour the amino-acids. They have a bitter taste, although the amino-acids from which they are formed have a slightly sweet taste, in this way again resembling the natural peptones. The higher members of the series give certain reactions, such as the biuret reaction, which are regarded as characteristic of peptones, and like the latter are pre- cipitated by phosphotungstic acid. Their behaviour with trypsin depends on the optical behaviour of the amino-acids from which they are formed. If synthetised from the amino-acids identical with those occurring in the disintegration of natural proteins, they resemble the peptones in undergoing hydrolysis and disintegration into their constituent amino-acids. Trypsin, however, has no influence on poly- peptides compounded of the inactive amino-acids, or of the amino- acids which are the optical opposites of those which form the constituents of normal proteins. Though most of the amino-acids which occur naturally are Isevo-rotatory, the polypeptides formed from them are generally strongly dextro-rotatory. Thus in the building up of the protein molecule there is an almost indefinite coupling up of numerous amino-acid groups, the connecting element in each case being the nitrogen. Of the two or more optical isomers possible of each amino-acid containing more than two carbon atoms, only one is made use of for this purpose. A still further flexibility in its reactions to its environment is conferred on the protein molecule by changes occurring with great readiness in the intra- molecular grouping of its constituent atoms. Thus, if we take the simplest member of the class of polypeptides, glycyl glycine, four structural formulse are possible, namely : (1) NH2CH2CO - NH . OH2 . COOH (2) NH.CH2.COX (3) NH2 . CH2 . C(OH) = N . CH2 . COOH (4) N.CH2.CO C(OH)CH2.NH (2) and (4) being the intramolecular form of the 100 PHYSIOLOGY (3) and (4) are sometimes spoken of as the enolic form. If we con- sider that perhaps some hundred of the amino-acid groups may go to making up a single protein molecule, it is possible to form some con- ception 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 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 pro- tein 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 proteins, the differences between the latter being deter- mined 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 are given in the following Table : Serum albumin Egg albumin III (3<5 & Gliadin Caseinogen S 3 e 3 Salmin a *n _3 00 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 T5 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 T39 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 T5 — — 0 3-2 Tryptophane present present present 1-0 1'50 present — — 0 — Cystine 2-3 0-2 0-25 0-45 ? 0-3 — — — ov. 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 JheStf results ^sjaow that all the proteins contain a very considerable THE PROTEINS 101 proportion of the total number of ami no- 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. Thus, glutamic acid, which forms 8 per cent, of egg albumin and only 1-7 per cent, of globjn (derived from haemoglobin), amounts to 31-5 per cent, in gliadin, the protein extracted from wheat flour. Striking differences are also noticeable 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 prota- mines, 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 pro- tein 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 haemo- globin would come out at about 14,000, a figure which agrees with that derived from a study of the amounts of sulphur and iron respectively 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 recogni- tion of the individual amino-acids. may yet throw some b'ght 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 deci- normal 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- acids 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 102 PHYSIOLOGY TABLE I. Group Protein Source N per cent. Amide N Amino N Basic N Humin N Protamines fSalmine \Sturino Salmon-roe Sturgeon -roe — 0 0 87-8 83-7 Histories Histone Thymus — 3-3 38-7 Albumins ) and 1 Ovalbumin Egg-white 15-51 8-64 68-13 21-27 1-87 phospho- | Caseinogen Milk 15-62 10-36 66-00 22-34 1-34 proteins I Globulins f 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 Wheat and rye 16-13 17-66 18-40 23-78 77-56 70-27 3-03 5-54 0-99 0-79 fProt- Witte's Albumoses 1 albumose Hetero- peptone Witte's — 7-14 68-17 25-42 — (_ albumose peptone — 6-45 57-4 38-93 — TABLE II. — DISTRIBUTION OF THE NITROGEN IN VARIOUS PROTEINS (VAN SLYKE) Gliadin Edestin Hair (dog) Gelatin Fibrin Hsemo- cyanin Ox 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 Arginine N Histidine N . 1-25 5-71 5-20 1-49 27-05 5-75 6-60 15-33 3-48 0 14-70 4-48 0-99 13-86 4-83 0-80 15-73 13-23 0'' 7-70 12-70 Lysine N Amino N of the 0-75 3-86 5-37 6-32 11-51 8-49 10-90 nitrate 51-98 47-55 47-50 56-30 54-30 51-30 57-00 Non-amino N of the nitrate (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 lOO'OO determined by Kjeldahl's method. Table I. (above) gives some of the results obtained in this manner, and shows that there are con- siderable differences in the distribution of the different kinds of THE PROTEINS 103 nitrogen among the various classes of proteins. The method is, however, only a rough one as compared with the separation of the individual amino-acids. 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., p. 102. 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 hydrated 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.NH2 CO.NH2 CO.NH2 CO— NH2 CO— NH2 and the group (NH2)C— CO— NH— C 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 pro- duced 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. 104 PHYSIOLOGY (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 solution 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 precipitation 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 Adamkiewicz 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 : Glyoxylic 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 essen- tially 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 concentrated hydrochloric acid, when a blue colour is produced, glyoxylic acid being derived from the alcohol and ether. (6) REACTIONS INDICATING THE PRESENCE OF CARBO- HYDRATES. 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 molecule, 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. THE PROTEINS 105 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 alka- loidal 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. (6) 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, 106 PHYSIOLOGY 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. (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 pro- duced at the junction of the two fluids. A similar coagulative effect is produced 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 are 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. Sodium chloride. Sodium sulphate. Sodium acetate. Sodium nitrate. Magnesium sulphate. Class II. Potassium acetate. Calcium chloride. Calcium nitrate. The two calcium salts are, however, rarely employed, as they tend to render the precipitated protein insoluble. Class III. Ammonium sulphate. Zinc sulphate. The salts of the first class require much higher concentration for the precipitation of the albumins than those of the second, and these than those of the third. Since the degree of concentration of any THE PROTEINS 107 salt necessary for the precipitation of any particular protein is charac- teristic 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 disintegra- tion 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 constitution. At the present time it is obviously impossible to admit any classification on such a basis, since the neces- sary knowledge is wanting, and we have therefore to make use of a purely artificial classification, such as that adopted by the Chemical and Physiological Societies in 1907, based chiefly on the 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 occur. (1) THE PROTAMINES. These occur in the body only in com- bination with other groups. They are obtained from the ripe sperma- tozoa of certain fishes, where they occur in combination with nucleic acid. They are characterised 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 -amino -acids may also be obtained from their disintegration (v. Table, p. 100). 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 hsematin. They may be obtained from red blood corpuscles, 108 PHYSIOLOGY 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 pre- cipitated by complete saturation with ammonium sulphate, zinc sulphate, or sodio-magnesium sulphate. EGG ALBUMIN 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 laovo-rotatory, its specific rotatory power being — 35-50. SERUM ALBUMIN occurs in large quantities in the blood plasma, serum, lymph, and tissue fluids of the body. It coagulates at 75° C., and is distinguished from egg albumin by its greater specific rotatory power, — 56°, and by the fact that it is not precipitated by ether and sulphuric acid. Some vegetable proteins belong to this class, e.g. the lemosin 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 satura- tion 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. 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 constituent 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 THE PROTEINS 109 obtained from hemp seeds, cotton seeds, and sunflower seeds, zein from maize, leyumin 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 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 to nearly neutralise the solution of acid albumen, 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 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 albumen. It is precipitated on neutralisation of its solution. In close association with this group may be included the proteins as they occur in 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 of 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 the 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 compounds 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 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. 110 PHYSIOLOGY These hydrolytic changes proceed, however, 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, pro- teoses 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 peptones 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 mole- cule determines a great similarity of composition between its various disintegration 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 forma- tion, and this heterogeneous character of the molecule renders possible a much greater variety of intermediate 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 repre- sented 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 polypeptides. 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 proteoses and pep- tones are, however, ill-defined bodies. We have at present no satis- factory means of isolating the different members of these groups and obtaining them in a state of chemical purity. Their classification is therefore, like that of the proteins generally, a conventional one, depending on their solubilities and their precipitability by neutral salts, especially ammonium 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 phosphotungstic 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 THE PROTEINS 111 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, 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 solu- tions 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.g. fibrin peptone, gluten peptone. These are all soluble in pure .water, diffuse fairly readily through animal mem- branes, but otherwise 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 phos- phorus-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 the 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 libera- tion of phosphoric acid, and do not contain purine bases. The phos- phorus of the nucleoproteins is not split off by alkali (1 per cent.), 112 PHYSIOLOGY and on hydrolysis the nucleic acid constituent gives rise to purine (9) 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 com- bined with some other body, often spoken of as the prosthetic group.* (a) CHROMOPEOTEINS. Of this class, consisting of a colouring matter combined with a protein, the most important is Jicemoglobin. This substance, which is the red colouring matter of the red corpuscles of the blood and plays an important part in the processes of respiration, acting as an oxygen carrier from the lungs to the tissues, is com- posed of the protein, globin, united with an iron -containing body, hsematin. Oxyhaemoglobin contains from 4-5 per cent. ha3matin (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 Kespiration. (6) THE NUCLEOPROTEINS. These are formed by the combina- tion 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 * 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 to the Germans as ' Eiweisskorper. ' On account of the con- fusion which has arisen from this double use of the term ' proteid,' I have attempted to avoid it altogether in this volume. THE PROTEINS 113 that the imclein obtained from this source contained GO'S per cent, nucleic acid and 35-56 protamine, and was in fact a nucleate of prota- inine. The nuclein derived from the spermatozoa of echinoderms has been found to be a compound of nucleic acid and hist one. From organs rich in cells, such as the thymus and the pancreas, and from nucleated red blood corpuscles, nucleoproteins 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 separating 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 fibrinogens are highly unstable bodies and undergo changes in the mere act of precipitation 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 in 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 nucleoprotein may be represented therefore by the following schema : Nucleo-protein Protein Nuclein Protein Nucleic acid (generally histone or protamine) Since we have already dealt with the chemical constitution of the proteins, it remains only to discuss the nature of nucleic acid. 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, C6H5N'5, and 114 PHYSIOLOGY guanine (C5H5N50). These substances, with the products of their oxidation, xanthine, C5H4N402, hypoxanthine, C5H4N40, 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, 5C— NH7 II II >H8 SN_ 4c— N9 r 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 >0 — C— NHX It can be synthetised by fusing together in a sealed tube trichloro- lactamide and urea. Thus : NH2 CONH2 NH— CO II II CO + CHOH + NH2X = CO C-NHX + NH4C1. + 2HC1 II >co I || .)co NH2 CC13 NH/ NH— C— NH/ The relation of xanthine, hypoxanthine, guanine, and adenine to uric acid is shown by the following formulae : NH— CO HN— CO CO C— NH\ II >co 1 CO — C-NHv NH— C— NHX HN— C— N Uric acid 2 - 6 - 8 -tr ioxypurine Xanthine 2-6-dioxypurine HN— CO N = C.NH2 1 1 1 NH— CO 1 HC C — NHx HC C— NHN NH2C C— NHv II II >H || N — C — N '• N— C^-N )CH || || \C N— C— N ' Hypoxanthine Adenine 6-oxypurine 6-amino-purine Guanine 2-amino 6-oxypurine Closely allied to this group of bodies are the chief constituents of tea, THE PROTEINS 115 coffee, and cocoa, namely caffeine, which is trimethyl dioxypurine, and theobromine, which is dimethyl dioxypurine. From the structural formulae given it will be seen that the purine radical contains two nuclei. The nucleus N— C c c N— C is spoken of as the pyrimidine nucleus, pyrimidine having the formula HO 5CH The other is the radical which we have met with already in histidine, a disintegration product of proteins, namely iminazol : HC— NH II >H HC— N r Besides the purine bases proper, we meet among the disintegration products of nucleic acid with a series of bases derived from the pyrimi- dine ring. These are uracil, thymine, and cytosine. URACIL is 2- 6- dioxy pyrimidine, NH— CO CO CH I II NH— CH THYMINE is 5- methyl uracil, NH— CO CO C.CH3 1 II NH— CH while CYTOSINE is 6-amino-2-o2ypyrimidine, NH— C.NH2 CO CH I II N =CH 116 PHYSIOLOGY Besides these two groups of nitrogenous compounds derived from the purine and pyrimidine rings, many nucleic acids yield on hydrolysis a carbohydrate. Thus, Hammarsten has isolated a pentose, xylose, from the nucleoproteins 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 : Nucleo -protein on digestion yields nuclein proteoses and peptones dissolved in alkali and precipitated with hydrochloric acid yields nucleic acid acid derivatives of protein, histones or protamines hydrolysed yields phosphc )ric acid reduci Qg sugar purine bases pyrimidine bases pentose or adenine uracil hexose guanine thymine cytosine Tt must not be imagined, however, that all these disintegration pro- ducts 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 thymine 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 con- stituent radicals from those which distinguish the proteins of the cell protoplasm. Further importance is lent to 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. (c) THE GLYCOPROTEINS. 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 THE PROTEINS 117 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 acids, and after precipitation need the addition of alkalies for their re-solution. They are not coagulable 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 albumin and albumoses and glucosamine. From the mucin of frogs' eggs a similar treatment results in the produc- tion of galactosamine. With the mucins may be classified certain bodies which have been derived from ovarial cysts, namely, pseudomucin and paramucin. Pseudomucin occurs as a constituent 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 which 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. Chondromucoid 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 sub- stance chondrosin, which is certainly an amino-derivative of a poly- saccharide 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 ' lardaceous 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 pre- cipitated 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. 118 PHYSIOLOGY (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 tissues seems to be determined by their insoluble character. On this account it is practically impossible to speak of purifying them. Jn 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 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 polypeptides which present considerable resistance 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 con- stituent 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 solution. 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 support- ing network of adenoid tissue, and has also been described in the spleen, the mucous membrane 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 PROTEINS 119 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 prepared, 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 ex- tracted from keratin. They also yield, on acid hydrolysis, tyrosine in larger quantities than is the case with the ordinary proteins. Neurokeratin, which forms the basis of the neuroglial frame- work 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 the tissue to prolonged tryptic digestion, which leaves the neurokeratin unaffected. Fibroin of silk Elastin Keratin from horn Keratin from horsehair Keratin from feathers Gelatin Glycine .... 36-0 25-75 045 4-7 2-6 16-5 Alanine 21-0 6-6 1-6 1-5 1-8 0-8 Amino-valerianic acid 0-0 1-0 4-5 0-9 0-5 ro Proline .... present 1-7 5-2 Leucine .... 1-5 214 15-3 7-1 8-0 21 Phenylalanine 1-5 3-9 1-9 0-0 0-0 04 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 04 04 Tyrosine 10-5 0-34 3-6 3'2 3-6 0-0 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 — 04 Oxyproline ~ ~ _ ~ 3-0 Elastin is a constant constituent of the connective tissues, where it forms the elastic fibres. In some localities, as in the ligamentum nuchae, practically 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 xantho- proteic and Millon's tests. 120 PHYSIOLOGY Other members of this group are fibroin, which forms the main substance of silk, spongin, the horny framework of sponges, concJiiolin, 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 differences in their qualitative and quantitative composition in amino-acids. Their proximate composition is shown in the Table on the preceding page (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 com- position 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 substances. In every case the substance is characterised necessarily according to its place of origin, little or nothing being known 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 unorganised 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 carbohydrates, 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 nitrosomonas, described by Winogradsky, grows on a medium devoid of all organic constituents, 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 produc- tion of oxygen by the green plant was first discussed 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 chloro- 121 122 PHYSIOLOGY phyll corpuscles, and regarded these as the first products of assimila- tion. 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 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 4- 5H20) = (C6H1005)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 atmo- sphere, 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 purpose 784 ccm. 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 men- tioned. It is also increased by raising the percentage 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 THE MECHANISM OF ORGANIC SYNTHESIS 123 •03 per cent, carbon dioxide at 100, the assimilation in an atmosphere containing 1 per cent, was 237, and was not increased by raising the percentage of carbon dioxide to 7 per cent. Owing to the decomposi- tion 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 conditions 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 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 trans- mitted 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 chloro- phyll 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 pronounced 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 physiologists are still undecided. There can be no doubt that an early product of the process is a hexose, which is rapidly converted into car e 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 algae, 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 deriva- tives of formaldehyde as sodium oxymethyl-sulphonate, or from methylal. The difficulty in these cases is that possibly a spontaneous 124 PHYSIOLOGY formation of sugar from the formaldehyde had taken place in the solu- tion and that the plants were using up the sugar rather than the for- maldehyde as the source of their starch. One must assume, with Timiriazefr", 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 for- maldehyde. Usher and Priestley, on treating a solution of carbon dioxide with 1*5 per cent, uranium acetate or sulphate in bright sun- light, obtained uranium peroxide and formic acid, but no formaldehyde. The formation of peroxides in these conditions suggests that the first change in the chloroplast may be as follows : C02 + 3H20 = 2H202 + CH20 Such a reaction must be regarded as reversible since the hydrogen peroxide 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 corpuscles 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. formaldehyde, 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. THE MECHANISM OF ORGANIC SYNTHESIS 125 (6) 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 photo- lysis 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 diagrammatically expressed thus : Carbon dioxide + Water i f (// not removed, destroys) •> CHLOROPHYLL Hydrogen peroxide + Formaldehyde .-(// not removed, poisons) ENZYME LIVING PROTOPLASM >1^ ^X' 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 pro- cess. 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 optically 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 produc- tion 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 126 PHYSIOLOGY 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 sub- stances such as glyoxylic acid and other derivatives of the fatty acid series. Such by-products might play an important 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 carbo- hydrates, 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 forma- tion. 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, provided only that they possess chlorophyll corpuscles and so are able to utilise 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 bacteria of the soil acquire so great an importance for agriculture. From the carbon dioxide of the atmo- sphere or from the hexose formed by the assimilation 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 c THE MECHANISM OF ORGANIC SYNTHESIS 127 of these constituent groups. Just as in digestion the protein mole- cule is taken to pieces with the formation of the different amino- acids, so in the synthetic action of protoplasm the reverse process of dehy- dration occurs, resulting in a coupling up of the different groups, as has been effected by Fischer in the case of the poly pep tides. 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 break- down 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 inter- ference 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 that obtained in 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 necessary from one organ to another. We shall later on have to discuss the possibility of synthesis of the different amino-acids in animals. We need, therefore, at present only deal 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 CH.OH + NH3 = CH.NH2 + H20 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 assimila- tion of carbon dioxide. If a solution of glucose together with lime be 128 PHYSIOLOGY exposed to sunlight for a considerable time it undergoes decomposition with the formation of lactic acid. Thus : C6H120, glucose 2C3H603 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 : H.C.< OH.C.H H.C.OH OH or OH.C.H When either of these arrangements reacts with water, thus : CH2OH CHOH OH.C.H OH H DOH I 2HOH CH2OH CHOH COH + H20 CH2OH CHOH COH COH we obtain two molecules of glycenc aldehyde, which then by a further shifting of the OH and H groups becomes CH3 I CH.OH COOH lactic acid Lactic acid with ammonia and some dehydrating agent will give amino- THE MECHANISM OF ORGANIC SYNTHESIS 129 propionic acid or alanine. The formation of the higher amino-acids involves a process of reduction of the sugar first formed in the chloro- phyll granules. It is possible, however, that the starting-point for the amino-acid synthesis may be, not a hexose itself, but some other sub- stances, 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 CH, CH3 N}H/ CO + CH.OH + NH3 + H2 CH2 + 2H20 CH3 COOH CH.NH2 COOH As an intermediate product in the synthesis of starch, glyoxylic acid CHO has been described as occurring in the green parts of plants. COOH This substance with ammonia gives f ormyl glycine, and by the splitting oil 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 substance derived from the carbon dioxide assimilation to form amino- compounds. In general we may say that the probable mechanism of formation of amino-acids is the production of a-oxyacids, which then react with ammonia to form the amino-acids of the protein molecule ; but of the exact steps in this process we are at present ignorant. Knoop's work would point to the ketonic acids as forming one step, and as interacting with ammonia, with simultaneous reduction, to form amino-acids. 9 130 PHYSIOLOGY 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 or ni thine by a process of con- densation with the loss of ammonia. Thus : CH2NH2 . CH2 . CH2 . CH . NH2COOH becomes OH2.CH2.CH2.CH.COOH NH or, as it is generally written : CH2 CH.COOH \/ 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 hsematin, 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 CH— N It is interest ing to note that if we attach to this compound carbamide or urea we obtain a body belonging to the class of purines. Xanthin, for instance, would have a formula NH— CO CO C— NHX >CH NH— CH— ] ' THE MECHANISM OF ORGANIC SYNTHESIS 131 Thus by the action of simple catalytic agencies on sugar and ammonia we can obtain the iminazol nucleus, and by easy transitions pass through this to the purine nucleus with its contained ring, the pyrimidine nucleus, found in the bases cytosine, uracil, &c.; 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, C6H12Ofl, but is a saturated ring compound : CHOH CHOH /\ CHOH CHOH I jj CHOH CHOH and may be expected to be formed as a result of polymerisation of formaldehyde. 