Principles of General Physiology

The function of zinc in the growth of Aspergillus should also be referred to under the head of accessory food-stuffs, since it appears from the work of Bertrand and Javillier (1912, 2) that it enters as a constituent into the plant itself. Of course, it is not necessarily implied that it forms a part of the chemical structure of protoplasm itself. 2. As Hormones. — We have seen that gliadin, in the experiments of Osborne and Mendel, although lacking in lysine and insufficient for growth, appears to be adequate for maintenance, although the results of Hopkins and Neville (1913) throw some doubt on its adequacy for any length of time even for this purpose. Now there are other substances, of which a very minute amount only is required, but in whose absence a diet, otherwise perfectly adequate even for growth, is unable to preserve life. There are several sets of facts derived from different kinds of phenomena which prove this statement.

As many of these experiments were made on rats and mice, and some investigators hold that these small animals are inappropriate for metabolism experiments, it is well to refer to the circumstance pointed out by Hopkins (1912, p. 427) that for the kind of experiments in question such small animals are especially valuable ; a number can be dealt with at the same time, and the fact that their metabolic processes are rapid is important, especially for experiments on growth.

To turn to the experiments themselves, Hopkins (1912), one of whose curves is given in Fig. 72 (page 254), showed that a trace of fresh milk added to a diet, on which otherwise rats ceased to grow and ultimately died in about twenty-seven days, made it perfectly normal. We notice that even maintenance at the weight attained, except for a short time, is impossible without the addition. The active substance in milk must have been present in extraordinarily minute amount. Protein-free alcoholic extracts of milk solids, as well as the ether extract of thfe dry residue of the alcoholic extract, containing no inorganic constituents, as also the boiled watery extract of mangolds, were effective. The constituent is therefore neither protein nor salts ; lactose can also be excluded, since the addition of it to the diet is useless, and we have seen from Lunin's experiments that a diet containing casein, butter, lactose, and salts is insufficient as a diet.

We may make a rough calculation as to the maximum possible amount of the active constituent in 2 c.c. of milk thus: milk contains only about 4 per cent, of ether-soluble constituents, so that 2 c.c. would contain 0'08 g. Moreover Stepp's experiments (1909 and 1911) enable us to exclude the greater part of these. This observer fed mice with bread and milk which had been extracted with alcohol and ether ; he found that Buch a diet kept them alive for only three to four weeks, whereas if a minute amount of the dry residue from the extracts were added, they lived indefinitely and appeared to be in normal condition. In further work (1913) Stepp finds that ether alone does not extract the important constituent from dry food, although alcohol alone does so. He also found that the addition of the neutral fat of milk was ineffective ; as were also lecithin, cholesterol, cephalin, cerebron or phytin. We may therefore subtract from the above 0'08 g. in Hopkins' diet, that part consisting of fat, lecithin, cholesterol, etc. This would leave scarcely more than a few milligrams, if so much. Other evidence is that boiled watery extracts of mangolds, which would not contain more than a trace of lipoids, are active, as Hopkins found. We must therefore agree with Hopkins that a catalytic or stimulating influence of some kind is more likely than that an actual tissue constituent is supplied.

Stepp apparently holds that a " lipoid " of some kind is responsible for the result produced by the extracts in question. There are, however, other substances present in bread and milk which are soluble in alcohol and even to some extent in ether. Osborne, Mendel, Ferry, and Wakeman (1913) find that the "accessory factor" for growth, contained in milk, goes into the fatty part of the butter. This contains neither nitrogen nor phosphorus, so that the active substance is

