Principles of General Physiology

The Protozoa are apt to be considered as very primitive organisms, rudimentary ancestors of higher animals, because they are unicellular. But, although there is no doubt that higher animals have arisen in the course of evolution from simple creatures of this kind, one must admit that the protozoa, as we have them now, are complex, highly differentiated organisms. The Amoeba, apparently, cannot be grown on a culture medium, unless it is supplied with bacteria, although dead ones suffice.

As a general rule, we may say that animals require food which has been previously built up by the plant. They feed either on vegetable matter or on other animals. It was believed at one time that animals, at all events the higher ones, required nitrogen in the form of more or less complex proteins, but we have now definite proof that the products of hydrolysis of proteins, amino-acids, will prevent loss of nitrogen from the adult animal. Optical Activity. — In connection with the remark made above as to the preference of one form of carbon or nitrogen food before another, it is interesting to note that, of the amino-acids, it is the /-series only which is utilised for the building up of the tissue proteins, although there is evidence that the opposite optical isomers can be used for energy purposes, although not so readily. In the case of carbohydrates, again, it is only the (/-series that is easily utilised. The mistake is sometimes made, however, of stating this use of one series only as an absolute fact, whereas it is only relative. Pasteur (1860, p. 33 of the reprint in Ostwald's "Klassiker") in his classical work on the separation of the d and /-tartrates, used moulds to consume the dextro acid and leave the other intact. As soon, however, as all the c?-acid was exhausted, the mould proceeded to consume the /-acid, so that the rotatory power of the solution passed through a maximum. Other instances will be referred to when enzymes are under discussion, and the general question is treated in a later section of the present chapter. There is also preference for certain disaccharides, and here the utilisation is connected with the possession of particular enzymes which hydrolyse the disaccharide. The facts have been chiefly studied in the case of different species of yeasts. Emil Fischer has also shown (1884-1908) that, of all the possible carbohydrates of the general formula CuH2nOn, ordinary yeasts can only act upon those in which the number of carbon atoms is three or a multiple of three ; moreover, of those of the same constitution, but of different stereochemical configuration, a particular yeast will ferment one at a much greater rate than another.

One of the most striking examples is that of the sorbose bacterium, as studied by Bertrand (1896). Acting only on glycerol or on sugars with a terminal alcohol group (CH2OH), it attacks a CHOH group near this one, transforming it into CO and thus producing a ketone. Moreover, the OH of the group attacked must not be next to the H of a neighbouring CHOH. Glycerol is thus oxidised into dihydroxyacetone. Salts. — As already stated, these are necessary for all organisms, but the requirements as to particular salts vary considerably. This is one of the problems with which the agriculturist has to deal. Apart from nitrates, which do not come under the head of salts as such, potassium and calcium seem to be indispensable and other salts are more or less favourable. The reader is referred to the monograph by E. J. Russell (1912) for further information. As an illustration, we may refer to the pioneer work of Raulin (1870), some aspects of which have been mentioned above. By numerous experiments with different salts in different concentrations, it was found that for the growth of Aspergillus, a medium of the following composition gave better results than one in which any one of the constituents was omitted or present in another concentration : —

Note that, while some of these substances are foods in the narrow sense of the word, i.e., utilised as actual constituents of the cells, or for energy or growth, such as sugar, ammonium nitrate, phosphate, iron, and potassium, others have additional functions. Tartaric acid keeps the solution acid and prevents the growth -of bacteria; iron serves to neutralise, probably oxidise, injurious substances formed in the process of growth. The cane-sugar is first hydrolysrd by an enzyme in the mould and lactose will not replace it, since the enzyme

Fm. 69. DIAGRAM OF THE NITROOEX CYCLE, FROM THE ATMOSPHERE THROUOH required to hydrolyse lactose is absent. Glucose, of course, can replace it, and it is of interest that alcohol, while retarding the germination of the spores, serves as an excellent source of carbon to the grown plant. It will have become sufficiently obvious that the continued supply of carbon and hydrogen to living organisms in general is sufficiently provided for by the activity of the green plant in forming sugar from the carbon dioxide evolved in

