Principles of General Physiology

Another mode of oxidation of glucose should be referred to, namely, that to glucuronic acid, in which the CH.,OH group of glucose is converted into COOH. Further stages of oxidation would yield saccharic and oxalic acids. Camphor, administered to an animal, is excreted in combination with glucuronic acid (Musculus and von Mering, 1875). That this acid arises from oxidation of glucose is shown by the experiments of Paul Mayer (1902), who found that, in inanition, in which very little glycogen remains stored up, scarcely any glucuronic acid was excreted on administration of camphor ; whereas, if glucose was administered at the same time, the usual amount was obtained. It is doubtful whether, normally, further oxidation takes place along this path, siace oxalic acid is only oxidised with great difficulty in the organism (Dakin, 1912, p. 45). At any rate, this mode of oxidation of glucose is not the chief or normal one.

Diabetes. — If the pancreas be removed, and in some pathological conditions, large quantities of glucose are excreted by the kidneys. A remarkable fact is that, even after all carbohydrate stores are used up and none is given in the food, the organism breaks down body-protein in order to form glucose. From the experiments of Cathcart (page 269 above) we have seen the necessity of carbohydrate for protein synthesis and the facts of diabetes suggest that the cells imperatively demand carbohydrate. It is interesting to note that, even after a prolonged fast, sugar is never absent from the blood of the normal animal.

The experiments of Lusk (1910) have shown that glycine, alanine and three of the carbon atoms in aspartic and glutamic acids are converted into glucose in the organism and that 100 parts of meat can give 58 parts of glucose. Since the reaction is, doubtless, reversible, the possibility of production of various amino-acids from glucose is shown. For further information on the question of diabetes see Starling's "Textbook" (1912, pp; 903-914). According to Parkin (1911), saccharose is the sugar of most importance in the plant, both as reserve carbohydrate and as circulating sugar. It serves, in fact, as regards carbohydrate, much the same purpose as asparagine in respect of protein metabolism (Horace T. Brown, 1906). Saccharose has properties that fit it especially for such purposes. It is very soluble and yet easily crystallises. It is easily hydrolysed by acids and by invertase. It- has no reducing properties, since the aldehyde group is not functional. It appears that it can be condensed to starch, without previous hydrolysis, and probably also to cellulose.

The fats taken as food, or found in various situations in the body of the organism, are the tri-glycerides of the higher fatty acids, sometimes accompanied, as in milk, by small amounts of the glycerides of the lower fatty acids, butyric, caproic, etc. The acid may be either a saturated one, as stearic, or an unsatu rated one, such as oleic, in which there are carbon atoms united by double bonds ("ethylene linkage"). The substances known as "lipoids," which were described above (page 130), are also found in the tissues. The function of these in the formation of the cell membrane has also been discussed.

1. From Fat in the Food. — If not oxidised for energy needs, fats taken as food appear to be deposited in the tissues without change. Lebedev (1882) fed dogs, which had lost the greater part of their fat from inanition, either on a diet containing mutton fat in considerable amount, or on a similar diet containing linseed oil in place of the mutton fat. After some weeks, it was found that the fat of the dog which had received mutton suet was solid at 50° C., whereas that of the dog fed on oil was still liquid at 0° C. Although fats are hydrolysed in the lumen of the intestine, their constituents are resynthesised in the wall of the intestine, and are carried in the chyle to the blood as neutral fats. The part played by enzymes in this process will be discussed later.

2. From Carbohydrate in the food. — Although the ^formation of fat from carbohydrate in the process of fattening animals for food seems obvious in the ordinary practice of farmers, complete evidence was wanting until the experiments of Lawes and Gilbert (1852, p. 350) on young pigs, fed on barley, in which it was shown that the amount of fat laid on was .considerably greater than could possibly have come from the protein in the food, after deducting that used for tissue formation in the body. The amount of fat in barley food is very small.

Until the recent work of Miss Smedley (1912) the chemical mechanism of such a transformation was unknown. As regards the glycerol component, the facts described in the previous section show how it is readily obtained from glucose. As regards the fatty acid component, the first fact to be noticed is that the fats of the organism are limited to those whose fatty acids contain an even number of carbon atoms. There must obviously be some reason for this. Another fact is that the process of formation from carbohydrate is, as Leathes has shown (1906, p. 85), an exothermic one. This might, at first sight, seem surprising, since the heat of combustion of a gram-molecule of fat is so much higher than that of a gram-molecule of glucose. But we must remember that several molecules of sugar are required to form one of a higher fatty acid ; for example, stearic acid contains eighteen carbon atoms.