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 occur- rence, 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 ammo-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 phos- phoproteins 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 chick. In the same way the ovaries and testes of the salmon are formed during their sojourn in 132 PHYSIOLOGY 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 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 con- sist 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 mole- cules, 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 com- mon fats, tristearin, tripalmitin, and triolein, we find the glycerides of caproic, capryllic, 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 THE MECHANISM OF ORGANIC SYNTHESIS 133 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. CH. CH. CHOH = COOH Aldehyde undergoes condensation to form aldol. CHO-f H COOH CHO aldehyde CHOH CH2 CHO 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 : CH, HO H 0 gives CH2 0 C;H OH CH3 CH2 CH2 I COOH 134 PHYSIOLOGY 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 formation 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 prepared in this way, proof is still wanting that a continuous series of syntheses 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 continuous building up of fatty acids by the addition of aldehyde obtained through lactic acid from the disintegration of hexose molecules only requires 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 syn- thesis of the higher fatty acids from sugar is carried out in this way, the energy equations would be as follows (Leathes) : 1 g. mol. glucose ) (2 g. mols. aldehyde + 2 g. mols. formic acid. 677-2 cals. ft 2 x 275-5 + 2 x 61-7 - 674-4 cals. 2 g. mols. aldehyde ) fig. mol. aldol V__ : fig- mol. butyric acid. 551 cale. J " > t 546-8 cals. / ~ ^\ ~ 517-8 cals. Or, tracing the same change on as far as palmitic acid : 4 g. mols. glucose ) / 1 g. mol. palmitic acid + 8 g. mols. formic acid. 2708 cals. / \ 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 available 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. The glycerin which enters into the formation of the ordinary THE MECHANISM OF ORGANIC SYNTHESIS 135 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 con- version of glucose into lactic acid the first step is the formation of glyceric aldehyde, CH2OH CH2OH CHOH CHOH CHOH CHO CHOH CH2OH CHOH CHOH I I CHO CHO and it is easy to understand how by a process of reduction the alde- hyde 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 absorption 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 trans- port 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. 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 to be sought in 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 machine, that is to say, a system for the conversion of one form of energy into another. Thus the steam-engine converts the potential energy of overheated steam into mechanical work ; a gas-engine the chemical energy of an explo- sive mixture of gases into heat and mechanical energy ; in a battery there 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. The protoplasm, which is the seat of all these changes in both plants and animals, is active only within fairly narrow limits of tem- perature, approximately 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 conveyed with the food, it follows that all the energy with which we have to deal is the energy of molecules in watery solution, the playground of whose activities is a jelly-like mass of colloidal material, heterogeneous yet structurally continuous. It is important, therefore, at the outset to inquire into the nature of this energy and the methods by which it may be measured. 136 THE ENERGY OF MOLECULES IN SOLUTION 187 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 some heavier gas, such as oxygen or carbon dioxide, within a very short time 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 respon- sible 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 mole- cules 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 CO 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 FIG. 19. 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 continuous translatory movement of the dissolved molecules. Since the molecules 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 138 PHYSIOLOGY the fact that the molecules of sugar pass through it only with diffi- culty, and therefore in their passage outwards towards the confines of the water exert a pressure on the walls, driving them 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 sugar molecules, since the bladder wall itself is not absolutely impermeable 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 this process of diffusion will cease only when the concentration has become the same in all parts of the solution. Sup- posing, 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 involves the performance of an amount of work determined by the initial and final osmotic pressures of the solution. If, on the other hand, a weight be applied to the piston which is less than the osmotic pressure exerted by the sugar solution, the piston with its weight will be moved upwards, and the solution 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 that represented in the diagram, for the performance of work. THE MEASUREMENT OF THE OSMOTIC PRESSURE. By a method differing but little from the one just sketched out, Pfeffer succeeded directly in measuring the osmotic pressure of certain solu- tions. For this purpose Pfeffer took advantage of the fact, discovered by Traube, that various precipitates, if deposited in the form of mem- branes, 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 ferro- cyanide suspended to a glass rod be introduced carefully into a more dilute solution of copper sulphate, it will be observed that at the FIG. 20* THE ENERGY OF MOLECULES IN SOLUTION 139 junction of the drop and the surrounding fluid there is a brown mem- branous 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 of slight currents in the fluid set up by accidental vibrations. Sugar introduced 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 conceived the idea of depositing such a semi-permeable membrane within the interstices of a clay cell. Strengthened in this way, it is able to afford a resistance to pressure, and therefore to permit of the contained fluid reaching its full osmotic pressure. For this purpose a porous jar carefully cleansed and containing a solution of sugar mixed with a little copper sulphate is 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 passage of water, is impermeable to the sugar. The tube is then fitted with a cork provided with a closed mercurial manometer 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 temperatures : Pressure in atmospheres Temp. °C. Calculated. Atm. Atm. 6-8 0-664 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 140 PHYSIOLOGY 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 C^H^Ou = 342), if it could be converted into a gas at 0° C. and 760 mm. Hg, would have a volume of 22-41itres. One gramme of sugar therefore at the same temperature and pressure would have a volume of 22-4 -^r litres = 65-5 c.c. In Pfeffer's experiment the gramme of sugar was dissolved o4J in 100 grammes of water, making a total volume at 0° C. of 100-6 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 J. v/\J*O 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 hydro- chloric acid. Other indirect methods have therefore been applied to the comparison of the osmotic pressures of different solutions. DETERMINATION OF THE OSMOTIC PRESSURE BY PLASMO- LYSIS. 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. 24). 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 protoplasm, the plasmolysis, just occurs, and another smaller concentration at which plasmolysis 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 0'60 per cent, sodium chloride and is absent in a solution containing 0*59 per THE ENERGY OF MOLECULES IN SOLUTION 141 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 TOl per cent, solution of KN03 is found to be isotonic with a 0'58 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, haemo- globin, 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 haemoglobin 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 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 solutions of sugar. The osmotic pressure 142 PHYSIOLOGY 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 other hand, the smallest trace of salt added to distilled water enormously increases its conducting power. As Arrhenius has shown, this increase of the osmotic pressure of a salt solution is deter- mined by the dissociation which all these salts undergo in watery 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. solution. A dilute solution of sodium chloride contains, not the mole- cule NaCl, but an equal number of the ions Na and 01, 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 electro- lysis) 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 disso- ciation, a 0'58 per cent, solution of NaCl would be isotonic or isosmotic with a T8 per cent, solution of glucose. On account of the ionic disso- ciation or ionisation, it is actually isosmotic with a glucose solution of about 3*6 per cent. INDIRECT METHODS OF MEASURING OSMOTIC PRESSURE. Equimolecular solutions have the same osmotic pressures. Since the osmotic pressure of a solution is therefore directly dependent on the THE ENERGY OF MOLECULES IN SOLUTION 143 number of molecules it contains in unit space, any method which, will give us information 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 temperature to 100° C. On the other hand, Barger has suggested an ingenious method in which the alteration of vapour- tension is made the basis of a method for determining the osmotic pressure of small quantities of fluids at ordinary temperatures. And this method may find important applications in physiology. 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 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 are equal. A series of trials is made with different strengths of salt solution until this equality is established. In this method only minimal quantities of material are required, and the determination of the aqueous tension is made at ordinary temperatures. The method, however, which is of greatest value in physiology is the measurement of the depression of freezing-point. The determination is carried out in a Beckmann's apparatus with a thermometer reading to T^o° C. (Fig. 22). A solution freezes at a lower temperature than pure water, 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 in no wise altered by the process of freezing, and it can be applied to solutions containing coagulable proteins which would be irretrievably altered by any considerable rise of temperature. The depression of freezing-point can be converted directly into osmotic pressure by multiplying the depression of freezing-point observed by the factor 122*7. Thus a 1 per cent, solution of sodium chloride with A = O61 will have an osmotic pressure of 0-61 x 122-7 = 74-847 metres of water. 144 PHYSIOLOGY Every substance in solution possesses, therefore, a certain amount of potential energy in the form of osmotic pressure. This pressure is independent of the nature of the substance dissolved and is deter- mined 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 re- garded 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 con- ditions 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 sub- stance on its complete combustion with oxygen to carbon dioxide and water. In the inter- mediate 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 de™nl- the chemical changes which it is undergoing tion of freezing-point. 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 multiplication 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. SECTION II THE PASSAGE OF WATER AND DISSOLVED SUBSTANCES ACROSS MEMBRANES WE have already seen that if, in a solution, the concentration of the dissolved substance or solute is not uniform, there is a movement of the substance from the place of higher to the place of lower concen- tration, and this 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 intermixture of gases is attained. The move- ment in the case of dissolved substances, 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 squaie 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 — differences 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 infinitesimally 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 diffusion coefficient of urea is 0*810 at 7*5° C. denotes that if A be continually filled with a 1 per cent, solution of urea, while in B a constant current of distilled water is kept up so as to maintain the concentration at zero, in the course of a day 0*810 gramme of urea will pass from A to B through the cylinder of one centimetre in length 145 10 146 PHYSIOLOGY and one square centimetre in cross- section. The determination 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 sub- stances, and to maintain a constant 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. B <- . _ -4U4- i/i *s. - "«Mt 1 cm. FIG. 23. 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, therefore, 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 jto produce a movement of PASSAGE OF WATER AND DISSOLVED SUBSTANCES 147 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 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. m A B FIG. 24. If we suppose two vessels, A and B (Fig. 24) separated by such a membrane, A containing a solution of a and B a solution of /3, water will pass from A to B so long as the osmotic 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 transu- dation (the hydrostatic pressure) is equal to the force causing absorp- tion into B (the difference of osmotic pressures). Under no circum- stance will there be any transference of salt or dissolved substance between the two sides. Such semi- permeable membranes as this, however, rarely occur in the body over any extent of surface. The external layer of the cell protoplasm may resemble the protoplasmic pellicle of plant cells in possessing this ' semi-permeability ' ; but in nearly all cases where we have a membrane made up of a number of cells, it can be shown to permit the free passage of at any rate a large number of dissolved substances. Let us now consider what will occur when the two solutions A and B are separated by a membrane which permits the free passage of salts and water. 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 the dissolved substances from B 1 48 PHYSIOLOGY 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 movement 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 isotonic solutions of a and /3. It is evident that the movement of water into A will vary as A.p — Bp* = 0. But diffusion also occurs of a into B and of ft into A. Now the amount of substance 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.ak (when k is the diffusion coefficient). In the same way the amount of /3 diffusing into A will vary as By. /3k'. Hence, if ok is greater than /3k', 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 -f- ft in B will be greater than the number of molecules of a + ft in A, and this difference 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 atmo- sphere 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 * Ap = osmotic pressure of A, &c. PASSAGE OF WATER AND DISSOLVED SUBSTANCES 149 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 atmo- spheres' 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 com- parable to that of water ; so that the passage of salts through the membrane depends merely on the diffusion rates of the salts. There can b'e no doubt, however, that we might get analogous movements of fluid against total osmotic pressure determined, not by the diffu- sibility 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 a familiar 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 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 membrane, 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 sub- stance 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 * 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. t 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. 150 PHYSIOLOGY 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 they regard this absorption as pointing indubitably to an active intervention of living cells in the process. This argument requires examination. Let us suppose the two vessels A and B (Fig. 25) to be separated by a membrane which offers free passage to water and a difficult passage to salts. Let A contain 0-5 per cent, salt solution and B a solution isotonic with a 1 per cent. NaCl, but con- taining 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). m B FIG. 25. If, however, the membrane permitted passage of the dissolved sub- stances, 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 the 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 imper- meability of the membrane. As the NaCl in A reaches a certain concentration it will pass over into B, and this will go on until equili- brium is established between A 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 salt in any fluid above that of the same salt in the plasma, nor the passage of a salt from a hypo- tonic 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 -permeable. It is permeable to salts, but presents rather more resistance to their passage than to the passage of water. Hence on injecting 0-5 per cent. NaCl solution into the pleural cavity, water passes PASSAGE OF WATER AND DISSOLVED SUBSTANCES 151 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 impermeable. Since the osmotic pressure of B is higher, by the partial pressure of x, than that of A, fluid will pass from A to B by osmosis. But the consequence 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 attraction 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. 26), 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. Thus the transference of fluids and dissolved substances across 152 PHYSIOLOGY 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 membrane vary as their diffusibilities, and are therefore probably some function of their 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.) 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. In a recent paper Bayliss has drawn attention to certain other factors which may determine permanent inequality of distribution of a salt on the two sides of a membrane permeable to the salt. If Congo red, which is a compound of an indiffusible colloid acid with sodium, be placed in an osmometer which is immersed in water, a certain osmotic pressure is developed, On 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 PASSAGE OF WATER AND DISSOLVED SUBSTANCES 153 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 Chlorine uye Inside Outside 30 52 30 30 465 73-6 30 <5500 180 100 32-9 29-5 the litres to which each gramme molecule of the salt is diluted. Appa- rently the difference depends on the fact that the non-dissociated salt must be equal on the two sides of the membrane and that the dissocia- tion 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 case 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 active 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 con- ditions is still very imperfect, but the important part played by col- loids in the processes of life renders it necessary to discuss in some detail their properties and modes of interaction. The term colloid, from KoXXrj, 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 crystal- lised 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 sepa- ration of crystalloids from colloids. Although the broad distinction 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 salt, which are very diffusible. Graham pointed out that colloids exist under two conditions : (1) In a state of solution or pseudo-solution, in which they form sols, and are distinguished as hydrosols, when the solvent is water ; and (2) In a solid state, in which a relatively small amount of the colloid 154 THE PROPERTIES OF COLLOIDS 155 sets 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 is obtained on dissolving a little gelatin in hot water and allowing the mixture 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. Another 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 mole- cular 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, a 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 A1203. 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, however, was not precipitated, but remained in sus- pension or pseudo- solution, giving a deep red * or a blue liquid, accord- ing to the conditions under which the reaction was effected. This * Ruby glass is a colloidal ' solid ' solution of gold in a mixture of silicates. 156 PHYSIOLOGY solution was homogeneous 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 of all sizes. The larger settle at the bottom of the vessel, the smaller — which are ultra-microscopic in size, fc.e.from 5 JULJUL to 40 JULJU* — -remain in suspension, 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. PROPERTIES OF GELS. A typical hydrogel is the firm mass in which a solution of gelatin sets on cooling. It is clear, hyaline, appa- rently structureless, and possesses considerable elasticity, i.e. resist- ance 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 inter- stices of which is embedded the second phase, consisting of a very weak solution of gelatin. If the process be observed under the micro- scope, 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 concentrated 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 alco-gel. In a dry atmosphere the gel loses water and becomes shrivelled and dry, but in some cases, e.g. gelatin, 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 * One p is one -thousandth of a millimetre ; one pp. is one-thousandth p, t.e, one-millionth of a millimetre. THE PROPERTIES OF COLLOIDS 157 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 of wood ; water was poured on the wood, and the swelling of the wedges split the rock in the desired direction.* It is on account of the extent of surface that it is practically impos- sible to wash out the inorganic constituents from a gel. The diminu- tion of the osmotic pressure of many dissolved substances at surfaces causes the concentration at the surface of a gel 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 ferro- cyanide, are not only impermeable to colloids, but also to many crystalloid substances. These membranes, therefore, were used by PfefEer for the determination of the osmotic pressure of such crystal- loids 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 becomes 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 applica- * According to Rodewald, the maximal pressure with which dry starch attracts water amounts to 2073 kilo, per sq. cm. 158 PHYSIOLOGY tion of energy, to separate one from the other. Thus filtration, gravita- tion leave the composition of the solution unchanged. It is true that, by the employment of certain kinds of membrane, e.g. the semi-per- meable 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 colloid solutions also display an osmotic pressure ? Sabanejeff has attempted to decide this question in an indirect manner, i.e. by the determination of the depression of freezing-point caused by the addition to water of various colloids. The depressions observed by this author were so small that they might be regarded as 'falling within the limits of experi- mental error. Assuming that the depression in each case was due to the presence of the dissolved colloid, Sabanejeff arrived at the following molecular weights for certain colloids : Tannin . . . 1,322 Egg-albumm . . . 15,000 Starch . . . over 30,000 Silicic acid . . „ 49,000 I have shown, however, that it is possible to determine the osmotic pressure of colloidal solutions directly, taking advantage of the fact that colloidal membranes, 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 is obtained, perfectly 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 osmometer, the filtrate being used as the inner fluid. The construction of the osmometer is shown in the diagram (Fig. 27). 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 cigarette. 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 prevent 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 peri- toneal 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, THE PROPERTIES OF COLLOIDS 159 One of these, (), is for filling the outer tube ; the other is fitted with a mercurial manometer, M. Two small reservoirs, CO, 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, CO, 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 removed, 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 corre- spond to a molecular weight of about 30,000. A more convenient form of osmometer has been devised by B. Moore, using parchment paper as the membrane. With this osmo- meter, the existence of an osmotic pressure in colloidal solutions has been definitely established both by Moore in the case of haemo- globin, proteins, and soaps, and by Bayliss in the case of colloidal dyes, such as Congo red. The osmotic pressure of haemoglobin was found by Hiifner to correspond to a molecular weight of about 16,000, i.e. a molecular weight already deduced from its composition and its combining powers with oxygen. Often, however, the osmotic pressure shows a molecular weight which is very 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 condi- tions 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 the 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 160 PHYSIOLOGY 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 only form 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 substances, 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 /u, 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 mole- cules. As a matter of fact we find that such solutions present an amazing mixture of properties, some of which betray them as mechani- cal suspensions, while others partake of the nature of the chemical reactions such as those studied in the simpler compounds usually dealt with by the chemist. OPTICAL BEHAVIOUR OF HYDROSOLS. Nearly all colloidal solutions 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 illu- minated, acts as a centre of dispersion 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 dis- persed by them at right angles to the beam was polarised. This THE PROPERTIES OF COLLOIDS 161 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 posi- tion the light is bright, in the position at right angles to this it becomes dim or is extinguished. The production of the Tyndall phenomenon may therefore be regarded as a test for the presence of ultra-microscopic particles, varying in size from 5 to 50 /UL/UL. 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 raffinose (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 X or -3 /m, 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. The arrangement for this purpose was first devised by Zsigmondy and Siedentopf . 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 Brownian movements. The smallest particles which can be seen show a combined movement, consisting of a translatory move- ment, in which the particle passes rapidly across the field in one- sixth to one-eighth of a second, and a movement 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 HJL. 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 phases. 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 .11 162 PHYSIOLOGY the direction of their movement 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 we obtain a colloidal suspension of albumin. When thoroughly FIG. 28. Movements of two particles of india-rubber latex in colloidal solution, recorded by cinematograph and ultra-microscope. (HENRI.) dialysed, this protein is insoluble in pure water, but is soluble in traces of either acid or alkali. In acid solution the protein particles carry a positive charge, whereas in alkaline solution their charge is negative. The charged condition of these particles must play a considerable part in keeping them asunder and therefore in preventing their aggrega- tion and precipitation. This is shown by the fact that any agency which will tend to discharge them will cause precipitation and coagu- lation. Among such agencies is the passage of a constant current, just mentioned. To the same action is due the coagulative or pre- cipitating effects of neutral salts. Thus any of the colloids we have mentioried, 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 precipita- tion of the electro-positive ferric hydrate the acid ion of the salt b the determining factor, the coagulative power increasing rapidly THE PROPERTIES OF COLLOIDS 163 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 Bax/, 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/7, is 400 times as effective as BaCl2. AMOUNT OF SALT NECESSARY TO PRECIPITATE COLLOIDAL SOLUTIONS To coagulate Fe.20 K2SO4 1 g. mol. in 4,000,000 c.c. MgS04 „ „ „ 4,000,000 „ BaCl2 „ „ „ 10,000 „ NaCl „ „ „ 30,000 „ To coagulate Gold BaCl2 1 g. mol. in 500,000 c.c. NaCl „ „ „ 72,000 „ K2SO4 „ „ „ 75,000 „ 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 the 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 combinations, 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 absorption compounds. Since, how- ever, 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 aggregates of molecules which distinguish the colloidal state form a system with a considerable inertia, so that we have a tendency to the establish- ment 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. 