combustion processes, including those of plants and animals, water being taken into the molecule in the process. Water and salts are also readily available and suffer no degradation of energy in passing through the organism. Nitrogen, on FIG. 70. ROOT TUBERCLES OF LEGUMINOS^E. —About natural size. the other hand, must be presented to the green plant in the form of nitrate, in order that it may be further synthesised into a suitable form for the needs of the animal. Atmospheric nitrogen is useless for this purpose, and although ammonia, which is formed from animal excreta and from the debris of plants, chiefly by bacterial agency, can be utilised by the higher plant as a source of nitrogen, it is by no means the normal and efficient one (see Russell, 1912, pp. 30-31); moreover, in the conversion of the residues from plant and animal into ammonia, a certain amount is always lost in the form of free nitrogen. It is therefore a matter of fundamental importance for the continued existence of life on the earth that some means should be present for the conversion of nitrogen gas into a form available for the growth of the green plant, and also that an effective mechanism should exist for the transformation of ammonia into nitrates.

The diagram given in Fig. 69 will serve to elucidate the process by which these requirements are met and will enable a shorter verbal account to be given than would otherwise be necessary. Additional details may be found in the monograph by Russell (1912, chap. iv.). Following the direction of the arrows in the figure, and starting from atmospheric nitrogen, we notice that there are two ways in which this is "fixed" in a form available for the use of plants. In the first place, there

are bacteria in the soil which are able to obtain their nitrogen from the atmosphere. Their existence was clearly shown by Vinogradsky (1895). The chief forms are a Clostridium, anaerobic,|isolated by the observer named, and Azotobacter, aerobic, discovered'by Beijerinck (1901). Pure culture. Stained with methylene blue. Size of organisms, 1'3 A1 x 37 fJ- Vinogradsky recognised that the process required energy to be supplied, since it is endothermic ; in experimental work, glucose is added, and a considerable amount is consumed ; each milligram of nitrogen fixed requiring the oxidation of 500 mg. of sugar. In the soil, decomposition products of cellulose apparently take the place of the glucose as sources of energy.

The second process is peculiar to the leguminous plants, together with a few others. Russell points out (1912, p. 84, footnote) that it was known to the Romans that the growth of vetches (a leguminous plant) on ground afterwards used for wheat caused an increased crop of this latter. In Vergil's " Georgics," Book I., lines 73 and following, the farmer is recommended, "before sowing his yellow wheat, to take off a crop of beans, with their rattling pods, or of the frail offspring of the vetch, or of lupins, with their brittle stalks and rustling straw." All of these are leguminous plants, be it noted.

The word translated " straw" in this passage is " silva" ; but it is difficult to see in what sense a field of lupins could be called a " wood." The reason of the beneficial effect of such plants was discovered by Hillriegel and Wilfarth (1888). They showed, in the first place, that, adding together the nitrogen of the soil in a particular culture pot to that of the plants grown in it, there is, in the case of oats, always a little less than that originally present, but, in the case of peas, always more. This could only come from the nitrogen of the atmosphere. At this time it was already known that the nodules on the roots of leguminous plants contain bacteria, and the hypothesis was a natural one that these organisms were able to fix nitrogen and hand it over to the plant in some way. Beijerinck (1888) isolated the organisms from the root nodules, but, although they must be present in the soil, since extracts of soil on which leguminous plants have been grown will infect the roots of other plants of the same order, it was found impossible to discover them therein. After entry into the root hairs, they multiply rapidly and presently form a nodule on a part of the root. Inside the nodules they change to Y-shaped "bacterioids." Fig. 70 shows roots with nodules and Fig. 71 (from the paper by Miss Dawson, 1900) gives the appearance of the bacteroids. The chemistry of the process is unknown. The final product is supposed to be soluble protein, which is passed on to the plant. From what we know as to the nitrogen supply to the tissues in animals,'-it seems more likely that it is an amino-acid or amide. In any case, the facts are of great practical importance, since leguminosae are among the commonest plants, and the process is independent of organic matter in the soil. The carbohydrate required to afford energy for the work of the micro-organisms is obtained from the plant on which it grows. The growth of these plants, then, always leads to increase of organic nitrogen in the soil.

Owing to the two processes named, the green plant has been enabled to form proteins. If eaten by an animal, these proteins serve as nitrogen food for it. The waste products containing nitrogen, from both animal and plant, some of them of simple composition, such as urea, others more or less insoluble solids, on return to the soil, are converted into ammonium salts, mainly by the agency of bacteria, although it is said that the process may take place slowly in the presence of antiseptics. The reaction probably consists, in the case of the more complex compounds, in the production of amino-acids and subsequent hydrolysis or oxidation of these. During the process, however, a considerable loss of nitrogen in the gaseous form occurs, as presented by the thin line in the diagram. This loss is supposed to be due to oxidising bacteria, but the question is not yet decided.