Miss Smedley and Miss Lubrynzka (1913, 1 and 2) have brought forward good evidence to show that the process of fat synthesis in the organism takes place in the following way : We have seen that pyruvic acid is formed as a stage in the oxidation of glucose. Moreover, there is reason to believe that pyruvic acid is converted by a further process, probably enzymic, into acetaldehyde and carbon dioxide. This aldehyde may then condense with another molecule of pyruvic acid to form a higher ketonic acid, thus : —

The investigators named have succeeded in obtaining this reaction with butyl aldehyde and pyruvic acid. The next stage is the conversion of the ketonic acid thus obtained into its aldehyde and carbon dioxide, by a similar process to that by which the acetaldehyde was originally obtained from pyruvic acid : — This aldehyde, which has two more carbon atoms than that from which we started, condenses with another molecule of pyruvic acid, forming a ketonic acid of still longer carbon chain, and so on.

From the unsaturated ketonic acid formed at any stage we obtain, by oxidation, an unsaturated fatty acid with one less carbon atom, thus :— and from this, by reduction, the fatty acid containing two more carbon at«'m> than the aldehyde from which we started in this particular stage. For example :— that is, we have obtained butyric acid starting from acetaldehyde. The process may be repeated many times, and fatty acids with a long chain of carbon atoms obtained. It is necessary to remember, however, that branched chains are not formed in this comparatively simple manner.

The reverse change from fat to carbohydrate is known to occur in the germination of fatty seeds, where starch and cellulose are formed from the fat. In hibernating mammals, as Pembrey (1903) has shown, there is an extraordinarily low respiratory quotient of 0'3-0'4. This means that there is a conversion of substances containing a small amount of oxygen into others containing a larger amount of oxygen, that is, fat into carbohydrate. It can be shown by the increase of weight that oxygen is actually retained. When a marmot converts the carbohydrate of its food into fat, preparatory to hibernation, the respiratory quotient is high.

This numerical quantity, the respiratory quotient, has not yet been explained in these pages?. It is simply the ratio of the volume of the carbon dioxide given out to that of the oxygen taken in. Carbohydrate may be looked upon as consisting of carbon plus water, so that, if this were the only material oxidised in the organism, the oxygen would have been entirely used to combine with the carbon and the respiratory quotient would be unity. In fat, on the other hand, besides the carbon there is also hydrogen to be oxidised, as easily seen by the formula, say of palmitin, CjjH^Oe. Part of the oxygen taken in is used for the oxidation of hydrogen, so that there is less carbon dioxide given out than that equivalent to the oxygen taken in and the respiratory quotient is less than unity. The determination of the respiratory quotient enables us to see what is being oxidised, when different diets are given.

3. Fat from Protein in the Food. — Although ammo-acids are de-aminated in the organism, so that pyruvic acid is formed from alanine, and,* from pyruvic acid, as shown in the preceding section, higher fatty acids can be synthesised, it is a remarkable fact that all evidence tends to show that no fat is laid on by the organism, however large a diet of pure protein is taken. The effect is simply to increase the nitrogenous and general metabolism. In certain toxic conditions there appears at first sight to be a change of the protoplasm of the cells, especially of the liver, into fat. But careful investigation has shown that there is no actual increase of the total fat of the body ; what happens is that fat from other parts migrates to the liver and there is also some kind of aggregation of the lipoids of the protoplasm, so that, from being invisible as a distinct phase, they become particles or droplets, readily seen under the microscope.

That fats can be used as sources of energy, in muscular contraction, for example, is shown by the respiratory quotient. We have just seen that, if fat is being consumed, this value falls. When muscular work is performed with a diet consisting almost entirely of carbohydrate, the respiratory quotient is 0*9 ; when fat is exclusively taken, it falls to 0'72. This shows that in the latter case a substance containing oxidisable hydrogen as well as carbon, that is, fat, is being consumed.

The chemical processes taking place in the utilisation of fat are not definitely known. But it is altogether probable that the process of synthesis from lower fatty acids, described above, goes also in the reverse direction and, when arrived at, these acids are quickly oxidised. There is also direct evidence of the breakdown of fats into aceto-acetic and /3-oxy-butyric acids, together with acetone, in diabetes, where they appear on a diet formed exclusively of fat and protein, In the normal organism, an exclusively fatty diet, continued for some days, has been found to give rise to large quantities of these partially oxidised products.

