If it is true that all primary effects of stimulation consist either in an excitation or depression of the metabolism, and that all other effects of stimulation secondarily follow this primary alteration of the metabolism of rest, then every thorough analysis of the mechanics of reaction must have its beginning in the investigation of these primary processes. I desire to adopt this method here and will analyze somewhat further the primary process of excitation and its immediate and remote sequences. This will be followed later by the analysis of the process of primary depression and its results.

The investigation of the more obscure processes in the living substance places us in a difficult position, for their details cannot be observed by the unaided senses. That which we can perceive is merely the grosser vital action, consisting of a complex combination of the individual processes, the total result of a multitude of different components. For this reason the conception of excitation can only be established by observations based upon the combined vital actions, which are produced by the effect of stimulation upon the complex system. In the beginning, the process of excitation was studied exclusively on the muscle and nervous system. A physical factor served as indicator, such as muscle contraction or production of electricity. These showed, besides the direct and primary effect of stimulation, the secondary process of conductivity. Even graphic registration is merely an expression of the phenomena composed of a great mass of individual elements. The visible course of the phenomena, as shown, for instance, by the latent period by the ascent and descent of the curve of contraction, represents as it were a reflected picture of the actual excitation processes similar to an object seen in a distorting mirror; the first and the last parts of the process are not even perceptible. Later, when organ physiology was extended into a cell physiology the processes of excitation were studied in numerous simple organisms, such as the plant cell, the rhizopoda, the infusoria, etc. Later, in this way, by the use of comparative methods many essential facts were discovered. However, even the single cell, in spite of its minuteness, is, compared with the size of a molecule, a gigantic system, and it would be a grave error if we should consider this system even in its simplest aspect as homogeneous. In order, therefore, to analyze the vital activities in the cell, cell physiology must endeavor to penetrate into molecular conditions. For this purpose the indicators employed must be essentially of a chemical nature, capable of magnifying the processes of molecular dimension to such a degree that we are enabled to base conclusions upon these not otherwise directly perceptible phenomena. To obtain a sufficient magnification we must necessarily place somewhat larger quantities of living substance under observation and apply a stimulus of such frequency or length of duration that the chemical alterations as a result of excitation are so increased as to be plainly perceptible with the aid of our chemical indicators. Unfortunately, we do not possess specific chemical indicators for every individual molecular constituent process of the cell and so cannot dispose with the help of indicators of the combined happenings in a greater quantity of living substance. It remains for us to obtain data concerning the cycle of excitation processes in the living substances by the aid of the combined employment of the most varied kinds of physical as well as chemical indicators. If we use the most varied types of living substance of widely differing properties, showing us the greatest variety of vital manifestations, we may hope by the use of comparative physiological methods, even though with difficulty, to separate more and more the essential details of the general processes of excitation. At present we are still at the very beginning of this task and vast fields of unexplored regions are yet before us. But it is the unknown which has a particular fascination, especially if we succeed from time to time in making new advances.

If we suppose a living system in a state of metabolism of rest influenced by an instantaneously excitating stimulus, the entire course of excitation extends from the first alteration produced by the stimulation until the complete restitution of the metabolic equilibrium, and we will, therefore, differentiate individually the successive stages of this whole process.

The very beginning of the chain of alterations produced by the excitating stimulus cannot be studied by any indicator. The changes must first reach a certain dimension by conduction from the point of stimulation before they influence even the most delicate indicators. The application of the stimulus is, therefore, followed at first by a measurable “latent period,” in which the living substance remains apparently at rest. This latent period has been particularly studied in muscle. After its discovery by Helmholtz55 it was made the object of innumerable investigations and met with an interest which can only be explained by the exactness of the methods employed. Among others Tigerstedt56 has made the most thorough study of the influence of various factors on the duration of the latent period. These experiments have established the fact that the duration of the latent period varies according to the intensity of the stimulus, temperature, loading or fatigue. This is apparent when it is understood that the amount of the alterations produced by the stimulus must ascend from the value zero to a certain height before the changes are perceptible, and that under various conditions this amount is, on the one hand, attained in different lengths of time and, on the other, must reach a varying amount before it is perceptible by means of the indicator.

The facts concerning the whole latent period and its dependence on various factors would be incomprehensible if it were assumed that no alterations whatever take place during the latent period although the stimulus is already operative. In reality, the alterations following a stimulus occur with imperceptible rapidity in the form of a molecular interchange, and the latent period is simply an expression of the fact that the primary alterations, being limited in nature, are not registered by our indicators.

The question first arises, In what do these first imperceptible alterations consist? Nernst57 has evolved the theory for electric stimulus, that the primary effect produced by the electric current is an alteration in the ion concentration on the surface of the living substance. In fact, we know that the surfaces of all protoplasm possess the property of semi-permeable membranes and that changes in the concentration of ions invariably occur when an electric current flows through two electrolytes separated by a semi-permeable membrane, in which the anions and cations have a different rapidity of movement. It is apparent, therefore, that such an alteration in the ion concentration must be followed by further chemical processes in the living substance. According to the theory of Nernst the first impetus for all further alterations, which the electrical stimulus brings about in the metabolism of rest, is the alteration in the concentration of the ions on both sides of the semi-permeable membrane, which represents the surface of the protoplasm. In view of the present findings of physical chemistry, objections can hardly be made to this theory of Nernst’s. It is a question, however, in how far this theory, especially established for the electric stimuli, can be applied to other forms of stimuli and their action. It cannot be denied that the degree of dissociation of an electrolyte can be altered by very different factors, such as heat, light, chemical processes, etc., and in that the surfaces of the protoplasm, acting as semi-permeable membranes, bring about a selective action on the passage of the ions, there arises the opportunity for the development of difference of electrical potential on both sides, and for further chemical alterations in the protoplasm. These observations, however, require further experimental investigations in many fields, before we are justified in extending the Nernst theory of the manner of action of the electric stimuli to a general explanation of the primary alterations produced by all stimuli in the living substance. For the present we must confine our observations to those alterations which are known to be responses to an excitating stimulus; these are the chemical alterations in the metabolism of rest in the living substance.

