The Dynamics of Living Matter

We understand how it happens that in times of abundant fat supply our tissues are able to store up fat, while in times of want fat disappears from them. If the blood receives no fat from the intestine, and if the other sources of fat formation, which we shall mention later, cease, the digestive effect of the lipase in the cells must outweigh its synthet- ical action. The experiments of Kastle and Loevenhart were not tried with the fats occurring in the body. A. E. Taylor f has filled this gap by showing that the lipase extracted from the castor bean, which digests fats, is able to produce synthetically the triglyceride of oleic acid. The pro- cess is a very slow one, inasmuch as in six months only 3.5 g. of the fat were formed. Taylor concludes that in the body other agencies than mere enzymes must contribute toward the acceleration of the hydrolytic, as well as the synthetical processes. These conditions are, however, not of a vitalistic character, but may be due to the pres- ence of certain other substances. Thus Hewlett has recently found in Taylor's laboratory that the addition of lecithin to lipase accelerates the hydrolysis of fat considerably. Taylor found also that the lipase from the castor bean cannot synthetize every fat, but only the triglycer- ide of oleic acid. Experiments with palmitic and stearic acid and glycerine as an alcohol gave negative results, as gave also experiments with oleic acid and mannit or dulcit as alcohol. This is in harmony with the theory of intermediary reactions, which will be discussed later.

It is, however, worth mentioning that fat may be produced in the body from carbohydrates, and that lipase. has, as far as we can tell, nothing to do with this mode of fat formation. The most striking case of such an origin of fat is found in the leaves of the olive tree, which synthetize it from the carbohydrates formed from the CO2 of the air. The fact that a reduction must form part of the process of the forma- tion of fat from carbohydrates may explain why so often a hypertrophic heart has a tendency to fatty degeneration, inasmuch as the hyper- trophic heart is as a rule an overworking heart, and is thus liable to suffer from lack of oxygen.

Neilson* has shown that the catalytic action of lipase on ethylbuty- rate can be imitated by platinum-black. The latter not only acceler- ates the hydrolysis, but also the synthesis of ethylbutyrate. Kastle and Loevenhart found that certain poisons like hydrocyanic or sali- cylic acid weaken the action of lipase. Neilson found that these poisons act similarly on the digestion of ethylbutyrate by platinum-black. The study of the reversible action of enzymes in the carbohydrate group is complicated by the fact that the digestion of starch to sugar occurs in a series of successive stages, and that apparently each stage requires a different catalyzer. According to Duclaux,f we possess specific enzymes for the transformation of solid into liquid starch ; the liquid starch is then split by amylase or diastase into a disaccharide, i.e. maltose. Maltose is split by the enzyme maltase into d-glucose. In case another disaccharide is formed in the place of maltose, other enzymes are required, e.g. in the case of cane sugar, invertase, whereby dextrose and laevulose are formed. In the animal body, glycogen is formed in the place of starch. It is obvious that the synthesis of dex- trose into glycogen or starch requires the presence of several catalyzers. The action of these catalyzers must be studied individually.

Hillij: found that the hydrolysis of maltose in the presence of the enzyme maltase is retarded if dextrose is added to the maltose; that, moreover, the hydrolysis of maltose under the influence of maltase is complete only in very dilute solutions, while the reaction otherwise comes to a standstill before all the maltose is transformed into glucose. The following table shows the point at which the hydrolysis comes to a standstill at various concentrations: —

Hill convinced himself that if the enzyme maltase is added to a solu- tion of glucose, maltose is formed, and that, moreover, equilibrium is reached at the same point as in the case of hydrolysis. When he added fresh maltase to a 40 per cent solution of dextrose in one experiment, 14.5 per cent, and in another 15.5 per cent, maltose was formed. It has since been shown that the synthetical product formed in this case was isomaltose instead of maltose, but this slight deviation does not alter the principal result.

Hill raises the question, as to whether or not a synthetical forma- tion of maltose under the influence of maltase may occur in the living cell. He points out that for such a result a high concentration of dextrose in the blood is by no means necessary; that it is sufficient if the product of the synthesis is removed immediately, possibly through a further synthesis into a higher carbohydrate by another enzyme in the cell. In this way the concentration of the maltose is kept at zero, and the tendency toward the establishment of the chemical equilibrium must favor the further synthesis.