164 PHYSIOLOGY The factors involved in the formation of adsorption or absorption combinations 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 with a surface of 22 sq. cm., if reduced to a fine powder consisting of spherules of -00000025 cm. in diameter, will have a surface of 20,000,000 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 deter- mined 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-conducting 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 Jit 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 compounds 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 solution Proportion of dye 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 0-002 trace trace practically all practically all If put into the form of a curve, where the ordinates represent the THE PROPERTIES OF COLLOIDS 165 percentage of dye left in solution, and the abscissae the original con- centration of the solution, the curve only approaches the axis (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 electrolytes. By continuously washing a protein or gelatin with distilled water, 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, therefore, 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 recog- nised 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 indeter- minate, the effect of adding either acid or alkali to the neutral globulin being to cause a gradual conversion of an oqaque, 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 ampho- teric substances, such as the amino- acids. An amino-acid, such as glycine, can react as a basic anhydride with other acids, thus : NH2 NH2HC1 CH/ + HC1 = CH<( XC02H C02H or as an acid anhydride with bases : CH2.NH2 CH2.NH2 | +NaHO= | +H20 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 166 PHYSIOLOGY 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 01 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 quantitative relations of its components, but also by the past history of the system. COMBINATIONS BETWEEN COLLOIDS Besides the compounds between colloids and electrolytes, com- bination, or at least interaction, takes place between different col- loids. 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 re- actions between toxin and antitoxin, between ferment and substrate, which we shall study later, are of this character, and that the com- pounds 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 par- ticles aggregating to form larger complexes. These aggregations may settle to the bottom of the fluid as a precipitate, or may form a species THE PROPERTIES OF COLLOIDS 167 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 agencies may lead to the production of chauges 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 concentrated 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 floccu- lent 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 phenomenon. It depends on the fact that a large number of substances in solution (viz. any which lower the surface tension of their solutions) undergo concentration at the free surface of the fluid. Such substances are proteins, bile- salts, quinine, saponin, &c. In the case of proteins the concentra- tion 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 con- nected with these by all possible grades. In a solution of an ordinary crystalloid or electrolyte the molecules of the dissolved substance are 168 PHYSIOLOGY distributed equally and homogeneously 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. The ' dissolved ' molecules now begin to 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 surface 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 in many cases it is impossible to distinguish from the process of solution. This phenomenon, which was long ago studied by Chevreul and has lately been the subject of a series of careful experiments by Overton, is exhibited by aft animal tissues and all colloids. Thus elastic tissue dried in vacua 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 pres- sure 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 increases 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 THE PROPERTIES OF COLLOIDS 169 pressure, as a -67 per cent. NaCl solution. A 67 per cent, cane-sugar solution has, however, the same osmotic pressure as an 18J per cent, solution of common salt. It is impossible, therefore, to draw any hard line of distinction between imbibition pressure and osmotic pressure- In the same way it is impossible 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 substances in question. Thus all the crystal- line 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 condensation 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 case 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 tur- pentine often to an indefinite extent, while they are untouched by water. With this behaviour we may compare the easy solubility of the hydrocarbons, the aromatic acids, and esters in ether and benzol, and their insolubility in water. As Overton 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 substances 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 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 tem- peratures. (2) The specific direction of the process, which is therefore almost complete, with a surprising absence of the side reactions which inter- fere to such an extent with the yield of the methods employed in a chemical laboratory. This second characteristic may, however, be regarded as a con- sequence of the first, since an increase in the velocity of any given 170 CHEMICAL CHANGES IN LIVING MATTER. FERMENTS 171 reaction will determine a preponderance of this reaction over all other possible ones. A fundamental 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 dehydration, 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 simul- taneous dehydrolysis at different parts of the molecule determine a number of chemical transformations; 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. 128. (2) DEAMINATION. This process involves the splitting off of an NH2 group from an amino-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 suggested that here also the process of splitting off ammonia is a hydrolytic one and that the NH2 group is replaced by OH. Thus- CH3 CH3 CH.NH2 + H20 = CH.OH + NH3 COOH COOH (alanine) 172 PHYSIOLOGY Recent work by Neubauer tends to show that deamination is accompanied in the first place by oxidation, so that the first inter- mediate product formed is not an a oxy-acid, but an a ketonic acid. A second atom of oxygen is then taken up, and carbon dioxide is split off, with the production of the next lower acid of the series. We might represent these changes as follows : (1) CH3 CH3 GOGH COOH (2) CH3 CH3 CO +0= I +C02 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 ammonia. 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 con- tinuously 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 benzylpyrotartaric acid (C6H5. CH2.CH2.CO.COOH) to a dog, a certain amount of benzylalanine (C6H5.CH2.CH2.CHNH2.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 ! CHNH2 + 0 C CO + NH3 COOH COOH COOH (3) DECARBOXYLATION. Many amino-acids when subjected to CHEMICAL CHANGES IN LIVING MATTER. FERMENTS 173 the agency of bacteria lose a molecule of carbon dioxide and are converted into a corresponding amihe. For instance, lysine, which is diamino-caproic acid, is converted into pentamethylene diamine or cadaverine. Thus : CH2.NH2 CH2.NH2 CH2 CH2 I I CH2 becomes CH2 CH2 CH2 CH.NH2 CH2.NH2 COOH In the same way ornithine derived from the breakdown of arginine is converted 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. /3 phenylethvlamine from phenylalanine, C6H5.CH2.CH2.NH2. Para, oxyphenylethylamine from tyrosine, OH.C6H4.CH2.CH2.NH2. A similar process has been supposed to take place as a step in the successive oxidation of the carbon atoms in the long chain fatty acids or carbohydrates, 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 /3 position. On the other hand, decarboxyla- tion 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 . 0 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 reactions of the living body, but may lead to the produc- tion of the most complex substances known — are performed with little 174 PHYSIOLOGY expenditure or evolution 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 Heatofcom- Maltose .... 1350 Glucose .... 677 Hippuric acid . . . 1013 Heat Final of substance combustion 2 Glucose . . . 1354 2 Lactic acid 659 (Glycine . 235 \ \Benzoic acid . 773 J ' 1008 (2) DEAMINATION Initial , Heat of substance combustion Alanine .... 389-2 Leucine . . . . 855 Aspartic acid . . . 386 Final Heat of substance combustion Lactic acid . . 329-5 Caproic acid . . 837 Succinic acid . 354 (3) DECARBOXYLATION Initial Heat of substance combustion Alanine .... 389 Leucine 855 Final Heat of substance combustion Ethylamine. . . 409 Amylamine. . . 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 oxidation 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. CHEMICAL CHANGES IN LIVING MATTER. FERMENTS 175 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 means 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 relationships 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 in- creasing 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 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 presented by a precipitate. A common method of isolat- ing, 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 f 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 represents some of the ferments whose 176 PHYSIOLOGY 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.) 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 Lactase ..... Milk sugar Glucose and galactose Invertase or sucrase . Cane sugar Glucose and levu- lose Arginase ..... Arginin Urea and ornithine Urease . . Urea Ammonium carbo- nate Lactic acid ferment . Glucose Lactic acid. Zymase ( ? present in the body) . Glucose Alcohol and C02 Deaminating ferment (?) v. p. 171 Amino-acids Oxy-acids (?) 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 monosaccharides. 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 hydration 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 CHEMICAL CHANGES IN LIVING MATTER. FERMENTS 177 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 spon- taneous 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 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 12 178 PHYSIOLOGY are converted 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 oxida- tion of hydriodic acid by bromic acid, but not for the oxidation of the same substance by iodic acid. Iron and copper salts in mi mite 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 oxidation 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 reac- tion increases rapidly with rise of temperature, in the case of ferments this increase 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 con- siderably below that of boiling water. Thus ferment actions, like cata- lytic 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 l 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, coagulated, 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 tran- scriptions in 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 CHEMICAL CHANGES IN LIVING MATTER. FERMENTS 179 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 catalytic 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 sub- division, and is best marked when the metal is reduced to ultra- microscopic dimensions, 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 involved. The same process of condensation occurs with dissolved substances. Just as the pressure of a gas in immediate contact with a solid body is diminished, so the osmotic pressure of a substance in solution is diminished at the surface. There is therefore 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 com- pression 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 re-trans- formed 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 contain- ing 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 180 PHYSIOLOGY 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. by the formation of an intermediate product. Thus, in the ordinary process for the manufacture of sulphuric acid, the nitric oxide may be supposed 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 hypo- thesis. 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 suffi- cient explanation of a catalytic process only when it can be demon- strated 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 perinolybdic 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 interaction of hydrogen per- oxide 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 inter - * Quoted by Mellor, " Chemical Statics." CHEMICAL CHANGES IN LIVING MATTER. FERMENTS 181 mediate 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 intro- duction 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 the edge, 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 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 infinitely 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 depends 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 decomposition into hydrogen and arsenic. This decom- position is not immediate, but takes a certain time, and the velocity with which the change occurs depends on the temperature. At any given temperature the amount of substance changed in the unit of time varies with the concentra- tion 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 tempera- ture, and C represents the concentration of the substance. The equation 0 = 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(CX x Cy). In the case of the unimolecular reaction, halving the con- centration 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 182 PHYSIOLOGY 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 molybdift acid on the interaction 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 : 0=K(CHaoz x Cm). After the addition of molybdic acid, the equation becomes : 0 = K(CH2o2 + y C molybdic acid)Cm, when y is another constant depending on the molybdic acid. If fermenl s 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. Various methods may be adopted for the study of the velocity of ferment in action. If. for instance, we are investigating the action of diastase upon starch, we should take solutions of starch and of diastase of known concentrations, 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 with- drawn 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 only necessary 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 per- centage 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 wil precipitate all the unchanged protein, but will leave in solution the products of hydration of the protein. From the amount of nitrogen CHEMICAL CHANGES IN LIVING MATTER. FERMENTS 183 in the filtrate from the precipitate can be determined the total amount of protein which has undergone hydration in the sample under observa- tion. 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 intensity of the reaction will first rise to a maximum and then gradually disappear. 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 disintegra- tion 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 disintegration. By such methods it has been found that, when the quantity of ferment employed is very small in comparison with the substrate (the substance acted upon), the amount of change in a given time is proportional to the amount of ferment present, and is (within limits) independent of the concentration of the substrate. This is well shown by the two following Tables representing the action of lactase upon lactose (E. F. Armstrong) : PROPORTIONS HYDKOLYSED 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 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 2-21 184 PHYSIOLOGY Moreover, if we take only the earlier stages of the ferment action, it is found 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 LACTASE Time Amount hydrolysed J hour 3-2 I „ 6-4 1 „ 9-6 2 hours 164 3 „ . . . . . . . . 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 molecules 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 to 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. 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 $ = KG, where C is the concentration of the ferment. This concentration is always being renewed, and kept constant by the breaking down of the inter- mediate 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 rate 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) : CHEMICAL CHANGES IN LIVING MATTER. FERMENTS 185 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 7th 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 retarda- tion can be augmented by adding to the digesting mixture the boiled end-products of a previous digestion. The retarding effect of the end-products resembles in many ways that observed in a whole series of reactions which are known as reversible. As an example of such a reaction we may take the case of methyl acetate and water. When methyl acetate is mixed with water, it undergoes decom- position with the formation 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 : MeC2H3O2 + HOH ;=± MeOH + HC2H3O2. methylacetate water methylalcohol acetic acid Each of these changes has a certain velocity constant, and, since they are in opposite directions, there must be some equilibrium point where no change will occur, and 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 inter- action 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 designated as isomaltose or revertose. To this reverse action may be due a certain amount of the retardation observed in the action 186 PHYSIOLOGY 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 altera- tion of the substrate. 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 o-glucosides, it has no power on the /3-glucosides ; that is to say, maltase will fit into a molecule of a certain configuration, but is powerless to affect a mole- cule which differs from the first only in its stereochemical structure. On the other hand, emulsin, which breaks up />glucosides, has no influence on a-gluco- sides. This specific affinity of the ferments for optically active groups of bodies suggests that the ferment itself 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 experiments 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 and Isevo-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 Isevo-rotatory. Thus the rate of hydrolysis of the dextro- component of the ester is greater than that of the Isevo-component, a result which can be CHEMICAL CHANGES IN LIVING MATTER. FERMENTS 187 best explained by the assumptions (a) that the enzyme or a substance closely associated with it is a powerfully optically active substance ; (6) that actual combination takes place between the enzyme and the ester undergoing hydro- lysis. 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 sub- stances, 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 colloid or semi-colloid character, cannot be dealt with in the same way as the catalysts of definite chemical composition, 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 sur- faces 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 belongs, therefore, to that special class of inter- actions, 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 pro- ceeded 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 188 PHYSIOLOGY that the hydrolysis of esters by lipase is a reversible reaction, the action of lipase being simply to hasten the attainment of the equili- brium 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 appearance 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 only obtained with distinctness 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 inter- preted 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, addition 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 whichever 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 THE 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 dis- sociation 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 knowledge of the changes in the distribution of charged ions responsible for the 189 190 PHYSIOLOGY response ought to throw important light on the intimate nature of excitation generally. It may be therefore advisable to consider more closely the 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 — In ®Zn - ®2n Zn© - ©Zn 2n © Zn ©2" i i FIG. 29. FIG. 30. from B to A through the cell. A 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 dis- solve. 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 only possible 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 discharged by combining with the S04 ions passing to the zinc from ELECTRICAL CHANGES IN LIVING TISSUES 191 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 contact 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 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 therefore 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 refrigerating 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 concentra- tion 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 192 PHYSIOLOGY in diffusibility of the substances in solution, especially if the two solutions be separated by a membrane. Very large differences may be produced if this membrane be practically impermeable to one or other of the dissolved substances. In the same way a semipermeable membrane, i.e. a membrane with different permeabilities for the different ions of the two solutions, may suffice to bring the differences OL potential of a concentration cell up to and beyond the extent which is observed in living tissues. Supposing we have (Fig. 31) two solu- tions, A and B, each containing an electrolyte, UV, in different con- centrations separated by a membrane m. If u represents the velocity m UV B UV FIG. 31. of transmission of U through m, and v the velocity of V, then the electromotive force of the cell is given by the formula u — v 0-0577. log.10--! Volt. If v is taken as very small, the membrane may be regarded as semi- permeable 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 ^a 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 solution 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, U^, U2V2 (U being the cation in each case), separated by a membrane with varying permeability for the different ions, the electromotive force of the cell is given by the following formula : 0-0577 log. 10 ELECTRICAL CHANGES IN LIVING TISSUES 193 where UjVJt u2v2, 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 w2 and % very small, the expres- sion log.10 — may be made to attain anv quantity, and in the «V+?1 same way by making ut + v2 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 ultramicroscopic 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 Vj 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 accumu- lation of the ion at the surface of the membrane, so that this will become polarised. Such an accumu- lation 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 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 connected 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 con- nected with the positive pole, its charge will be increased and its surface tension correspondingly diminished, so that the meniscus 13 FIG. 32 194 PHYSIOLOGY will move towards the point of the capillary. The movement of the meniscus to or away from the point may thus be used, as in the capil- lary electrometer, to show the direction and amount of any moderate electrical change occurring in a tissue, two points of which are con- nected 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. BOOK II THE MECHANISMS OF MOVEMENT AND SENSATION CHAPTER V THE CONTRACTILE TISSUES SECTION 1 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 mechanisms 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. Tt 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 environment 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 contraction 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 197 198 PHYSIOLOGY of the body, or of the afferent nerve passing from the surface to the central nervous 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 each 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 reaction forms an end link in the reflex chain, namely, the muscle, Sensory Surface \ \ Cenfra/ Nervous System FIG. 33. Diagram of a reflex arc. 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 intestine, 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 tissues have been most fully investigated in the voluntary muscles, almost exclusively on the THE STRUCTURE OF VOLUNTARY MUSCLE 199 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 incidentally 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 FIG. 34. Muscular fibre of a mammal, examined fresh in serum, highly magnified. (SCHAFER.) 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 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 striaa, arranged at right angles to its long axis, and enclosed in a structureless sheath— the sarco- lemma. 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 trans- parent 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. 200 PHYSIOLOGY The development of this regular cross and longitudinal striation 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 amceba, can effect only slow and weak contractions. 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 striation. 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 differentia- tion affects only part of the cell, so that while one part presents the ordinary granular appearance, the other half is finely and longitu- THE STRUCTURE OF VOLUNTARY MUSCLE 201 dinally striated, the striation being apparently due to the develop- ment of special contractile fibrillae. 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 with and often apparently subordinated to a transverse striation, due to the regular segmentation of the contractile fibrillae or sarcostyles. Every muscular fibre, which presents any trace of histological differentiation, may be said to consist of contractile fibrillse (sarcostyles), each composed of FIG. 37. Transverse sections of the pectoral muscles of a, the falcon, b, 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.) 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 largely conditioned by the varying relations, spatial and quantitative, of the sarcoplasm to the sarco- sfcyles. Thus in the higher vertebrates, two types of voluntary muscular fibre are distinguished, according to the 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 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 sarco- styles. The connection between structure and activity of the muscle- fibres is well shown by Fig. 37. 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 202 PHYSIOLOGY 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 knowledge 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 demonstrably visible. It FIG. 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 of Hensen. c, an uncontracted fibril, showing the porous structure of the sarcous elements. 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 varying elements of the muscle fibre. All observers are agreed that the essential contractile element is the row 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. S.E, Diagram of a sarcoinere 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 element. (SCHAFER.) THE STRUCTURE OF VOLUNTARY MUSCLE 203 Most histologists agree in assigning to the middle part of* the sarcous element a denser structure than to the two ends. According to Macdougall, however, the lighter appearance at each end of the sarcous element 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 exten- sible, and is folded longitudinally, so that it can bulge out and produce a shortening and thick- ening of the whole sarcous ele- ment if by any means the pres- sure be raised in its interior. In FlG- favour of a differentiation within the sarcous element itself is the fact that under certain conditions it is possible to produce a preci- pitate, limited only to the central part of the sarcouselement, i.e. the part to which Schafer assigns a tubular structure. When a muscle fibre, killed by osmic acid or alcohol, is examined under the microscope by polarised light, it is seen to be made up of alternate bands of singly and doubly refracting material. The doubly refracting (anisotropous) substance corresponds to the dark band, and the singly refracting (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 refracting, 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 apparent 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 strise appear dark, and the dark striae by contrast lighter than they were before. That there is no true reversal of the strise is shown by examining 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 204 PHYSIOLOGY 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 undifferentiated protoplasm, con- taining nuclei, and lying in contact with the contractile 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 con- siderable length of the muscle fibre. The sole plate in this case seems to be limited to scattered 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 connec- tion with the contractile substance itself. Most of our knowledge on the subject of muscle has been derived from the study of the gastroc- nemius and sartorius muscles of the frog. The position of these muscles is shown in the accom- panying diagram (Fig. 41). The gastrocnemius, which, with the attached sciatic nerve, is most fre- quently employed as a nerve- muscle preparation, forms a thick belly immediately iinder 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 only be employed 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. FIG. 40. Motor end-organ of a lizard, gold preparation. (KtifiNE.) n, nerve fibre dividing as it ap- proaches the end-organ; r, ramifica- tion of axis cylinder upon 6, granu- lar bed or sole of the end -organ ; m, clear substance surrounding the ramifications of the axis cylinder. THE STRUCTURE OF VOLUNTARY MUSCLE 205 The sartorius 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 pubis 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 conditions of a muscle fibre accompanying its activity. Tib. ant. long. - Tendo Achillis FIG. 41. Muscles of hinder extremity of frog. (After ECKBR.) When a greater mass of approximately 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- preparation of the frog is a useful one for the study of the action of different substances on muscle fibres. SECTION II EXCITATION OF MUSCLE A MUSCLE may be caused to contract in various ways. Normally it contracts 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 stimulated, 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 to effectually 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 ure 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 ; 206 EXCITATION OF MUSCLE 207 so we must conclude that the curare paralyses the muscles by affecting the terminations of the nerve within the muscle, and probably the end-plates themselves. 