If it is asked, which members of the entire metabolic chain are increased primarily by the stimulating excitation of a vital system, we should not be able to answer this question generally for all living systems. To begin with, it appears highly probable that the various forms of vital substances in this respect act quite differently. It is to be regretted that, up to the present, this question has not been treated from a comparative standpoint. This inquiry should be extended to the greatest possible number of organisms. Still there is enough material at hand, obtained from the muscles, glands, ganglion cells, nerve fibers and plants, to show that the complexity is by no means so great as one might at first assume.

In considering the two stages of metabolism, assimilation and dissimilation, in their entirety, it appears as a very remarkable fact, that nearly all stimuli produce primarily a dissimilative excitation. We are only acquainted with a primary assimilative excitation, that is, an augmentation of the building up processes, in short, the formation of living substance, occurring as a primary result of stimulation, following increased introduction of foodstuffs extending over a prolonged length of time. With this exception it cannot be proved that any other stimuli, either especially those operative in the activity of the animal organism or any of the physiological nerve impulses which regulate the actions of the different organs and tissues, bring about primarily an assimilative excitation, which leads to an increase of new formation of living substance. The much-discussed teaching of the existence of the trophic nerves has not given us a single case in which there was positive proof that a nerve impulse brought about a primarily assimilative excitation. I have endeavored for nearly fifteen years to discover such a case. My efforts have been, however, without avail. In the most recent critical review by Jensen58 on the subject of the trophic nerves, the same conclusion is reached although certain facts, as, for instance, the excitation of assimilative processes in the green plant cell, produced by light, seems at the first glance to clearly demonstrate a primary excitation of the building up processes resulting from a stimulation. Nevertheless closer observation invariably shows that these conditions are much more complicated and that primarily assimilative excitating reaction of the stimulus cannot be conclusively shown. There remains, therefore, as a primary assimilative excitating stimulus only the increased introduction of nutrition in a living organism. This excitating effect on the assimilative portion of metabolism is, as we shall see later, a simple manifestation of the law of mass action.

As a result manifold effects of excitating stimulation, which seemed possible at a first glance, are already considerably restricted. The great mass of excitating stimuli produce an acceleration of the dissimilative processes of the metabolic chain. But here our former observations have already shown that certain constituent processes are especially responsive and very readily increase as a result of the most varied adequate and inadequate stimuli. These are the “functional” members of metabolism. These members are particularly labile, so that they are always affected by every influence to which the system is subjected in the form of a stimulus. The functional portion of metabolism of the muscle, which is particularly labile and is always primarily affected by stimulation, consists as demonstrated in increase of formation of carbon dioxide and water, and in the disintegration of the nitrogen-free groups. The innumerable observations on metabolism during the stage of the activity of the muscle, as those of Hermann, v. Frey, Fletcher, Johannson, Thunberg, and many others on the individual muscle, and those by Voit, Fick and Wislicenus, Pflüger, Rubner, Zuntz, Lehmann and Hagemann, Bernstein and Löwy and others on the muscle of the entire organisms, have sufficiently proved this fact. However, we should not apply in detail the conditions existing in the muscle to all living substance. Comparative methods show us, rather, that the functional portion of metabolism is very differently involved in various forms of living substance. The formation of carbon dioxide and water is constant in nearly all forms of living substance. We must, however, exclude certain micro-organisms, which have adapted themselves to unusual vital conditions. Further, there appear in some forms manifold special constituent processes consisting in a disintegration of living substance which are in part converted into very complex combinations. In the gland cells this type is represented in an especially high degree. Here the functional disintegration leads to excretion of proteins, glycoproteins, nucleoproteins, cholic acid, enzymes of various kinds, all of which are complex and at the same time nitrogenous organic combinations. This fact must not be lost sight of. The origin of these special members, however, for the present is completely unknown, while on the other hand, it is self-evident that the general and constant constituents of the process of excitation must claim a first place in our interest. It is just at this point, therefore, that we must endeavor to penetrate somewhat more deeply into the mechanism of the excitation process and analyze in greater detail the acceleration of the functional constituent parts of metabolism produced by the stimulus bringing about the formation of carbon dioxide and water.

The question arises: By what means is the particular labile state of just this constituent part of functional metabolism conditioned? The lability of the functional portion of metabolism, excitated by the stimulus, resembles the processes in the disintegration of explosive combinations. Iodide of nitrogen, for instance, in a manner similar to the living substance in the state of the metabolism of rest, constantly disintegrates even without the influence of an impact. The disintegration is suddenly enormously increased by the result of a jar. An explosion follows. In a like manner the functional metabolism of rest is explosively excitated by the stimulus, the transformation of the energy involved likewise bears a similar relation.

