Principles of General Physiology

With regard to the first step in the process, it has been shown by Dakin and Dudley (1913, 3) that an a-amino-acid in solution in water undergoes spontaneous dissociation into the corresponding a-ketonic aldehyde and ammonia and this fact makes it probable that the process is accelerated in the organism by an enzyme. Alanine becomes in this way pyruvic aldehyde and ammonia. The ketonic aldehyde may undergo further change in three modes, oxidation, hydrolysis, or reduction, thus : —

Incidental^', it may be noted that, as we shall see later, there are three series of enzymes, known to be present in cells, capable of effecting these three processes of oxidation, hydrolysis, or reduction, respectively. De-amiiiation. — The whole of the blood from the intestines, containing aminoacids, in the mammal, passes through the liver before reaching the various other organs and tissues. In other vertebrates, a part of it goes this way. The liver has the power of converting ammonium salts into urea, as was first definitely proved by Schroder (1882 and 1885). We might expect, then, that the main body of the amino-acids would be first de-aminated in the liver, the resulting ammonia converted to urea, while the fatty acid remainders would be sent on to

the tissues. A certain fraction of the amino-acids must be supposed to escape the action of the liver, in order to afford the nitrogen required by the tissues for growth and maintenance. There are, however, certain difficulties in this view. In the first place, although these acids in de-amination lose very little energy (see the work of Leathes, 1906, p. 154), so that, as far as their actual content of energy is concerned, the fatty acids are very nearly as valuable, yet there must be for this purpose alone some way in which, as it appears, the amino-acids are superior to the simple hydrocarbon acids. The taking of protein food causes a greater increase in the total metabolism than a corresponding number of calories taken as fat or carbohydrate. This is sometimes spoken of as the " specific dynamic energy " of Rubner. It seems possible that the products of the de-amination reaction are more available in the course of the reaction itself, that is, in the " nascent " state. For this reason, it appears to be of advantage that the de-amination reaction should take place in the tissue cells generally, and the ammonia sent on to the liver for conversion to urea. The small amount of energy given off in de-amination would also be available. At the same time, too much stress must not be laid on this point of view, since the chief use of nitrogen food is to repair waste of tissue and not to supply energy, and the actual amount demanded by the adult animal is not great.

With regard to the "specific dynamic action " of proteins, Williams, Riche, and Lusk (1912, p. 374) hold that the effect is due to the large influx of amino-acids causing increased metabolism by their mass action on the cell protoplasm and that the view of Rubner does not explain the facts, nor does that of increased intestinal activity. In the second place, certain experiments tend to show that the liver has not much more power of de-amination than the other cells of the organism. The experiments of Lang (1904) and of Miss Bostock (1911) have shown that tissues in vitro are capable of de-aminating amino-acids to some extent, but that the process is not quite the same as that in the living organism, since amides are more readily acted on in vitro than are amino-acids, while the contrary is the case in the organism. That the main part of the amino-acids absorbed by the intestine escape immediate de-amination by the liver is also shown by the results of van Slyke and Meyer (1912, p. 408). The blood of the femoral arteyy contained in one experiment, before feeding, 3*7 mg. of amino-acid nitrogen per 100 c.c., and after feeding, 8 '6 mg., while that of the portal vein, at the same time, contained very little more, namely 9 -5 mg. This means that there was a loss of only 0'9 mg. in traversing the liver, not more than would be expected if the liver only de-aminated as much in proportion to its size as other organs.

After large doses of amino-acids the de-aminated products can be detected in the urine. Lactic acid from alanine (Neuberg and Langstein, 1903), glyceric acid from diamino-propionic acid (Mayer, 1904) may be referred to. Moulds, bacteria, yeast, and the larvae of flies have also been shown to split off ammonia from amino-acids. We may conclude that amino-acids are supplied to the tissues, and, with the exception of that small part used for repair or growth, are de-aminated there.

