A chemical sign of life

Front matter

A

SHIRO TASHIRO

THE Roswell P. Flower Library THE GIFT OF

Preface

The present work is an attempt to apply facts discovered during the study of the physiology of nerves to living processes in general. ‘That mechanism characteristic of all living matter which enables it to respond to the external world is best developed in the nervous system. This mechanism may be called the most characteristic thing in life. The chemical accompaniment, or basis, of this mechanism, discovered by the author in nerve fibers, he has hoped to show exists also in all forms of living matter, both of plants and of animals. It gives a chemical method of distinguishing living from dead tissue, and of measuring the quantity of life.

This book, therefore, contains somewhat.in detail all the essential facts which he with his students has discovered from studies of the chemical changes in nerves accompanying functional change. In the presentation of this work, however, many important references and discussions have been omitted in order that the reader may not lose the main trend of the argument. The facts themselves are nevertheless given in the form of accurate numerical data so that the book may be useful also to the specialist whose interest lies more directly in the general physiology of the nervous system.

In an appendix the detailed method for the use of the biometer is added in response to frequent requests of many friends and students who wish to use it for various biological and chemical researches.

The author is deeply indebted to Professor A. P. Mathews for his criticism, scientific and literary, during the preparation of the work. Cee one

January, 1917 vii

CONTENTS CHAPTER PacEe I. IRRITABILITY ASA SIGNOFLIFE. . .. 2 4 The Characteristics of Living Matter. Irritability.

Functional Changes as a Sign of Irritability. Functional Changes in the Nerve Fiber.

II. CHEemicaL SIGNS OF IRRITABILITY IN THE NERVE FIBER... * * #@ » TO Signs of Metabolic Activities in 4 Tissue. Indirect Evidence for Presence of Metabolic Activity in the Nerve Fiber. Direct Evidence. Experimental Methods with Non- Medullated Nerve Fiber. Medullated Nerve Fiber. Is Carbon Dioxide Produced by Living Processes? Discussion.

TIT. CHemicaL SIGNS OF IRRITABILITY IN THE NERVE: FiIneR—Continued » a « 34

Increased Metabolism on Stimulation. Experimental. Electrical Stimulation. Other Stimulations. Discussion.

IV. EXcITATION AND CONDUCTION : a See

Excitability and Metabolism. Degree of Excitability € Compared with Metabolism. Effects of Narcotics. Lower Concentration. Higher Concentration. Direction of the Nerve Impulse and Metabolic Gradient. Velocity of the Nerve Impulse and Carbon Dioxide Production.

V. CHemicaL SIGNSor LIFE. . . . wae 189 Resting Metabolism in Seeds. Increased Metabolism in peas on Injury. Other Tissues. A Chemical Sign of Life.

VI. Conciustons eK OK d Ce Siow 2. NOs Summary. Other Criteria of Life. Functions of Resting Metabolism. Metabolism and Irritability. Chemical Conditions in the Living Processes. Quantity of Life.

APPENDIX. THE BIoMETER: Howto UseIT . . . 109 ix

Chapter I Irritability As A Sign Of Life

In the pages which follow we shall consider par- ‘ticularly the question of the chemical processes which take place in nerves when nerve impulses pass over them. There is scarcely a subject in the world more interesting than this one, for the question of what is the nature of that disturbance in nerve tissue which shows itself in our thoughts has attracted men from the earliest days. We must first find out the changes of a material kind—if there are any such changes— which occur in the brain when we think before we can form any probable idea of the relation of these changes to the psychical changes which accompany them. Obviously, we must first try to solve the simplest problem in this field and discover what are the changes of a chemical or physical kind when a nerve impulse flashes over a nerve before we can form any conception of the relation of the material to the psychic world. The following pages do not contain, of course, the solution of this problem of such absorbing interest, but they do present the first accurate information we have had of the chemical changes which accompany the nerve impulse; they have in them, therefore, the foundation upon which a solid structure of fact can be based.