In both instances the transformation of energy, constant in the resting state, is by the impact of the stimulus suddenly increased. The dynamic method of investigation of the excitation process with its physical indicators, forms, therefore, in many respects an excellent addition to the chemical analysis. A development, that is, exothermic formation, of energy can only occur in a chemical process when the chemical affinities which are to be combined are stronger than those which have been separated. When this process is brought about by a simple impact, the energy value of which bears no relation to that of the quantity of energy in the process itself and which occurs with explosive rapidity, then it can be simply a question of a liberation process, that is, a process by which the impact brought about a conversion of latent chemical energy into that of kinetic energy. The comparison of the functional excitation process with that of an explosion does not, therefore, consist in a merely superficial analogy, but is founded on the same dynamic principles.

When we study the chemical process which occurs in the explosive transformation of potential into kinetic energy we find two types of chemical processes. The first type includes the synthetic processes. For this, the synthesis of water from explosive gas may serve as a simple example. Here the weaker affinities in comparatively simple molecules (H + H and O + O) are separated and stronger affinities are combined in the formation of more complicated molecules (H + O + H). The second type represents the process of cleavage. As example for the latter, the explosive disintegration of nitroglycerine may be quoted. Here the atoms, held together in a complex molecule by weaker affinities, are changed by transposition of nitroglycerine. For instance, the hydrogen atoms loosely combined with carbon enter into strong combinations with oxygen and the oxygen loosely combined with the nitrogen enters into strong combination with carbon, so that water and carbon dioxide are formed and nitrogen and oxygen set free.

In the functional disintegration of living substance, the last type is realized. Living substance contains loose complex combinations, and we know that functional disintegration is accompanied by the consumption of these organic combinations. In the functional disintegration of muscle substance the nitrogen-free groups are concerned, and we must, consequently, first consider the carbohydrates. However, without further study we should not generalize from that which is true in the case of muscle. There are other forms of living substances which contain different combinations, which disintegrate as a result of the contact of a stimulus and yield carbon dioxide. A clue as to which combinations in individual cases undergo disintegration as a result of excitating stimulation, is furnished by the metabolism of rest in the particular substance. Plants and micro-organisms have been investigated more thoroughly in this connection than animals. Plant physiology has demonstrated that the material employed for the CO₂ formation and with it the production of energy is carbohydrate, but that, on the other hand, various plant organisms and protistæ also use a quantity of other substances, such as fats and protein, indeed even such comparatively simple organic combinations as alcohol, formic acid and methane. It may be accepted that in all these various instances of excitation of the functional metabolism as a result of stimulation, the specific respiratory material of the substance concerned is used in greater amount in the decomposition and likewise invariably yields carbon dioxide.

The point of most essential interest for the analysis of the excitation processes is, above all, the mechanism of the organic combustion and the associated energy production. Here we may base our observations on the disintegration of carbohydrates, which is most extensive in the animal as well as in the vegetable kingdom. We may now ask how dextrose, for instance, disintegrates in the living system into carbon dioxide, for it is this, or a sugar of similar chemical nature, which is generally concerned. Plant physiology, which here, as in many other respects, is in advance of animal physiology, has indicated two ways by which this can be accomplished in the living substance. One is oxydative, the other, anoxydative disintegration.

In the oxydative disintegration of dextrose, taking place in aërobic organisms, if sufficient quantities of oxygen are present, there occurs a splitting up of the carbohydrate molecule, as a result of the introduction of oxygen, into simpler substances and finally into carbon dioxide and water, just as the dextrose molecule, when subjected to oxydative processes, is split up into simpler molecules. In the living substance the oxydases play the important rôle of oxygen carriers. It cannot be denied, however, that up to now no carbohydrate splitting oxydases have been obtained from living substance. This, of course, does not prove its nonexistence. But this deserves consideration in connection with an assumption very widely spread among plant physiologists in regard to the aërobic disintegration of the carbohydrate molecule, which I shall touch upon presently. If we suppose that oxydases exist, which bring about primarily the oxydative disintegration of the dextrose molecule, its first point of attack must obviously be sought in the aldehyde group. Here would be situated the activator, as it were, for the whole carbon chain, from which, as by a spark, the entire series of links would be ignited.

In an anoxydative disintegration of dextrose as observed in anaërobic as well as in aërobic organisms, provided the latter have an insufficient supply of oxygen, the dextrose molecule, by enzymic action as a result of the splitting off of carbon dioxide, is converted into substances having a comparatively large carbon content. The best-known example of this anoxydative disintegration is the formation of alcohol by fermentation in which the dextrose molecule is split up by the yeast into alcohol and carbon dioxide. (C₆H₁₂O₆ = 2C₂H₅OH + 2CO₂.) Instead of the production of alcohol and CO₂ we may have other enzymic actions with the formation of other carbon-containing disintegration products, such as lactic acid, fatty acids, hydrogen, etc. Of course in such an anoxydative disintegration, which does not lead to the formation of such simple combinations as carbon dioxide and water, the quantity of energy set free is much less in amount than in complete oxydative decomposition, the energy production of the alcohol fermentation being only 11 per cent of the latter. In order to produce the same amount of energy as in the former, a much greater number of molecules is required. We find, therefore, that the anoxydative type of disintegration develops either only where the respiratory substances are present in sufficient amounts, as for instance, in the case of yeast cells, existing in nutritive solutions rich in sugar; or where the chemical and energy transformations occur only to a limited extent, as, for example, in the presence of low temperature. In this respect Pütter59 has demonstrated in the leech that at a higher temperature, the oxydative, at a lower, the anoxydative, decomposition predominates. These are important facts in that they show us the superiority of oxydative to that of the anoxydative disintegration in the cell economy. This is of particular interest when we consider those organisms in which great demands are made upon the capability of movement, above all, in homothermous forms, the metabolism of which takes place on a continuously high level. For this reason, in homothermous animals the respiration of oxygen is the almost exclusive source of energy production.