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 with- out 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 effect 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 con- tains 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 FIG. 42. The ramification 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 that we have mentioned as 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 may be applied many times to any point on the muscle or nerve with- out killing the part stimulated, whereas with other forms of stimulus it is difficult to obtain excitatory effects without injuring to a greater or less extent the part stimulated. 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 inten- sity 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 element. Hence the current flows (in the cell) from zinc to copper, within the sartorius muscle of the frog, showing the freedom of the lower portion of the muscle from nerve fibres. (KtJHNB.) 208 PHYSIOLOGY 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, 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 very convenient form of battery, though not so constant as the two forms just described, is the bichromate battery, with a single fluid. This consists of a plate of zinc between two plates of carbon. The whole are arranged so that they can be immersed in or drawn out of the fluid at pleasure. The fluid used is a mixture of sulphuric acid and potassium bichromate. The wire attached to the carbons is the positive pole and the current in the outer circuit flows from carbon to zinc. Another useful type of cell is the Leclanche cell. This consists of a glass jar containing 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 surrounded 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 connected 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 polarisation 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, par- ticularly 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 14 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 Daniell's 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 completing 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 anode, and the point at which the current leaves the nerve is called the cathode. The wires by which the current is conducted to and from the nerve are called the elec- trodes. As electrodes we generally employ two platinum wires mounted together on a piece of vulcanite. EXCITATION OF MUSCLE 209 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. Muscle. Jfathode. FIG. 43. Anode. Ntrve. Du Bois Raymond's key consists of two pieces of brass, each of which has two binding 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 show the way in which the key is arranged for short-circuiting. 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 the nerve however is about 100,000 ohms, whereas that of the bridge is not the thousandth 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 FIG. 44. Du Bois key, closed. Du Bois key, open. practically infinite compared with that in the brass bridge, and so all the 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 insulating 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 consisting of two wires joined by an 14 210 PHYSIOLOGY 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 the 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 FIG. 45. Diagram of 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 between 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 be 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) 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 con- taining many turns of wire. The smaller coil (%, Fig. 46), consisting of a EXCITATION OF MUSCLE 211 few turns of comparatively thick wire, is the primary coil, and is put into connection 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 inducing secondary currents. The secondary coil, R2, of a large number 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 connected with the nerve or other tissue that we wish to stimulate. Since the electro- motive force of the 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 current 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. FIG. 46. Diagram of inductorium. By primary ; R.2, secondary coil. m, electro -magnet of Wagner's hammer, w, Helmholtz's side wire. 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) j (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-induction. At break of the current, an extra current is also produced in the primary coil in the same direction as the battery 212 PHYSIOLOGY 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 disappear- ance 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 b corresponding to the make and c d to the break. The lower line represents the momentary currents induced in the secondary circuit, m being the current of low intensity and long duration produced by the make, and B 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 in FIG. 47. attached to the coil, known as Wagner's hammer (Figs. 48A and 48B). In this case the wires from the battery are connected to the two lower screws (a and &, Fig. 46). Fig. 48A shows the direction of the current when Wagner's hammer is used. The current 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 BJ. From the primary coil it passes up the small coil m, and from this to the terminal b and back to the battery. But in this course the coil m is converted into an electro-magnet. The hammer h attached to the spring is 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 broken 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 in the primary will be much stronger than the inter- vening 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 usqd. EXCITATION OF MUSCLE 213 In this arrangement the side wire w, shown in Fig. 46, and diagrammatically in Fig. 48B, 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 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's wire w to the screw c, thence through the primary coil %, 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 arrange- ment is insufficient to magnetise this, and the hammer springs up again ; thus t e IT 1 __=£_- J h -*~R fj C _n_ i Jy i — i : m i j ,i 1 /^ ^r^ j -fl i ^ ^ — ' A 11 I1 ' — i — ' ]_ i FIG. 48A. Diagram showing course of current in inductorium when Wagner's hammer is used. T Cr:±:7 FIG. 48s. Diagram showing course of current when the Helmholtz side wire is used. 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 in the primary current when the current is short-circuited instead of being broken, and & represents the effect produced in the secondary coil. It will be seen that the currents m and 6 are practically identical in intensity and duration. When the induction-coil is used for stimulating, it is usual to graduate the strength of 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 pro- duced by make or break of the primary current is very small, and on moving the secondary from 20 up to 10 cms. the increase in strength of the current will not be 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 214 PHYSIOLOGY 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 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. 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 FIG. 49. Diagram to show number of plates of tinfoil, separated by discs of the mode of construction of mica or paraffined paper. Alternate discs are a condenser. connected together : 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 6 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 pre- paration that 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 6, it is evident that if c is pushed close to 6, the E.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 TTRT v°l** 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-tiviteh. 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 EXCITATION OF MUSCLE 215 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- con traction. 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 constant 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 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. 216 PHYSIOLOGY 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. This contraction is effected by a change of form. 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 contrac- tion or the muscle-twitch, and to study 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 in fact use the graphic method either for registering the changes in volume or for registering changes in tension of a muscle which is prevented from contracting. In order to record the muscle-twitch on 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 inser- tion 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 217 218 PHYSIOLOGY 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 FIG. 51. Arrangement of apparatus for recording simple muscle-twitch. arranged that it knocks over a key as it shoots across, and so breaks the primary circuit and excites the nerve or muscle cf 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. In the figure (Fig. 52) the upper line is the curve drawn by the lever of the FIG. 52. Curve of single muscle-twitch taken on a rapidly moving surface (pendulum myograph). (¥EO.) muscle as it contracts ; the small upright line shows the point at which the muscle was stimulated ; and the second line is the tracing of the chronograph, every vibration representing ^ of a second. In the pendulum myograph (Fig. 53) a smoked glass plate is carried on a heavy iron pendulum. At each side the pendulum is armed with a catch, which fits on to other catches at the side of the triangular box, from the apex of which the pendulum is 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 is THE MECHANICAL CHANGES OF MUSCLE 219 sent into the muscle or nerve, which contracts, and a curve is obtained similar to that shown in Fig. 62. Since the rate of the pendulum is constantly K\ FIG. 53. Simple form of pendulum myograph. 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. FIG. 54. Diagram of spring myograph, or ' shooter.' 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 slides 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 220 PHYSIOLOGY the catch holding it in this position is released by the trigger, the spring, which only acts for a short space, 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. The 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 relative positions at which the circuits are broken can be altered by a con- venient adjustment. A tuning-fork vibrating about 100 per second fixed to the base of the instrument marks the time ; its prongs are sprung apart by t' Cl — 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 connection 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. 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 instru- mental 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 (mv^\ = j, 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 load the THE MECHANICAL CHANGES OF MUSCLE 221 muscle with 40 grams 1 millimetre from the axis than with 1 gram 40 millimetres from the axis, though the tension put on the muscle will be the same in both cases. In the first case the energy of the moving mass will be proportional to = 800, and it is this energy which 40 x (I)2 1 x ; = 20, and in the second to determines the overshooting of the lever and the deformation of the curve. Since throughout the contraction the lever follows the muscle in its movement, , , TO DRUM 3SCM -». u Q D FIG. 55A. Myograph for optical registration of muscular contraction. (K. LUCAS.) the tension on the muscle remains the same throughout, and the method is therefore known as the isotonic method. In many cases it is of importance to be able to record the development of the energy (i.e. the tension) of the active muscle apart from any changes in its length. For this purpose the muscle is allowed to contract against a strong spring, the movements of which are magnified by means of a very long lever. Thus the shortening of the muscle is almost entirely prevented, but the in- crease in its tension causes a minute but proportionate movement of the spring, which is recorded by means of the lever. Since in this case the length or measurement of the muscle remains approximately constant, while the tension is continually varying throughout the contraction, it is known as the isometric method. The great magnification necessary in this method introduces serious sources of error ; but it seems that, if all due precautions be taken to avoid these errors, the isometric curve differs very little in form from the isotonic, displaying only a somewhat quicker development of energy at the beginning of contraction. It would probably be 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. 55 A). 222 PHYSIOLOGY 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. (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 -^ second, the phase of shortening T£Q, and the relaxation Tfo second. Thus a single muscle-twitch is completed in about TV second. It must be remembered, however, that this number is only approxi- mate, 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 deformation of the curve due to defects of the recording instruments used. Thus the relative period during which no visible mechanical changes are taking place in the muscle must always be shorter than is apparent from a curve obtained by the foregoing method. The elasticity and extensibility of the muscle must prolong the 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 move the tendon to which the lever is attached. Thus if we have a weight supported by a rigid wire, and suddenly pull the upper end of the wire so as to raise the weight, the latter will rise instan- taneously. If, however, the weight be suspended by a piece of elastic, it will not follow the pull exactly, but will lag behind, the first part of the pull being occupied with stretching the india-rubber, and only when this is stretched to a certain degree will the weight begin to rise. The same retardation 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. Thus in the experiments of Sanderson and Burchthe thickening of the muscle at the point stimulated was recorded graphically by photographing the movement on a slit (Fig. 56), behind which was a moving sensitive plate. 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. 57). This figure we can take as the average latent period for the skeletal muscle of the frog at the ordinary temperature of the laboratory (about 16° C.). We shall have occasion later on to consider the changes which occur in the muscle between the application of THE MECHANICAL CHANGES OF MUSCLE 223 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 Fia. 56. Burdon Sanderson's method for photographic record of muscle- twitch. The exciting shock is sent into the muscle by the wires d and d'. normal condition is complete. It is not active — that is to say, is not due to a contraction in the transverse direction — but is a passive effect of extension and elastic rebound. This may be shown by FIG. 57. Photographic record of muscle-twitch. (B. SANDERSON.) The upper curve is the movement of the muscle, the middle curve the signal showing the moment of excitation, and the lower curve is that of a tuning-fork vibrating 500 times a second. allowing a muscle to contract while floating on mercury. The sub- sequent 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 224 PHYSIOLOGY over-shoot of the lever whenever, as at high temperatures, the con- traction is sufficiently rapid. The effect of this is that one cannot assume the existence of an actual pull on the lever during the whole FIG. 58. V. Kries' apparatus for taking ' after-loading ' and, contraction ' curves. arrested 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 actually pulling on the lever, which will occupy only a part of the AAAAAAAAAAAAAAAAAAAAA FIG. 59.^ Curves of isotonic and arrested contractions of an unloaded muscle. (KAISEK.) ascent of the curve. The duration of this period of contractile stress may be shown by™recording what is known as ' arrested ' contractions. One mechanism for this purpose is shown in the figure (Fig. 58). The stop Su is 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 regulated so that it suddenly checks the movement of the lever at any desired height above the base line. We may thus get a THE MECHANICAL CHANGES OF MUSCLE 225 series of contractions such as those shown in Fig. 59. 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 X 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. 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 FIG. 60. Diagram of arrangement for recording the contraction wave in a curarised sartorius. and spreads in both directions through the muscle. The rate of propagation of the contraction 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 sartorius muscle 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. 60). The difference between the latent periods of the two curves represents the 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 contraction in frog's muscle is 3 to 4 metres per second ; in the muscle of warm-blooded animals it may amount to 6 metres. 15 226 PHYSIOLOGY The actual duration of the shortening at any given point is neces- sarily 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 3000 X -05 (= 150) and 4000 X -09 (= 360) milli- metres. 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. SUMMATION OF CONTRACTIONS If a muscle or its nerve be stimulated twice in succession so that the second stimulus becomes effective before the state of activity due to the first stimulus has come to an end, we get a combination of the FIG. 61. Muscle curves showing summation of stimuli, r and r't the points at which the stimuli were sent into the nerve. From the first stimulus alone the curve a b c would be obtained. From r' the curve def is obtained. These two curves are summated to form the curve aghik when both stimuli are sent in at the interval r i'. effects of the two stimuli, and the resulting contraction of the muscle is as a rule greater than that which can be evoked by a single stimulus. If the interval between the two stimuli is so far apart that the second becomes effective just as the contraction due to the first has commenced to die away, the second contraction seems to start from the point to which the muscle has been raised by the first (Fig. 61). If the second stimulus becomes effective at the height of the first contraction, the shortening of the muscle may be almost doubled. By repeating these stimuli the contraction may be made three or four times as extensive as that due to a single maximal stimulus. This increase in height due to summation is best marked when the muscle has to overcome the resistance of a considerable load. If the muscle is extremely lightly loaded, the contraction evoked by a single stimulus may be as high as that which can be brought about by repeated stimuli. The phenomenon of summation is due to the fact that by the first THE MECHANICAL CHANGES OF MUSCLE 227 contraction the muscle is, so to speak, after-loaded for the second. The period of contractile stress in a frog's gastrocnemius at the ordinary temperature is only -03 to -04 sec. This sudden jerk is applied through an elastic tissue, the muscle itself, to overcoming the inertia of the weight which has to be raised. If this latter is at all considerable, the moving mechanism is obviously ill-adapted for the purpose. The energy contained in a rifle bullet is very large, but firing a rifle bullet at a door would not be the best way of shutting the door. Even if the door were made of steel the bullet would flatten itself against it and its energy would be transformed for the FIG. 62. Contractions of a frog's muscle. Two single twitches are followed by a tetanus, which is almost twice as high as a 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 increased. It can be seen that the curve obtained in this way is as high as the original tetanus. (V. FREY.) most part into a heat, only a small part being utilised in moving the mass of the door. The contractile stress acting through the muscle for a period of -03 sec. is only sufficient to impart a certain velocity to the weight, and therefore to raise it to a certain height. Before the muscle has had time to accomplish its maximum shortening the period of contractile stress has passed away. That this is the case 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 a 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 raise the weight if the stimuli were repeated many times (Fig. 62). In summated contractions the apex of the second contraction occurs rather sooner than would be the case if the second curve had exactly the same course as the first curve. The latent period of the second twitch is also often found to be shorter than that of the first twitch. Both these results might be expected from what we know of the deforming effects of elasticity of the muscle on the graphic record of the mechanical events which occur in the muscle. If summation is to occur at all, the single stimuli must not be applied in too rapid succession. The smallest effective interval depends in any given preparation on the temperature and on the strength of stimulus. It 228 PHYSIOLOGY differs also according to the nature of the tissue which is being stimu- lated, and will be shorter in the case of the frog's nerve than in the case of the frog's muscle. The reason for this we shall have to consider later. TETANUS If a muscle be stimulated so many times in a second (e.g. with the interrupted current of an ordinary induction-coil) that it has no time to relax between each stimulus, we get a prolonged steady contraction, which in a loaded muscle is much stronger than the maximal muscle-twitch, owing to the summation of the rapidly follow- ing stimuli. This condition is called tetanus. The rapidity of stimulation needed to produce an unbroken tetanus depends on the duration of a single muscle-twitch, and varies therefore according to the kind and condition of the muscle. Thus the rapidity need only be small in the case of cooled and tired muscles, or of the red muscles of the rabbit and tortoise. The rate varies from about 15 in the case of red muscles to 30 or 40 for white muscles. For the much more highly differentiated muscles of insects the rate is probably very much greater. FIG. 63. Curves showing forma- tion of tetanus (from frog's gastrocnemius). a. Six sti- muli per sec. 6. Ten stimuli per sec. c. Thirty stimuli per sec. CHANGES IN THE MUSCLE ACCOMPANYING ACTIVITY EXTENSIBILITY. Besides the change of form, we find changes in the elasticity and extensibility of muscle taking place during contraction. Living muscle in a perfectly normal condition is distinguished by its slight but perfect elasticity ; that is to say, it is considerably 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 muscle is already stretched. The accompanying 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 muxjle will not stretch it so much as THE MECHANICAL CHANGES OF MUSCLE 229 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 FIG. 64. Extensibility of india-rubber (a) compared with that of a frog's gastrocnemius muscle (6). muscle at rest. A gram applied to a tetanised gastrocnemius will cause greater lengthening than if it were applied to the same muscle at rest. At the same time the elasticity is more perfect — that is to say, when the weight is removed the muscle returns more quickly to its original length. 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 effect 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 contraction 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 indi- vidual muscle fibre of which the muscle is composed. It seems more probable, however, that, when a minimum or subminimal response is obtained, not all the fibres making up the muscle are contracting. A minimal con- traction 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 o concerns each individual muscle fibre every contraction FIG. 65. 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 enunciated for heart-muscle is probably true for every contractile element. The difference between skeletal and heart muscle lies in the fact that in the former the excitatory pro- cess 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-hand side of the muscle, while a stimulus applied to B is in the same way limited to the right-hand side. If a piece of ventricular or auricular 230 THE MECHANICAL RESPONSE OF MUSCLE 231 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. It was shown by Gotch 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-cutaneus muscle of the frog, the contraction of the muscle increases, not gradually, but by a series of steps. This can only be explained by assuming that the smallest effective stimulus 100 150 200 FIG. 66. Curve showing relation of height of contraction of dorso-cutaneus muscle to strength of stimulus. Ordinates = height of contraction ; abscissa = strength of stimulus. (K. LUCAS.) 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). • LOAD. The height of contraction of a muscle diminishes as the load is increased. This diminution in height is at first very slight and is not proportional to the load, so that the work done by the muscle, which is measured by the product of the weight lifted and the height to which it is raised, w X h, with increase of weight rises at first quickly, then more slowly to a maximum, and then, on further increasing the load, sinks. This will be rendered clearer by reference to the diagram (Fig. 67) representing the lengths of the resting and contracted muscle with various loads. The lines h0, hl5 &c., are the actual height of contrac- tion of the muscle when loaded with weights of 0, 10, 20 grm., &c. The work in each case is given by h0 X 0, ht X 10, h2 X 20, h3 X 30, &c. By inspection it will be seen that — O.h0 <10.h1 20.h2 30.h3> 40.h4> 50.h5. In this case therefore the maximum of mechanical work is obtained 232 PHYSIOLOGY when the muscle is loaded with about 30 grm. This increase of work with increased load shows that the amount of external work performed by a muscle is not a constant quantity, nor one determined solely by the strength of stimulus, but is essentially conditioned by the tension under which the muscle contracts. The muscle is in fact endowed with a certain power of adaptation, so that it can respond with increased efforts or expenditure of energy when it has more work set it to do. It might be thought that the increased mechanical energy evolved under these conditions had its origin at the expense of some other form of energy, such as heat or electrical changes, but it is found that increased tension augments all the processes of muscle, FIG. 67. Curve showing the length of a muscle under various loads in the contracted condition B, and uncontracted condition A. The double lines a &, &c., represent the contracted muscle, while the long single lines a c, &c., show the length of the inactive muscle. including chemical changes and the production of heat. This excita- tory effect of tension on skejetal muscle is aided in all the higher animals by impulses which pass through the central nervous system, the nature of which we shall have to discuss later on when dealing with the question of so-called " tendon reflexes." The phenomenon, however, is common to all forms of contractile tissues, and is indeed much better marked in such forms as the heart-muscle and the unstriated muscular fibres of the viscera. One may occasionally find that the application of a slight load to a skeletal muscle actually increases the height of the contraction, especially if the muscle be not after-loaded. In the heart-muscle an increase of tension within physiological limits causes invariably increased contraction — a fact of very great importance for the physiology of compensation in heart disease. This excitatory influence affects not only the strength of contraction but also the automatic, rhythmic, and conducting power of the muscle ; and in some cases, as in the snail's heart, the rate of beat is absolutely determined by the tension, the heart stopping altogether if the tension be reduced to nothing. THE MECHANICAL RESPONSE OF MUSCLE 233 TEMPERATURE. Speaking generally, the effect of warming a muscle 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 FIG. 68. 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.) 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. 68). If a muscle be heated gradually (without stimulation) up to about 45° C., it begins to contract slowly at about' 34° C.. and this contrac- tion 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 the contraction period are prolonged. The action of cold on the excitability of muscle is to increase it, so that any form of stimulus is more effective at 5° C. 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 234: PHYSIOLOGY 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 shortening which remains is spoken of as FIG. 69. Muscle curves showing fatigue in consequence of repeated stimu- lation. The first six contractions are numbered, and show the initial increase of the first three contractions. (BRODIE.) ' 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. 