The ammonium salts thus formed are capable of serving to a certain extent as nitrogen food for the green plant, indicated by the interrupted line in the diagram leading back to plant proteins ; but they are not efficient in this respect and, according to Russell (1912, p. 31), plants fed only on ammonium salts as source of nitrogen, really suffer from nitrogen starvation. A means of converting ammonia into nitrates is clearly an essential requirement. This is actually provided in the following way. The first step is the formation of ammonium carbonate, by simple chemical reaction with alkaline carbonates, so far as not already present in this form. This ammonium carbonate is rapidly

converted by a special organism, Nitrosomonas, into nitrite and this nitrite again into nitrate by another organism, Nitrobacter. These bacteria are always present in normal soil and act with such rapidity that only traces of either ammonia or nitrite can be detected in the soil. The fact can readily be observed by adding about half a gram of soil to 50 c.c. of a culture and adding to 50 c.c. about half a gram of solid magnesium carbonate to preserve neutrality.

After four weeks or so, the ammonia will be found to have disappeared and nitrate to have taken its place. The existence of the latter can be shown by the reaction with diphenylamiue The chemical process occurring is unknown, but the bacteria concerned have rather extraordinary properties. Carbon dioxide will serve as source of carbon, and in fact it seems that, in cultures in vitro, other more complex carbon compounds are injurious, especially glucose or peptone. But, in order- to synthesise cell stuffs from carbon dioxide, a supply of energy from without is necessary. Light is out of the question, since the organisms do not possess chlorophyll and, in point of fact, light is actually fatal to them. It has been suggested that the oxidation of ammonia and of nitrite might afford sufficient energy. The injurious effect of organic matter only applies to artificial cultures ; in the soil, glucose has a beneficial effect, although other sugars are inert and nitrogen compounds injurious. The organisms are killed by a temperature of 45° C. or by the absence of oxygen.

Preparations containing nitrifying bacteria and called "nitragin" have been sold for the purpose of improving the soil, but their beneficial effect is doubtful (see Miss Dawson's investigations, 1898 and 1900). Probably most soils already contain abundance of the organisms, and infertility is due to other causes (see Russell's monograph, 1912, p. 98). The normal use of the nitrates is to form plant proteins, but, in the absence of oxygen, they rapidly disappear if not made use of. They are converted back to nitrates and ammonia on the one hand, as shown by the dotted lines on the diagram, and to free nitrogen, on the other hand, as shown by the thin line. Many various forms of bacteria are concerned in the process, producing also a number of substances other than those named.

On account of the importance of nitrates as food for plants, and indirectly for animals, any means of obtaining them from the atmosphere by non-living agency is of value. It has long been known that the oxygen and nitrogen of the air, in presence of water vapour, can be caused to combine by the electric spark, forming nitric acid. Of recent years, the process has been developed on a commercial scale by the use of large electric arcs, spread out by magnetic action ; considerable quantities of nitric acid are made yearly in situations where there is abundant water power. In Birkeland's process, worked chiefly in Norway, the arc is produced by an electromotive force of 5,000 volts and is spread out into a disc of 2 m. in diameter, having a temperature of some 3,000°. Again, Calcium cyanamide is formed when nitrogen is passed over calcium carbide, heated to 1,000°. Calcium carbide itself is produced in the electric furnace from lime and carbon. Calcium cyanamide gives ammonia when acted on by water and was, at one time, advocated for the purpose of supplying nitrogen to plants. Another process, apparently of great efficiency, has recently been discovered by Haber (1913). By compressing a mixture of hydrogen and nitrogen and causing it to be acted on by a certain catalyst at a high temperature, ammonia is formed by direct combination, in large proportion. By passing this over another catalyst, it is oxidised to nitric acid.