The previously mentioned facts make it clear that in one and the same form of living substance both oxydative and anoxydative decomposition processes are found, depending upon the conditions. This does not apply merely to the individual organic forms, such as the facultative anaërobic organisms, but generally to all aërobic living substance. If oxygen is withdrawn from an aërobic organism the disintegration does not cease in consequence. In place of the oxydative we have anoxydative decomposition. The various aërobic organisms are, however, adapted in very different degrees to the possibility of an anaërobic existence. While the facultative anaërobic organisms can continue to exist without oxygen, the homothermous animals become asphyxiated in a very short time in the absence of oxygen, in that they are poisoned by the products of the anoxydative decomposition, which are eliminated with much more difficulty than carbon dioxide and water. The fact, however, that disintegration also continues in an anoxydative form, if oxygen is withdrawn, has given rise to the thought, which has been accepted especially by plant physiologists with great readiness, that the decomposition of organic respiratory substances of the aërobic organisms invariably takes place in two stages; in that the dextrose molecule—to again use this as an example—is split up first by an enzyme into larger fragments, which then in the second stage of the process undergo combustion to the formation of carbon dioxide and water. Such a possibility cannot be repudiated. I wish, however, to state that one should be very reluctant in generalization of this assumption for all aërobic organisms. The types of metabolism in the different organisms are so manifold and of such immense variety that we should be very careful in our generalizations before being in possession of material extending over a great number of groups of organisms. Above all, it does not seem justifiable to also accept this type for life existing at higher temperatures, and still less to apply it to those instances in which the production of energy following stimulation is suddenly increased to great amounts. Let us suppose that the disintegration process occurs in two phases, the first of which after the type of the fermentation of dextrose separates the molecule into larger fragments, while in the second phase these fragments are split up through oxydation into the formation of carbon dioxide and water. We can then say with certainty that in the first stage only a comparatively small amount of energy production occurs, for energy production by enzymic processes of this kind is never great; the second phase, on the other hand, must be associated with a very considerable energy production, for by the addition of oxygen and the formation of carbon dioxide and water the strongest affinities possible are combined. With this assumption in certain cases, as, for instance, in the sudden production of energy in muscle contraction, which necessarily occurs in the purely oxydative phase of the whole process, the view is forced upon us, that, in these cases, the entrance of oxygen into the molecule from the very beginning, even the first impact, produces oxydative decomposition of the whole molecule. The view that, in the reactions of warm-blooded animals, which occur with great rapidity and considerable energy production, the oxygen primarily explosively breaks up the whole carbon chain, certainly presents no more difficulties than the supposition that the simpler substances are attacked secondarily, provided sufficient oxygen be present. This method would be obviously the simplest. This is, however, mere speculation and a definite decision between the two possibilities cannot be made at present. However, whether the process takes place in two phases, an anoxydative and an oxydative, or simply in an oxydative phase, in any case, the sudden discharge of energy in the aërobic organism set free by the stimulus, is brought about by the addition of oxygen.

This is a highly important fact and as such requires the most thorough confirmation, and is best accomplished by the investigation of the state of excitation of aërobic substances on the withdrawal of oxygen. Experience gained by observation in this respect on a great number of living substances shows that excitability decreases upon the withdrawal of oxygen. In this connection I should like to cite some particularly significant instances.

During a sojourn at the Red Sea in 1894–95 I was able to establish this dependence in the single-celled organism, the Rhizoplasma Kaiseri, a large naked orange-colored rhizopod. (Figure 10, A.) Mechanical stimulation, which under normal vital conditions of these organisms brings about contraction in the long-branched pseudopods, becomes ineffective with a cessation of the movement of protoplasm, when oxygen is removed and is replaced by a stream of hydrogen. (Figure 10, B.) With renewed introduction of oxygen there is a return of the protoplasmic movement and entire recovery takes place.

This dependence of irritability upon oxygen is most clearly demonstrated in the nerve centers. For this purpose I have employed the spinal cord of the frog.60 A canula is introduced and fixed into the aorta of the animal and the blood is replaced by a current of oxygen-free saline solution. The centers of the spinal cord are thereby wholly isolated from the supply of oxygen. The indicator for the irritability here used is reflex excitation from the skin to the gastrocnemius, or better, stimulation of the central stump of the sciatic nerve with single induction shocks, bringing about reflex response of the triceps. The reflex may be considerably augmented by increasing the reflex excitability of the spinal cord by poisoning the animal with strychnine. On testing the reflex excitability at the beginning of the experiment it will be found that the reaction to each individual stimulus consists, in consequence of the strychnine poisoning, of a long-continued maximal tetanus. The longer the deficiency of oxygen continues, the briefer become the tetanic reflex contractions following a single stimulus. Soon reflex tetanic responses are merely short single contractions, which decrease more and more with the continuance of oxygen deficiency. Finally, the same stimuli which previously produced strong tetanic contractions of long duration are altogether without effect. Although by increasing the intensity of stimulation brief contractions can again be brought about, irritability decreases more and more, until at last even the strongest stimuli remain without result. If the oxygen-free saline solution is now replaced by one saturated with oxygen, or blood of the ox, rendered arterial, the excitability returns within a few minutes and soon reaches the maximal height which it possessed under the influence of the strychnine poison. Even the weakest single stimuli now again produce tetanus. The same process reoccurs, if the fluid used for transfusion containing oxygen is again replaced by an oxygen-free saline solution. In this way, by repeated change of the perfusing fluid, we can demonstrate in the most positive manner this alteration in irritability, the result of the alternate presence and removal of oxygen. This is perhaps the best example of the close dependence of irritability on oxygen.