It will have been noticed how frequently this compound makes its appearance in various metabolic processes. It is interesting to collect together these facts. It is converted into alanine by a reversible reaction. It is a stage in the oxidation of glucose, so that all the substances contained in the scheme on page 273 above can be obtained from it. Further, Miss Smedley's work has shown how higher fats can be synthesised by starting from pyruvic acid. Fats, carbohydrates, and proteins, therefore, come into connection at this meeting place.

We saw above that the living cell of Aspergillus is able to form several different amino-acids from nitrate. It was pointed out, also, that if the appropriate a-ketonic or hydroxy-acid were available, the corresponding aminoacid could be formed by the liver. But the only such acids known to be present in the organism are pyruvic and lactic, from which alanine alone is formed directly. Miss Smedley's work, however, has shown. how an unsaturated a-ketonic acid with two more carbon atoms can be obtained from pyruvic. From this, by addition of hydrogen, the saturated ketonic acid may be formed, and therefore the amino-acid. But the process has its limitations, since it only gives us a straight chain of carbon atoms, while leucine, for example, has a branched chain. See also Lusk's results, page 277 above.

The ivailt-r will have noticed that many of the reactions described as stages in the metabolism of proteins, fats, or curbc (hydrates rest upon evidence derived either from perfusion of isolated organs or from experiments with extracts of tissues in rifro. Some investigators appear to doubt whether legitimate conclusions, as regards the processes taking place in the organism as a whole, can be drawn from experiments of this kind. It does not seem to me that sucli criticism is justified. Investigation of the excreta compared with the ingesta, valuable as the information is for certain purposes, gives us very little knowledge of the chemical reactions by which the latter are converted into the former. The comparison of the organism to a town, where various occupations are carried on, is often made. If we notice that a large quantity of milk goes into the town and that a corresponding amount of cheese comes out, we conclude that the milk has been used to make the cheese, but we learn nothing about the method employed. Still less is learned by such methods with regard to the more intellectual occupations, such as that of the poet or musician. Hopkins has made use of the simile of a conjuror, who puts a loaf into a hat and takes out a rabbit. What we want to know about are the intermediate stages between the loaf and the rabbit.

Looking at the question from another point of view and taking, for example, the oxidation of glucose, it is surely permissible to consider what are the possible chemical changes that might take place. Suppose then that we find certain of the possible reactions to be brought about by extracts of tissues, while others, also chemically possible, are not; we are, it seems to me, justified in stating that the former is the way in which the organism works, at all events until the contrary has been actually proved.

Again, as to perfusiou experiments : when pyruvic acid is added to blood passing through the liver and alanine is found in the issuing blood, it cannot be denied that, even in the whole organism, if pyruvic acid be present in the portal blood, alanine will be found in the blood of the hepatic vein. If it be objected that alanine might come from the substance of the cells themselves, it may be pointed out that, when the related a-ketonic acid of butyric acid is perfused, u- amino-butyric acid is formed. It seems extremely improbable that the permeability of the cells should be affected by closely related ketonic acids in such a way that the corresponding amino-acid, and this only, is washed out of them.

Since the reactions under discussion are reversible, take for example that between lactic acid and glucose, it is scarcely credible that lactic acid should wash out or cause the cells to give up glucose, while glucose causes them to lose lactic acid. The organism is capable of storing, in some form or other, the chief classes of food-stuffs, although in different degrees. Carbohydrates. — These are stored, mainly in the form of glycogen, in the liver and muscular tissues for the most part. Glycogen, being an insoluble substance, is kept out of the risk of undesirable participation in chemical reactions ; on the other hand, while soluble carbohydrate is required, the action of the enzyme, amylase, converts it into maltose or glucose. In the plant, starch is the iimst common form of stored carbohydrate, but saccharose is very frequently met with. Soluble carbohydrates do not appear to be stored in the animal, at all events in more than very small amounts.

Fat is stored in practically all cells in larger or smaller quantity, frequently in the form of lecithin, or related substances. The main store of neutral fat is in the subcutaneous connective tissue. In the plant, neutral fats are found in some fruits and practically only in fruits. Proteins. — It is impossible to name any particular form in which nitrogen is stored. Protoplasm in general is capable of increase and, in starvation, as we have seen, the less important tissues give up amino-acids for the benefit of the more vital organs, thanks to the presence of autolytic enzymes. Whether there

is any special form particularly adapted for the purpose of storage is at present doubtful, although certain observers believe that they have evidence of some such substance (see Cathcart's monograph, 1912, pp. 58 and 79). Berg (1914), and Berg and Cahn -Brenner (1914) describe the presence of granules of protein material (not protoplasm) in the liver cells of well-fed animals, particularly after feeding with amino-acids. These are regarded as storage of nitrogenous substance.