The fatty acid part is utilised for supply of energy and the next question is the fate of the ammonia. The great activity of the liver in the conversion of ammonia to urea makes it probable that the main part of the ammonia from the tissues is converted into urea in this organ. When an Eck's fistula is made, that is, when a connection is made between the portal vein and the vena cava, so that the liver is practically cut out of the circulation, there is a great increase of ammonia in the blood. Normally, also, there is much more ammonia in the portal blood going to the liver than in that of the hepatic veins coming from it. The investigations of Nencki and of Salaskin may be consulted (see Cathcart's monograph, 1912, p. 51).

On the other hand, Folin and Denis (1912) hold that the tissues themselves have the power of converting ammonia into urea. They find that the urea content of the blood of the hepatic vein, after the injection into the lumen of the intestine of various proteins and amino-acids, is not greater than that of the femoral artery. This implies that the liver has not added any more urea to the blood than was already in that arriving to it from the other parts of the animal. Should this be so, all tissues must take part in the formation of urea. In the words of these authors (1912, p. 161), "the food protein reaches the tissues in the form of amino-acids, and those amino-acids which are not needed for the rebuilding of broken-down body material are not rebuilt either into protein or protoplasm, but are broken down and their nitrogen converted into urea."

The results of van Slyke and Meyer (1913, 2), contrary to those of Folin and Denis, are in favour of the first view, that the liver is the chief, if not the only, situation where de-amination occurs. Amino acids, as we s.-i\v, air taken up rapidly by all tissues. Those taken up by the liver disappear again in a comparatively short time, but, during the time required for this disappearance from the livei1, no appreciable diminution has occurred in that stored in the muscles. From other organs also they disappear less rapidly than they do from the liver. This diminution of amino-acid content of the liver is accompanied by increase of urea in the blood. The liver, therefore, continually tends to decrease the amino-acid content of the blood, and, since there is always an equilibrium between the amino-acid concentration in the blood and that in the tissues, as the liver removes these acids more passes from other tissues to restore equilibrium. We see then that it is not neress.n v that de-amination should be performed by any tissue other than the liver. In a further paper (1913, 3) the same investigators show that in starvation the ami no-acid content of the tissues does not decrease. It is most probably renewed by autolysis of protein, so that the amino-acid protein system appears to be that of a reversible chemical reaction. This view is supported by the fact that feeding with large quantities of protein does not increase the aminoacids of the tissues, so that any nitrogen stored beyond the normal amount of amino-acid must apparently be in the form of protein.

In any case, we may conclude that nothing is left of the old discussion bet\v"<>n Pfliiger and Voit as to the necessity of food protein becoming living protoplasm before being utilised. If the view taken be correct, we see also that, so long as the "accessory factors " are present, there is no necessity for the food proteins to be of similar constitution to the tissues. In fact, experimental evidence confirms this deduction ; except for differences in degree of digestibility, and so on, there does not seem to be any particular preference for one protein rather than another.

A further consequence is that feeding with pure amino acids should be possible. The evidence that this can be done has been referred to above (page 256). As to the chemical mechanism of de-amination we have little information. Probably all the three reactions given above (page 264) are concerned. An old observation hy Streckcr referred to by Bach (1911, pp. Io7, 158) is interesting a- n possibility of the formation of hydroxy-aeids and aldeh3'des from amino-acids. Alloxan n M< i- in -Mijes with amino-acids thus : —

The general facts of protein metabolism may be looked at from a slightly different point of view, as in the original expression of his theory by Folin (I !><>.'», who carried out a large number of analyses of urine on two kinds of diets, rich and poor in nitrogen, but both practically free from purines, creatine or creatinine (see later, page 270). Comparing the two series, we note that there are some products of metal>olism which maintain a nearly constant figure, while others are much greater under rich nitrogen food than under food poor in nitrogen. The constant products

are chiefly creatinine and neutral sulphur ; to a less extent, uric acid and ethereal sulphates. The variable products are urea and inorganic sulphates, not creatinine and probably not neutral sulphur. The former obviously represent some constant form of metabolism always proceeding, to which Folin gave the name of tissue or endogenous metabolism, and to the variable type, that is, to the proteins used for de-amination and giving of energy, the name intermediate or exogenous metabolism. This view has been generally accepted, although some minor points as to the complete distinction of the particular products in each case have been taken exception to. Folin himself admits that urea is probably an end product of both. According to Cathcart (1912, p. 95), the output of creatinine is itself subject to small changes when the protein ingested is altered.