This same fact can be observed with equal clearness in the nerve. At my suggestion H. v. Baeyer61 showed as the result of investigations made in the Göttingen laboratory the dependence of irritability of the nerve upon oxygen for the first time. By employing as the method the ascertainment of the threshold of stimulation I then made a closer study of the alterations in irritability during asphyxiation. These observations were soon after continued by Fröhlich.62 The method is as follows: the nerve of a nerve-muscle preparation of the frog is drawn through a glass chamber which is made completely air-tight and containing platinum electrodes. The air in the chamber is then displaced by a stream of pure nitrogen. (Figure 11.) On testing that part of the nerve situated within the glass chamber with single break induction shocks it can be observed that its irritability, measured by the threshold of stimulation for muscle contraction, decreases more and more, until after the lapse of some hours, the stimulation required is so strong as to reach the region of the “Stromschleifengrenze.” If in place of the stream of nitrogen, air or pure oxygen is now allowed to flow through the chamber, the nerve recovers almost instantaneously. Within the space of a minute its irritability has risen again to its full height and the same experiment, with the same result, can be repeated. Finally, as Fillié63 has shown, the like result is obtained when the nerve is asphyxiated in a fluid medium.

All these facts, the number of which indeed could be increased greatly for other aërobic forms, suffice to establish the fundamental importance of oxygen to the maintenance of irritability of living substance. Oxygen is of greatest importance for a high degree of irritability in all aërobic organisms. All living systems which are characterized by a great capability of activity and evince strong responses under the influence of stimulation, such as the vertebrates and insects, are necessarily aërobic, whereas the living organisms of pronounced anaërobic character, as some bacteria, yeast cells, parasitic organisms, etc., manifest on the average much less capability of activity.

Finally, to briefly summarize the foregoing, the following picture presents itself of disintegration produced by a momentarily acting stimulus. It is immaterial how the stimulus produces an excitating effect in the given case, whether through changes in the ion concentration of the living system, by increase of intramolecular atomic movement or in any other manner, it invariably accelerates the disintegration of the complex molecules concerned in functional metabolism, the nature of which varies in the special cases. In the great majority of instances nitrogen-free organic combinations serve as material for the functional constituent members of metabolic processes. In the anaërobic organisms this decomposition takes place anoxydatively with the coöperation of enzymic processes, and as larger fragments generally result from the disintegration of the complex molecule, the production of energy is accordingly smaller. The disintegration of aërobic organisms, on the other hand, occurs in the form of an oxydative splitting up of the complex molecules into carbon dioxide and water so that the production of energy attains a high value. The details concerning the manner in which the individual stages of this decomposition take place and the interactions by which its end products are reached is at present beyond our knowledge. It would be a mistake to generalize in this connection from the behavior of certain groups of organisms. The assumption that under certain conditions the disintegration occurs in two phases, the splitting up resulting from enzymic action of the complex molecule into larger fragments, followed by an oxydative splitting up of these into carbon dioxide and water, can in no case as yet be justifiably applied to all conditions and all aërobic organisms. This is more or less the impression which we derive of the functional excitation process as seen today.

Under normal conditions the functional excitation is at once followed by a succession of secondary processes, the “self-regulation of metabolism.” Self-regulation after a functional excitation is a fact demonstrated by experience. But in what manner does it take place in detail?

As the functional constituent members of metabolism involve a disintegration of the nitrogen-free atom groups, the functional self-regulation must necessarily furnish in sufficient quantity and in proper form the carbon, hydrogen and oxygen atoms, which have been removed in the production of carbon dioxide and water. This is accomplished, as is well known, by the food and the intake of oxygen. It is of importance to the maintenance of living substance that after every functional activity it is as soon as possible again capable of reaction. Therefore, it is absolutely necessary that this material is in the proper place, where building up is essential, and is at the same time constantly in proper form. Indeed, the restitution of the original state follows under favorable conditions with lightning rapidity, although varying in different forms of living substance. This occurs in the nerve in an extremely short time. From this it might be supposed that the living system by accumulating a store of the necessary compensation substances in suitable form, had made itself independent to a certain degree of the frequently varying supply of material obtained from the medium.

This may be held as the proper view, first with regard to compensation substances. The fact that living organisms can under some conditions remain for a lengthened period in a state of starvation, without losing their capability of activity, can only be explained by the presence of a great quantity of reserve supplies of compensation substances. In the course of work in the laboratory every physiologist has become acquainted with the fact that frogs which have been kept without food for a year, although much reduced in weight, are still capable of some muscular activity.

