Principles of General Physiology

It will be noticed that in the purine nucleus the two component rings have two . carbon atoms in common. For convenience, each constituent of the purine nucleus is numbered, as in the formula. Purine itself is represented as : — and is described as 2-6-8 trioxy-purine. A large number of important derivatives are known, in which amino-, oxy-, or methyl groups occur in various positions. The substances called nucleins are compounds of a protein with nucleic acid. This latter is itself a compound of phosphoric acid with a pentose (five carbon sugar) and a purine or pyrimidine derivative. There is a whole series of enzymes concerned in the metabolism of nucleins, according to the nature of the particular purine derivative present (see the monograph by Walter Jones, 1914).

Puriiie Metabolism. — Like that of proteins, is exogenous and endogenous. If we look at Folin's table reproduced on p. 94 of Cathcart's monograph (1912), we -may note that, although the excretion of uric acid, taken as representing the purine metabolism, increases somewhat on a diet rich in nitrogen, the relative increase is much less than that of urea. Thus, while urea rises from 2-2 g. to 14'7 g., uric acid only rises from 0'09 to 0'18 g. This indicates, as Folin points out, that the chief source of uric acid is endogenous. In these experiments, purines were, of course, excluded from the diet, as far as possible. At the same time, if purine derivatives are given in the food, in excess of the amount required for maintenance, they are excreted. The data of Hamill and Schryver (1906) show that, on ordinary diet, there is a constant ratio between the uric acid and total nitrogen output. The organism can also form purines from ordinary proteins, as shown by their increase in the developing chick ; before incubation, there are practically no purines in the egg. Although we have no evidence of such synthesis in the adult mammal, it cannot be excluded as a possibility. Moreover, the question is complicated by the fact that there are oxidising enzymes in various tissues, whose action results finally in the conversion of uric acid into urea and oxalic acid. One of the intermediate substances formed is alloxan, whose possible intervention in the process of de-amination we have seen above (page 266). Prof. Hopkins informs me that he has obtained evidence that arginine and histidine together serve as sources of the purine ring.

As regards endogenous uric acid, there are two states in which increased excretion occurs, fever and severe muscular work. Both are associated with breakdown of muscular tissue, so that the uric acid seems to be chiefly derived from this tissue. For further information, the reader is referred to Starling's book (1912, pp. The increased production of uric acid in severe muscular exertion leads us next to consider the question of protein metabolism in work.

Since the endogenous output of nitrogen is to be regarded as the expression of wear and tear of the tissues, it would naturally be expected that muscular work would lead to a marked increase. But it is a remarkable fact that, so long as the work is not excessive and does not lead to pathological conditions, there is practically no change in the nitrogen output, assuming also that the supply of carbohydrate and of oxygen are in sufficient amount. For the various evidence bearing on thia point, the reader is referred to Cathcart's monograph (1912, pp. 109-121). The work of Higgins and Benedict (1911) on the urine of the runners in one of the Marathon races may be added. They were unable to determine the absolute amounts of the various constituents,

but point out the importance of the ratio of carbon to nitrogi-n and of caloric.-, to nitrogen, as indicating normal or perverted protein metabolism. In twelve out of eighteen, the values were normal, in six they were high. In these six, tlinv was practically no lactic acid and no reducing power to indicate disturbance of carbohydrate oxidation, so that the result must be considered to be due to abnormal protein metabolism. Such substances as creatinine, uric acid, aminoacids would account for the increase of the carbon to nitrogen ratio above the normal, where it is given chiefly by urea and ammonia.

There is general agreement that the source of the energy in muscular work is the oxidation of carbohydrate, which will be discussed in the next section of this chapter. At the same time, it is extraordinary that there should be so little evidence of increased wear and tear of the nitrogen-containing machinery of the cell. What explanation can be suggested for this fact? To begin with, although excessive work may be looked upon as pathological, the fact that uric acid is increased in such a condition suggests that there is always an increase of the endogenous protein breakdown due to wear and tear, since the result of excessive work is probably to be regarded merely as an exaggeration of a particular phase of the chemical reactions involved in the whole process of contraction and restitution. Moreover, analysis of muscle itself after work has shown that the purine content is increased (Burian, 1905, M'Leod, 1899), while Brown and Cathcart (1909) and Pekelharing and Van Hoogenhuyze (1910) found an increase of creatine.