Noel Paton (1910) has brought evidence, depending on the proportion of creatine to total nitrogen excreted, to show that, in fasting, after an abundant protein diet, it is mainly non-muscle protein that is first used up. The value of the evidence depends, of course, on the constant percentage of creatine contained in muscle. Other investigators hold that there may be a storage of nitrogen in some form which lias a simpler constitution than protein. In making this distinction between protein nitrogen, and that in simpler form ("extractives"), itr may be noted that we assume that any nitrogen stored otherwise than as cell protoplasm does not form an integral part of a "giant" protoplasmic molecule, or " biogen," as a purely chemical system.

It was mentioned above that van Slyke and Mayer found that amino acids, introduced into the blood, disappeared rapidly therefrom. In a further paper (1913, 1), they show that these substances are taken up by the tissues in a form such that they can readily be washed out again, even by cold water. There is, moreover, a definite equilibrium established, so that the blood, even in starvation, still contains 3-8 mg. per 100 c.c. This points to an adsorption process, especially since the alternative hypothesis mentioned by the authors, that compounds like those supposed to be prepared by Pfeiffer and Modelski between amino-acids and neutral salts, is improbable, on account of the fact that there is no evidence for the existence of such compounds, as I have shown (Communication to Biochem. Society, not yet published).

A word may be said here with respect to the experiments of Grafe and Schlitpfer (1912), already referred to, in which, on feeding animals with a diet containing ammonium salts as the only source of nitrogen, a diminution of nitrogen output occurred. The conclusion drawn seems to be that protein can be synthesised from carbohydrate and ammonia. We have seen, indeed, that, from pyruvic acid and ammonia, alanine can be formed in the organism, but evidence is wanting as to other necessary amino-acids. Admitting the possibility of the requisite a- ketonic acids being formed from carbohydrate, the retention of nitrogen from ammonium salts, if utilised for synthesis of protein, should not be observed if. the diet is free from carbohydrate. Accordingly, Taylor and Ringer (1913) made the experiment. They found that, even in these conditions, nitrogen was retained from ammonia and they are of the opinion that the evidence points to the reversal of the process of de-amination as the explanation of the phenomenon. This reaction, if an oxidation, may be presented thus :

where K is the equilibrium constant and the factors in brackets are the concentrations of the four components of the system. Increase of ammonia leads to diminution of ketonic acid and this again involves increase in amino-acid, which may be utilised in the organism. Alanine, for example, obtained in this way, may be used for the same purposes as that derived from proteins, so that the latter are spared from breaking up. Support is given to this view by the later experiments of Grafe (1913), in which it is shown that retention of nitrogen can be obtained when urea is given as food. It is only necessary to assume that the reaction by which ammonia is converted into urea is also reversible, and we see that excess of urea involves increase of ammonia, and we have the same phenomenon as when ammonia itself is given. The fact that when either ammonia or urea is injected subcutaneously, it is entirely excreted by the kidneys, without giving rise to any retention of nitrogen, suggests that the concentration in which it arrives at the de-aminating tissues is too small to result in any perceptible mass action.

It is well known that compounds containing one or more carbon atoms A, B, C, D represent four different groups, attached to the four angles of a tetrahedron, which stands for a carbon atom. The two figures are mirror-images of one another, and cannot be made united to four different atoms or groups rotate the plane of polarised light, on account of their asymmetry. FIG. 75. PHOTOGRAPH OF A MODEL OF AN ASYMMETRIC CARBON ATOM, TOGETHER WITH ITS IMAGE IN A MIRROR. — The four solids of different shape represent the four different chemical groups. The reflected image can be detected by the double outline in places.

The theory of the asymmetric carbon atom is due to Le Bel (1874) and van't Hoff (1874). If we take lactic acid, we see that the central carbon atom is united to four different compound is accordingly optically active. Since a large number of the compounds of physiological interest are of this nature the method by which the changes of their concentrations are investigated by measurement of their power of rotating, the plane of polarised light is a very valuable one. The instrument used is called polanmetKr and details of its construction will be found in Findlay's book (1906).

Returning again to lactic acid, we find that, when prepared from muscle, it rotates the plane of polarised light to the right and is hence called dextro- or (/lactic acid. On the other hand, when cane sugar is fermented by certain bacteria, a lactic acid is obtained which rotates to the left and is hence called Isevo- or ^-lactic acid. It will be obvious that a mixture of the two in certain proportions will rotate equally in both inactive ; such a mixture is called inactive or oW-lactic acid.