The practical interest and importance of the question rests on the fact that the amount of nitrogen which it is absolutely necessary to take in the food, is, in theory, limited to that required bo form new tissues or replace wear and tear ; that is, the endogenous fraction. Now the nitrogenous food is the most costly part of a diet, so that it is of some importance to know how far it can wisely be reduced. The value of the nitrogen minimum is therefore a question requiring discussion.

Firstly, what is the excretion of nitrogen in starvation 1 This may be taken as the index of the waste of tissues, with certain qualifications. But it will be clear that, for our present purpose, what we want to know is the loss of nitrogen when sufficient carbohydrate is supplied for energy purposes, nitrogen being absent from the food. The tissue proteins begin to break down in complete starvation in order to afford the energy demanded by certain organs of vital necessity, such as the heart, so that the issue becomes confused. If a particular excretory product, under normal diet, were definitely known to be a product of endogenous metabolism and of this alone, it would be more satisfactory to determine the amount of this substance excreted. We cannot, as yet, be quite certain as to the existence of such a product, although, according to Cathcart (1909), creatine, a constituent of muscle tissue, is such a product, present only in starvation, so that the study of its excretion gives valuable information, to which reference will be made later. Some doubts, however, have been thrown by Graham and Poulton (1913) on the cogency of the method used to estimate creatine in the urine. According to these workers, there is no satisfactory evidence of the presence of this substance in the urine, under any circumstances. According to Cathcart and Orr (1914), however, these results do not affect the conclusions drawn by Cathcart from his experiments. But, in any case, there is, according to M'Collum (1911, 1), another index in the output of creatinine, a product obtained from creatine by removal of water (see below, page 270). This is a constant fraction of the total nitrogen eliminated after a long-continued diet free from nitrogen. In the pig, the creatinine nitrogen is, under these conditions, 18 '5 per cent, of the total nitrogen excreted.

So that if the creatinine nitrogen be multiplied by 5 -5, the total nitrogen resulting from endogenous metabolism is obtained. This conclusion rests on the fact that creatine or creatinine is a characteristic product of the breakdown of muscular tissue. In a later paper M'Collum and Hoagland (1913) show that this conclusion requires certain modifications, which must be taken into account in attempts to make use of it. There are, they say, at least two types of endogenous protein metabolism, one which can be stimulated to increased production of ammonia by feeding with mineral acids, or to hippuric acid production by glycine, while the other, which is represented by creatinine, remains unaffected by these agents.

From Cathcart's experiments (1909) it appears that, in man, the total output of nitrogen on a carbohydrate diet, free from nitrogen, is about 5 g. per day. Now Voit had laid it down that the daily intake of protein should be 120 g., equivalent to 18 g. of nitrogen. Chittenderi (1905) regards this as far too much and was able to maintain nitrogen equilibrium on 6 g. of nitrogen (40 g. of protein) in various classes of men engaged in different kinds of work. There is no doubt that Voit's amount is considerably in excess of that taken by a large number of men. For example, Hamill and Schryver (1906) determined

the nitrogen output in the urine of seven of us who were working in the Physiological Laboratory of University College, London, at that time. No alteration was made in our occupations nor in the food taken, except that a dinner of the Physiological Society occurred on one day, which tended to increase the general average. The values obtained were from 0-16 to 0'2 g. of nitrogen per kilogram of body weight, or an average of 13*5 g. per individual, equivalent to 93 g. of protein ; a value only three-quarters of that given by Voit, although rather more than twice that regarded by Chittenden as adequate.

A point of interest is that in Rowntree's " Poverty, a Study in Town Life," the author has adopted Atwater's standard of 125 g. as the minimum protein and consequently finds that 27 per cent, of the population of York are living in poverty, because their protein consumption is below this figure. Jn point of fact, the lowest value found was 89 g. , very little below that of the lalxiratory workers, and this applied only to those whose weekly wage was lwlo\\ twenty-six shillings. Caution must then be exercised in drawing conclusions as to social conditions from protein consumption. One would have to conclude that physiologists as a class are living in poverty.