The synthesis of sugar into glycogen is of general importance, inas- much as glycogen is the form in which the carbohydrates are stored in our liver and muscles. Max Cremer* immediately after the appear- ance of Hill's paper published the important observation, that the juice pressed out from yeast, which had previously been rendered free from glycogen, is capable of forming glycogen from sugar. When 10 per cent of a fermentable sugar was added to the juice, the latter gave the glycogen reaction after from twelve to twenty-four hours, but this result was not obtained in all cases.

It may perhaps not be unnecessary to call attention to the fact that in all the cases we have discussed the enzymes are soluble substances, which can be extracted from the cells, and therefore can exist inde- pendently of the life or structure of the cell from which they are obtained. In the group of proteins we not only meet with the same difficulties which are found in the group of carbohydrates, but also with the addi- tional difficulty, that we know considerably less about the constitution and configuration of the various protein molecules than of the carbo- hydrates. These two conditions probably account for the fact that a direct reversion of the action of a hydrolytic enzyme has not yet been satisfactorily proven for proteins.

It is hardly necessary to mention especially the fact that hydro- lytic enzymes, e.g. of the type of trypsin, acting on proteins, are found not only in the intestine, but also in tissues, probably generally. Thus Salkowski has shown that if yeast cells or muscles are kept aseptically in an incubator, an autodigestion occurs in which leucin and tyrosin, i.e. typical end products of proteolysis, are formed. Kutscher f has completed the proof by showing that, in addition to the acids, the other end products of proteolysis are formed in the autodigestion of yeast; namely, the hexonbases, e.g. arginin and lysin. He found, moreover, that in starving yeast the above-mentioned end

products of tryptic hydrolysis are found in considerable quantities, while these products could be obtained in fresh and well-nourished yeast only in minimal quantities. In this he sees, and probably cor- rectly, an indication of the reversible action of the proteolytic enzymes. There is, however, one essential link missing, i.e. the proof that the hydrolytic action of the proteolytic enzymes is retarded, and finally inhibited, when the products of digestion are not removed.

R. O. Herzog has recently made an attempt to prove the reversible action of enzymes in a very interesting way upon the discussion of which, however, we cannot enter here. 3. RESPIRATION AS A CATALYTIC PROCESS. OXIDATION AND OXIDASES By respiration we mean the taking up of oxygen and the giving off of CO2. We shall see later that the latter process can exist indepen- dently of the taking up of oxygen. Since the days of Lavoisier and La Place the real problem of oxida- tion has consisted in the explanation of the fact that at the body tem- perature our food stuffs are not oxidized at all, or only infinitely slowly, outside the body, while in the body they are oxidized rapidly. The solution of the problem was found in the discovery of "oxidizing fer- ments" in living organisms. This conception is chiefly due to Moritz Traube,* who was also the first to recognize that the oxidations occur in the cells, and not, as had been assumed before, in the lungs or the blood.

Traube's idea was that there exist in the cells autoxidizable sub- stances, i.e. substances which bind loosely the free oxygen at a com- paratively low temperature, and which are capable of giving off their oxygen to disoxidizable substances, such as our food stuffs. It is obvious that Traube's idea of the action of an oxidizing ferment was that of intermediary reactions. He realized also that these oxidizing ferments exhibited no effects which could not be produced in inanimate nature, as the following quotation shows: "The ability to transfer oxygen . . . is found in many, even inorganic bodies. There are substances like nitrogenoxide, platinum, various coloring matters, copper salts, which are capable of transferring free oxygen upon neighboring substances." f

* M. Traube, Ueber die Beziehiing der Respiration zur Muskelthatigkeit und die Bedeu- tung der Respiration uberhaupt, 1861. Gesammelte Abhandlungen von M. Traube, p. 157, Berlin, 1899. (In this paper Traube showed also that the work of the muscle is normally done at the expense of carbohydrates. His arguments induced Pick and \Vislicenus to try the classical experiment by which this theory was proved.) t M. Traube, Die Chemische Theorie der Ferment wirkttngen und der Chemismus der Res- piration, 1878. Gesammelte Abhandl., p. 384, Berlin, 1898.