Hermann (1867, p. 100 of the first part) distinguished between two processes in muscle, the one the contraction process, by which energy is given out, associated with the production of carbon dioxide, lactic acid and a nitrogenous compound, called provisionally " myosin " ; the other process is associated with a using up of the tissue itself, giving rise to carbon dioxide and creatine. The restitution of the energy-affording material of high chemical potential is effected by the aid of oxygen, and makes use of the nitrogenous product of the breaking-down process " myosin " and probably also of the lactic acid. The restitution of the tissue structure itself requires the supply of some nitrogenous material from without — Hermann says " protein," we should now prefer to say amino-acids or purines. Oxygen is of course required. The resemblance of this view to that associated with the names of Fletcher and Hopkins, which they have established by a large number of experiments, and shown that the contraction itself is a double pr< >• is great, as we shall see later ; the point to be noticed here is the difference between the nitrogenous metabolism in the two kinds of change; the normal contraction results in the separation of a substance which is used up again with the aid of energy derived from an oxidation process of some kind, whereas the wear and tear of the machine itself gives off such nitrogenous compounds as creatine or uric acid, etc., which are excreted and must be replaced by new material.

The name "ntogen" for the complex substance of high energy content was first used l>v Hermann, and will be found on p. 79 of the third part of the book above referred to. The statement is made there that it was suggested in the second edition of the same author's " Grundriss der Physiologic des Menschen," Berlin, 1867. In starvation, the heart while continually at work, does not lose weight ; so that it must be able to utilise nitrogen derived from the other tissues. In Cathcart's experiments (1909), already mentioned, the appearance of creatine in starvation was made use of to investigate the problem of resynthesis. It was assumed that its escape was due to the absence from the tissues of some material which normally caused its retention. It was found to disappear if carbohydrate food was given, but not if either protein or fat without carbohydrate was given. The appearance of creatine is, according to this worker, to be regarded merely as an index of failure of resynthesis, which process only takes place in the presence of carbohydrate.

We saw above (page 269) how important the function of carbohydrate is in the synthesis of protein, so that the hypothesis in its application to muscle is in accordance with other known facts. As to the nature of the chemical changes concerned in this synthesis of protein under the influence of carbohydrate we have, at present, little more than suggestions. We know that simple aldehydes form compounds with ammonia, and it seems more than likely that amino-

acids combine in a similar way with reactive aldehydes or ketones, in the organism. The formation of pyruvic and glyceric aldehydes in carbohydrate metabolism, as we shall see later, is practically certain. Knoop (1910) showed that an a-ketonic acid injected into an animal was converted into the corresponding amino-acid. This is the reverse process to one of the modes of de-amination of amino-acids, as we saw above, and is, apparently, together with the similar conversion of a-hydroxy-acids, the first step in resynthesis of protein.

Many facts relating to this part of our subject have been referred to previously, in an incidental manner. It will have become clear that the great function of carbohydrate food is to afford energy. This applies not only to that given off by muscle in contracting, about which more details will be given in Chapter XIV., but also to that required to bring about endothermic reactions, an example of which we have met with in the case of nitrifying organisms.

The consumption of sugar in the active heart has been shown by Locke and Rosenheim (1907), Rohde (1910), and confirmed by others ; in the intestine by Rona and Neukirch (1912). In these cases, the particular kind of sugar supplied is not a matter of indifference. Dextrose, mannose, and galactose are utilised by the intestinal muscle, and increase its activity. Fructose is not consumed, and has no effect in increasing activity. In what follows I must assume that the reader is familiar with the elementary facts relating to the properties and stereochemistry of the ordinary carbohydrates ; they will be found in the book of Bunge and Plimmer (1907, pp. 106-130) and from some aspects in that of L. J. Henderson (1913, pp. 222-232). The work of Emil Fischer (1884-1908) has been the chief means of our information of the constitution of the sugars ; as we have seen, that of the purines and proteins is also due to him.