The observations which we have to present are not confined to nerves, however, for psychic phenomena are

I not confined, we believe, to animals which have welldeveloped nerves. The course of evolution from the simplest to the most complex shows us very clearly that the complex psychic life of man and the higher animals did not suddenly spring fully formed into existence. In every child, in fact, it can be seen to appear very slowly and gradually and to increase as the child develops. We cannot say at what point psychic life begins, for the simplest organisms show some signs of it. Indeed, as living originates from lifeless, we are led to conclude that the simplest rudiments of psychic life must be found also in the lifeless. And perhaps the universe as a whole, inert as it appears to us to be, may have a psychic life of its own. So it is not necessary to confine our studies to nerves, for we find the same phenomena which nerves show, phenomena corresponding to those of nerve impulses, even in plants, and indeed in the simplest kinds of plants. The differences between animals and plants are superficial differences. Plants, in general, are sessile; they cannot move freely from place to place as animals do; and they have a green ' pigment in them—chlorophyll—while most animals have a red pigment in their blood. This green pigment enables plants to make their food from simpler substances than can be done by animals. But these differences are superficial, and fundamentally plants and animals are alike. We must suppose, therefore, that even so humble a living form as a small plant seed has a psychic life of its own. Impulses pass through it like nerve impulses; it may be anesthetized as in the case of man; it sleeps as does man; and, indeed, many of the fundamental properties it shows resemble those which

IRRITABILITY AS A SIGN OF LIFE 3 we possess. Thus a part of the study which follows has been made upon such simple things as seeds and garden peas. It is surprising how closely the results obtained with these parallel those obtained from nerves.

The really important and peculiar property of living things is the psychic life they show. And if we actually had an accurate means of testing the degree or amount of life, it would be some kind of a reagent or instrument for testing “‘psychism,” as we may call it. But unfortunately we cannot at present find any means of testing this property. We do not know what its physical basis is, and, until we discover that, we cannot make a psychometer which we can apply to all kinds of living and non-living things, and thus measure the arnount of psychism, and hence of life, which they possess. In the absence of any such psychometer we have to do the best we can, and take as a measure of this property those physical and chemical changes which experience or experiment demonstrates to us always accompany the psychic change. The situation is very much as it was in the realm of electricity before the galvanometer was invented; an idea of the quantity of electricity produced by a battery could be obtained only indirectly by measuring the amount of chemical change which the current produced, since Faraday found that that amount was always a measure of the amount of electricity.

There are material changes which occur in living things as long as they are alive and show psychic life of any kind. The changes which we may rely upon to measure the amount of life, and thus indirectly the amount of psychism, are partly visible changes, but in part they are invisible and have to be detected by special apparatus. The visible changes consist in some reaction of the organism when its surroundings change. If it moves when it is touched, the degree of movement not being related to the impulse given it; if it breathes; if it changes its rate of growth under changing conditions, we say that it is alive. It is irritable, and it has the property of irritability. Another thing noticed in a living thing is that the impulse which arouses it to action may cause reaction in a part of the organism distant from the point of stimulation. In other words, the change, whatever its nature, set up in the organism by the stimulus is propagated to a distance from the point of irritation. We can see the results of this propagation. All things which show this conduction and response we say are living things. These are physical processes which apparently always accompany the psychic process. But sometimes these changes, although they occur, produce no visible result; consequently we must have methods which will detect conduction and irritability, even though there are no visible signs of them. One cannot see, for example, that anything has happened to a seed when it is pricked by a pin; it does not say “ouch!” loud enough for us to hear it, or in a language we understand; but nevertheless it jumps when it is pricked as if it did say “ouch!” as we can show by appropriate methods.