Organs and tissue, which are cut off from all food supply through the blood and lymph, may remain active for many hours. H. v. Baeyer64 has shown that the ganglion cells in the frog, in which saline solution was transfused at room temperature and containing no trace of organic substances and where irritability has been increased to the maximal by means of strychnine, were capable of strenuous work for nine or ten hours before losing responsivity. The nerves and muscles of the animal retain their excitability for even a longer period under the same conditions. Indeed, we have histological evidence of the existence of organic reserve material in the various cells in the form of embedded bodies in the protoplasm. As for instance the disappearance of the Nissl granules in the ganglion cells following great activity,65 (Figure 12), or that of the granules in infusoria cells during starvation.66 (Figure 13.) We assume that a certain amount of organic foodstuffs in a state properly prepared is present in the cell. As the amount of these prepared substances is consumed, new quantities of stores, having undergone various preparatory processes, among which the enzymic actions may be considered to play a chief rôle, are brought into that form in which they appear suited to fill the gap produced by disintegration. Plant physiologists in particular have here again furnished us with some essential data for the assumption of the existence of such processes which regulate the transformation of reserve substances as well as its extent. Pfeffer67 has found in several fungi and bacteria that there exists a compensation between the diastatic breaking down of the carbohydrates stored as reserve material and the quantity of dextrose introduced. He further found that the more the reserve substance is split up into dextrose the less of the latter is introduced from without and vice versa. De Bary68 some time ago also observed in the bacillus amylobacter an analogous relation between the enzymatic cellular digestion and the quantity of dextrose introduced with the food. An equilibrium, therefore, exists between the required amount of dextrose and the extent of enzymic splitting up processes of the reserve material. A great number of similar processes have been observed. Even though the details of the whole preparatory assimilative processes are beyond our knowledge we can still say with certainty that, on the one hand, everywhere great quantities of organic reserve substances are always present in the cell, and on the other, that these substances are subjected to a transformation into suitable material for building-up processes, the extent of which is controlled according to need, by the processes of self-regulation.

Entirely different is the question if the cell also possesses a reserve store of oxygen. In this respect views have widely differed, and even today no conformity of opinions has been arrived at. The fact that many purely aërobic organisms and tissues can exist under complete exclusion of oxygen for a longer or shorter period, retaining their excitability and producing carbon dioxide, has for a long time led a great number of investigators, such as Liebig, Matteucci, Engelmann, Pettenkofer and Voit, Claude Bernard, Verworn, H. v. Baeyer and others, to the supposition that a reserve store of oxygen must exist in the living substance which maintains its excitability for a time. More recent information, however, of the transition of the oxydative to the anoxydative disintegration under a deficiency of oxygen, as can be observed in plants and certain invertebrate animals, indicates that here also there is the possibility of another explanation of these facts. Various attempts have been made to solve the problem if reserve oxygen is present in the cell or not. The experiments of Rosenthal,69 carried out with his respiration calorimeter, seemed to point directly to an oxygen reserve in the organism of the mammal. He observed that during respiration in an atmosphere rich in oxygen the respiratory quotient (CO₂ : O₂) became lower than in ordinary air, that is, that oxygen, and that indeed in considerable quantity, must be retained in the organism. Nevertheless Falloise70 found that when rabbits, which had been kept in an atmosphere containing 80 per cent of oxygen, were asphyxiated, the time necessary to produce death was no longer than in animals which had been kept previously in ordinary air. The correctness of the observations of Rosenthal have been disputed by Durig.71 Winterstein72 also, employing the microrespiration methods of Thunberg upon the spinal cord of the frog, believed that he had found proof that an oxygen reserve cannot take place. He reasoned thus: If the cells of the spinal cord contain reserve oxygen, which is used up when pure nitrogen only is breathed, then it necessarily follows that after reintroduction of oxygen, following asphyxiation, a definite quantity must be stored up again as reserve. In consequence, the respiratory quotient following the intake of oxygen after asphyxiation should be smaller than when the animal is in air. He found, however, that the respiratory quotient does not essentially change and concluded from this that storage of oxygen does not take place. In these experiments, however, there exists no certain indicator as to the state of the spinal cord during asphyxiation and recovery in the given case. The spinal cord may be severely injured and even undergo degeneration during asphyxiation, and the recovery following the reintroduction of oxygen may be either incomplete or nil, without there being a method for its determination. Apart from this, Lesser73 has already emphasized, in opposition to these experiments, that the respiratory quotient in recovery is no criterion to guide us. It is immaterial whether during asphyxiation oxygen respiration occurs following a reserve supply, or that an anoxydative formation of carbon dioxide has taken place, for in both instances the respiratory quotient would be less after asphyxiation when there is again an oxygen supply. It is, therefore, quite impossible to decide the question by the employment of this method. For this reason Lesser has attempted to solve the problem by means of quite another method, and was convinced that he had refuted finally the belief in the existence of reserve oxygen. His method consists in the employment of the Bunsen ice calorimeter, by which he determines the heat production of frogs, kept first in air, then in nitrogen, and at the end of each experiment ascertaining the amount of output of carbon dioxide, respectively in air and nitrogen. He found that the quantity of heat, calculated in terms of 100 grms. body weight per hour, produced in nitrogen was considerably less than that under corresponding conditions in air, but that the production of carbon dioxide, on the other hand, during the first hours in nitrogen was doubled in amount, as compared to that in air. From this he concludes that the carbon dioxide formation in nitrogen must be different from that in air, as it is associated with a reduced heat production. In other words, carbon dioxide formation, while the animal is in a nitrogen atmosphere, does not have its origin in oxydative processes at the cost of stored up oxygen. I regret that I am unable to accept these arguments as conclusive evidence against the assumption of an oxygen reserve, as this question cannot be decided by the use of such methods. Lesser does not measure the amount of carbon dioxide until the end of his experiments, that is, he learns merely the entire carbon dioxide production during a period of many hours. No conclusions can be drawn from this as to the conditions existing in the first period of time, directly after the animals have been subjected to an atmosphere of nitrogen. It is quite possible that subsequent to the change to nitrogen an oxydative carbon dioxide formation may have continued in decreasing degree, without this being shown in the final result. The problem of the existence of a reserve supply of oxygen is in no way solved by these experiments.