Since the organism must have a supply of material for energy purposes, if the more appropriate carbohydrates and fats are absent, protein is used up. This function of carbohydrates as sparers of protein is shown even in starvation, where the nitrogen output falls to one-third of its previous amount if cream and starch are given (Cathcart, 1909). But carbohydrate is more effective than fat; the nitrogen output, diminished by carbohydrate, goes up again on fat only. The fact is probably one aspect of the essential function of carbohydrate for protein synthesis.

Although the ultimate products of carbohydrate metabolism in the organism are, of course, carbon dioxide and water, it is a matter of interest, as well as of importance, to know the stages passed through, since these result in the production of reactive substances, which play an essential part in various physiological phenomena, including the processes of synthesis. The knowledge we possess is due mainly to the work of Embden with his co-workers and of Dakin with his co-workers. To simplify description, a diagram, taken in the main from the results of these investigators, will be of service : —

It will be noticed that most of these reactions are marked as being reversible ; we shall find evidence of this by direct experiment in most cases and, as is pointed out by Embden and Kraus (1912), the processes of hydrolysis, oxidation, and synthesis are all intimately connected iu carbohydrate metabolism. Carbohydrate food, when stored, takes the form of glycogen and this, hydrolysed, becomes glucose as required by the organism. Here we have clearly a reversible reaction and the fact that glucose is produced warrants our taking glucose as the starting point of our investigation.

We may now inquire what experimental evidence there is for the series of changes represented as occurring in the organism. It will be noted that some of the reactions in the numbered paragraphs include more than one step. They are numbered for convenience of future reference and the letter R directs attention to the fact that the reaction so marked is the synthetic aspect of the reaction with the same number. 1. Glucose to Lactic Acid. — Embden and Kraus (1912) showed that the liver, when poor in glycogen, produces lactic acid when blood containing glucose is perfused through it. If the liver contains much g4ycogen, lactic acid is given off, without the necessity of adding glucose.

1. R. Lactic Acid to Glucose. — The previous reaction reversed. In the above experiments, if the liver was poor in glycogen and blood containing lactic acid was perfused, lactic acid was found to disappear. Further, lactic acid is converted to glucose in the dog, made diabetic by removal of the pancreas (Embden and Oppenheimer, 1912, p. 196), (Mandel and Lusk, 1906). The various changes with which we are dealing are, in all probability, some of them certainly, carried out by the agency of enzymes. The conditions in which enzymes favour the synthetic side of reversible reactions will be discussed in the next chapter.

2. Glyceric Aldehyde to Lactic Acid. — Why is glyceric aldehyde instead of dihydroxy-acetone (CH2OH.CO.CH2OH), indicated as the intermediate stage between glucose and lactic acid ? From the action of alkali on glucose (see Dakin's monograph, 1912, p. 86) it is most probable that one or the other of these is the correct one. Embden, Baldes and Schmitz (1912) showed that washed blood corpuscles readily form lactic acid from the former, as they do from glucose, but that from dihydroxy-acetone very little is formed, Jess in fact than from glucose, so that it does not appear to be the normal process. It is remarkable that the unnatural /-lactic acid is formed in larger proportion than the eMactic acid. The liver, when poor in glycogen, has the same effect.

It is probable that the /-lactic acid appeared in these experiments because the racemic glyceric aldehyde was used and the rf-component is used by the liver to form glucose more rapidly than is the /-component, with which the lactic acid reaction has to be content, so to speak. On the other hand, there is evidence that di-hydroxy-acetone is more readily fermented by yeast than is glyceric aldehyde, so that, in this case, it may be the intermediate stage ; although lactic acid itself does not seem to be so (see Harden's monograph, 1911 pp. 90-94).