There are two signs, or tests, which all living things show and which are an index of life. One of these is an electrical disturbance. This was discovered a very long time—a hundred years—ago, and its discovery was the basis of the development of knowledge of electricity. The

IRRITABILITY AS A SIGN OF LIFE 5 other is a chemical sign, which has just been discovered and which will be discussed in this book. The electrical sign of life was discovered by Galvani when he found that animal tissues are a source of electricity. He discovered animal electricity. It is now certain that whenever the response to a stimulus takes place in animals or plants—the response which is the sign of life—an electrical change accompanies it. By placing a galvanometer on the animal or plant we can study this electrical response. Life. and electricity are thus shown to be related. Electricity and psychism have something in common, although just what the connection is cannot at present be said. The English physiologist Waller has recently introduced as a measure of life a particular kind of electrical response which he has discovered and which he calls the “blaze’’ current, because it is as if the electrical display suddenly blazed up when the living matter was disturbed; this he calls an electrical sign of life. By it he can tell whether a dry seed is alive or not without putting it in the ground and letting it sprout. It is very hard to know whether this electrical disturbance which living things show is due to physical or to chemical changes in their substances.

It is therefore a matter of very great interest that I have recently found that there is always and everywhere an accompanying chemical change of a particular kind which is as sure a sign of life and as invariable an accompaniment of the vital reaction as the electrical change. This chemical sign is the sudden outburst of carbon dioxide which all living things show—plants as well as animals, dry seeds as well as the nerve tissues of the highest mammals—when they are stimulated in any way. The instrument which I have made to detect this carbon dioxide I have called a “‘biometer”’ because, as will be appreciated from this short discussion, it is an apparatus for measuring or detecting the amount of life possessed by different things. I shall show in the following pages that the increment of carbon dioxide produced by living things when they are irritated, or stimulated in any way, is a sure measure of the amount of life they have; and we may hope that it is to be an indirect measure of the amount of psychism they possess, although of course we cannot be sure of this as yet. It will be noticed that it is not the absolute amount of carbon dioxide which is the measure of life, but the increase above the usual production which occurs when a definite amount of stimulus is applied to the living thing, which is the real measure of life. Anesthetized or sick things do not show the normal increase; those abounding in life show a remarkable increase.

The first results to be presented will be the proof that carbon dioxide production is the sign of life of a nerve fiber. And it will be well before going into this to say a few words about the scientific opinion concerning the nature of the nerve impulse generally prevailing before the work recorded here was done.

The main function of a nerve fiber is to transmit a state of excitation from one place to another. It serves for the conduction of the nerve impulse, which it transmits in the most efficient manner. The nerve is also excitable at all points, since it can be stimulated by a variety of methods at any point along the fiber. When physiologists investigated what takes place in nerve fibers during the passage of nerve impulses, many

IRRITABILITY AS A SIGN OF LIFE 7 peculiar results were brought out. In the first place, if there are no other organs attached to the nerve it is impossible to determine by casual observation whether or not the nerve has been stimulated, for there is positively no visible physical sign of the vitality init. Not even with a microscope can any structural change in the tissue be seen. There is, also, no heat change detected with a method which is sensitive to a millionth of a degree Centigrade. There was, before this work was published, no apparent production of carbon dioxide, or any other chemical change in the tissue. These facts seemed to indicate that the functional activity of nerve fibers was in no way associated with any chemical change. This failure of a nerve to show any chemical or structural changes similar to those of muscles had a decisive influence in the formation of ideas concerning, not only the nature of the nerve impulse, but also the nature of irritability in general. For nerve fibers not only show the highest type of irritability of protoplasm, but they also possess, as stated before, the power of transmitting the state of excitation in the most perfect manner. And all attempts to explain the nature of irritability in general must necessarily account for the ‘peculiarities of the nerve fiber where we find that property in its highest development. If irritability, excitation, and conduction do not involve chemical changes in nerves, it may be concluded that neither do they in any other tissues. Thus, on account of the absence of evidence of any chemical changes accompanying irritability in nerves, we have gradually drifted away from the notion that the fundamental condition for protoplasmic activity is chemical.