I think Jacquet* was the first to separate a "ferment of oxidation " from the living organism, and to obtain it in a watery extract from tissues. The oxygen of the air oxidizes benzylalcohol (C6HSCH OH) only slowly to benzoic acid (C6H5COOH) at body temperature; the same is true for the oxidation of salicylaldehyde C6H4rn-H-to salicylic tissues accomplish this oxidation comparatively rapidly. Jacquet proved that this energetic oxidation of benzylalcohol is not dependent upon living protoplasm, as he found that it occurred also in dead tissues. Tissues poisoned with carbolic acid continue to accelerate these oxida tions, and even tissues which have been preserved in alcohol are capable of so doing. Nor are these oxidations dependent upon the structure of the cells, as watery extract from the cells also had oxidative effects upon benzylalcohol. The action of the oxidizing enzymes is annihi- lated when they are heated to a temperature of about 100° — pos- sibly through a hydrolysis of the enzyme itself.

Engler and Wild f have found that there exists a group of substances which behave like Traube's autoxidizable substances. These sub- stances have the peculiarity of easily forming peroxides of the following These peroxides are capable of giving off one atom of oxygen to dis- oxidizable substances. Through the loss of this atom of oxygen the peroxides are transformed into oxides. This view is supported by an important observation which was first made by van't Hoff and Jorissen. If the quantity of oxygen which disappears in the oxidation of a known quantity of a disoxidizable substance is measured, it is found to be in most cases exactly twice as large as the quantity required for the oxida- tion of the disoxidizable substance. % This finds its explanation in the fact that for every molecule of oxygen which is taken up by the autoxi- dizable substance, only one atom is transferred to the disoxidizable substance.

The view of Engler and Wild is also supported by the investigations of Kastle and Loevenhart § on the oxidizing effects of plant tissues, e.g. the potato, and their watery extracts. They found that organic peroxides, e.g. benzoylperoxide, phthalylperoxide, and succinylperox- ide, or inorganic ones, like lead peroxide and manganese peroxide, pro- duced the same blue color in the tincture of guaiacum as the tissues of plants, or watery extracts from the same. The production of this blue color is due to oxidation. The same authors showed, moreover, that the same poisons which in plants prevent the action of oxi- dases - - this is the name given to the enzymes of oxidation — prevent also the oxidizing action of the above-mentioned organic and inorganic peroxides upon tincture of guaiacum. Kastle and Loevenhart there- fore conclude that the oxidases, or oxidizing enzymes, in the tissues of animals and plants, are organic peroxides.

Here we meet with a difficulty, however. Oxidations occur inces- santly on a large scale in the living body, especially at the temperature of the warm-blooded animals. The peroxides, however, are not capable of transferring oxygen in unlimited quantities to disoxidizable sub- stances, but as soon as a peroxide molecule has given off one atom of oxygen, its oxidizing power is at an end. This difficulty can be over- come in the following two ways: it is possible that new autoxidizable substances are formed incessantly in the body; the second possibility is the existence of a second class of oxidizing enzymes which act more in the sense of true enzymes than the peroxides, inasmuch as they are able to take up and give off oxygen indefinitely. Haemoglobin is capable of binding and setting free oxygen indefinitely, but it is not capable of transferring its oxygen to disoxidizable substances, and hence does not act as an oxidase.

The question of localization of the oxidizing enzyme in the cell was raised by Spitzer.* He found that the proteins which can be extracted from the cells do not possess the qualities of Jacquet's oxidase, but that these qualities are found in such extracts as contain nucleoproteids. The nucleoproteids are typical constituents of the cell nucleus, and they differ from the proteins proper in that they contain PO4 and Fe. Spitzer was able to show, moreover, that of the products of cleavage of nucleoproteids only those constituents were able to act as oxidases which contained the Fe group. Two years ago I pointed out that if Spitzer's researches are correct, the cell nucleus must be regarded as the essential respiratory or oxidizing organ of the cell.f

It is possible that we have two groups of oxidizing catalyzers in the tissues : first, those of the type of peroxides which are possibly present in the protoplasm; and second, substances which can act indefinitely as oxidases, and are found in the nucleus. There are, indeed, a number of facts which seem to harmonize with the idea that the nucleus is the main oxidizing organ of the cell. Processes of regeneration demand oxygen. Nussbaum* has shown that if an Infusorian be cut in such a way as to divide it into two pieces, one containing the nucleus, and one without any nuclear matter, only the former is capable of regenerat- ing the lost parts. The other piece lives but a comparatively short time and is not capable of regeneration ; it dies under symptoms which are rather similar to death from lack of oxygen.