2. R. Lactic Acid to Glyceric Aldehyde. — I am not aware that this change has, directly, been shown to occur. But, of course, if glyceric aldehyde is an intermediate stage, it must do so, since lactic acid is converted to glucose, as we have seen. 3. Glucose to Glyceric Aldehyde. — This reaction also has not actually been shown to happen, but the same argument as above applies. 3. R. Glyceric Aldehyde to Glucose. — Embden, Baldesand Schmitz (1912, p. 127) have brought evidence to show that the liver performs this reaction.

4. R. Glycerol to Glucose. — Confirmatory evidence of the importance of glyceric aldehyde is afforded by the behaviour of glycerol. Liithje (1904) showed that the diabetic animal can form glucose from glycerol, and Schmitz (1912) found that glycerol, added to blood perfused through the liver, diminished ; although if the liver were rich in glycogen, this did not occur. 5. Lactic Acid from Glycerol. — Oppenheimer showed that lactic acid is formed from glycerol by perfusion through the glycogen-free liver. The obvious way from glycerol to lactic acid is by glyceric aldehyde, as a stage of oxidation, so that the way to glucose is also, no doubt, through the same substance.

5. R. Glycerol from Lactic Acid — Embden, Schmitz and Baldes (1912, p. 185) showed that the liver, perfused with glyceric aldehyde, forms glycerol, so that this reaction, again, is a reversible one. 6. R. Alanine from Pyruvic Acid. — As already mentioned, the formation of alanine from pyruvic or lactic acid has been shown by Knoop (1910), and by Embden and Schmitz (1910). 6. Pyruvic Acid from Alanine. — Neuberg and Langstein (1903) obtained lactic acid from alanine, so that the reaction is reversible and undoubtedly passes through the stage of pyruvic acid. We are, therefore, justified in placing pyruvic acid as a stage further on than lactic acid in the oxidation of glucose.

7. Pyruvic Acid to Lactic Acid. — Paul Mayer (1912) found that sodium pyruvate in excess, administered subcutaneously, gave rise to both glucose and lactic acid in the urine. Embden and Oppenheimer (1913) found that large amounts of lactic acid were produced by perfusion of the glycogen-free liver with pyruvic acid. 8. Pyruvic Acid to Glucose. — See number 7 above. A. I. Ringer (1913) also found that pyruvic acid yields glucose in the organism ; but in certain cases it was found that the amount obtained was much less than when corresponding amounts of lactic acid or alanine were given. Pyruvic acid, apparently, is not a necessary intermediate stage in the conversion of alanine into glucose. What the intermediate stage is will appear presently. Dakin and Janney (1913) state that pyruvic acid is only converted to glucose when the conditions are such as to favour its initial reduction to lactic acid, which is the necessary intermediate stage.

9. Pyruvic Aldehyde to Lactic Acid. — Pyruvic aldehyde is sometimes, not quite correctly, called methyl-glyoxal, but it cannot chemically be regarded as derived from glyoxal (| ) by replacement of a hydrogen atom in an aldehyde group by methyl. Although Embden and Oppenheimer (1913) do not think that this substance is an intermediate stage between glucose and lactic acid on account of its not being optically active, recent work by Dakin and Dudley (1913, 1, 2, 3) indicates that it has, to say the least, considerable importance. These observers find that there is present in nearly all tissues, especially in the liver and muscles, an enzyme, glyoxalase, which acts with great rapidity on "glyoxals" of various composition, transforming them into lactic acid compounds. The presence of this enzyme strongly suggests that pyruvic aldehyde is an intermediate stage between glucose and lactic acid and it might well come in between glyceric aldehyde and lactic acid in the scheme given above. The fact that it does not possess an asymmetric carbon atom and that, on this account, there are not two optical isomers, as in lactic acid and in glyceric aldehyde, is no serious objection to the