When it was found that an electrical change occurred in a nerve when it conducted an impulse, the problem was considered to be settled. The nerve impulse was supposed to be electrical in nature. This idea was soon questioned, however, when the speed of the conduction of a nerve impulse was found to be so slow in comparison with that of an electrical current. The speediest nerves, such as those of human beings, conduct impulses only at the rate of a hundred meters per second, whereas electricity travels in a wire at a speed of thousands of kilometers per second. One thing seemed to be certain— that the nerve impulse can pass through a fiber without consuming any material. It was found that some nerves could not be fatigued even on prolonged stimulation. This fact supported the idea that certain quickly reversible physical conditions must exist in the nerve, and that the changes in these conditions, rather than chemical changes, must determine the phenomena of irritability and conductivity. Ultimately physiologists settled down to the view that the physical and fundamental changes concerned in irritability were either a change of colloidal state, of surface tension, or in the permeability of the nerve to salt, or changes in the distribution of electrically charged particles in the nerve.

Although such physical changes as these in nerves have never been demonstrated experimentally, biologists generally have tried to explain the nature of a nerve impulse and the phenomena of excitation purely on the basis of these hypothetical physical changes; and they have neglected the chemical changes. They have also attributed many other important physiological functions, such as secretion and contractility, to these

IRRITABILITY AS A SIGN OF LIFE 9 purely physical conditions, and this in spite of the fact that this point of view is obviously incomplete, if not fundamentally erroneous, because the source of all energy of living things is chemical. The chief reason, as has been stated, for the adoption of this hypothesis was the fact that the most irritable tissue—nervous tissue—had shown no sign of chemical changes when it functioned, and also the fact that some other living tissues, such as seeds, seemed to maintain their vitality without any chemical change. The erroneous character of this view will be apparent from what follows, where it is shown that chemical changes are, indeed, an invariable accompaniment of nervous activity, and of all life.

Chapter II Chemical Signs Of Irritability In The Nerve Fiber

There are various chemical processes which occur in all forms of living matter and which we might examine in order to see whether they are associated with the property of irritability, but we naturally seek to make use of that one which is the easiest to detect. Among these chemical processes there are, in the first place, the processes concerned in growth. All living matter has the power of building up complex proteins, fats, and carbohydrates as long as it is vigorously alive. But it is clear that this process would be very hard to measure quantitatively without killing the living matter and determining how much substance it has produced. And there are also other objections to using growth as a measure of vitality. Another chemical process found in all, or nearly all, forms of matter is respiration. By ' respiration we mean the gaseous exchange of living matter with its environment: the taking on of oxygen and the production of carbon dioxide. This is a very much more promising line of experiment to follow in measuring life and metabolism, for, in the first place, it is universal, as I shall presently show, and, in the second place, the oxygen may be measured, or the carbon dioxide given off may be determined, without injuring the living matter. It was for this reason that the carbon dioxide was selected for study as probably

Io

CHEMICAL SIGNS OF IRRITABILITY II being the easiest of quantitative determination and undoubtedly correlated with the most fundamental vital processes.

The idea that respiration is one of the most fundamental of vital phenomena is by no means a novel view. Even in the earliest times breathing was supposed to be the process most intimately connected with life. When a man stopped breathing he died. As early as the second century Galen had the notion that there must be a pneumatic spirit in the air which kept up life, and he predicted that some day it would be discovered. It was after the lapse of fifteen hundred years that this prediction was verified or fulfilled when Mayow, an English physician, discovered that there was a gas, or spirit, in the air which was essential to life and combustion. Later, oxygen was discovered by Priestly, and it was Lavoisier who first showed that this oxygen after entering the lungs came out again as carbon dioxide; and he proved that animal heat was due to the combustion of the materials of the body by the oxygen to form water and carbon dioxide, and that the sole source of energy of living things was this combustive change. In selecting respiration as the chemical test of life we are, therefore, selecting that most fundamental reaction by virtue of which living things get their energy. It is clear that it is this reaction, rather than any other chemical reaction, which touches most closely the phenomena of irritability; for, to move or to think, we must have energy. It is much better to take this reaction, rather than those chemical changes which are related to growth or the repair of waste, as a criterion of living, for the very essence of a living thing is that by chemical transformations it sets free energy and moves itself.