69). The fact that the relaxation part of the muscle curve is affected by various conditions, especially 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 relaxation. (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 C02, with a consequent relaxation. A retardation of this second phase would cause the prolonged curve with ' contraction remainder ' observed in a fatigued muscle. The absence of any appreciable evolution of heat in the conversion of glucose to lactic acid shows, however, that the formation of lactic acid cannot account for the whole of the energy involved in the phase of shortening. If left to itself, the muscle which has been exhausted by repeated THE MECHANICAL RESPONSE OF MUSCLE 235 stimulation 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 available for the supply of potential energy to this material. (2) The accumulation of waste products of contraction. Among these waste products the lactic acid is probably of great importance. Fatigue may be artificially induced in a muscle by ' feeding ' it with a dilute solution of lactic acid, and again removed by washing out the muscle with normal sab'ne solution containing a small per- centage of alkali. After a certain time the mere removal of waste products by means of an artificial circulation of salt solution becomes inadequate to restore contractile power to the muscle. In this case the muscle can be made to contract once more by supplying it with fresh food material, as by the circulation of serum or diluted blood. 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, so that single induction shocks may cause tetani- form contractions. The same excitatory effect is still better marked with solutions of Na2C03. If a thin muscle, such as a frog's sartorius, be immersed in a solution containing O5 per cent. Nad, 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 labora- tory should always be made with tap water, containing calcium salts. Potassium salts, although forming so important a constituent of 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 potassium 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 per cent. 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. 236 PHYSIOLOGY THE ACTION OF DRUGS Of the drugs that have a direct action on muscle, the most remark- able is veratrin, which causes an excessive prolongation of a muscular FIG. 70. 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 'oft') It will be noticed that the two curves are practically identical. (Miss BUCHANAN.) contraction (produced by a single stimulus). Thus the c twitch ' of a muscle poisoned with veratrin may last fifty or sixty seconds, instead of the normal one- tenth of a second (Fig. 70). 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 hours. We get then on a single stimu- Excitation. jus a response lasting many seconds and exactly similar in FIG. 71. Tracing of the contraction of a height and form to a tetanus muscle poisoned by the injection of a , . . -, , -, . strong solution of veratrin, showing the obtained by discontinuous stimu ^™^tcaotiondiietoimeqiialpoi«m. lation. If stronger solutions be mg of different fibres. (BIEDEEMANN.) ' , , 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 contraction (Fig. 71). If the muscle be excited immediately after the prolonged contraction has passed away, it responds with a single twitch like a normal muscle, but if allowed to rest a few minutes, stimulation is again followed by the peculiar long-drawn-out contraction. SECTION V THE CHEMICAL CHANGES IN MUSCLE CHEMICAL COMPOSITION OF VOLUNTARY MUSCLE IT is impossible to speak with certainty about the chemical com- position of any living tissue, since in the act of analysis we destroy the life of the tissue ; all we can do in most cases is to find the proxi- mate principles present in the dead tissue. But, by using certain precautions, we may learn some interesting facts about the chemistry of living muscle. Muscle of cold-blooded animals may be cooled below 0° C. without losing its irritability on re- warming, and therefore we may say without its life being destroyed. If the living muscle of frogs be frozen, then minced with ice-cold knives as finely as possible and pounded in a mortar with four times its weight of snow containing 0-6 per cent, of common salt, and the mixture thrown on to a filter and kept at a little over 0° C., an opalescent fluid filters through. The filters soon get clogged and therefore must be frequently changed. Their temperature must not be allowed to rise over 2° or 3° C. This fluid is called muscle-plasma. If its temperature be allowed to rise to that of the room, it clots, and the clot soon contracts, squeezing out a serum, just as in the case of blood- plasma. The muscle-plasma is neutral or slightly alkaline. When coagula- tion takes place, however, it becomes distinctly acid, and this acidity has been shown to be 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 con- verted when clotting takes place into myosin. The exact nature of the proteins in muscle-plasma, as well as of the pro- tein constituent 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 MgSo4, precipitated by complete saturation with MgSo4, 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), 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, 237 238 PHYSIOLOGY 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 Furth 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. These proteins are extremely unstable, and are gradually transformed on standing into insoluble 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 on dialysis. Transformed 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. myosin or paramyosinogen. ^myogen (myosinogen of Halliburton, albumate of Kiihne). I Soluble myogen fibrin. 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 instan- taneous clotting of frog's muscle-plasma on warming to 40° C. The residue left after the expression of the muscle- plasma consists chiefly of connective tissue, sarcolemma, and nuclei, and as such contains gelatin (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 con- stituents of muscle. These are : (A) COLOURING-MATTERS. All red muscles contain a considerable amount of haemoglobin. In many, a special pigment, probably allied to hiemoglobin, is also present. This has been named myohcematin (MacMunn). (B) NITROGENOUS EXTRACTIVES. Of these, the most important is creatin (C4H9N302 -j- H20), which occurs to the extent of 0-2 to 0-3 per cent. This substance is found only in muscular and nervous THE CHEMICAL CHANGES IN MUSCLE 239 tissues. Its significance we shall discuss- later on when inquiring into the history of the proteins in the body. Other nitrogenous extractives are : Hypoxanthine or sarcine, xanthine (both bodies allied to uric acid), and a trace of urea. (c) NoN-NlTROGENOUS CONSTITUENTS. Fats. Glycogen. The amount of this is very variable. In the embryo the muscles may contain large quantities, but in the adult they contain only from 04 to 1 per cent. Inosit (C6H1206 + 2H20), or ' muscle-sugar/ which occurs in minute traces, is non-fermentable, does not rotate polarised light, and does not reduce Fehling's solution. It does not belong to the group of carbohydrates at all, being a derivative of benzene. Dextrose. It is doubtful whether this is present in fresh resting muscle. (D) INORGANIC CONSTITUENTS. Muscle contains about 75 per cent, of water. The ash forms 1 to 1-5 per cent, and consists chiefly of salts of potassium and phosphoric acid. There are small traces of calcium, magnesium, chlorine, and iron. RIGOR MORTIS All muscles, within a short time of their removal from the body, or if left in the body after general death, lose their irritability, and this is succeeded by an event which occurs rather suddenly, and is known as rigor mortis. The muscle, which was before translucent, supple, extensible, becomes more opaque, rigid, and inextensible, and shortens. The shortening is not very powerful, and can be prevented by loading the muscle moderately. Chemical changes also take place. The muscle, which was previously alkaline, becomes distinctly acid, the acidity being due to the formation of sarcolactic acid. There is also production of C02 with evolution of heat. It is generally believed that this change is identical with the clotting of muscle- plasma, and that the rigidity as well as the contrac- tion of the muscle is due to the coagulation of the muscle- proteins. That there is at any rate a close connection between the two sets of phenomena is shown by Brodie's experiments. This observer found that, if a living muscle be lightly loaded and then warmed very gradu- ally, a series of stages in the heat-contraction could be distinguished corresponding to the coagulation temperatures of the different proteins described by von Fiirth in muscle -plasma. It seems likely, however, that the main contraction at all events, that which comes on sponta- neously after death or immediately on warming the muscle to 45° C., has another component. In the coagulation of the separated muscle - proteins there is no evidence of any appreciable formation of sarco- 240 PHYSIOLOGY lactic acid or of C02, whereat the formation of these bodies seems to bear an important relation to the occurrence of rigor. Thus after severe muscular fatigue, as in hunted animals, where there has already been a considerable formation of these waste products of muscular contraction, rigidity may come on almost immediately after death. If living muscle be plunged into boiling water, it undergoes instant coagulation, but no chemical change. The reaction of the scalded muscle, like that of fresh muscle, is slightly alkaline to litmus. No sarcolactic acid or carbonic acid is produced. On the other hand, in surviving muscle, after the cessation of the circulation, there is a steady formation of lactic acid which accumulates in the muscle. The actual coagulation of the muscle -proteins occurring in rigor is largely, if not entirely, determined by the increasing acidity of the muscle thereby produced. In fact, it is the production of the acid which causes the onset of rigor, and not the rigor which causes a sudden formation of acid. Hence if the accumulation of lactic acid be pre- vented by perfusing the muscle with salt solutions, the onset of rigor may be postponed indefinitely, and the muscle may begin to putrefy without having undergone rigor. The lactic acid formed in muscle (sarcolactic acid) is a physical isomer of the lactic acid formed in the fermentation or souring of milk. They both have the formula CH3.CH(OH).COOH, i.e. they are ethylidene lactic acids. The lactic acid of fermentation is optically inactive ; sarcolactic acid rotates polarised light to the right ; while a third isomer which is laevo-rotatory is produced by the action of various bacilli and vibriones on cane sugar. THE CHEMICAL CHANGES WHICH ACCOMPANY ACTIVITY The principle of the conservation of energy teaches us that the energy of the contraction of muscle must be derived from chemical changes, probably processes of decomposition and oxidation, occurring in the muscle itself. In seeking out the nature of these changes three methods are open to us : (1) We can examine the changes in the muscle itself, avoiding so far as possible reintegrative changes by working on excised muscles. (2) We can investigate the changes in the medium surrounding the muscle. Muscle may be exposed in a vacuum or in a confined space of air, and its gaseous interchanges during rest and activity compared. Or we may lead a current of defibrinated blood through excised muscles, and determine the change in the composition of the blood before and after passing through the muscle Under various conditions. (3) A method which, although apparently complex, has rendered the utmost service to the physiology of muscle is to use the changes in the total metabolism of the animal during rest and muscular work as a clue to the muscular metabolism itself. In such a case the THE CHEMICAL CHANGES IN MUSCLE 24! respiratory exchanges of the animal are determined (viz. its oxygen intake and its C02 output), and the urine and faeces are carefully analysed, in order to judge of the action of muscular work on the carbon and nitrogen metabolism of the body. By one or other of these methods it has been found that the main products of muscular activity are the same as those which are pro- duced during the death of a muscle, viz. sarcolactic acid and carbon dioxide. It was shown long ago by Helmholtz that when a muscle was tetanised to exhaustion, the total amount of its watery extractives diminished, while the amount of its alcoholic extractives increased ; and there is no doubt that part of this difference is due to the formation of lactic acid. The souring of muscle during activity can be easily demonstrated by stimulating the muscle for some time and then crushing a fragment of the excised muscle on litmus paper. The litmus is at once turned red. Or we may inject a solution of acid fuchsin under the skin of a frog, and the next day expose a sciatic nerve and stimulate it for fifteen or twenty minutes. On skinning the hind-legs a difference in colour will be at once apparent, the leg which has been active being of a deep rose colour, owing to the action of the acid on the fuchsin. Sarcolactic acid is not present in a free state in muscle, the acidity being, like that of urine, due to the presence of acid phosphates. The sarcolactic acid can be extracted from the muscle by means of alcohol. It is generally separated in the form of the zinc sarcolactate, by boiling its partially purified solution with zinc carbonate. Its presence may be tested for by means of Uffelmann's reagent, which is made by the addition of ferric chloride to dilute carbolic acid. The purple solution thus produced is at once changed to yellow by the addition of even traces of lactic acid. A much more definite colour reaction for lactic acid has been introduced by Hopkins. The test is carried out in the following way. About 5 c.c. of strong sulphuric acid are placed in a test-tube together with one drop of saturated solution of copper sulphate, which serves to catalyse the oxidation that follows. To this mixture a few drops of the solution to be tested are added, and the whole well shaken. The test-tube is now placed in a beaker of boiling water for one or two minutes. The tube is then cooled under a water-tap, and two or three drops of a very dilute alcoholic solution of thiophene (ten to twenty drops in 100 c.c.) are added from a pipette. The tube is replaced in the boiling water and the contents immediately observed. If lactic acid is present the fluid rapidly assumes a bright cherry red colour, which is only permanent if the tube be cooled the moment after its appearance. We get a similar formation of lactic acid in excised mammalian muscles which are kept alive by an artificial circulation. We do not know how far the formation of lactic acid -occurs under normal circum- stances in the living body. At all events, if lactic acid is produced by the muscle in any quantity during some phase of its activity in the normal animal, the greater part is further transformed (to C02) before it leaves the body. If actual dyspnoea is present during muscular 16' 242 PHYSIOLOGY exercise, lactic acid is certainly formed and may escape from the body. Normal urine has been shown by RyfM to contain about 3-4 mg. of lactic acid per hour. In one case the urine passed thirty minutes after running one-third of a mile with the production of severe dyspnoea contained 454 mg. of lactic acid. The lactic acid under these conditions can be shown also to be increased in the blood. Thus, in one experiment, the blood obtained before running contained 12'5mg. per 100 c.c., that obtained immediately after running one-third of a mile contained 70 '8 mg. per 100 c.c. On the other hand, the examination of the urines of competitors in a twenty-four hours' track walking race showed no increase in the output of lactic acid above the normal 4 mg. per hour. The excretion of lactic acid was also observed many years ago by Araki in cases where the oxidation processes of the body were interfered with in consequence of CO poisoning. The second substance, carbon dioxide, is continually being formed by all living tissues, and is the end-product of practically all the carbon metabolism of the body. If a muscle be hung up in a confined space, it will be found to take up oxygen and give off C02 ; and these inter- changes are quickened by causing the muscle to contract. It has been shown by Fletcher that the effect of activity is dependent on the composition of the gas surrounding the muscle. If it be hung up in a vacuum or in an atmosphere of nitrogen or hydrogen, there is a slow evolution of C02, which is not appreciably quickened during contraction, and seems to be conditioned by a gradual driving off of C02 from the alkaline carbonates in the muscle, as a result of the steady production of lactic acid which precedes the onset of rigor. If, however, the muscle be suspended in an atmosphere of pure oxygen, the formation of acid is diminished or abolished ; but now each con- traction of the muscle is followed by an increased evolution of carbon dioxide. We see therefore that, according to the environment of the muscle, its activity is attended by the formation either of lactic acid or of carbon dioxide, the latter substance being the sole product if sufficient oxygen be supplied to the muscle. If the supply of oxygen be inadequate, both substances are produced, the proportion of lactic acid varying according to the relative inadequacy of the oxygen supply. This relation holds good both in rest and activity, the effect of activity being merely to increase the chemical changes which are going on spontaneously in the surviving resting muscle. It is an interesting point to determine whether we have here really two alternative chemical mechanisms for the production of energy. We know that sugar can be utilised by muscle as a food and source THE CHEMICAL CHANGES IN MUSCLE 243 of energy. It has been suggested therefore that, in the absence of oxygen, the energy for contraction is derived from, a process of dis- integration, each molecule of grape sugar breaking down into two molecules of lactic acid, thus : C6H1206 = 2C3H603. sugar lactic acid On the other hand, in the presence of sufficient oxygen the sugar would be entirely oxidised with the formation of C02 and water, thus : C6H1206 + 602 = 6C02 + 6H20. sugar The change from sugar to lactic acid involves, however, practically no evolution of energy — so that in the absence of oxygen the energy of contraction must be derived from some other source. It seems more probable that we are dealing here with two stages of one process, and that in the muscle under normal conditions (i.e. richly supplied with oxygen) the first chemical change is one of disintegra- tion, leading to the formation of lactic acid (and probably other substances), and that this is followed by a process of oxidation, in which all the products of the first stage are converted into C02, which can be rapidly eliminated from the muscle. If the supply of oxygen is deficient, the products of the first stage remain in the muscle, giving rise to the phenomena of fatigue, and finally inducing the coagulation of the muscle-proteins which determines rigor mortis. That lactic acid is a normal metabolite, and not simply the result of an alternative chemical change occurring only under abnormal conditions, namely, want of oxygen, is indicated by "the fact demon- strated by Hopkins and Fletcher, viz. that the muscle possesses in itself a chemical mechanism for the removal of lactic acid when once formed. These observers have shown that if a fatigued muscle be exposed to pure oxygen, 30 per cent, of the lactic acid produced by the fatigued muscle may disappear within two hours, and 50 per cent, within ten hours. Thus even apart from the circulation, which of coarse would remove large quantities of any lactic acid which might be produced in the muscles, the muscles themselves can deal with this metabolite locally. Since the main products of muscular activity are C02 and lactic acid, and no change has been found to occur in the creatine or other nitrogenous extractives of the muscle during contraction, it has been thought that the sole source of muscular energy is the combustion of carbohydrate or fatty material, the proteins of the body taking no part in the process. In dealing with the general metabolism of the 244 PHYSIOLOGY body, we shall see that it is impossible to draw any qualitative distinc- tion between the metabolism which results in muscular work, and the metabolism of the resting animal. Thus the relative proportion of CO the C02 produced to the oxygen taken in, the ' respiratory quotient '- -?, ^2 will vary according to the food that is being consumed, being unity with carbohydrates, less than unity with proteins, and still less with fats. It is found that muscular work does not alter the respiratory quotient, i.e. during work the qualitative metabolism of the whole body is the same as during rest. We must conclude therefore that the muscle derives its energy from the combustion of all three classes of food-stuffs, although in the absence of food it will perform its work at the expense of stored-up fat or carbohydrate, proteins not under- going any storage in the body. The absence of change in the respiratory quotient during exercise shows moreover that, in a muscle under normal conditions, the two processes, viz. the taking in of oxygen and the giving out of C02, keep pace one with the other. In warm-blooded animals the shutting of! of the oxygen supply rapidly induces paralysis and loss of irritability of the muscles. This result, coupled with the fact that, as mentioned above, the final results of muscular activity differ according as the muscle is or is not supplied with oxygen, suggests that the oxygen takes part in the process of activity only after the disintegration of the complex living molecule has already begun. Such a conclusion is, however, opposed to the generally accepted views on the nature of the-oxidation processes in the cell. According to Hermann, Pfliiger, Verworn, and others, there is during rest a building up both of oxygen and food material into the living molecule. Activity consists in a rearrangement of the molecule (spoken of by Hermann as the inogen molecule), with the assumption of more stable positions by the oxygen and carbon atoms, and a consequent production of C02. (Compare the explosion of gun-cotton or nitro- glycerin.) The presence of this intramolecular oxygen in an unstable position would be a necessary condition both for the irritability as well as for the activity of all forms of living tissue, especially muscle and nerve. If the muscle can use all classes of food-stuffs in its metabolism, one would expect to find some change in the nitrogenous constituents as the result of activity. Physiologists have searched in vain, however, for any evidence of the formation of creatin or urea in excised muscle during contraction. Cathcart and Brown have found an insignificant increase of creatin in excised frog's muscle during contraction and a more significant decrease if the circulation has been maintained intact THE CHEMICAL CHANGES IN MUSCLE 245 during the stimulation of the muscle. SchondorfE has shown that if excised muscle be kept alive by perfusion of defibrinated blood, its activity is associated with slightly increased formation of ammonia. The formation of ammonia is, however, the natural mode of protection of the whole organism against acid poisoning, and it seems quite probable that in SchondorfTs experiments the ammonia formation was simply a secondary result of the lactic acid formation and not a direct expression of the metabolism of the active muscle. SECTION VI THE PRODUCTION OF HEAT IN MUSCLE THE experience of everyday life teaches us that muscular exercise is associated with increased production of heat. Thus a man walks fast on a frosty day to^keep himself warm. In large animals the production of heat in muscular contraction can be easily shown by inserting the bulb of a thermometer between the thigh muscles, and stimulating the spinal cord. The rise of temperature produced in this way may amount to several degrees. This observation is confirmed when we investigate the contraction of an isolated muscle outside the body. If a frog's muscle is tetanised, its tempera- ture rises from 0*14° to 0'18° C., and for each single twitch from 0-001° to 0-005° C. It is evident that such small changes in temperature as 0-001° cannot be estimated by ordinary thermometric methods. By converting a heat change into an electrical change, however, we can estimate differences of temperature with much greater accuracy and fineness than by the use . . of a thermometer. Two main principles are ANTIMONY | employed in measuring temperature by elec- trical methods. The thermo -electrical method depends on the fact that, when the junc- tions of a circuit made of two metals are at different temperatures, a current of electricity generally flows through the circuit. This current can be measured by means of a galvanometer, and is proportional to the difference of temperature between the two junctions. Thus in the circuit (Fig. 72) composed of two metals, antimony and bismuth, if the upper junction be cooled, there will be a current flowing from antimony to bismuth in the direction of the arrow, and this current will within limits be proportional to the difference of temperature. To measure the production of heat during muscular contraction, a small flat thermopile (containing four or six elements composed of iron and German silver) is fixed with one of its ends between two frog's gastrocnemii. Another exactly similar pile, but reversed, is placed between two other gastrocnemii, which are kept resting and at a perfectly constant temperature. So long as the two piles are at the same temperature no current flows ; but, with a sensitive* galvanometer, the slightest difference of temperature, such as that caused by the contraction of one pair of muscles, at once causes a deflection of the galvanometer, the extent and direction of which enable us to estimate exactly the seat and amount of heat produced. When we are using such delicate detectors of temperature difference, we are 246 THE PRODUCTION OF HEAT IN MUSCLE 247 met by the difficulty that every junction in the circuit tends to become the seat of an electromotive force in consequence of slight changes of tempera- ture due to currents of air, &c. It is therefore advisable to use a plan adopted by Blix, of placing all the apparatus, the muscle included, within the galvano- meter case. The arrangements of such an experiment as employed by A. V. Hill are shown in the diagram (Fig. 73). In this instrument the junction of copper with the alloy constantan constitutes a thermo-electric couple. The magnet and mirror chamber is entirely separated off from the rest of the instrument by the walls of the tube containing the magnet. The grooves are usually filled with plasticine, Magnet & Mirror Chamber I Quartz Fibre Constantan Muscle Electrode Broca Magnet System Copper Coil Celluloid Plate Electrode FIG. 73. and into them fit the edges of an outer case of brass constituting the walls of the muscle chamber. The inside of this case is lined with wet blotting-paper. The copper coil consists of many more turns than are shown in the figure : its ends, AA and BB, are separated by the celluloid plate, and are connected by the constantan plug ; the points where the copper meets the constantan constitute the thermo-electric junctions. The tube containing the magnet hangs down through holes bored in the broad copper coil. The two semi- membranosus muscles ride astride of the celluloid plate, one in contact with each end of the constantan plug. The small piece of bone at their upper ends which has been left connecting the two muscles is placed exactly on the top of the celluloid plate at x and held in position by a clamp (not shown in the figure). The copper terminals of the coil are coated with celluloid varnish to prevent short-circuiting of the thermo-electric currents, and to prevent poisoning of the muscles. Each muscle is in contact with a pair of electrodes, made of fine platinum wire ; the muscle lies over the upper and beneath the lower of these electrodes, as shown in the figure. The tendons at the lower ends of the muscles are tied to silk threads which pass through holes in the base of the instrument. These are then attached to recording levers which write on a drum beneath 248 PHYSIOLOGY the table. When all is ready three heavy soft-iron cylinders are placed over the instrument : the latter is screwed to a wooden block which is fixed to a thick iron plate attached to the table. In the cylinders holes are bored to admit and let out after reflexion the light from a Nernst lamp. The lamp, which is about three metres away, shines upon the mirror, and a line in it, after reflexion, is focussed on to the screen, which is also three metres away. The line is brought on to the scale by the small field exerted by a control magnet placed outside the cylinders at a suitable position on the table : its position may be read easily to half a millimetre, and the movement due to a twitch is usually of the order of 80 mm. These soft-iron cylinders cut off entirely all external magnetism sufficient to cause harmful disturbances during an experiment. They lower very largely the strength of the constant external field in which the magnet lies, and leave it chiefly supported in any position by the quartz fibre. Thus all the movements set up in the magnet by the 1.1 FIG. 74. Arrangement of apparatus for measuring small differences of temperature. thermo-electric currents are working against little more than the torsion of a quartz fibre only 6 p thick. This explains the great sensitivity of the instrument. A second method depends on the fact that rise of temperature increases the resistance