For the food of the higher animals, it is usually of advantage to make up the requirements by a combination of certain proportions of fat, carbohydrate, and protein. Fat is of value on account of its high potential energy, being less oxidised than carbohydrate. It is not, of course, a necessary article of diet, since it can be formed in the organism from carbohydrates. A certain amount of carbohydrate appears to be a matter of necessity, as we shall see later. And, although the whole of the energy requirements could be supplied by protein, it would be very wasteful, since only a comparatively small amount of nitrogen is actually required. The point is that a minimum total energy value, usually expressed in heat units, must be supplied to an animal, in order to prevent loss of body substance.

As organisms increase in complexity in the course of evolution, it appears that their capacity of synthesising the innumerable compounds of which they consist Lower curve (as far as the 18th day)— eight male rats on pure diet, free from Upper curve — eight similar rats taking, in addition, 3 c.c. of milk per day. On the 18th day, marked by vertical dotted line, the addition of milk was is diminished. There are certain differences as regards requirements for growth, maintenance, or energy, so that a diet which is adequate as a supply of energy, that is, one which has a sufficient calorie value, may be inadequate for replacing wear and tear, while a diet which is adequate for this purpose may be unable to allow growth to take place. Hopkins (1912) showed, for example, that voung rats if fed on mixtures of pure caseinogen, fat, carbohydrate, and salts rapidly ceased to grow, although the energy value of the diet was amply sufficient for the purpose. On the other hand, if a minute quantity of fresh milk (3 c.c. per day) was added to the diet, growth recommenced and went on rapidly. Fig. 72 is a representation of one of these experiments. It is to be noted that these amounts of milk merely added some 4 per cent, or less to the total solid eaten and are altogether inadequate to account for the increased growth, which amounted to about half a gram per rat per day, whereas the total solid content of the milk

added would not be more than about 0'08 g. The experiment is referred to at this point merely as an illustration and will be discussed further later on. We may note, however, that the active constituent of milk is not one of the known ones. Milk freed from protein and salts is equally effective and it was shown some time ago by Lunin (1880, p. 37) that a "synthetic" milk, containing all the known constituents, will not serve as a complete diet. We will now proceed to attempt some kind of analysis of the different sorts of those special constituents of food, of which only a minute amount is required, but which is essential. At the outset, it is clear that such substances cannot be required for energy purposes directly, so that the part they play must be either in growth or maintenance of cells, or else as hormones or catalysts, the function of which will be explained presently. As regards growth and maintenance, there is evidence that a particular substance may be necessary for the former but not so for the latter.

The tissue proteins, as we have seen (page 103), are composed of a considerable number of different amino-acids, so that this question resolves itself into the capability of the organism to synthesise for itself all these constituents, or whether it has to depend upon the supply of some of them from the outside. The green plant needs no further nitrogenous food than nitrates, so that the constituents of its proteins, which are identical with those of animal protein, must be formed in the organism itself. One of these plant proteins, gliadin from wheat, contains alanine, valine, leucine, phenylalanine, tyrosine, serine, cystine, proline, aspartic and glutamic acids, tryptophane, arginine and histidine. For the chemical constitution of these, the reader is referred to the monographs by Plimmer

An interesting direct proof of synthesis of some of them has been given by Abderhalden and Rona (1905), who grew the mould, Aspergillus, on a culture fluid containing only potassium nitrate as source of nitrogen and cane-sugar as source of carbon. The amino-acids were then separated by the esterification method of Emil Fischer (see Plimmer's monograph, 1912, pp. 22, etc.). Glycine, alanine, leucine, aspartic and glutamic acids were separated and identified. The absence of aromatic derivatives is notable.

Although the plant is capable of such varied synthetic processes, it is remarkable that the power has been lost to a large extent by the animal organism. A certain limited capacity is, however, known to exist, and it is possible that further instances may be found in the future. Glycine can be formed in the higher organism, as was shown by Magnus-Levy (1907). Two lines of evidence may be cited. The proteins of milk contain only about 0'3 per cent, of glycine, but a sucking calf can build up 78 g. of tissue protein out of 100 g. of milk. Now this animal tissue protein contains at least 2 '5 g. of glycine. Again, when benzoic acid is given to an animal, it becomes conjugated with glycine to form hippuric acid (benzoyl-glycine), which is excreted by the kidney. A rabbit which was estimated to contain 6*6 g. of glycine, excreted 8 g. of this amino-acid in combination with benzoic acid, when the latter was administered to it. This experiment is, perhaps, not altogether convincing, on account of the uncertainty in the actual content of the rabbit in glycine, although it is improbable that all the glycine-containing tissues should be decomposed in such a way as to give up the whole of their glycine.

