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 Falloise[70] 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] Winterstein[72] 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, Lesser[73] 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 ago[76] 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 Lemberger[77] and Ishikawa[78] 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.