of a wire to the passage of an electric current. A current detector consists of a small grid of fine platinum wire which is placed against the muscle between two muscles. This grid is then made one limb of a Wheatstone's bridge (Fig. 74). A small current is passed through the circuit, and the resistances are so adjusted that no current flows through the galvanometer. Any alteration in temperature of the grid will alter the balance of the resistance and will cause a current to flow through the galvanometer in a direction which will vary according as the resistance in the grid is increased or diminished. It is possible to calibrate the arrangement so that a deflection of the galvanometer over one degree will correspond to a certain fraction of a degree of difference in tem- perature of the grid. This method is employed in Callender's recording thermo- meters, and has been made by Gamgee the basis of an arrangement for the continuous record of the temperature of the human body. The discovery of exact means of measuring the heat production during contraction was naturally utilised to determine the relation between the heat produced and the work done under varying con- ditions. In the muscle, as in a steam-engine, we have a conversion of potential energy stored up in carbon compounds into kinetic energy, which may appear as work and heat. In the engine there THE PRODUCTION OF HEAT IN MUSCLE 249 is a definite ratio between work and heat. Only a certain small proportion of the total energy can be utilised as work, the rest being dissipated as heat. The exact proportion depends on the difference of temperatures that is available in the machine, and in the best engines at our disposal amounts to one-tenth. If the machine does no work, the heat production is increased by the amount corresponding to the work. The same is true to a certain extent in muscle. If a muscle be allowed to contract and relax twenty times when loaded by a weight, the total external work done will be nothing. If, how- FIG. 75. Diagram of Fick's Arbeitsammler or muscle crank, a & is a counter-balanced lever, attached to the muscle M at ra. When the muscle contracts, the catch c carries round the circumference of the wheel D and so coils up the weight w round the axle of the wheel. When the muscle relaxes, if c.2 is in the situation of the^lotted line, the weight pulls the wheel and lever back to its original position. If, however, c2 be applied to D, the backward movement of the wheel is prevented, and the muscle is extended simply by the weight of the lever ab. Thus at each contraction the weight is drawn a little higher, and external work is performed by the muscle. ever, the weight be attached to the axle of a wheel, which is provided with a catch so that the weight can only be drawn up (Fig. 75), and the muscle be allowed to pull at each contraction on the circum- ference of the wheel, at each contraction work is done. It is found that in the latter case the muscle is less heated than in the former, and the difference is equivalent to the work done in raising the weight. But as soon as we begin to alter the work by altering the weight, we are at once met by the difficulty that increased tension augments all the properties of the muscle, and with the same stimulus both work and heat production are raised by increasing the load. In fact the maximum amount of heat is produced when the muscle 250 PHYSIOLOGY is made to contract against a strong spring, so that it cannot shorten at all (isometric contraction). In view of the comparison of the muscle to a heat engine, it becomes interesting to inquire into its efficiency, i.e. the relation of the work to the total energy expended. This amount is found to vary within very wide limits. In a fresh muscle the heat energy may be twenty- five times as great as the work energy, but the heat evolved with each contraction diminishes with fatigue more rapidly than the work done, so that the proportion may fall to as low as three to one. In the intact animal, in the dog fed on a pure flesh diet, Pfliiger has calculated that the efficiency may be as great as 48 per cent. The experiments made by Atwater and Benedict on man point to a mechanical efficiency of about 12 to 20 per cent. The efficiency of a heat engine is determined by the difference of absolute tempera- tures obtaining on the two sides of the machine ; and since we cannot imagine even minutely localised changes of temperature in the animal body of more than a few degrees Centigrade, we must discard altogether the analogy of the steam-engine, and seek some other explanation of the mechanism by which the muscle is enabled to transmute the chemical energy of its food into work or heat. It seems probable that the two products, heat and work, are simultaneous and independent in their origin, and that any proportion between them, therefore, is accidental. The muscle is, in fact, not a heat engine, but a chemical engine. This conclusion is borne out by the observations of A. V. Hill. He finds that the heat production of a single twitch is usually of very short duration, i.e. less than O'l sec., but that it is prolonged by depriving the muscle of oxygen, and may outlast the mechanical effect. No constant ratio is found between work done and heat evolved. The important factor is the tension, as first pointed out by Heidenhaim. In an isometric muscle twitch — ({ e . enslonN\ -g H \ ' heat / constant whatever the number of fibres contracting, the strength T of contraction, or the initial resting tension on the muscle ; i.e. — H is the same for every fibre. The tension is the measure of the potential energy which is thrown suddenly into being at the moment of excita- tion, and this is proportional to the heat, and therefore to the total chemical change evoked. On the basis of these results, Hill suggests that there are three stages in the process of muscular contraction : (i) The liberation of certain molecules following an excitation. (ii) The action of these molecules on certain local structures, in producing a local tension. (iii) The removal or replacement of these molecules, under the action of oxygen, with evolution of heat. , SECTION VII 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 hydrogen appearing on the positive plate (anode), and bubbles of oxygen 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 FIG. 76. Diagram of non- become a galvanic cell, the platinum covered polarisable electrode. .,, , ,,, , . . a, covered wire; 6, amal- Wlth OX7gen bubbles belng tlie POSltlve gamated zinc rod ; c, element, and that covered with hydrogen lX±t\on;tplugod£ bubbles the negative eknxnt. Exactly the zinc sulphate clay;/, plug same process of electrolysis or polarisation of normal saline clay. -111 ,11 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 tissues, it is necessary to have some form of electrodes in which this polarisation will not occur. The ' non-polarisable ' elec- trodes which are most generally used for this purpose are made in the following way. A glass tube (Fig. 76) is closed at one end with a plug of kaolin made into a paste with a satu- rated solution of zinc sulphate. The rest of the tube is filled with a similar solution. Dipping 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 tube, 251 FIG. 77. U-shaped non-polarisabh electrodes. 252 PHYSIOLOGY 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. hydrogen 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. 77). 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 current through the nerve or muscle to the metallic part of the circuit may be represented as follows : In Zn 504. a a so* FIG. 78. If a muscle such as the sartorius be removed from the body, and two non-polarisable electrodes connected with a delicate galvano- meter 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 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 mole- cules, 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 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 contractile 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 pait is negative towards the points of the uninjured surface." Fig. 79 shows the direction of the current in a muscle with two cut ends. When the whole muscle is quite dead, this current of rest, ELECTRICAL CHANGES IN MUSCLE 253 FIG. 79. Current of rest. or ' demarcation current ' (Hermann), 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. containing 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. 80) be allowed to fall on an excised muscle b, 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 electrical change occurs in a muscle when it contracts. To show this change, we may lead off two points, one on the cut end and one on the surface of the muscle of a muscle -nerve preparation, to a galvanometer. We shall then obtain a deflection of the mirror of 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.' FIG. 80. Rheoscopic frog. 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 dia- gram. Two non-polarisable 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 connected with a Pohl's reverser p, and this in its turn with the shunt s. 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 254 PHYSIOLOGY 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 -jTjVo °^ ^e total current, and then by means of the shunt, xcro> Y^> 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, stimu- lation 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. FIG. 81. 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 ordinary galvanometer. For this purpose we may employ either the capillary electrometer 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 pointsjif 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 sul- phuric acid, at the bottom of which is a little mercury. Two platinum wires melted 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 oi> a screen with the aid of the electric light. If now the qapillary and acid be connected with two points, it will be observed that any difference in the potential of these two points causes a movement of the ELECTRICAL CHANGES IN MUSCLE 255 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 i? free from instrumental vibrations, it is extremely useful in recording the quick changes in potential occurring in the diphasic electrical 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 instru- ment is an electrometer (measurer of difference of potential), and not a galvanometer (current measurer). When the electrometer is connected with two points at different potential, no current passes through it. Hence the use of non-polarisable electrodes is not so essential in experiments with this instrument as when we make use of the galvanometer. In the D'Arsonval galvanometer (Pig. 83) 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 Capillary electrometer. (BURGH.) galvanometer of Einthoven (Fig. 84). In this a very delicate thread of silvered quartz <3r of platinum is stretched between the poles of a strong magnet. The poles of the magnet are pierced by holes so that FIG. 83. 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 256 PHYSIOLOGY thread may be thrown upon a screen. Whenever a current passes through the thread it moves laterally, and the lateral movement may be photographed on a moving photographic screen. Owing to the extremely minute dimensions 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. 85), such as the sartorius, be stimulated with a single induction shock at one end, x, FIG. 85. Diagram showing diphasic variation of uninjured muscle. and two points, a and b, be led off to a capillary electrometer, each stimulus applied at x gives rise to an excursion of the meniscus of the electrometer, known as a ' spike,' and shown in Fig. 86. FIG. 86. A typical electrometer record from a sartorius muscle excited by a single induction shock. Time-marking = 200 D.V. (KEITH LUCAS.) 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 stimulated at x, a contraction wave commences which travels down the muscle through a and b. The electrical investigation of the muscle shows that excitation of x arouses an electrical change which ELECTRICAL CHANGES IN MUSCLE 257 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 the stimulus, i.e. there is no latent period to the electrical change. On leading off from a and b there is a latent period between the stimulus and the first change, representing the time taken for the change to travel 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 repre- senting negativity of a to b, and the second phase 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 diphasic variation of such a direction that the point stimu- lated 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 6, the change accompany- ing 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 "0025 sec. to attain its culminating-point. At this point the mechanical change or contraction of the muscle begins. * 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 mis- conception. 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 electro- positive to the copper, and in the same way we must assume that the excited portion of a muscle is really electropositive to the unexcited portions. When, therefore, we speak of any part of a tissue being negative, we are using a con- ventional 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 confusion which might result from an attempt to replace the loose expression ' negative ' by the more correct expression ' electropositive,' Waller has suggested the employment of the term " zincative " to indicate the electrical condition accompanying excitation. This term also serves to emphasise the fact that the excited portion, like the zinc in a zinc-copper cell, is the chief seat of chemical change, 17 258 PHYSIOLOGY These time-relations vary with the temperature of the muscle. We have already seen that the effect of lowering the temperature is to increase the 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. 87, 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 sec FIG. 87. Diphasic response of uninjured sartorius (obtained by analysis of curves such as Fig. 86). A, at 8° C. ; B, at 18° C. (KEITH LUCAS.) 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 excita- tory condition may be propagated without the presence of a visible contraction, Thus, if tjie middle third of the sartorius be soaked ELECTRICAL CHANGES IN MUSCLE 259 for a time in water, it passes into a condition known as ' water rigor,' in which, it is incapable of contracting, although capable of trans- mitting 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. 88 A is a photographic record of the variation obtained from the tortoise ventricle, which is led FIG. 88. 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.) 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 1J 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 Jjne, Her§ it stays for 260 PHYSIOLOGY 1J to 2 seconds. During this time the whole heart is in an excited condition. Both base and apex are equally excited, and there 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 photo- graph 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. 88 B. It is evident from this figure that the electrical sign lasts practically 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. FIG. 89. Superimposed photographs of the electrical variation of the sartorius in response to a single stimulus. (BunDON SANDERSON.) 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. 85) has ceased to be negative before the negativity of b has attained its full height, and there is thus no prolonged equipotential 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 therefore the wave character of the electrical change, considerable difficulty is experienced sometimes in recognising that the ' spike ' record of the electrical change in voluntary muscle or in nerve is also due to a diphasic variation. In this case the electrical change at any spot lasts only about -g-jjij 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. 89, the upper shadowy tracing being that obtained from the injured muscle. It will be seen that the distinguishing character of an electrometer record of a diphasic variation in the rapidly contracting striated muscle consists in the fact that the downstroke of the image of the meniscus is as rapid as the upstroke, whereas the monophasic variation of the injured muscle presents a slow fall produced by the ELECTRICAL CHANGES IN MUSCLE 261 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. 90, which represent the 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 77^- 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 direc- tion, but that a change in one direction at one electrode dies away and is succeeded by a similar change in the same direction, which also dies away, at the second FIG. 90. Monophasic variations of an injured sartorius. A, at 18° C ; B, at 8° C. (KEITH LUCAS.) electrode : that is to say, a diphasic variation implies the progression of a wave of electrical change between the leading-off points. The electrical variation obtained by leading off a heart beating normally is a much more complex affair, and even now physiologists are not agreed as to its interpretation. Gotch has suggested that the complex character of the variations obtained both from the spontaneously beating frog's heart as well as from the mammalian heart is due to the twisting and alteration in direction of the fibres and of the course of the contraction wave which have occurred in the evolution of the heart from a simple tube. The question will te discussed more fully in chapter xiii. 262 PHYSIOLOGY THE DEMARCATION CURRENT OR CURRENT OF INJURY According to Hermann, 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 con- clusion, only paradoxical in terms, that the so-called currents of rest are really currents of action and are due to excitation around the injured spot.* We shall see later, in dealing with the electrical changes which accompany the excitatory state, that the two conditions of injury and of excitation are really attended with similar molecular changes in the muscle or the nerve. 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. 91) be laid so as to touch at two points the cut end and surface of the muscle 6, and the nerve of b then stimulated with single induction shocks, every contraction of b will be attended by a con; traction of a, excited by the negative varia- tion of the current passing through its nerve from the point touching the cut end to that FIG. 91. r 7 Rheoscopic frog. m contact with the equator of b. If the nerve of b is tetanised, a as well as b enters into a continued contraction. JThis ' 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 only due 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 demar- cation current of a sartorius equals about 0-05 of a Daniell cell. The action current of the same muscle may attain to an E.M.F. = 0-08 of a Daniell 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 proportion 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 T — T' heat engine is expressed by the formula E = , where T is the highest temperature (in absolute measurement) obtained by the working substance and T' 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 tem- perature 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 transformation of heat into mechanical energy. He has found that non-living substances, 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 263 264 PHYSIOLOGY 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 imbibe the surrounding water so that they change 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 temperature it would be impossible to attain the efficiency of 12 to 20 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 100 per cent, of the total chemical energy available, 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 every- thing 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 may, 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 sarco- plasm. We may imagine the excitatory process to consist in a sudden chemical change, either of disintegration or of oxidation, occurring in the contents of the muscle prism. As a result there is a production of a number of new molecules within the muscle prism (e.g. of an acid or of carbon dioxide), which raises the osmotic pressure within the prism and occasions a rapid inflow of water from the sarcoplasm. As a result the pressure in the muscle prism rises and causes a bulging of its lateral wall and a shortening of the whole element. The subse- quent phase of relaxation may be due either to a secondary change in the products of 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 differences 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 INTIMATE NATURE OF MUSCULAR CONTRACTION 265 condition of combination with the proteins of the sarcous element. We have indeed certain 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 modification of the surface will alter the tension, and therefore state of expansion, of 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. 92. If B represents the shape of the globule lying ABC FIG. 92. 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 represent its shape when it is connected with the positive pole of a battery, the other pole in each case being connected with the acid. If we 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 deter- mined 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. It is impossible at present to decide between these different theories. They have their use, however, in showing the possibility of ' explaining ' a muscular contraction, i.e. of bringing it into a series of phenomena the other members of which are already familiar to us. They may therefore serve to point the direction which future researches into the intimate nature of muscular contraction must take. 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 circum- stances 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 physiologists 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 con- tinued 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 a 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 266 VOLUNTARY CONTRACTION 267 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 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 only occurs 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 excitation 1'iG. 93. 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 cathodal end having been moistened with a weak solution of Na2C03. of a nerve, which is apparently continuous, may excite a corre- spondingly continuous state of excitation in the muscle attached. During the passage of a constant current through muscle there is a continuous contraction in the neighbourhood of the cathode. If the irritability of the muscle at this point ^i»e 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. 93). More- over in frogs, the excitability 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 ascending current through the nerve. The question however can only be decided by experiment. If a voluntary 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, 268 PHYSIOLOGY 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. Helm- holtz 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 the 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 muscles of the body is caused^by^strong excitation of the cerebral cortex, as in epilepsy. On taking a record of such contractions, Schaefer 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 con- tractions of the whole muscle. Von Kries has found that the duration of a muscle twitch may be lengthened by lengthening 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 VOLUNTARY CONTRACTION 269 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. Each discharge produces, not a twitch, but a con- tinued 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 only be compared to a short tetanus. The most recent investigations of the question we FIG. 94. Electrical variations produced by voluntary contractions of human muscle. (PIPER). owe to Piper, who made use of the string galvanometer, an instru- ment 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 con- tracted 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. 94). Piper obtained similar records on leading ofl; 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 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. 1 [This matter has been taken up by Dittler, who 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 nerve. He finds that both in the muscle and in the nerve there is evidence that 270 PHYSIOLOGY each contraction is a fused series of single contractions, evoked by the discharge along the nerve of between fifty and seventy excita- tions per second. So far therefore the evidence is in favour of the view that voluntary contraction, and, one must add, the tonic con- tractions 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 con- taining 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 intimately 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 un differentiated 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, * 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 back- wards 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. 071 272 PHYSIOLOGY presents in many cases rhythmic contractions, and can carry out a peripheral adaptation to its environment. These rhythmic contrac- tions are almost invariably observed if the muscular tissue be subjected to a certain amount of tension, after separation from the central nervous system. The rhythm of the contractions 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 instances a single induction shock, even if very strong, is powerless to excite contraction, and the make- induction FIG. 95. At the cathode K there is a small line 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 obser- vation it would be thought that contraction occurred at the anode itself. 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 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 definite sheets, one consisting of longitudinal, 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 contraction of the circular coat at the anode, which spreads up and down the intestine, and a linear contraction of the longitudinal coat at the cathode. The same result is observed in the 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 accom- panying diagram (Fig. 95) will serve to show the condition of the circular coat at each electrode. As a matter of fact, in consequence of the arrangement of the fibres, we have in OTHER FORMS OF CONTRACTILE TISSUE 273 the neighbourhood of the anode a number of places (virtual cathodes) where the current is leaving the muscle- cells to enter inert conducting tissues, and in the same way there will be in the neighbourhood of the cathode a number of virtual anodes (Fig. 96). 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 moistened with normal saline FIG. 96. 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 elec- trodes 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/X, E,,,, E,,,, due to the current leaving the muscle to flow through indifferent tissues. (BIEDERMANN.) 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. SUMMATION. If two stimuli be sent into a voluntary muscle within a short interval of time, there is a summation of effect, the contraction due to the second stimulus being piled, so to speak, on the top of the first contraction. That a maximal twitch is not as high as a tetanus, the production of summation of many twitches, is due to the fact that the relaxation processes of a muscle begin before it has time to overcome the inertia of the mass moved, and so accomplish its maximum shortening. If therefore we support the muscle in any way, whether by screwing up the lever (after-load- ing) or by sending in a previous stimulus, the contraction due to a stimulus will be more pronounced, until the shortening of the muscle attains that observed in tetanus. For the same reason the height of a single twitch in relation to a tetanus of the same muscle increases as we slow the contraction, until, with a prolongation such as is produced by veratrin, there is no difference at all between the height of a maximal single contraction and the height of a tetanus. These considerations would lead us to expect no trace of any process analogous to summation of contraction in the slowly moving smooth muscle. In the heart muscle this is the case, no increase in the height of a contraction being produced by sending in one, two, or more shocks in quick succession. When, however, we record the contractions of a muscle, such as the retractor penis, which is more closely under the control of the nervous system, and excite with a series of induction shocks, we get results which at first sight are 18 274 PHYSIOLOGY exactly analogous to the summation of contraction in a voluntary muscle. It may be noticed, however, that the first three or four stimuli are ineffective, and that there is in this case a 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 con- tinually increasing height of contraction, the so-called ' staircase.' It will be remembered that the same initial increase of effect was observed when voluntary muscle was excited by continually recurring stimuli (v. Fig. 69, p. 234). 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 con- tractions just as in the case of skeletal muscle. Many drugs, such as physostigmin, 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 increased 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 disten- sion. 