Ralph Lillie t tried to test the idea that the nucleus is the main oxi- dizing organ with the aid of staining substances which diffuse into the cell, and change color when they are oxidized. He worked with the cells of the blood, the liver, and the kidneys of frogs, and found that the oxidation seems to occur most rapidly in the nucleus and on its surface. He found, moreover, that the oxidations were most rapid in those organs and those regions of organs where the nuclei were densest.

It has been noticed that if cells containing chlorophyll are deprived of their nucleus, they keep alive longer if exposed to the light than if kept in the dark. This may be connected with the fact that in the light the chlorophyll is capable of liberating the oxygen from the CO2. It is stated, as a rule, that the role of the oxygen is to supply the energy for the production of heat and of mechanical work, but it is evident that this statement does not take into consideration the fact that sessile plants, in which the loss of heat does not need to be com- pensated by a production of heat, and in which no energy is spent in mechanical motion, are in need of oxygen and possess oxidases. With the exception of a limited number of anaerobic bacteria, the statement can be made that oxidations and the presence of oxidases is a general characteristic of living matter. There are, however, other vital processes which are more general than those of locomotion and heat production, which also require oxygen, i.e. cell division and growth. Pasteur made the fundamental discovery J that with lack of oxygen the yeast cells continue to produce lively fermentations of sugar, - - in fact, Pasteur stated that their fermentative action is more energetic than in the presence of oxygen, - - but that they grow and multiply very little or not at all. If, however, oxygen is added freely, the yeast cells multiply and grow considerably, provided the necessary nutritive salts are present. According to Hoppe-Seyler and Duclaux, the absence of oxygen favors the formation of the catalyzer for the alcoholic fermenta-

tion in the yeast cell, the zymase ; * but we are here chiefly concerned with the fact which nobody has failed to confirm, that the presence of oxygen favors cell division and growth in yeast cells; that without oxygen these processes soon come to a standstill. The same is true for animals. I made a large number of experiments on the effects of lack of oxygen on the newly fertilized eggs of sea urchins and fishes (Fundulus and Ctenolabrus).t The eggs were kept in small Engel- mann gas chambers through which a current of hydrogen was sent to drive out the oxygen and the CO2 formed by the eggs. In the eggs of sea urchins and Ctenolabrus, the segmentation stopped in less than an hour after the beginning of the current of hydrogen. When the air was again admitted, the eggs began to divide, provided they had not remained too long without oxygen. The eggs of Fundulus, also a marine fish, do not respond as quickly, inasmuch as it required about twelve hours before they stopped segmenting in the current of hydrogen. Similar results were obtained by Godlewski | on the eggs of frogs. As far as growth and regeneration are concerned, I have found that without oxygen both are impossible in Hydroids (Tubularia).§ In plants, conditions are the same; seeds require a comparatively abundant supply of oxygen for germination. || The question arises, as to what connection exists between the oxidations in living tissues, and cell divi- sion and growth. We cannot answer this question as we do not know into which form of energy chemical energy must be transformed in order to produce cell division and growth. But another point may be settled. For the process of growth an increase in the quantity of living matter is required, and this requires synthetical processes.

Schmiedeberg has called attention to the fact that oxygen is especially fitted to serve as a connecting link between organic radicals, and that through the intervention of oxygen a great many syntheses in the body may occur. He mentions, as an example, the combination in which sulphuric acid may appear in the urine. When a dog is fed with benzol, the benzol appears in the urine as benzolsulphate, provided that enough free oxygen is present. According to Schmiedeberg, this synthesis occurs in the following way : —

* H. Buchner denies that lack of oxygen increases the rate of alcoholic fermentation by yeast, although the facts seem to speak in favor of Pasteur's statement. E. Buchner, H. Buchner, und M. Hahn, Die Zymasegarung, Miinchen und Berlin, 1903. § Loeb, Untersuchungenzur physiologischen Morphologic der Tiere, II, Wiirzburg, 1891. It is quite possible that the idea of Schmiedeberg will prove ex- tremely fertile in further work in this direction.* But even this addition does not exhaust the role of oxygen in life phenomena; there are indications which make it appear as though the oxygen acted as a protective substance. When respiration is interrupted for but a short time in mammals or birds, loss of conscious- ness, and very soon death, follow. Lack of oxygen therefore affects first the cerebral hemispheres, and especially the ganglion cells. The blood supply to the nerves is either lacking, or is so meager that we must conclude that the functions of the nerves require very little oxygen. The experience in drowning shows that lack of oxygen leads in a limited number of minutes to death. In the case of death through lack of oxygen, the respiratory ganglia evidently undergo irreversible changes, which make attempts at revival futile. The heart retains its irritability much longer than the respiratqry ganglia. Kuliabko has recently shown that the heart of a child can be caused to beat, or to show fibrillary contractions, eighteen hours after death. f This shows that death was not due to the inability of the heart to resume its beat, but to the inability of the respiratory ganglia to work properly. It has often been observed in my laboratory that in dying larvae of fish or frogs the respiration stopped sooner than the heartbeat. The irreversible changes which mark death occur with unequal rapidity in the various tissues of an animal.