not u lecithin like substance, nor like Funk's " vitauiines," to be mentioned presently. The fact that it is soluble in fat does not, of course, necessitate the conclusion that it is of fatty nature. Funk's vitamine must belong to another class of substances, or, more probably, the nitrogenous substance which lie has analysed is not the really active one. The experiments of Osborne, Mendel, and Ferry (1912, 1) prove undoubtedly that a protein as unlike the tissue proteins as gliadin is, can .serve for the purpose of forming new tissues, through the intervention qf the adult animal. The experiments given on pp. 485 and 486 of their paper are instructive. A rat was fed for 178 days on a diet consisting of gliadin only as protein, along with protein-free milk, carbohydrate, and fat ; at the end of this time, four young rats were born. Of these, after thirty days' feeding by the mother's milk, three were put on a normal diet while the fourth received gliadin food similar to that which the mother had been receiving all the time. The three former grew well, but the latter quickly began to fail. The diet which sufficed for the adult, not only for its own maintenance, but also for production of young and secretion of normal milk, was insufficient for the growth of the young animal. It will be noticed that the requisite substances were present in the milk secreted by the mother, since, while fed on this, the young rats showed normal growth. The adult, therefore, has the power of forming some substances out of others in the diet, which power is not possessed by the young animal, so that they must be supplied to it from without. It will be seen that these experiments do not altogether decide the question as to whether the failure to grow was due to want of lysine in the diet or to want of some " accessory factor." The fact that when casein was substituted for gliadin, the diet was adequate, indicates that the former was the case.

In a later paper, Osborne, Mendel, and Ferry (1912, p. 242) come to the conclusion that maintenance is possible in the absence of any " hormone " element, since the diet was extracted to remove such substances. Whether this conclusion is justified seems doubtful from the experiments of Hopkins and Neville (1913), who repeated the experiments in question. The rats fed in this way ceased to grow almost at once and, after a brief period in which no loss of weight occurred, steadily declined and, with the exception of four out of twenty-four animals, died before the fortieth day. These observers are inclined to think that a trace of the active substance may have remained in Osborne and Mendel's diet. It is clear ttiat conclusions must be drawn very cautiously from results which might be due to such extraordinarily minute quantities of unextracted matter.

We must not forget that there is no reason to suppose that the diet, containing amino-acids as sole source of nitrogen, which was found by Loewi and others to be capable of maintenance, or even a certain degree of laying on of protein, was free from the " accessory factors " in question, since uuextracted carbohydrate was added to the food. There is, on the whole, considerable evidence that a diet, wanting in something necessary for yrwvlh, may nevertheless be capable of keeping up the general condition in a normal manner. An interesting and, if confirmed, pract it-ally important application of this fact is reported by Sweet, Ooraon- White, and Saxon (1913). Since tumours, such as cancer, are growths, it seemed that it should be possible to retard their increase by a diet insufficient for growth, while adequate for maintenance. The experiments showed that mice, fed on well-washed gluten from wheat, together with starch, lard, lactose, and salts, could be kept at a constant weight, that is, without growth, for thirty days or more. Comparing animals on this diet, inoculated with a rapidly growing tumour at the same time as those on normal diet, a considerable dift'ereiu -e \\as found in the rate of growth of the tumour, which was much slower in those on the restricted diet. One mouse, for example, on restricted diet, at fifty-two days after inoculation, had at this time a scarcely visible tumour of 4 mm. in diameter. The mouse was then put on normal diet of bread, corn, etc., and, thirty days later, the tumour was nearly as big as the mouse itself. Prof. Hopkins informs me that he had already, before 1913, obtained similar results.

We may next consider shortly the results obtained by various observers, with respect to certain diseases produced by the absence of some substances of a similai kind to those which we have discussed in the preceding pages. It has been clearly shown by Fraser and Stanton (1911) that the disease known as " Beri-Beri" is due to the exclusive feeding on rice which has had the pericarp and most of the underlying layer removed by " polishing." Fowls fed on such rice develop a polyneuritis (inflammation and subsequent degeneration of peripheral nerves), which is similar to that occurring in beri-beri, and can therefore be used to study the disease experimentally. If the polishings be added to the white rice, the animals remain healthy and those suffering from the disorder can be cured. As to the nature of the protective substances whose absence entails the disease, it was found that heating to 120° for two hours destroys them. They are soluble in slightly acid 91 per cent, alcohol. Further evidence on the nature of these substances has been brought forward by Funk (1911, 1912), who has separated a substance from rice polishings, and also from yeast, milk, ox-brain, and lime-juice, which, in very small doses, 0-02 to O04 g. by the mouth, cures polyneuritis in fowls. Funk states further that it has the properties of a pyrimidine base. Pyrimidine is : —