It is much better, too, to take the carbon dioxide produced, rather than the oxygen consumed, as the measure of the metabolism associated with irritability, for the reason that sometimes organisms get their oxygen from sources other than the air, whereas their carbon dioxide production is always something positive and universal.

Indirect evidence of the presence of metabolic activity in the nerve fiber —The search for some kind of metabolism, such as the production of carbon dioxide, in nerves had been made by many physiologists on many occasions, but it was impossible for them to discover this substance because their methods were not sufficiently delicate. No carbon dioxide could be found, and for this and other reasons the conclusion was incorrectly drawn that there was none produced, or that, if it were produced, it had no connection with the vital functions of thenerve. Most physiologists were accordingly of the opinion that the conduction of the nerve impulse was a physical process and involved no transformation of energy and no consumption of material. There was one exception to this rule. Professor A. D. Waller, the eminent English physiologist, maintained that, because of their electrical behavior, nerves certainly produced carbon dioxide. In 1896 he showed that carbon dioxide when applied to a nerve produced a very characteristic change in the electrical response which a nerve exhibits when it is irritated. It will be remembered that when a nerve or, in fact, any kind of protoplasm is irritated in any way, if one applies two electrodes to the living tissue in such a way that one electrode is on a part which is in activity and the other on a part which is less active, it will be found that there is a current which flows in the tissue from the more active to the less active part, from the more to the less excited, and outside in the galvanometer from the less to the more excited part. This electrical current was discovered by Galvani and is called the current of action, or the action current. Generally, when an impulse sweeps along a nerve to which two electrodes are applied, first one electrode and then the other becomes negative, so that the current is diphasic, running first in one direction and then in the other. Now, Waller observed that when a nerve was exposed to carbon dioxide this diphasic current showed a characteristic change, the negative phase being first increased by small amounts of carbon dioxide and then diminished. He then discovered that just the same kind of a change occurred in the electrical response if he stimulated a nerve repeatedly at very short intervals of time. He concluded from this that on stimulation of the nerve carbon dioxide was produced, and that this caused the characteristic alteration of the electrical response which occurred in the tetanized or repeatedly stimulated nerve. This conclusion was not generally accepted by physiologists for the reason that it was possible that the same change in the electrical response might be produced in other ways than by carbon dioxide, and while the experiments were regarded as circumstantial evidence of value, showing that a chemical change accompanied the nerve impulse, they were not regarded as conclusive.

Waller supported this conclusion by another discovery, namely, that when he stimulated the nerve at regular intervals, not too long or too short, by a strong induction shock, he obtained a series of electrical responses, each of which is the greatest the nerve can give at the time, but each of which is a little greater than its predecessor. Such a series of increasing responses is known as a staircase, the negative phase increasing steadily while the positive phase decreases. This Waller explained by supposing that small amounts of carbon dioxide were formed by each nerve activity, and that this augmented the negative response and diminished the positive response, just as does carbon dioxide applied to the outside of the fiber. He considered that our failure to find the gas was due to the inadequacy of the chemical methods then in existence. That this criticism of Waller’s was a just one and that there may be carbon dioxide produced by nerves, but too small in amount to be measured by the ordinary chemical method, is shown by the following calculation: A frog (Rana temporaria) gives off 0.355 g. of carbon dioxide per kilogram per hour at 19° to 20° C. A small piece of the nerve fiber of the same animal, say 1 cm., or threeeighths of an inch, in length, will weigh, probably, not more than 10 mg. Now, if this mass of the nerve fiber respires at the same rate as the whole animal, it will not give off more than about 0.000,000,7 g. of carbon dioxide during ten minutes. This calculation at once suggested that the failure to detect the evolution of carbon dioxide in nerves was very probably due to the limitation of the methods for the estimation of the carbon dioxide, and that it was not at all conclusive evidence that carbon dioxide was not produced. It was evidently necessary to devise methods for the detection of very minute quantities of carbon dioxide.