In assuming the presence of a reserve supply of oxygen in the cell we must above all entertain no false conception as to its amount. This must be, as I have often had occasion to emphasize, exceedingly small and in no way comparable with the great masses of organic reserve substances contained in the cell. The assumption, especially for the nerve centers of the frog, that the excitability remains after complete exclusion of oxygen must be looked upon as demonstrating a reserve supply of oxygen, would oblige one to suppose the presence of such a small store of oxygen that it would be completely exhausted by continued activity in room temperature within ten to twenty-five minutes. Strychninized frogs, in which the blood has been replaced by an oxygen-free saline solution, lose, as I have shown,74 their excitability completely within ten to twenty-five minutes after the blood has been displaced. Nevertheless the assumption of the existence of a small oxygen supply in the cell can hardly be evaded. It must not be imagined that the moment the blood of the frog has been replaced with an oxygen-free solution, there is not a trace of oxygen left in the organism. Were such the case, the irritability, if measured by the extent of the response, would sink momentarily to a very low level, for the anoxydative disintegration processes are associated with an incomparably smaller production of energy than those of oxydative disintegration. We see, however, that the irritability in the muscles, nerves and nerve centers of the frog even after the complete withdrawal of oxygen at first remains practically at the former height and only very gradually decreases. Above all it would seem to me to be in the interest of the preservation of the organism and especially of those parts in which there is a high energy production and particularly those substances in which energy production predominates, that the material necessary for its formation is always at its disposal in sufficient quantity. Otherwise the capability of action of the organism would be impaired at every moment or at least suffer great fluctuations.

In accordance with this we must suppose that under physiological conditions all those substances required to replace the disintegrated molecules are always present in the cell in sufficient quantity and suitable form to replace at once those lost by excitation. Further, without doubt, in the organism which is always aërobic, oxygen must be present in certain quantities to assure at any moment oxygen replacement following oxydative disintegration, to guarantee sufficient amount for succeeding stimulation.

A further question arises: How is it that the material lost in disintegration is always replaced in just sufficient quantity to establish the metabolic equilibrium? In short, how are we to understand in a mechanical sense the self-regulation of metabolism?

In the preservation of metabolic equilibrium, we have a process before us, the principle of which is nowadays restricted to living substance. In my “Biogen hypothesis,”75 I have associated the self-regulation of metabolism with the chemical equilibrium in interreacting masses. I have considered the metabolic self-regulation as the expression of the formation of a mass equilibrium between the quantity of foodstuffs and the quantity of a hypothetical combination of living substance, the biogen, which continuously disintegrates and builds up again of its own accord. In fact, however, we have in the chemical equilibrium of reacting mixtures in the non-living world, a principle which is completely analogous to the self-regulation in living substance. The chemical facts are, indeed, well known. If we take the classical example of the formation of ethylacetat from acetic acid and alcohol, we have a case of an inanimate system, in which the amounts of the reacting substances are in constant equilibrium. The reaction following the mixture of equal amounts of alcohol and acetic acid is as follows:

¹⁄₃ Mol. C₂H₅OH + ¹⁄₃ Mol. CH₃COOH
= ²⁄₃ Mol. CH₃COOC₂H₅ + ²⁄₃ Mol. H₂O.

In this reaction there is an alteration only in the absolute quantity of the individual constituents but never in the relative amount. In the living system we have a completely analogous instance, which apart from its course differs from the inanimate example merely in the following points: In the first place, certain quantities of substances reacting on each other are continually introduced into and certain reaction products continually removed from the living system. Secondly, the reacting mixture of the living substance is not homogeneous, and at the same time is more complicated than that of the inanimate example. Thirdly, the sum total of the reaction is not reversible in its entirety. The question arises, should any essential difference between metabolic self-regulation and the maintenance of chemical equilibrium be assumed upon this statement? I must confess that this does not appear to me to be the case. The fact that organisms exist in a stream of substances by which their nutrition is introduced and the metabolic products removed, cannot have any influence on the state of equilibrium so long as the conditions are again and again replaced in the same manner. The equilibrium can only be influenced when the introduction of foodstuffs or the output of metabolic products is changed in value. Then they occur as the inanimate example, when various amounts of material are brought together. A new equilibrium takes place, having a higher or a lower mass level. This is also true in the living substance, in growth and in atrophy. The equilibrium is disturbed as happens in the inanimate reacting mixture, where different quantities of reacting substances are brought together. In both instances we have in principle a conformity of behavior of the inanimate and the living system. Secondly, as far as the greater complexity and inhomogeneity of the living reacting mixture is concerned, it is self-evident that this likewise does not constitute an essential difference, for we are acquainted with conditions of equilibrium in chemical reactions possessing a number of members and in inhomogeneous mixtures. Finally, the fact that the reaction in the living system is not totally reversible, forms no barrier to the assumption in principle of metabolic self-regulation as a chemical equilibrium. It is quite possible to conceive of a chemical equilibrium in a reacting mixture, of which only certain constituent processes are reversible, without the totality of the reactions as a whole being necessarily so. Let us assume, by way of example, that the assimilative processes of the metabolic chain are reversible, then under constant quantitative relations of foodstuffs, following every disintegration of assimilative products with removal of the decomposition products, the same amount of assimilatory processes is required for building up. And this is just that which we observe in metabolic equilibrium. Accordingly, we may look upon the metabolic equilibrium as a special, although a very highly complicated, instance of chemical equilibrium, and we may explain the metabolic self-regulation following a dissimilative excitation of the same, by those principles on which the rebuilding of chemical equilibrium is founded. It is true that the special details of this process can be differentiated in only that degree in which it is possible to penetrate at all into the details of metabolism of the given cell form. In this, as is well known, the advance is extremely slow.