the characteristic constituent of the nucleins. Marked improvement was, in fact, obtained by giving certain substances related to the nucleins, such as thymus, nucleic acid, guanosin, etc. Funk suggests the name " vitamines " for the class. They are remarkably active ; a quantity containing only 0-4 mg. of nitrogen cures a pigeon. An interesting point is that pigeons severely affected with polyneuritis recovered in six to twelve hours, and frequently seemed quite well after three hours. It appears that the paralysis can have been due to a functional disturbance only of the axis cylinders, although the medullary sheath was degenerated. It is impossible that recovery of degenerated axones could take place in so short a time. It appears that a normal medullary sheath is not absolutely necessary for nerve conduction. It should be mentioned that the constitution assigned by Funk to these " vitamines" is not generally accepted. We have seen above (page 257) that the active substance in butter contains no nitrogen, so that it seems probable that the substance investigated by Funk was not the active one, and that this latter was present only in traces. Indeed, Funk himself appears to have come to the conclusion later that this is the case, although he holds that the active substance is of basic nature, and precipitated by phospho-tungstic acid. The possibility has not yet been excluded, however, that the precipitate might carry down the active material by adsorption.

Scurvy has also been shown by Hoist and Frolich (1912) to be due to a deficiency of some essential constituent in preserved foods, but present in all kinds of fresh material, animal or vegetable. It will be remembered how Captain Cook, in his second voyage, was successful in avoiding this disease, although the voyage lasted 1,000 days. He says (1776, p. 405): "We came to few places where either the art of man or nature did not afford some sort of refreshment or other, either of the animal or vegetable kind. It was my first care to procure what could be met with of either by every means in my power, and to oblige our people to make use thereof, both by my example and authority ; but the benefits arising from such refreshments soon became so obvious that I had little occasion to employ either the one or the other."

The extreme sensitiveness to the want of some particular substance in small amount does not seem to be limited to the higher animals. Wildiers (1901) stated that if a normal artificial culture medium is inoculated with yeast in too small an amount, there is no growth, whereas, if the same quantity is added to sterilised beer wort, the growth is vigorous. The effect of adding larger quantities is said to be due to the presence of a substance which Wildiers calls " bios," provisionally, until its chemical nature is known. This bios is found in all cultures of growing yeast. If the culture medium is inoculated with an insufficient quantity of yeast, and, at the same time, with small quantities of boiled yeast extract, even if filtered through porous clay, growth is ensured. The beneficial effect of phosphate on fermentation by yeast is well known, so that it is necessary to note that the artificial culture fluid used contained this salt already. A culture medium may sometimes become infected with yeast from the air, and it seems difficult to understand how bios could be taken with it ; however, according to the observations of Kossovics (1896), it seems possible that bacteria might produce some substance of this nature. Since this work seems to have been somewhat overlooked, it is advisable to give some further details. It is interesting to note that the work was done at the University of Louvain, whose tragic fate has aroused the indignation of the whole civilised world.

As to the chemical nature of bios, we may note that it is soluble in 80 per cent, alcohol, not in absolute alcohol nor in ether. It is not precipitated by lead acetate, phospho-molybdic nor phospho-tungstic acid, nor by mercuric chloride. It is present in Liebig's meat extract and commercial peptone as well as in decoction of germinated malt (beer wort) before the action of yeast. It is not contained in the products of peptic or tryptic digestion of pure proteins. Thymus nucleic acid does not give the result. Whatever it may be, it is evidently not the same substance as that of Hopkins, since it is insoluble in ether, nor is it Funk's " vitamine " since it is not thrown down by precipitants of organic bases. An important point is that it is not produced by the yeast plant itself in the course of its growth, in fact it seems to disappear. If the culture to which originally a certain amount of bios was added be boiled and concentrated, it is found necessary to add at least that amount of the concentrated extract which would contain the original quantity of bios in order to ensure growth in a new experiment. Wildiers calls attention to the circumstance that the results seem to give a possible explanation of the famous contest between Liebig and Pasteur. The latter inoculated his cultures with a fragment of yeast of the size of the head of a large pin. This was probably about the lowest limit of "bios," so that, if Liebig took a smaller amount, he would not get growth. As Wildiers remarks (p. 328), if Liebig had accepted Pasteur's invitation to see his experiments, the discrepancy would probably have been explained. We see also how minute is the amount of bios required.