We must admit the possibility of the formation of alanine also in the following way. Embden and Kraus (1912) showed that lactic acid is formed by the liver from glycogen, and Knoop (1910) that the liver can synthesise a-hydroxy-acids with ammonia to form the corresponding u-amino-acids ; from lactic acid, alanine is therefore obtained thus : — Moreover, Embden and Schmitz (1910) actually found that a liver rich in glycogen showed a considerable formation of alanine when ammonium chloride was added to the perfusion fluid. The liver can also use the ketonic acid for Synthesis of the

corresponding amino-acid. It is only necessary then to supply this organ with the appropriate hydroxy or ketonic acid in order that an amino-acid may be formed by it. However, we know as yet of no a-hydroxy or a-ketonic acid produced by the organism with the exception of lactic and pyruvic acids, both of which give alanine. Dakin and Dudley (1913, 1) have shown that many amino-acids, in fact all examined by them, namely glycine, alanine, valine, leucine, phenyl-alanine and aspartic acid, undergo spontaneous dissociation at low temperatures into the corresponding a-ketonic aldehyde and ammonia. The reaction is no doubt reversible and probably catalysed by an enzyme, so that the formation of an aminoacid from the hydroxy-acid seems to pass through the intermediate stage of the corresponding ketonic aldehyde. Thus: —

The reader may be reminded that it is the a-amino-acids that are wanted by the organism, that is, those in which the NH., group is next to the carboxyl. The experiments of Henriques and Hansen (1904) are of interest in this connection. They found that rats could be maintained in nitrogen equilibrium, that is, without loss of nitrogen, if they were fed on the mono-amino-fraction of the products of digestion of proteins. Since the tissue proteins contain diamino-acids, there must have been synthesis of these, if the results are correct.

Hindhede (1914) finds that the minimum nitrogen required with a diet of bread is the same as that with potatoes ; it seems that we must conclude that no special kind of amino-acids is necessary, at all events for maintenance. On the other hand, we have to remember that it is possible for a diet to contain all that is necessary for repair of adult tissue, although it may not contain some constituent required for new growth. If this constituent cannot be formed in the organism itself, it is clear that the diet will be inadequate for growth. This fact has already been insisted upon. Osborne and Mendel (1912, 2) have maintained adult rats for as long as 530 days on gliadin and protein-free milk. Now gliadin is wanting in glycine and lysine, which are necessary as constituents for the growth of new tissues, and, in fact, experiment showed that the diet in question was actually inadequate for growth. It seems probable that the wear and tear of the cells does not involve disintegration of that particular protein which contains lysine, or that the group containing lysine may be left intact when other parts are split off.

There is another way in which the want of a particular constituent in diet may be of importance. As we shall see in more detail later, there are numerous substances produced by various organs which act upon other organs, such as adrenaline. These are essential for the normal functions of the organism, so that if they require for their production some particular chemical grouping, which the organism itself is unable to supply, this grouping must be present in the food, otherwise normal life is impossible.

There is no doubt that Tryplophane is of especial importance. This substance is found in most proteins used for food, and has the constitution : — that is, skatol-amino-acetic acid. Now the protein of maize, zein, is peculiar in containing no tryptophane. Hopkins and Wilcock (1906) found that mice were unable to live for more than about twenty days on a diet of zein, carbohydrate, and fat. Whereas, if tryptophane were added, the animals not only lived much longer, but were obviously in better condition.

Further evidence is afforded by the experiments of Henriques (1907) in conjunction with those of Abderhalden and Frank (1910). It has been mentioned above that the products of complete enzymic hydrolysis of proteins are sufficient as protein diet. Further, if the proteins are hydrolysed by the action of acid, provided that the hydrolysis be only carried on for six hours, it was found by Henriques that the products were also able to maintain nitrogen equilibrium.

Whereas if the hydrolysis were allowed to proceed for seventeen hours, the resulting product was useless. The only detectable difference was that the tryptophane reaction was still present after six hours, absent after seventeen hours. Abderhalden and Frank completely hydrolysed horse flesh by boiling with sulphuric acid and then added 0-5 per cent, of tryptophane. This mixture was adequate for dogs. It is impossible to say definitely what the function of tryptophane is, perhaps for the elaboration of some internal secretion, as suggested by Hopkins.