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. OTHER FORMS OF CONTRACTILE TISSUE 275 PROPAGATION OF THE EXCITATORY STATE, OR WAVE OF CONTRACTION. On stimulating any part of a voluntary muscle fibre, 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 by simultaneous excitation of all its fibres. It is doubt- ful whether this isolation of the excitatory state is found in smooth muscle. As a 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 propagation 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. Indeed by FlG- 97- clamping two curarised sartorius muscles together, as in the diagram (Fig. 97), it is found that stimulation of the muscle A causes contraction 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 fibres, and probably the same is true for many kinds of involuntary muscle. INFLUENCE OF TEMPERATURE. Smooth muscle is extremely susceptible 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 does not depend 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 warm- ing 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 276 PHYSIOLOGY 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 depen- dent for its activity on the central nervous system. Cut off from this it is flabby and motionless. Its sole function is to contract efficiently and smartly on receipt of impulses arriving along its nerve. It is only necessary therefore that these impulses should be of one character — motor, and we know that each fibre of a muscle, such as • FIG. 98. Tracing from the retractor penis muscle of the dog, showing lengthening (inhibition) on stimulation of the nervus erigens, and a smart contraction on stimulating the pudic (motor) nerve. (Move- ments of muscle reduced |.) the 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 inner va- tion may be at one time in a state of relaxation, at another in a state of tonic contraction. 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. 98. OTHER FORMS OF CONTRACTILE TISSUE 277 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 contracted muscle (Fig. 99). AMOEBOID MOVEMENT Amoeboid movement is seen in the unicellular organisms such as the amoeba . i i * I i i i * I i.j and in the white blood corpuscles. It can FlG- f99- Tracing of contraction of adductor muscle of claw of crayfish, showing inhibi- tion resulting from stimula- tion of its nerve (at 6) by means of a constant current. The break of the current causes a second smaller in- hibition. (BlEDERMANN.) occur only within certain limits of tem- perature (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 con- traction 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 genera- tive 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. 100) are delicate taper- ing 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. In action the cilia bend suddenly down into a hook or sickle form, and then return more slowly to the erect position. This movement is FIG. 100. Ciliated columnar epithelium from the trachea of a rabbit; ml, m2, ra3, mucus-secreting cells. (SCHAFER.) 278 PHYSIOLOGY 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 is. however, a functional connection between all the cells of a ciliated epithelial surface, 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 as those for amoeboid 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. CHAPTER VI NERVE FIBRES (CONDUCTING TISSUES) SECT] ON I THE STRUCTURE OF NERVE FIBRES ON stimulating the nerve part by electrical, thermal, FIG. 101. Diagram of a motor nerve- cell with|its nerve fibre. (After BARKER.) «.&, axon hillock; d, deudrites; a.x, axis cylinder; m, medullary sheath ; n.R, node of Ranvier. of a nerve- muscle preparation at any or mechanical means, the stimulus is followed, after a very short interval, by a contraction of the muscle. This obser- vation illustrates the two functions of nerve fibres, irritability and conducti- vity— that is to say, a suitable stimulus can set up changes in any part of the nerve, which are transmitted down the nerve without any visible effects occur- ring 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 nor- mal 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 essential that there should be vital continuity along the whole length of the fibre. Damage to any part, such as by crushing, heat, or any other in- jurious condition, infallibly causes a block to the passage of an impulse. A nerve fibre is essentially a long process or arm of a nerve-cell (Fig. 101). The cell may either be situated on the surface of the body or, as in most cases in the higher animals, may be withdrawn 279 PHYSIOLOGY from the surface into a special collection of cells such, as the posterior root ganglion, or may be one of the mass of cells and interlacing processes making up a central nervous system. All nerves are alike in possessing 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 fibrillae or neuro- fibrils, embedded in a more fluid material (Fig. 102). These neuro- FIG. 102. Medullated nerve fibres, showing continuity of the neuro-fibrils across the node of Ranvier. (BETHE.) a, longitudinal ; &, transverse section. fibrils are supposed to be continuous throughout the cell and the axis cylinder and to represent the essential conducting constituents of the nerve. In the course of growth the nerves develop certain histo- logical 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, THE STRUCTURE OF NERVE FIBRES 281 and staining black with osmic acid, supported in the interstices of a network formed of a horny ubstance known as neurokeratin. The medullary sheath is surrounded by a structureless membrane, the primitive sheath or neurilemma. At legular 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 dis- tributed 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 FIG. 103. Non-medullated nerve fibres. (SCHAFER.) or peripheral ganglia, the impulses being carried on to their destina- tion by a fresh relay of non-medullated nerve fibres. The non-medullated fibres (Fig. 103) 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 are 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 branch- ing always occurs at a node of Ranvier. 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 re- generation 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 con- duction within any given nerve fibre. We have, however, 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 movements is required in the skeletal muscles than in the case of the visceral unstriated muscles. Moreover in the central 282 PHYSIOLOGY nervous system the main tracts cannot be shown to be functional before the date at which they acquire their medullary sheaths, sug- gesting that previously 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 out- growth. 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 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 gastrocnemius is prepared, with a long piece of sciatic nerve attached. The muscle is arranged (Fig. 104) so that its con- traction may be recorded on a rapidly moving surface, on which are FIG. 104. 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 s, the break shock in the secondary circuit can be sent through the nerve n, either at 6 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 surface by the automatic opening of the key k. (The time- marker is not shown.) also recorded, by means of electro- magnetic signals, the moment at which the stimulus is sent into the nerve, and also a time-marking showing -j-J-tf 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 stimu- lus is sent into the nerve and the point at which the lever begins to rise, is rather longer in the first 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 velocity of propagation in frog's nerve is about 28 metres per second. In man and in warm-blooded animals the velocity has been variously 283 284 PHYSIOLOGY estimated at from 60 to 120 metres per second. The higher of these figures is probably nearer the truth. On the other hand, in invertebrata the velocity of propagation along nerve fibres may be quite slow. The following Table represents the velocity of trans- mission along a number of different fibres, as determined by Carlson, compared with the duration of single muscle-twitch in the same animal. Muscle . Nerve Species Contrac- Rate of Muscle tion !N"crv6 the impulse time 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 determine 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 contraction. There is another method of deter- mining 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 imme- diately 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 same 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 PROPAGATION ALONG NERVE FIBRES 285 transmit impulses in either direction is shown further by the experi- ment known as Kiihne's gracilis 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 cylinders themselves. If the section a in the diagram (Fig. 105), which is quite isolated from the rest of the muscle, be stimulated, as by snipping it with scissors, 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 down 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 .. FlG/ 105- . . not the case. As a matter of fact we find in the experiment/8 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 root 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 therefore not so much on the structure of the nerve fibre itself as on the con-, nections of the fibre. We can show this experimentally by grafting 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 286 PHYSIOLOGY 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 Mutter's ' law of specific irritability ' in the chapter on Sensations. 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 mechani- cal 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 pro- pagated 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 iso- electric. On making a cross-section of the nerve at one leading-off 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 287 288 PHYSIOLOGY 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 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 galvanometer. 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 1 8 mm. 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 temperature at which this occurs varies according as we use a warm- or a cold-blooded animal. In the frog it is necessary to cool the nerve below 0° 0. 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 nerve does not excite it, this procedure forms a convenient method for blocking the passage of impulses along a nerve without using the irritating pro- cedure of section. On warming the nerve again the conductivity returns. The rapidity with which the excitatory pro- cess is propagated along either a nerve or a muscle fibre depends on the tempera- ture. Thus the mean rate of conduction in the frog's nerve at 8° to 9° 0. is about 16 metres per second. The temperature coefficient of the velocity of nerve propa- velocity at fn + 10 , gation, i.e. - — has been velocity at Tn found by Lucas to be about 1-79. The same value was found by Maxwell for conduction in molluscan nerve, and in frog's striated muscle Woolley found the temperature coefficient for conduction of the excitatory process to vary between 1-8 and 2. An ingenious method (Fig. 106) has been used by Keith Lucas for the determina- tion of the conduction rates in nerve at different temperatures. The glass vessel represented 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 289 19 290 PHYSIOLOGY vessels at c and D, and is attached to the thread. F, I, and G are three non- polarisable electrodes composed of porous 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 sensi- tive plate. The whole apparatus, with the exception of the glass rod at H, can be immersed in a water bath at any given temperature. Two records are FIG. 107. Curve of muscle-twitch obtained by foregoing method. (KEITH LUCAS.) A = moment of excitation. B = movement of muscle, c = time-marker. taken with the whole apparatus, first stimulating at c, and secondly stimu- lating 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 temperature. 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 muscular 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 demon- strate any phenomena of fatigue in the nerve-trunk.* This fact can * Unless it be asphyxiated by total deprivation of oxygen. CONDITIONS AFFECTING A NERVOUS IMPULSE 291 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 physostigmin, when the muscles will at once begin to react to the excitation. The same fact may be shown on the excised nerve-muscle prepara- tion of the frog. The gastrocnemii of the two sides with the sciatic A B FIG. 108. Arrangement of experiment for demonstrating the absence of fatigue in medullated nerve-fibres. EC, exciting circuit ; CP, polarising circuit. nerves are dissected out, and an exciting circuit is so arranged that the interrupted secondary currents pass through the upper ends of both nerves in series (Fig. 108). 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 constant polarising current is made, the muscle B may give a single twitch, and then remains quiescent. 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 subsides as 292 PHYSIOLOGY fatigue comes on. Since both nerves have been excited throughout, it is evident that the fatigue does not affect the nerve- trunk. We have already seen that a muscle will respond well 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 inhibi- tion of the centra] motor apparatus from the muscle itself. Thus after FIG. 109. 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 contraction 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 anaes- thetics. 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 may be studied by means of the simple apparatus represented in Fig. 109. The nerve 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 C02, or air charged with vapour of ether or other narcotic, can be passed through the tube. The nerve is armed with two pairs of electrodes which are stimu- lated alternately, the pair within the tube serving to test the action of the drug on the excitability, while the pair outside the tube shows 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 abob'sh the excitability and conductivity of the nerve fibres. The conductivity, however, persists after all trace of excitability has disappeared, before in its turn being also abolished. CONDITIONS AFFECTING A NERVOUS IMPULSE 293 On removing the gas or vapour by blowing air over the nerve, the conductivity and excitability gradually return in^the reverse*order to their disappearance (Fig. 110), FIG. 110. 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 (gastroc- nemius). 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, disap- pearance of conductivity ; D, reappearance of conductivity. (From a tracing kindly lent by PROF. GOTCH.) 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 ch cro- form 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, mechanical, 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 contrac- tions. Sudden warming of the nerve always gives rise to excitation. At about 45° C. 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 purpose we may use either the make and break of a constant current, the induced current of short dura- tion produced in a secondary coil of an inductorium by the make or break of the primary circuit, or the discharge of a condenser. The last-named method of stimulation is especially useful when we desire to determine the total amount of energy involved in the electrical stimulation of a nerve or muscle. The arrangement of such an experiment is shown in Fig. 111. By means of the switch S the condenser can be put into connection either with the battery from which it receives its charge or with the nerve through which it can discharge. By knowing the capacity of the condenser 294 THE EXCITATION OF NERVE FIBRES 295 and the electromotive force by which it is charged, we can estimate the energy of the charge sent through the nerve. E (energy in ergs)* = 5FV2 (F = capacity in microfarads ; V = electromotive force in volts). In this way it has been found that the energy of a minimal effective stimulus for frog's nerve is about T ^^ 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 expenditure of energy, which he calls the "characteristic" of the tissue in question. To this point we shall have occasion to refer later. When using the make and break of a con- stant current as a stimulus, the first fact of importance is the relation of the seat of exci- tation to the poles by which the current is led into or out of the excitable tissue. We have already seen that when a current is passed through a muscle r or nerve the muscle contracts only at make or at break of the current, Fl ?.h(™; *he f ect of * injury on the excitability of a muscle. kathode (injured) no contraction at make. &, battery ; ra, muscle. The arrows indicate the direction of the current. short duration no excitation is produced at break. Every induction shock can be therefore regarded as a 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 THE EXCITATION OF NERVE FIBRES 297 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, however, 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 consider- ably modified. This modification in the condition of the nerve is spoken of as electrotonus, and includes changes 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 FIG. 114. Arrangement of apparatus for showing electrotonic changes in irritability. e, exciting current ; p, polarising current ; r, Pohl's reverser. non-polarisable electrodes, and a pair of ordinary platinum electrodes. Fig. 114 represents roughly the arrangement 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 direc- tion 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 inter- posed, 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 con- traction can be recorded. We first find the position of the secondary coil, at which the break induction shock is a submaximal stimulus, and we employ this strength of stimulus throughout the experiment. The make induc- tion shock is prevented from acting on the nerve by closing a short- circuiting key in the circuit of the secondary coil. The nerve is now 298 PHYSIOLOGY 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 probably 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 contraction 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 contraction evoked by the induction shock. We now reverse the direction of the polarising current, so that the current of the nerve runs from Jc 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 Jc. On break of the polaris- ing current the condition of the nerve returns to normal, and the sub- maximal stimulus is once more submaximal throughout. This return to normal conditions, however, 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 anelectronus 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 appearance of catelectrotonus, or a sudden disappearance of anelectrotonus. I have said sudden because the steepness of the rise of irritability is a neces- sary factor in causing excitation. If the polarising current passing through a nerve be slowly and gradually increased to considerable THE EXCITATION OF NERVE FIBRES 299 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 FIG. 115. 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 ; yv effect of weak current ; y.2, medium current ; y3, 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 PFLUGER.) than in muscle, and 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 is neither increased nor diminished. We find experimentally ascending current [ make excitation blocked at anode. an. kath: break excitation at anode blocked at kathode. FIG. 116. Diagram to show the blocking effect of a strong constant current passed through the nerve of a nerve-muscle preparation. that this indifferent point is nearer the anode when the polarising current is weak, and gets nearer to the cathode as the current is strengthened, so that with very strong currents nearly the whole intra- polar length is in a condition of anelectrotonus (Fig. 115). When a strong polarising current is used, the depression of irritability at the anode is so marked that no impulse can pass this region. Thus if we send a very strong ascending current through the 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 muscle, its passage down the nerve being blocked in the region of the anode (Fig. 116, A). 300 PHYSIOLOGY The results of stimulating motor nerves by means of constant currents were studied by Pfliiger and, embodied in a Table, make up what is known as Pfliiger's law. The result of stimulating varies with the strength of a current. LAW OF CONTRACTION Strength of current Ascending Descending Weak Make c Break O Make Break c O Medium . c c C c Strong O CorT CorT 0 c = contraction. C = strong contraction. T — tetanus. 0 = no effect. With the weakest currents excitation occurs only at make, since a make- stimulus, i.e. the rise of catelectrotonus, is always more effectual than a break- stimulus, i.e. the disappearance of anelectrotonus. With currents of moderate FIG. 117. Arrangement of experiment to demonstrate Pfliiger's law of contraction. strength excitation occurs both at make and break, being better marked at make, especially in the case of descending currents. With very strong currents we get a contraction at make only when the current is descending, since, when the current is ascending, the excitation started at the cathode cannot pass the block at the anode. For the same reason a break contraction is obtained only with an ascending current, since at the break of a descending current there is a swing-back of the nerve at the cathode to a condition of diminished irrita- bility, which effectually blocks the excitation started higher up the nerve at the anode. The arrangement of the experiment for demonstrating Pfliiger's law is shown in Fig. 117. The strength of the current is graduated by means of the rheochord, the current being led into the nerve by means of non-polarisable electrodes. It is extremely important in these experiments to avoid any injury or drying of the nerves at either of the two electrodes, since the excita- tory effect either at make or break would be abolished by local injury. These results, worked out chiefly on motor nerves, have been confirmed as far as possible experimentally on sensory nerves, and on THE EXCITATION OF NERVE FIBRES 301 muscle and contractile tissues generally, and probably hold good for all irritable living tissues. It is said that an anelectrotonus takes some time to attain its full height, and a catelectrotonus reaches its maximum almost directly after the current is made, and that it is on this account that a current of very short duration excites only at the make, the break occurring before the anelectrotonus is deve- loped enough for its disappearance to cause a stimulus. Other things being equal, a current of given strength causes a stronger excitation the greater the length of nerve that it flows through. It must be remem- bered, however, that the nerve offers considerable resistance to the passage of the current, and so, to keep the current constant while increasing the length of intrapolar nerve, we must largely increase the electromotive force employed. A very convenient method of show- ing the effect of the length of intrapolar nerve on excitation has been suggested by Gotch. The two sciatic nerves of a frog are dissected out, one of them being in connection with the gastroc- nemius. These are first arranged as in Fig. 118. a, b, and c are three non- polarisable electrodes, the terminals of a constant battery being connected to a and c. The position of the rider on the rheochord is then ascertained at which make of the current just excites contraction in the muscle of nerve 2, the current in this case passing from a to b along nerve 1, and from b to c along a small piece of nerve 2. We will suppose that eleven units of current are necessary to produce excitation, b is then withdrawn and the nerve 2 laid on a (Fig. 1 18, B), so that the current can now pass from a to c entirely through a long stretch of nerve 2. On again seeking the minimal stimulus, it will be found that a smaller current is sufficient to excite, contraction being obtained with seven units. Since the length of nerve traversed, and therefore the resistance to the current, are the same in both cases, it is evident that a current is more effective the greater the length of excited nerve that it traverses. A nerve cannot be excited by currents passed transversely across it, since in such cases the anode and kathode lie so close to one another in a nerve-fibril, as it is traversed by a current, that their effects counteract one another. FIG. 118. 302 PHYSIOLOGY ELECTRICAL STIMULI AS APPLIED TO HUMAN NERVES When we attempt to apply the results gained on frog's nerves to man, we are met at once by the difficulty that we cannot dissect out the nerves and apply stimuli to them directly. So usually unipolar excitation is used, one electrode, either anode or cathode, being applied to the nerve to be stimulated, and the other to some indifferent point, such as the back. It is evident under these circumstances that the current is concentrated as it leaves the anode and reaches the cathode, and diffuses widely in the body, seek- ing the lines of least resistance. Thus it is impossible to get pure anodic or cathodic effects. If the anode be ap- plied over the nerve, the current enters by a series of points (the polar zone), and leaves by a second series (the peripolar zone). The polar zone will thus be in the condition of anelectro- tonus, and the peripolar zone in that of catelectrotonus. The current, how- ever, will be more concentrated at the polar than at the peripolar zone, and ^Vs&over™^ £ BO the former effect will predominate. A the polar area is anelectrotonic These restrictions in the application of and the peripolar catelectro- ,-, ,. -, , , • tonic. The former condition tne current cause slight apparent irre- therefore preponderates, since gularities in the law of contraction as the current here is more con- tested on man. centrated. In B the conditions are reversed, the polar zone corresponding in this case to the cathode. (WALLER.) In stimulating the nerves of man for the purpose of determining the conditions of the different muscles, we may use either induced currents (generally called faradic stimulation) or the make and break of a battery current (galvanic stimulation). It is usual to employ the unipolar method, in which one electrode is placed over the nerve at the point it is desired to stimulate, while the other electrode, spoken of as the indifferent electrode, is applied to the skin over a wide area, generally at the back of the neck. The current is then widely diffused as it passes through the indifferent electrode, but is concentrated as it passes between the skin and the stimulating electrode. The electrodes are covered with chamois leather moistened with salt solution in order to diminish the resistance of the skin. When it is desired to stimulate any given muscle, the stimulating electrode is brought as nearly as possible over the spot where the muscle receives its motor nerve. These ' motor points ' have been mapped out, and reference is generally made to a diagram in determining the point for any given muscle. By reversing the current the stimulating electrode may be made either anode or cathode. It is found that stimulation occurs most easily on closure of the current when the stimulating electrode is the cathode ; with the greatest difficulty when the current is broken and the THE EXCITATION OF NERVE FIBRES 303 stimulating electrode is the cathode. These different contractions are generally represented by capital letters, and the usual relationship is expressed by the statement that CCC is obtained most easily, then ACC and AOC, and finally COG. CCC = cathodal closing contraction. ACC = anodal closing contraction. AOC = anodal opening contraction. COC = cathodal opening contraction. When the motor nerve to a muscle has undergone degeneration the muscle also begins to degenerate, and we find certain alterations in its response to artificial stimulation. In the first place, the muscle may fail to respond to induction shocks, while it may show an increased irritability for galvanic shocks. In the second place, qualitative alterations in irritability may be present, so that ACC may be obtained with a smaller current than CCC. These alterations are spoken of as the ' reaction of degeneration.' SECTION VI THE CONDITIONS WHICH DETERMINE ELECTRICAL STIMULATION FOR every tissue traversed by a current there is a minimum rate of change at which the current through the tissue must be increased or diminished in order to cause excitation. If instead of suddenly making and breaking the current passing through an irritable structure we carry out the change gradually, no excitatory effect is produced, even although the current may finally attain a considerable strength. This fact may be demonstrated by the use of an apparatus known as the rheonome. A useful form of rheonome is that devised by Lucas (Fig. 120). Two zinc plates D and E, immersed in a saturated solution of zinc sulphate contained in a rect- angular cell, are separated from one another by a vulcanite diaphragm. In the diaphragm is a hole G by which the two sides of the vessels are connected. This hole can be closed at any desired rate by a shutter r. When the hole is closed no current can pass between the plates, and the amount which can pass will depend on the extent to which the shutter has been raised. By giving Dhe hole the right shape it is possible to diminish the resistance of the apparatus regularly. If this rheonome be placed in circuit with a battery and an excitable tissue, such as the nerve of a nerve-muscle preparation, we can make a current or break a current through the tissue at any desired rate. Thus the course of the current through the tissue will be represented, not by a vertical line, but by a sloping line which may be given any desired degree of steepness (Fig. 121). If the current be slowly increased through the nerve or be slowly cut off from the nerve, no excitatory effect takes place, while quickly opening or closing the shutter will cause excitation. It might be concluded that the excitatory effect of a current increases with 1. The intensity of the current. 