A smaller quantity than the optimal will allow of a very slow growth, but, under the microscope, there are to be seen, at one time, very few living cells, and it appears that, in order that a new cell may grow, it has to wait for the death of an old one in order to obtain the bios required. On the whole there appears to be some essential structural unit which the yeast cell is unable to form for itself. Abel Amand (1903) answers some possible objections to these experiments. We saw that the influence of the number of cells was eliminated in that the same number was ineffective in Pasteur's medium, effective in malt extract. Amand 's experiments were made to test the suggestion that the bios added was effective by counteracting some poisonous substance in the water used for the culture fluid, some " oligodynamic action" as described above (page 222). Copper was suggested. The water from glass stills, however, gave the same results. The chemicals used were also tested, and, although the details must be read in the original paper, the possibility of poisonous constituents seems satisfactorily excluded. Moreover, one may recall the experiments of Ringer, in which the poisonous action of distilled water was abolished by calcium, which is a constituent of the yeast culture medium. Amand, in a further paper (1904), shows that bios disappears and cannot be extracted from the cells, so that it is either used for synthesis of a more complex substance or undergoes spontaneous decomposition. According to Devloo (1906) the active principle is a base, precipitated by mercuric chloride in presence of barium hydroxide. It can be made to replace choline, at all events partially, in lecithin. It may naturally occur in the form of the base of a lecithin-like substance. It is not choline itself nor can it be prepared from choline.

Twort and Ingrain (1912) have made the interesting observation that Johne's bactthis, responsible for the pseudo-tubercular enteritis of oxen, will "only grow on a medium to which dead tubercle bacilli, or some other " acid-fast " bacilli, have been added. These authors have also been able to extract by alcohol this essential substance. The work of Bottomley (1914) is also of interest. He finds that substances which stimulate plant growth are formed in sphagnum peat when it is incubated with cultures of certain aerobic soil" bacteria. They are very active ; the watery extract of 0'18 g. of peat thus treated caused the growth of Primula seedlings in six weeks to be double that of control plants. They seem to be allied to the " accessory factors " of diet.

Thornton and Smith (1914) find that the chlorophyll-containing protozoon, Euglena, is unable to grow and multiply in a pure saline medium, although all the elements supposed to be necessary are present. If, however, a trace of tyrosine be added, growth is vigorous. It seems that the tyrosine does not serve as a foodstuff, since the active amount is so small (0'4 mgm. in 10 c.c.). Moreover, no growth occurs in the dark, so that the organism obtains its food from the carbon dioxide of the air and the mineral substances of the culture fluid. The tyrosine disappears, it is true, but this is due to the action of bacteria, unavoidably present in small numbers. See also E. J. Allen (1914) on the growth of diatoms.

The manganese and zinc required for the normal growth of Aspergillus may also be mentioned in the present connection. As to the mode of action of these "hormones" or " catalysts " we are as yet in the dark ; as indeed as to that of the similar substances in the "internal secretions." Some facts which bear on the question will be found in Chapter XXIV. and, as to the nature of catalysis, in Chapter IX. There is one possibility that has not been referred to as yet. We saw in Chapter V. how important a part the cell membrane plays in vital phenomena. Now, the actual amount of material contained in these membranes is almost infinitesimal, yet it might well be that there is something absolutely necessary to its proper constitution, a substance which may be gradually lost in the process of activity. One thinks of Clark's experiments (1913, 2) on the effect of prolonged per- f usion with a simple saline solution, which washes away something which can be replaced by an ether extract of the dry residue of an alcoholic extract of dry serum (see page 211 above). The properties of lecithin may occur to the reader, but this, as well as cholesterol, seems to be excluded by the experiments of Stepp, who found neither of these, nor indeed other known " lipoids," capable of replacing the active substance in milk.