Tn order to study the whole of the respiratory metabolism of a tissue, we should at least determine the oxygen consumption as well as the carbon dioxide production, and also generally the heat production. Inasmuch as the present problem, however, is concerned only with presenting direct evidence for the existence of metabolic activity in nerve fibers, we shall attempt to measure the carbon dioxide production alone; for while the lack of consumption of atmospheric oxygen may not necessarily indicate the absence of chemical changes, the production of carbon dioxide will surely prove the presence of metabolism, provided, of course, that we can prove that such carbon dioxide is formed by physiological processes. Furthermore, as carbon dioxide is the only universal expression of the respiratory activity in almost all anaérobic and aérobic plant and animal tissues in normal condition, metabolic activity is probably better represented by carbon dioxide production than by oxygen consumption, although we must restate here, of course, that the study of carbon dioxide alone will never reveal completely the nature of the metabolic activity.

Method.—The method which was finally devised to detect and measure quantitatively the very minute amounts of carbon dioxide which it might be expected would be formed consisted essentially in determining the amount of carbon dioxide which was just sufficient to produce a deposit of barium carbonate in a film of half-saturated barium hydroxide solution. Barium carbonate is- almost entirely insoluble in such a barium hydroxide solution, and a very small amount of precipitate can be detected with the aid of a small lens. The method is described in detail in the Appendix. The special apparatus, the biometer, as I have named it, which was constructed for the investigation, is shown in Fig. 1, and its use is detailed in the Appendix. It will detect one ten-millionth of a gram of carbon dioxide and estimate it with accuracy.

As is shown in the figure, the biometer has two respiratory chambers each provided with a small tube, at the top of which the hemispherical drop of barium hydroxide can be formed. Exceedingly minute amounts of carbon dioxide produced in the chamber by the small piece of nerve will be precipitated as barium carbonate on the surface film of these hemispherical drops and may beseen withalens. As the apparatus has two chambers, not only can we detect very small amounts of carbon dioxide which the nerve may produce, but we can also compare the output of carbon dioxide of different tissues under the same conditions, by placing one tissue in one respiratory chamber and another in the other.

To discover whether nerve fibers, as distinct from nerve cells, respire, particular care was taken to select at first those nerves which are known to be free from such cells and, as far as possible, free also from connective tissue. It was necessary to do this because the work of several investigators seemed to indicate that tissue oxidation was in some way dependent on the cell nucleus. Certain biologists even went so far as to believe that a nerve fiber ought not to respire at all, since it contained no nucleus. The fact that the blood supply to the brain, where most of the nerve cells are located, is so copious, whereas the blood supply to the nerve fibers is

Fic. 1.—The Biometer. One-fourth actual size so scanty, was partly responsible for this conclusion.” In order to test the correctness of such an assumption, we have studied quantitatively the output of carbon dioxide from various lengths of nerves which are known to be free from nerve cells and which have almost no connective tissue in them. For this purpose the claw nerve from the spider crab was selected.

Nerve fibers are of two kinds, called respectively medullated and non-medullated nerves. The essential conducting parts of these are alike, but the medullated fibers have lying about the conducting core of the fiber a white, glistening, fatty matter called the medulla, or myelin sheath. Most of the nerves going to voluntary muscle in the higher vertebrates are medullated; but the nerves to the viscera are often non-medullated and the nerves of the invertebrates are usually non-medullated. This medullary sheath is evidently something which is found in those nerves which it is important should conduct very quickly and which should not be fatigued by conduction, and it is clear that the medullation is an improvement which has not yet been universally introduced. The function of this sheath is probably nutritive. But in any case it is important, if we wish to avoid any complication which it may introduce into the physiology of the nerve, to examine both medullated and non-medullated nerves. And that we have done.