The rebuilding process following decomposition of living substance in response to an excitating stimulus consists not merely in compensation for the decomposed atom groups but also in the removal of disintegration products. This removal can be accomplished, in so far as simple chemical substances such as carbon dioxide and water are concerned, by diffusion. Observations have shown that the semi-permeable protoplasm surface is pervious to water and carbon dioxide. The latter can, therefore, depending upon the amount of concentration, be eliminated from the living substance. Output of water likewise takes place in so far as the specific water content of the living substance is exceeded and which is osmotically regulated by its amount of salt content. When, finally, osmotic pressure within the living cell and in the surrounding medium is equal, the interchange of water ceases. All these processes are explained by diffusion. Self-regulation takes place in this regard simply by osmotic means. The conditions in respect to those decomposition products consisting in more complicated organic combinations, such as lactic acid, fatty acids and nitrogen derivatives of protein disintegration, are somewhat different in that the protoplasm surface possesses the property of hindering the passage of these substances into the medium. These are, as is well known, first transformed by secondary chemical processes into transfusable substances. In this transference the oxydative decomposition with the formation of simpler substances plays the most important rôle, so that the substances thereby formed, namely, carbon dioxide, water and ammonia, are osmotically eliminated as the result of the selective permeability of the surface of the protoplasm. In this way the living cell rids itself of the useless products of metabolism.

Finally, the question remains, is the original state, as it existed before the influence of the stimulus, really completely recovered by metabolic self-regulation, or does even individual excitation of brief duration produce a continued change in the protoplasm? It is quite impossible to prove that such an effect follows the momentarily acting single stimulus, if stimulation has not exceeded the physiological limits of intensity. Should it exist, it must be imperceptible. Nevertheless, it ought to be possible by frequently repeated application of the stimulus to increase this which is imperceptible to an extent in which it is perceptible. This is, indeed, the case and is manifested as we have already seen in the increase of the volume of living substance by frequently recurring functional excitation. We can, therefore, assume with great probability that even the momentarily acting individual stimulus produces, although not perceptible per se, lasting effect in the cell. The functional excitation must be followed secondarily by an increase of the assimilative phase of the entire cytoplastic metabolism. Otherwise the taking place of the increase of volume of the living system following frequent excitation of the functional constituent members of metabolism, is unintelligible. But how are we to interpret these secondary results from a physical standpoint? First of all, it must be stated that we do not know of such hypertrophy following activity in unicellular organisms, but only in the tissues and organs of multi-cellular forms, in muscles, nerve cells, glands, etc. In the cell community of the vertebrates, however, the studies on the relations between activity and the blood supply of the particular tissue or organ furnish a physical interpretation for the existence of the functional hypertrophy. The active portions show a dilation of the blood vessels, therefore an increased supply of blood and consequently an increase in the circulation of lymph. In other words: the supply of nourishment to the individual cell and the removal of the metabolic products in a unit of time is increased. The preceding discussion of the dependence of the conditions of equilibrium upon the quantitative relations of the reacting substances makes it clear that under these conditions a metabolic equilibrium on a higher quantitative level must occur; that is, the living substance must increase in amount just as in the inanimate example the absolute amount of the æthylacetat increases if more alcohol and acetic acid are introduced to an equal degree. Some time ago76 I expressed the opinion that the increase of the blood supply in a functionally active organ must be based on a physical self-regulation, which takes place as a result of the fact that metabolic products of the tissue cells influence the cells of the vessel walls in that part, so that the vessels dilate and more lymph is formed. In the meantime this has been proved to be indeed the case. Schwarz und Lemberger77 and Ishikawa78 have shown that especially the weak acids, which are produced in larger amount as a result of strong activity of the cells, bring about vessels’ dilation. By the demonstration of this highly important process of self-regulation the last link has been added for the physical understanding of the hypertrophy of activity of the tissue cells by continued functional excitation. Whether or not the same applies to the single living cell, if the unicellular organism likewise undergoes a quantitative increase by a continuous functional excitation, and if the single cell possesses in itself a corresponding mechanism of self-regulation similar to the cell community in the vertebrates, cannot be answered, for concerning all these problems information is lacking for the present.