An rmportant conclusion with respect to the general theory of nutrition is to be drawn from the various facts given in the present section. As was pointed out by Starling and myself (1906, p. 696) and by Hopkins (1906, p. 395), independently, it is not sufficient to estimate the value of a diet, as to whether it is an adequate one or not, merely by its calorie value or its content in fat, carbohydrate, and protein ; the presence or absence of the small quantity of the " accessory factors " or " hormones " must also be taken into account.

Our next task is to discuss briefly the changes undergone by the three classes of organic food stuffs as they pass through the organism, commencing our study with proteins. Ttie Constitution of Proteins. — As was first definitely and completely shown by Emil Fischer (1899-1906), proteins, animal and vegetable, are composed of a series of amino-acids united by elimination of water, as described on page 103 above. This work is of such fundamental importance that I have thought it necessary to introduce a portrait of Fischer in Fig. 73.

The following is a list of all the constituents isolated from various proteins up to the present: — Glycine (amino-acetic), alanine (amino-propionic), amino-butyric, valine (aminoisovalerianic), leucine (amino-isobutyl-acetic), isoleucine («-armno-/3-methyl-/:?-etli\ I propionic), phenyl-alanine,, tyrosine (para-oxyphenyl-amino-propionic), serine (a- amino /i-oxy-propionic), cystine (condensation of 2 molecules of a-amino-^8-thiopropionic acid, the sulphur constituent of proteins).

Mono-amino-di-carltoxylic A cids. Aspartic (a-amino succinic), glutamic (a-amino-glutaric). Di-amino-mono-carboxylic A cids. Arginine (a-aimno-8-guanido-valerianic acid). Guanidine is NH = C-NH0. Histidine (/3-iminazol-a-amino-propionic acid). Proline (a-pyrrolidine carboxylic acid). Pyrrolidine is : — Oxy-proline (y- or /i?-oxy-a-pyrrolidine carboxylic acid). Tryptophane (constitution given on page 256). How far have all the constituents been accounted for ? The analysis of zein by Osborne and his co-workers account for 85'4 per cent, of the total nitroircn of the protein and, considering the inevitable losses in the mono-carboxylic acids, the result must be regarded as very satisfactory.

These amino-acids combine together in the way already indicated, that is : — In this way, Fischer has prepared a large number of peptides, containing two or more amino-acids, in great variety. The proteins of the tissues may, therefore, be regarded as made up by various selection out of the list given above, and in different relative proportion. Tims, while gelatine contains 16 per cent, of glycine and 0'9 per cent, of glutamic acid, gliadin of wheat contains no glycine, or extremely little, and 43 per cent, of glutamic acid.

We see that there are very te\vfree JV77, groups in a protein. Van Slyke and Birchard (1914), in fact, show that the only free NH2 groups in all the native proteins examined, a considerable number, amount to one-half of those of the lysine contained therein. All the other amino-groups are condensed into peptide linkings. Methods of estimating the amount of nitrogen combined in the form of NH9 are of value in determining approximately whether a given protein is a mixture of several simpler ones or is one single large molecule. We have also a means of following the degree of hydrolysis in the course of digestion or under the action of acid. There are two methods available for this purpose.

Sorenseris method depends on the fact that NH2 groups react with formaldehyde to form methylene-imino groups : — When proteins are acted on by formaldehyde, the basic groups are eliminated and the free carboxyl can be titrated with acid in the usual way. This fact was shown by Schiff (see Plimmer's monograph, 1912, 1913). Van Slyke's method depends on the fact that primary amino-groups react with nitrous acid thus : — By these methods we find that proteins do not contain more than one per cent, of their nitrogen in the NH0 form.