When the egg of a Fundulus is kept in an atmosphere of pure hydro- gen, segmentation comes to a standstill in about twelve hours, but permanent death occurs much later. The time required for permanent death to occur in the absence* of oxygen is the longer the younger the egg. When eggs were deprived of oxygen immediately after fertiliza- tion, they remained alive in the absence of oxygen for three or four days (at a temperature of about 22°). When embryos of three days were exposed to the same condition, death occurred in about thirty- four hours.

The higher the temperature the sooner lack of oxygen seems to cause death. I make this statement on the basis of casual observations, and I do not know whether or not any definite experiments exist on this point; should further investigation confirm this idea, it would seem to indicate that the changes in the tissues which cause death are produced by noxious substances formed in the absence of oxygen. Araki \ found that in the active muscle dextrose and lactic acid are found when the

muscle works in lack of oxygen, while these substances are not found in the presence of abundant oxygen. They are probably formed in the latter case also ; but if atmospheric oxygen is present, are immediately oxidized, while in the case of lack of oxygen they remain in the muscle. This may serve as an example of the fact that metabolism in the presence of abundant oxygen is different from that in lack of oxygen. Richet and Broca have shown that if an excised muscle is stimulated in the presence of oxygen until fatigue sets in, it will recover, but not if stimu- lated in the absence of oxygen. It stands to reason that in the latter case the recovery is prevented by noxious substances which would have been oxidized and rendered harmless in the presence of oxygen.

Bacteriology furnishes examples of the fact that in the case of lack of oxygen more virulent substances may be formed, or exist, than in the presence of atmospheric oxygen. Kastle quotes the statement that the toxin of the diphtheria bacillus is weakened under the influence of light in the presence of free oxygen, while the light has no such effect in the absence of oxygen. Pasteur observed that cultures of the anthrax bacillus and of chicken cholera become less poisonous when exposed to the air. Recent experiments by Kastle and Elvove* have shown that substances which have a high reducing power are especially toxic, and these authors are inclined to assume that many toxins belong to the group of reducing poisons.

The fact that lack of oxygen is capable of producing irreversible changes, and thus death, is rendered more easily comprehensible through the direct observation of physical changes of living matter under such conditions. I have made such observations in the segmenting egg of a teleost fish, Ctenolabrus.f When these eggs are deprived of oxygen at the time they reach the 8 or 16 cell stage, it can be noticed that the membranes of the blastomeres are transformed into small droplets within half an hour or more, according to the temperature. These droplets begin to flow together, forming larger drops. Figures i to 5 show the successive stages of this process. When the eggs are exposed to the air in time, segmentation can begin again ; but if a slightly longer time is allowed to elapse, the process becomes irreversible and life becomes extinct. Such clear structural changes cannot be observed in the eggs of other animals under the same conditions. Are these changes of structure (apparently liquefactions of solid elements) responsible for death under such conditions? In order to obtain an answer to this question, I investigated the effect of the lack of oxygen upon the heart- beat of the embryo of Ctenolabrus. The egg of this fish is perfectly trans-

parent and the heartbeat can easily be watched. When such eggs are put into an Engelmann gas chamber and a current of pure hydrogen is sent through, the heart may cease to beat in fifteen or twenty minutes; the heart stops beating suddenly before the number of heartbeats has diminished noticeably: it ceases beating before all the free oxygen may have had time to diffuse from the egg. In one case the heart beat ninety times per minute before the hydrogen was sent through; four minutes after the current of hydrogen had passed through the gas chamber, the rate of the heartbeat was eighty-seven per minute, three