2. The rate of change of the current. The second of these conditions needs, however, some correction. 304 FIG. 120. Rheonome of Keith Lucas. ELECTRICAL STIMULATION 305 As we increase the rate of change of current, by employing in the case of induced currents more and more rapid alternations, we find that the excitatory effect, instead of increasing, begins to diminish and finally disappears, so that high-frequency currents of enormous tension can, as in Tesla's experiments, be led through the body without any apparent physiological effect. On the other hand, by using more sluggish forms of irritable tissue, we may find that even induction shocks are too rapid for effective excitation. Thus the red muscles of the slow-moving tortoise react better to the slow make than to the sudden break induction shock, and many forms of unstriated muscle are unaffected by either make or break shock. There is in fact for each tissue an optimum rate of change varying with the character of the tissue, at which the current necessary to produce a response is at a minimum. This optimum rate of change is spoken of by Waller as the ' characteristic ' of an irritable tissue. FIG. 121. String galvanometer records of the change of current obtained by opening the diaphragm in the rheonome (Fig. 120) at different rates. (K. LUCAS.) A further investigation of the time relations of electrical stimuli by Keith Lucas has thrown important light on the character of the excitatory response itself. The difference between various excitable tissues is perhaps best brought out by finding the minimum strength of current which will excite at make and then determining how much this current must be increased when it is broken at a very short interval of time after it has been made. The following Table represents the relation between duration and strength of current necessary to stimulate in the case of the sciatic nerve of the toad : Duration of current (sec.) CO •0070 •0035 •00087 •00043 Strength of current (volts) •086 •091 •119 •179 •245 If we slightly alter the use by Waller of the word ' characteristic ' we may take as the characteristic of the tissue the duration of the stimulus at which the current necessary to stimulate was just double the minimum. ^In this case the minimal stimulating current was approximately doubled when the duration of the current was 20 306 PHYSIOLOGY limited to about '001 sec. From a number of experiments of this description Lucas gives the following as the characteristic, or what he terms the " excitation times," of muscle and of nerve : Muscle -017 sec. Nerve fibre -003 Nerve-ending (or intermediary substance) *00005 ,, Similar differences are obtained when we attempt to determine by means of the rheonome the minimal gradient of current necessary to excite a nerve. In the case of the toad's nerve the minimal gradient must be ten times as steep as in the case of the toad's muscle, and is such that in one second the current must reach a strength forty-five times the minimal strength which is necessary to excite when the current is made instantaneously. In the frog's nerve the minimal gradient is still steeper, so that in one second the current must reach sixty times the strength of the minimal exciting current. We may interpret these results as signifying that the excitatory state pro- duced in an irritable tissue involves the production of some change which passes away spontaneously. The rate of production of the change, and still more the rate of its spontaneous disappearance, differ from tissue to tissue. If the gradient of a current which is made through the tissue is too slight, the spontaneous disappearance of the excitatory change goes on as rapidly as the production in consequence of the rise of current. No excitation therefore takes place. The " excitation time " of the tissue will thus be proportional to the duration of the excitatory change produced in the tissue as a result of the stimulus. We may compare the excitation time of three tissues with the duration of the electrical change produced in the same tissues by a single stimulus. The excitation times were : Frog's nerve fibre "003 sec. Muscle fibre of sartorius . . . . '017 ,, Ventricular muscle of frog . . . 2*000 „ In the case of muscle, according to Burdon Sanderson, the electrical change reaches its culminating-point in '0025 sec., and may take perhaps eight times this interval before it dies away. In the cardiac muscle of the tortoise Sanderson found the electrical change to last between two and three seconds. SUMMATIO ; OF STIMULI. Closely associated with the excita- tion time of the tissues are the phenomena of ' summation of stimuli ' and ' refractory period.' If two subminimal stimuli are sent in within a sufficiently short interval of time, their effect is smnmated, so that two stimuli, each of which would be ineffective, ELECTRICAL STIMULATION 307 may together produce an excitation. In the case of striated muscles; in order that mechanical summation of contraction may take place, the second stimulus must become effective before the muscle has completely relaxed ; the second contraction, that is to say, starts from the height to which the first contraction has brought the muscle. A similar condition of things appears to hold for summa- tion of stimuli, if we substitute for mechanical change in muscle the molecular change which accompanies the excitatory state. For summation of two stimuli to take place, the second stimulus must occur at a time before the condition excited by the first stimulus has passed away. The maximum time at which summation of two stimuli can take place will therefore vary from tissue to tissue^and will bear a relation to what we have designated the ' excitation time ' of the tissue and also to the rapidity of current gradient necessary to excite the tissue. This will be evident if we compare the maximum summation intervals for different tissues with the excitation time of the same tissues. Stimulating current 5% above minimal stimulus Summation interval !' Excitation time " sec. sec. Frog's nerve . . . -0005 . . -003 „ sartorius .. . -0015 . . -017 „ heart . . . '0080 . . 2-000 REFRACTORY PERIOD. The phenomenon of a refractory period has long been known in connection with the heart muscle and has often been regarded as characteristic of this muscle. If, in the isolated ventricle, a beat be evoked by a single minimal stimulus, subsequent repetition of the stimulus during the course of the contraction is ineffective, and becomes effective only when the contraction has passed away. The heart is said to be refractory to stimuli during this period. The duration of the refractory period is a question of the strength of the stimulus used. With strong stimuli the heart may be made to contract when the relaxation has only pro- gressed half way, and with very strong stimuli one contraction may be made to follow the last at such a short interval that hardly any trace of relaxation is observable between the beats. The phenomenon seems to be common to all excitable tissues. Thus if two stimuli are applied to a nerve within a sufficiently brief interval, the second stimulus is ineffective, so far as can be determined by the response of an attached muscle or by means of a capillary electrometer. The period is longer the lower the temperature and varies from -0006 sec. at 40° C. to -002 sec. at 12° C. This critical interval is lengthened if the irritability of the nerve is depressed by narcotics. We may ascribe it to the second stimulus being applied before the excitatory change due to 308 PHYSIOLOGY the first stimulus has reached its culminating- point. If the first stimulus was maximal it is evident that no further addition to the molecular change could occur as a result of the incidence of the second stimulus. THE EFFECT OF TEMPERATURE ON EXCITABILITY. It was found by Gotch that the excitability of a nerve within certain limits was increased by cooling the nerve and diminished by raising its temperature (Fig. 122). Thus, if a frog be cooled to 2° C. or 3° C. for a day, it will be found that simple section of the sciatic nerve may suffice to send the gastrocnemius into continued contraction, and under these circumstances ' closing tetanus ' may be obtained with the greatest ease. This increase of excitability does not apply to all kinds of stimuli. In the case of nerve its irritability was found to be increased by warming, and diminished by cooling for FIG. 122. Tracing of muscle contractions to show effect of cooling a nerve on its excitability. The lower line indicates the changes in temperature of the excited part of the nerve. The muscle responded only when the nerve was cooled, the stimulus becoming ineffectual when the nerve was warmed. ( GOTCH.) induction shocks and for all galvanic currents of less duration than •005 sec. In skeletal muscle Gotch found the excitability for all forms of stimuli increased by cooling. Lucas has shown that these paradoxical effects in nerve, namely, increase of excitability towards currents of long duration and the simultaneous decrease towards currents of short duration, are conditioned by two opposed changes in the tissue. The fall of temperature delays the subsidence of the excitatory process, but at the same time renders more difficult the initiation of a propagated disturbance. The first of these effects reduces the current required for excitation in a ratio which is greater the greater the duration of the current. The latter increases the current required in the same ratio for all durations. If then the change of temperature is such that the two opposite effects are exactly balanced at a certain medium duration of current, it follows that for currents ELECTRICAL STIMULATION 309 of longer duration the net result will be to reduce the current required for excitation, while for currents of shorter duration the net result will be to increase the current required. The effect of temperature therefore on the minimum exciting current will vary from tissue to tissue according as the two factors, rate of subsidence of excitatory change and the initiation of a propagated disturbance as a result of the excitatory change, are relatively affected by change of temperature. THE EFFECT OF INJURY. The irritability of the nerve of a muscle-nerve preparation is not equal in all parts of its course, but is greater at the upper end, probably in consequence of the proximity of the cross-section. Some time after a motor nerve is divided the increased irritability at the upper end gives way to a decreased irritability, and this decrease goes on till the nerve is no longer excitable. The diminution in excitability gradually extends down the nerve fibre, so that the part of the nerve nearest the muscle remains excitable the longest. This progressive change in the irritability of a nerve after section is spoken of as the Hitter- Valli law. It is soon followed by definite } istological changes in the nerve, which we shall describe later. SECTION VII THE NEURO-MUSCULAR JUNCTION THE excitatory process travelling down a motor nerve has to be transmitted to the muscle by the intermediation of the nerve- ending or end- plate. We have learnt to regard the axis cylinder as the seat of the propagated excitatory process. In the end-plate, however, the axis cylinder comes to an end. When stained by methylene blue or by impregnation with chromate of silver or mercury, the axis cylinder, after passing through the sarcolemma of the muscle fibre, is seen to break up into a number of branches (in some cases forming a typical end- arborisation), which lie on or are embedded in a small amount of undifferentiated nerve protoplasm containing nuclei (the ' sole plate '). A similar break in structural continuity seems to occur in the central nervous system wherever an impulse is propagated from the axon process of one nerve- cell to the body or dendrites of another nerve- cell. The end- processes of the axon come in contact with the next member in the chain of neurons, but no anatomical continuity is to be made out, at any rate in the higher animals. In the central nervous system the area of contiguity, where an im- pulse passes from one neuron to another, is spoken of as a synapse. The presence of the synapse, or end-plate, between muscle and nerve imposes certain new conditions on the conduction of the excitatory impulse. One of the most important of these lies in the fact that the conduction across the end-plate, and probably across the synapse of the central nervous system, is irreciprocal. An excitatory process started in the nerve travels easily across the end- plate to the muscle. On the other hand, an excitatory process started in the muscle does not extend through the end-plate to the nerve fibre. This fact may be shown on the frog's sartorius. If the lower tibial end of the muscle be split, as in Fig. 123, a mechanical stimulus, such as a snip with the scissors, applied to the lower nerve-free end of one' of the limbs, e.g. at A, causes a 310 FIG. 123. THE NEURO-MUSCULAR JUNCTION 311 contraction of the corresponding half of the muscle, which does not extend to the other hah0. On snipping the muscle a little higher up at B, where nerve- endings are present, the resulting contraction involves the whole of the muscle, owing to the fact that the excitation started in the nerve-endings spreads in both directions through the branching nerve fibres. It is possible that this irreciprocity of conduction may be of comparatively late appearance in evolution. So far as we know, an excitatory process in a sheet of muscle and nerve fibres, such as we find in lower invertebrata, e.g. in medusa, may travel with equal facility in all directions. We are probably not warranted from our experiments on skeletal muscle in concluding that the contraction of a cardiac muscle- cell may not set up an excitatory process in the surrounding network of nerve fibres. It is impossible, however, to put such a suggestion to experimental test, since in the heart there is no portion of muscle fibre sufficiently removed from nerves to allow of an excitation being applied which might not at the same time affect the nerve fibres. There is evidence that the transmission of the excitatory condition across the end- plate, from nerve to muscle, involves a special excitatory process and the expenditure of energy. Thus there is a period of delay between the arrival of an excitatory impulse at the terminations of the motor nerve and the beginning of the electrical change which marks the moment of stimulation of the muscle fibre. If we compare the latent period of a muscle stimulated directly with its latent period when excited through the nerve, we find that there is an increased period of delay in the latter which is not wholly accounted for by the time taken for the impulse to travel from the stimulated spot down the nerve fibres to the muscle. The extra delay is due to the processes occurring in the end-plate. This end-plate delay has been found to amount to -0013 sec. We may take it roughly at a thousandth of a second. The end- plate seems to be the weakest point in the neuro- muscular chain. We have already seen that, when a nerve of a nerve- muscle preparation is stimulated repeatedly, the muscle very soon shows signs of fatigue, and that the seat of this fatigue is not in the nerve, nor in the muscle, but in the end-plate. It has been suggested that the excitation of muscle through nerve depends on an electrical change or discharge at the nerve- ending. This discharge must originate in the terminations of the axon and must influence, in the first instance, the substance which forms the intermediary between the axon and the contractile material of the muscle. We have indeed direct evidence of the existence of a third substance, neither nerve nor muscle, at the point of junction of these two tissues. Thus, curare is generally said to paralyse the end- plates. Evidence for this statement has been given in the 312 PHYSIOLOGY previous chapter. Kiihne has shown that when the irritability of the frog's sartorius is tested at different points it is greater in the situation of the end- plates. This might be ascribed to the presence of the more irritable nerve-fibres passing into the muscle-fibres at these points. The unequal distribution of irritability is not, however, changed when the muscle is fully poisoned with curare, so as to block entirely the passage of any impulse from the nerve to the muscle. We must therefore regard curare as acting, not on the axon terminations, but on the substance intervening between these terminations and the contractile substance of the muscle. Additional evidence of the existence of such a " receptor " substance, as he calls it, has been furnished by Langley. Nicotine resembles curare in blocking the passage of impulses from the motor nerve to skeletal muscle, though inferior to curare in this respect. If 4 mg. of nicotine be injected into the vein of an anaesthetised fowl, the hind limbs become gradually stiff and extended in consequence of a tonic contraction of all their muscles. The effect slowly passes off, but can be reinduced by a second dose of nicotine. It is worthy of note that the stimulating effect of nicotine occurs even when sufficient is given entirely to paralyse the motor nerves. It might be thought that the stimulating effect of nicotine was a direct one upon the muscle fibre, but experiment shows that curare has a marked antagonising action on the contraction pro- duced by nicotine. A sufficient dose of curare annuls the contraction produced by a small amount of nicotine and diminishes that caused by a large amount. The point of action of the nicotine must therefore be the same as that of the curare. After a muscle has been relaxed by curare it can be still made to contract by direct stimulation. On the other hand, nicotine will produce its stimulating effect when injected into a bird in which degeneration of all the nerve fibres of the muscle has been produced by previous section of the nerve- trunks. It is evident therefore that nicotine, like curare, acts, not on the axon terminations, but on a receptor substance, an intermediary substance intervening between the axon terminations and the contractile sub- stance of the muscle. Evidence in favour of such an intermediary substance has been brought by Keith Lucas from an entirely different standpoint. In determining the optimal electrical stimuli or the ' characteristic ' of muscle and nerve by the condenser method (v. p. 305), Lucas finds that, even after moderate doses of curare sufficient to abolish the possibility of excitation through the nerve-trunk, the muscles show two optimal stimuli, pointing to the existence in them of two excitatory substances, one of which is not paralysed by moderate doses of curare. This result was confirmed when the tissue was investigated by determining the relation of current duration to the liminal current strength THE NEURO-MUSCULAR JUNCTION 313 necessary to excite. In a normal sartorius he finds three substances, each distinguished by its own 'excitation time.' In the pelvic nerve-free end of the sartorius there is only one substance, with an excitation time of -017 sec. This may be regarded as the muscle substance proper. In the sciatic nerve-trunk there is a second sub- stance with a much steeper characteristic and with an excitation time of -003 sec. On experimenting on the middle region of the sartorius we find not only these two substances but a third substance, which Lucas calls the substance ft, with an extremely rapid excita- tory process. Its excitation time is 'OOC05 sec. The presence of these three substances in the middle part of the toad's sartorius is shown in the diagrams (Fig. 124), which represent the relation of FIG. 124. strength to duration of the currents necessary to evoke a contrac- tion. In this curve a represents the muscle material, y -the nerve material, and /3 the curve of the intermediary substance. Similar conditions are found in the visceral neuro- muscular system. Here the nerve fibres leaving the central nervous system do not pass direct to the muscle fibres, but end in arborisations round ganglion - cells, which are collected to form the ganglia of the sympathetic chain or ganglia situated more peripherally and nearer the reacting tissue. Relays of fibres, for the most part non-medullated, arise from these ganglion-cells and pass to the unstriated muscles of the blood-vessels and viscera, where they end in plexuses or networks among the muscle fibres, possibly connected by short branches with the fusiform muscle fibres themselves. No structure is present at the periphery exactly analogous to the end-plate, and it is possible that, as Elliott suggests, the end- plate is really homologous with the whole of the sympathetic ganglion with its post-ganglionic fibres passing to the visceral muscles. 314 PHYSIOLOGY At any rate, the action of curare and of nicotine on these peripheral ganglia is very similar to their action on the skeletal end-plates, nicotine, however, having a relatively stronger action than curare. Injection of nicotine stimulates and then paralyses the peripheral nerve- cells of the visceral system ; curare in sufficiently large doses para- lyses them. More instructive in relation to the presence of receptor substances is the action of adrenalin. This substance, which is pro- duced by the medulla of the suprarenal glands, has a specific action on all tissues innervated by the sympathetic system. It causes almost universal constriction of the blood-vessels, dilatation of the pupil, acceleration of the heart, and inhibition of the intestinal muscles, with the exception of the ileo-colic sphincter, which it causes to contract, all of which effects can also be produced by stimulation of branches of the sympathetic nerve. On the other hand, tissues which are not innervated from the sympathetic, such as the blood-vessels of the lungs, are unaffected by the drug. This fact, together with the opposite effects of adrenalin on different unstriated muscles, shows that its action cannot be a direct one on muscle-fibre. It presents a marked contrast, for instance, to barium salts, which produce a con- traction of every unstriated muscle-fibre in the body. On the other hand, we cannot ascribe this action to a stimulation of the sympathetic nerve-endings, since adrenalin is equally effective if applied after the whole of these nerve-endings have been made to degenerate by section of the post-ganglionic sympathetic nerve- trunks. Its action therefore must lie at the junction between nerve and muscle, and must be on some intermediate or receptor substance developed at the myoneural junction, and having for its function the transference of the excitatory process from the nerve fibre to the contractile substance of the muscle fibre. Similar receptor substances may act as intermediaries in every case of propagation of an impulse across a synapse of whatever description, and may by their properties determine the peculiar qualities of the synapse. We may compare them to the fulminating cap which in a shell is used to transfer the process of combustion from the slow-match to the bursting charge. Their existence is of especial importance when we endeavour to investigate the mode of action of drugs. It is probable that they will be found to play a great part in determining the differential action of drugs on various tissues in the bodv. SECTION VIII POLARISATION PHENOMENA IN NERVE ELECTROTONIC CURRENT. If a constant current be passed through a nerve fibre through the electrodes x and y— x being the anode and y the cathode — and the extrapolar portions of the nerve ab, cd be connected with galvanometers, the needles of both are deflected, and the direction of the deflection shows the existence of a current in the extrapolar portions of the nerve a to &/ and from c to d. ^ a 6 1 l-J, c d — - x y ~=-] t 1 . s~?\ , t I L ^7\ , £' G2 FIG. 125. Diagram showing electrotonic currents, p, polarising circuit ; G1, G2, galvanometers. The galvanometers will indicate, before the passage of the polarising current, the ordinary demarcation current of the nerve resulting from the cross-section at the upper end. This current flows, in the outer circuit, from equator to cut end, and therefore in the nerve-fibre from a to b, and from d to c. The effect of closing the polarising current will be to increase the current of rest between a and b, and to diminish that between c and d. We thus see that the passage of a current through a part of a nerve gives rise to a current flowing through a considerable portion of the nerve fibre on each side of the polarising current and in the same direction. This current is called the electrotonic current. It must not be confounded with the current of action, which originates at one of the poles, only at make or break of the current, and is trans- mitted thence in the form of a wave with a measurable velocity (in the frog) of about 30 metres per second. The electrotonic current is 315 316 PHYSIOLOGY developed instantaneously, and lasts the whole time that the current is flowing through the nerve. Its production is dependent on the occurrence of polarisation between the sheath and the conducting part of the nerve fibre and may be exactly reproduced on a model consisting of a core of zinc or platinum wire in a casing of cotton soaked with ordinary salt solution. Although thus physical in origin, its produc- tion is dependent on the vitality of the nerve, and so is not to be con- founded with the simple spread of current. M Glass tube containing 0-6%NaC|. Pt.wire FIG. 126. Apparatus for imitating the polarisation phenomena in medul- lated nerve (' Kernleiter ' model). The polarisation phenomena resulting from the passage of a constant current through a medullated nerve can be studied on a model made up of a glass tube filled with normal salt solution, con- taining a platinum or zinc wire stretched through it (Fig. 126). On leading a current through a and b, and connecting c and d with a galvanometer, a current will be observed in the extrapolar portion of the model in the same direction as in the intrapolar. That this spread of current is due to polarisation is shown by the fact that, if the model FIG. 127. Diagram to show polarisation at the surface between conducting core and electrolyte sheath in a ' Kernleiter.' be made of zinc wire immersed in saturated zinc sulphate solution, so that no polarisation can occur, the spread of current to the extrapolar area is also wanting. If we examine the phenomena taking place at the anode, we see that a current passes here through an electrolyte to the conducting core. Every passage of a current through an electrolyte must be accompanied by dissociation, the current being carried by the ions. We get therefore a movement of negative ions up into the electrode, and a deposition of electropositive ions on the core (Fig. 127, a). In the same way at the cathode there will be a deposit of electronegative ions on the core (Fig. 127, d), so we may say that POLARISATION PHENOMENA IN NERVE 317 the core is positively polarised at the anode and negatively polarised at the cathode. This polarisation, while opposing the primary current, will set up currents in the surrounding electrolytic sheath, as shown by the arrows in Fig. 128, the current passing from a to 6 and from b to c in the electrolyte, returning towards a in the core. Hence if we lead off from the sheath in the neighbourhood of the anode from a and c, a current will pass in the galvanometer from a to c, that is, along the core in the same direction as the intrapolar current. The same factors FIG. 128. Diagram to show polarisation currents in a ' Kernleiter,'1 or in a medullated nerve. will cause an extrapolar current in the cathodic area, the catelectro- tonic current. This polarisation will not disappear at once on breaking the polarising current. The nerve or nerve-model will still be positively polarised at the anode and negatively polarised at the cathode. On connecting therefore these two points with the galvanometer, we shall get a current in the opposite direction to the previous polarising current, viz. from anode to cathode (Fig. 129). This is the so-called Polarising vsV_^^/ Neqative polarisation. FIG. 129. Diagram to show direction of the negative polarisation current. negative polarisation of nerve. Similarly in the extrapolar regions of the nerve we shall have currents in the same direction as the previous polarising current, as shown by the arrows. So far then the nerve behaves exactly like the mechanical model. If, however, we pass a very strong current through a nerve, and then quickly switch the nerve on to a galvanometer, we find a momentary current through the galvano- meter in the same direction as the previous polarising current. This is known as positive polarisation of nerve. It is absolutely dependent on the living condition of the nerve, and is in fact an excitatory pheno- menon due to the strong excitation which occurs at break of the 318 PHYSIOLOGY current at the anode. Thus in the diagram (Fig. 130) a strong current is passing through the nerve from a to k. When this current is broken, excitation occurs, as we have already learnt, at the anode, and this excitatory state may, if the previous currents were strong, last two or three seconds. An excited tissue is, however, always negative towards adjacent unexcited tissue, and therefore if we connect a to k, there Polarising k ^^~{