Cathcart (1912, p. 69) regards 90 g. of protein as an average value, from his own experience. This author's discussion of the question will be found on pp. 66 to 72 of his monograph (1912). We may note that Siven (1901) found it possible to maintain nitrogen equilibrium on 4 '52 g. of nitrogen (=28'3 g. of protein) per day. But there seems some evidence that continued existence on so low a protein diet may entail low resistance to external influences, such as infection, although this effect is by no means clearly made out and the results of Hindhede, to be given immediately, show that it is not necessarily the case.

The degree of activity of the organism is naturally to be taken into account. We may recall M'Collum's experiments on pigs (1911), in which the total nitrogen required for maintenance appears to be only 2'6 g. for a pig of about the weight of a man. The recent work of Hindhede (1913) affords some valuable data on the question before us. In his experiments, care was taken that the total calorie value of the food was abundant, a point of essential importance, as Cathcart points out (1912, p. 70), and not sufficiently ensured in some of the experiments of Chittenden, in which it was too low. A further point of importance in Hindhede's experiments is that they were continued for a considerable time. A strong, healthy young man of t70 kg. weight, a laboratory servant in the Nutrition Institute of Copenhagen, was the chief subject. It was found that, while continuing to perform all his usual duties, he was able to live on a diet consisting only of potatoes, apparently new potatoes, together with margarine and a little onion for flavour, and containing, on the average, only 4 -425 g. of nitrogen per day. This experiment lasted 178 days and although 75 g. of nitrogen had actually been lost from the body, it was not possible to discover that the subject was otherwise in any different condition than at the beginning of the period. From the figures given, it appears that he was in nitrogen equilibrium during the actual time on which this diet was taken, and that the loss of nitrogen occurred in one or two short periods in which less nitrogen was taken, owing to replacement of the greater part of the potatoes by fruit. During the 1 50 days on which the potato diet was taken, nitrogen equilibrium was present on 5 g. of nitrogen per day. Taking one particular period of nineteen days, in which all the conditions were especially satisfactory, nitrogen equilibrium was maintained on only 3 '5 g. per day.

It is to be remembered that, on this potato diet, which seems to be the only one which can be put up with for so long a time, it was impossible to reduce the nitrogen further without diminishing the calorie value below that which was found to be essential, namely 4,000 calories per day. We may remark also that the method of cooking the food was found to be a matter of great importance, so that it should be sufficiently palatable to be taken with relish in large enough quantities to give the calorie value required, in fact, about 2-2 to 3-5 km., according to the severity of the work done. To assign proper value to the experiments, it is pointed out that the subject was really more than an ordinary laboratory servant ; he performed the duties of an assistant, working fourteen to sixteen hours a day, extremely active and taking great interest, not only in the experiments described, but also in the work as a whole. We may note the high calorie value of the diet ; that given by Voit for soldiers in war-time had an energy value of only 3,575

calories, although it contained 145 g. of protein, equivalent to 23*2 g. of nitrogen. A second experimental period was undertaken in which the subject performed hard work as mason and labourer for a term of ninety-five days. On a diet of about 5,000 calories, with an average of 7 '22 g. of nitrogen per day, a slight loss of nitrogen resulted, namely, 34 g. for the whole period. To get the nitrogen minimum for hard work, the last ten days of the period may be taken, in which nitrogen equilibrium was maintained 011 5'72 g. of nitrogen ( = 35'75 g. of protein).

An important question is, naturally, whether this subject was in any way the worse for this prolonged period of minimal nitrogen diet. It must be admitted that he had lost a certain amount of nitrogenous substance, although there was every evidence that his condition was just as good as at the beginning. No period of recovery was necessary and, indeed, he was anxious to begin a new experiment. Experiments were also made by Hindhede on himself and on a student with similar results. The former gave a protein minimum of 16 g., with a calorie value of 2,650, doing light work. The latter was doing moderate work on a diet of 3,700 calories and protein content of 25 g.

It appears that we must admit that, for a strong healthy man, the protein food actually necessary to replace wear and tear is very much less than that usually assumed. It is interesting to notice that, as would be expected, the wear and tear in hard work is greater than in moderate work, if we may judge by the rise in the protein minimum from 25 g. in the latter case to 35 g. in the former. But it is found to be the same fraction of the total intake in energy.