There are several classes of proteins, distinguished by their different properties, in addition to those conjugated with other substances, such as nucleins or carbohydrates. The nomenclature of these substances has been agreed upon by the Chemical and Physiological Societies of England and America and should be always used, if confusion is to be avoided. It will be found in Plimmer's monograph (1912, pp. 1 and 2). , We have next to inquire what becomes of the proteins taken as food before the end products of their metabolism are excreted. A word may first be said as to the meaning of this " metabolism." It was, as far as I can find out, first suggested by Michael Foster in his "Textbook of Physiology." It is used to express the chemical changes which take place in the various food-stuffs after they have passed from the alimentary canal into the blood. The changes produced by the digestive enzymes are usually excluded.

In the space that is available in such a book as the present one, it is impossible to describe in detail all the numerous facts which are known as to these phenomena, important as they are. For the subject of protein metabolism, the reader should consult the monograph by Cathcart (1912). There is strong evidence to show that the proteins of the food are completely hydrolysed into amino-acids, before being absorbed. For some time, however, it was held that re-synthesis to proteins took place in the wall of the intestine. This view was due to the fact that it had not been possible to detect amino-acids, nor even peptones, in the blood. It seems, on a priori grounds alone, that such an immediate resynthesis would be a very inappropriate one. Suppose that a particular tissue protein is to be built up and that this protein, while containing glycine, contains very little glutamic acid, also that another cell protein contains no glycine, but a large amount of glutamic acid. Now, if the synthetic protein supplied by the blood contained the right proportion of amino-acids for the one, a large quantity of it would have to be taken up by the other, in order to satisfy its requirements, and the remaining part of it, containing the excess of the particular amino-acid not wanted, would be wasted. Even if this last were utilised in some other way, it seems a useless process for a protein to be synthesised in the wall of the intestine, merely to be broken up again when it reaches the cells.

Direct evidence, moreover, is not wanting at the present time showing that amino acids do actually exist in the blood. According to van Slyke and Meyer (1912), in the first place, the blood of a dog which has received no food for twentyfour hours contains amino-acids equivalent to 4 mg. of nitrogen per 100 c.c. If meat is given, the value rises to 10 mg. during digestion. The reason why the quantity is so small is that these amino-acids are rapidly taken up by the tissues in some form, as we shall see later ; it was found, for example, that if 12 g. of alanine were injected into a vein of a dog during ten minutes, only 1'5 g. remained in the blood five minutes later, although only To g. had been excreted by the kidney.

Folin and Denis (1912) came to the same conclusions and could find no evidence whatever of protein synthesis in the intestine. Abel (1913), again, by the ingenious method of dialysis of the living blood, referred to above (page 83), has been able to collect as much as 20 g. of amino-acids from the blood of three or four dogs, so that it is possible to separate them and find out which are present. This is indeed being done. The fact that they have now been detected in the blood is due to the improved methods devised for their determination and we may conclude that they do actually, as such, reach the tissue cells. Confirmatory evidence is afforded by the experiments of Buglia (1912, p. 184) who found that sufficient nitrogen food to meet requirements can be injected, in the form of amino-acids, into the veins slowly without disturbance.

We have already seen evidence that the actual amount of nitrogen required for repair is very small and further details will be given later. How then is the remaining nitrogen of a considerable protein diet dealt with ? If we start from the other end, as it were, we find by experiment that the amino-acid nitrogen not needed for growth or repair appears in the urine as urea, while the rest of the molecule is ultimately converted into carbon dioxide and water.

From the chemical standpoint, the most obvious stages between an amino-acid and urea are, first, de-amination, by which ammonia is split off and some derivative of a fatty acid formed, and, secondly, the ammonia is converted into urea, while the hydrocarbon acid is burnt up for the purpose of affording energy. This view is, in fact, a part of the theory of protein metabolism associated with the name of Folin (1905). We have to inquire what evidence there is of such reactions occurring in the living organism, and, if so, the further question arises as to the organs in which they take place.