view of its importance as an intermediate substance between them. Dakin, indeed, thinks that the fact of its optical inactivity is favourable to the synthesis of dextrose through glyceric aldehyde. Suppose that both optical isomers of lactic acid be present, then, if converted first into pyruvic aldehyde, dextroglyceric aldehyde may be formed from both, by an appropriate optically active catalyst, and, from this, dextrose. In fact, ^-lactic acid, the unnatural form, readily yields glucose in the animal organism, when rendered diabetic by phloridzin (Dakin and Dudley, 1913, 2, p. 129). We shall see later that an optically active catalyst is able to form, from optically inactive substances, a preponderance of one optical isomer of an optically active product. When acting on pyruvic aldehyde, glyoxalase yields a mixture of the two forms of lactic acid, but in unequal proportion, and the authors think that two enzymes are concerned, since an enzyme preparation, giving, when fresh, a preponderance of the Isevo-acid, after standing, gave an excess of the dextro-acid, when acting on a new supply of the substrate. Glyoxalase appears to have a wide distribution ; it has been found in the oyster and in yeast. It is absent from the pancreas and a substance is present in extracts of this gland which has the power of actually inhibiting the action of glyoxalase (Dakin and Dudley, 1913, 3). These facts are significant in view of the profound relation between the pancreas and carbohydrate metabolism.

9. R. Lactic Acid to Pyruvic Aldehyde. — Dakin and Dudley (1913, 1) showed that lactic acid is readily converted into pyruvic aldehyde by digestion with nitro-phenyl-hydrazine. Further, that in faintly acid solution both lactic acid and alanine are decomposed with formation of pyruvic aldehyde : By the action of glyoxalase, then, lactic acid can be obtained from alanine through the intermediation of pyruvic aldehyde. With the exception of the direct conversion of pyruvic aldehyde to alanine, all the reactions involving the interconversion of glucose, pyruvic aldehyde, lactic acid, and alanine are shown to be reversible and the authors named have obtained the analogous synthesis of glycine from glyoxal.

Acetaldehyde. — Neubauer (1909) showed that a-ketonic acids are changed in the organism into the ordinary fatty acid with one less carbon atom ; so that pyruvic acid will go into acetic acid. In this process it does not seem possible that any intermediate stage other than that of acetaldehyde would be passed through. According to Neuberg and Karczag (1911) yeast juice ferments pyruvic acid with the production of carbon dioxide and acetaldehyde. Masuda (1912) found that the liver, perfused with blood containing alcohol, forms aldehyde and Enibden and Baldes (1912) that the reverse change from acetaldehyde to alcohol also takes place, even in the presence of oxygen.

There is no evidence that ethyl alcohol is a direct stage in the oxidation of glucose in the animal organism, but it appears that acetaldehyde may well be a stage in the formation of alcohol from sugar in fermentation, although it does not seem to be a necessary one. Alanine is fermented by yeast with formation of alcohol, carbon dioxide, and ammonia and the most likely stages seem to be pyruvic acid and acetaldehyde. Mention may be made of the fact that, under certain conditions, probably of deficient oxidation, ethyl alcohol may be obtained by distillation of various tissues, especially nni-rlr. The possibility of absorption from the alimentary canal seems to have been excluded in some of these experiments, although it must be admitted that it does not appear to be an easy matter to lie certain that it is so.

Aceto acetic-acid is produced by the liver from pyruvic acid (Embden and Oppenheimer, 1912). It must be formed by aldol condensation from acetaldehyde, through jft-oxy-butyric acid. " Aldol con(l<>i*<itif>n," the reader may be reminded, is simply the union of two molecules of an aldehyde, which may lie effected, for example, by the action of strong hydrochloric acid, thus : — Aceto-acetic acid is found in the urine in certain pathological states associated with distui'bed carbohydrate metabolism and has been found by Masuda (1912)

to be formed by the liver from ethyl alcohol through the intermediate stage of acetaldehyde ; so that acetaldehyde is, as it were, the meeting place of two reactions, both leading to aceto-acetic acid, the one from pyruvic acid, the other from ethyl alcohol. The further oxidation of aldehyde to carbon dioxide and water is probably through acetic acid, as suggested by Neubauer's change of pyruvic into acetic acid in the organism. This would then be the chief reaction ; those leading to alcohol or to aceto-acetic acid diverging at the acetaldehyde stage.