Effect of Carbohydrate. — In the experiments on feeding with the digestion products of proteins already referred to, it may have been noticed that, while Loewi (1902) was successful, certain other workers were unable to confirm his results. Cathcart calls attention to the fact that, in Loewi's experiments, carbohydrate was present to make up the proper calorie value, whereas in the experiments that failed, fat only was used. Further, Cathcart himself (1909) found that, if no carbohydrate was present in a nitrogen-free diet, creatine appeared in the urine, whereas it was absent when carbohydrate was given. The interpretation to be put on these experiments is that, in the presence of carbohydrate, resynthesis of creatine into some cell protein takes place, so that it would appear that some of the nitrogen lost in wear and tear can be made use of again by the aid of carbohydrate. It seems, however, from the results of Graham and Poulton (1913), that a repetition of these experiments is desirable, although Cathcart himself, with Orr (1914), points out that they do not affect his conclusions.

Other experiments confirm the necessity of carbohydrates for the synthesis of protein. It was shown by Hansteen (1899) that it applied to the higher plants and by Felix Ehrlich (1911) that amino-acids were incapable of acting as sources of nitrogen to yeast in the absence of carbohydrate. Maintenance. — Certain evidence has already been referred to which suggests that, in the wear and tear of active cells, it is not the whole of the large molecules of the nitrogenous constituents of the protoplasmic system that are broken up. One may state the fact either in the form that certain " side-chains " only of a giantmolecule or "biogen" are disintegrated, or that certain chemical individuals, forming part of the total reaction systems of the cell mechanism, are decomposed, perhaps by subsidiary reaction. Reasons have been given above (page 19) for regarding as doubtful the " biogen " view, and further evidence against it will be found on page 498, but, in the present state of knowledge, decision is impossible.

As to the fact that protoplasm itself does not break up, some additional evidence may be mentioned here. M'Collum (1911, 2) feeds pigs for a sufficient time on protein-free diet to obtain a constant ratio between the creatinine and the total nitrogen output; the total nitrogen is then taken as being that due to endogenous metabolism. The food protein to be tested is then introduced into the food in quantity equivalent to the nitrogen excreted, an isodynamic portion of the • carbohydrate food being withheld. The experiments of most interest in the present connection are those with zein and with gelatine. Zein contains neither glycine,

lysine, nor tryptophane, but an excess of glutamie acid ; gelatine contains neither tyrosine nor tryptophane, but an excess of glycine. The animal, however, utilises tinnitrogen of zein to the extent of 80 per cent., and that of gelatine to 50 or 60 per cent. This is shown by the fact that, instead of the extra nitrogen given appearing in the urine, as would happen if it were not utilised for repair, only 20 per cent, or 40 per cent, respectively is excreted. On the other hand, when zein is given, even in considerable excess over maintenance need, no evidence is obtained of the formation of new body tissue ; whereas, if casein is given, 20 to 25 per cent. increase in body protein results. It seems evident that the repair processes are of a different character from those of growth. The processes of cell wear and tear and their repair do not appear to involve the destruction and resynthesis of an entire protein molecule.

In the investigation of the endogenous nitrogen metabolism, the importance of creating has been pointed out, so that a few words as to its chemical nature are advisable. It may be looked upon as a substituted guanidine, in that one of the NH2 groups is replaced by methyl-glycine. Thus : — When boiled with dilute acids, it loses a molecule of water and is converted into creatinine, an internal anhydride, with basic properties, since the COOH group has disappeared : —

Creatinine is converted again into creatine in alkaline solution (see Bunge-Plinimer, 1907, pp. 153-155). The method used for estimation is that of Folin (1904), which depends on the colour reaction of creatinine with alkaline sodium picrate, as described by Jafle. As micleiiuf are important constituents of the cell nucleus, it is to be expected that their metabolism would be chiefly of the endogenous kind. Before discussing the question, the chemical nature of these substances must be indicated. As already described, their characteristic group is the purine nucleus, the chemistry of which has been completely worked out by Emil Fischer (1882-1906). It may be regarded as a fusion of the pyrimidine and imin-azole rings, thus : —