General Physiology: An Outline of the Science of Life

The globulins are soluble in water only when it contains neutral salts, but in less quantity than in saturation. If a solution of globulin be saturated with salts, the globulin is precipitated in a flocculent mass — a phenomenon that is termed " salting out." The globulin is likewise precipitated if the solution be wholly freed from salts by diffusion in a dialyzer. To the globulins belong serum-globulin, which is dissolved in the blood-serum ; fibrinogen, also a proteid of blood, which coagulates spontaneously into flakes and threads of fibrin when the blood is allowed to stand outside the blood-vessels ; myosin, the globulin of muscle, which likewise

coagulates spontaneously upon standing — a phenomenon that appears in dying muscle in rigor mortis ; and, finally, plantglobulin, which gives to kernels of grain their glutinous quality, and hence has been termed glutin. The mtellins are likewise soluble in neutral salt solutions only, but, in contrast to the globulins, they are not precipitated by saturation of the solution with salts. Among them are the so-called yolkplates of the yolk of eggs, and the already-mentioned aleurone grains of plant seeds, both of which are proteids capable of crystallization.

The above-mentioned proteids occur in a free state in living substance. A very large number of proteids, however, are not free, but are chemically combined with other substances. In these compounds, which have been termed combined proteids in distinction from the simple proteids, the proteid molecule behaves in general like a feeble acid, and by the addition of stronger acids it can frequently be forced out of its compounds/ the stronger acid taking its place. The proteid then becomes free. We have already become acquainted with one of these compounds, haemoglobin, which plays in blood so important a role and is a compound of proteid and iron. But the most important compounds, in which proteids appear without exception in every cell, are the nucleins. The nucleins, as Altmann ('89) has shown, are compounds of proteid with nucleic acid, an acid which is itself a compound of phosphoric acid with peculiar basic bodies, the so-called nuclein bases — guanin, adenin, xanthin and hypoxanthin. The nucleins are capable of entering into further combinations with a second proteid molecule, and these extremely complex compounds are termed nucleo-proteids or nucleoalbumins. Casein, a body which for a long time has presented difficulties to the physiological chemists, is such a nucleo-proteid, combined with calcium. Casein is the calcareous nucleo-proteid of milk that is manufactured into cheese ; it has the peculiarity of not coagulating when the milk is boiled, while it is immediately precipitated when separated, as by acetic acid, from the calcium. A fourth group of combined proteids is that of the glyco-proteids, in which proteid is combined with a carbohydrate ; prominent among these is mucin, which is contained in the cells of mucous glands.

Besides the genuine proteids which we have just described, there exist a number of bodies which behave in many ways similar to proteids and, therefore, have been termed albuminoids. The group of albuminoids is a true omnium gatherum ; it contains a very large variety of .bodies. These are partly compounds of proteids and partly bodies of similar constitution to the proteids, but which show in their chemical behaviour much less similarity and are much less known than the proteids themselves. Especially prominent among albuminoids are many of those substances that are produced by cells to serve as skeletal substances for the support

of the more delicate parts of the organism. A detailed examination of the known reactions which the numerous albuminoid bodies present would lead too far and be superfluous for our purpose.1 It is sufficient here to cite some of the most important members of the group, all of which occur in the solid undissolved state. Such are keratin, which is contained in most horny structures produced by the epidermis-cells of the skin (horns, hoofs, hairs, feathers, and nails) ; elastin, which composes the elastic fibres of the cells of connective tissue and the strong yellow ligamentum nuchce ; collagen, which composes the organic ground -substance of bones and cartilage, and in boiling passes over by hydrolysis into gelatine ; spongin, the skeletal substance of bath-sponges ; conchiolin, the organic substance of the shells of mussels and snails ; cornein, that of the skeletons of corals ; and many other substances that form skeletons, especially in invertebrates.

With the albuminoids also is classed a series of highly complex nitrogenous bodies which at least are derivatives of proteids and possess the greatest importance in the life of the organism, especially in digestion. These are the unformed ferments or enzymes, such as pepsin, produced by the gland-cells of the stomach ; ptyalin, by the cells of the pancreas and the salivary glands ; trypsin, produced likewise by the pancreatic cells ; and many others. Tihe properties of these bodies and their roles in the life of the cell will be considered more fully elsewhere.

There appear in living substance, as constant accompaniments of proteids, certain decomposition-products of them which can be divided into two groups — the nitrogenous and the non-nitrogenous cleavage-products. The former constitute a series of substances whose chemical constitution is more exactly known. They are the products of retrogressive proteid-metamorphosis. Among them belong especially the substances excreted in considerable quantity by the higher animals in the urine. Among them urea, (NH2)2CO, holds the first rank ; it is the richest in nitrogen of all the nitrogenous end-products of proteid-decomposition, and its artificial synthesis was accomplished by Wohler in the year 1828. Next to urea, uric acid, C5H4N4O3, contains the most nitrogen ; next to uric acid come in order hippuric acid, creatin, which originates in the muscles by the decomposition of proteid, and creatinin. Further, the nuclein bases, xanthin, hypoxanthin or sarkin, adenin and guanin are met with as end-products of the decomposition of nucleins in the living organism. Of these, especially the last in combination with calcium occurs very frequently in the skin-cells of Amphibia and of fishes, in the latter of which its crystals produce the well-known silvery sheen. Finally, there is one more group of nitrogenous bodies, the lecithins,

1 A review of the subject and the bibliography of it may be found in Neumeister : Lehrbuch der physiologischen Chemie. 2nd edition, Jena, 1897. which stand near the fats, but contain phosphorus ; they are probably present in every living cell and, according to Hoppe-Seyler, are to be regarded as cleavage-products of proteids, especially of nucleins, with which they occur. Among the non-nitrogenous end-products of proteid-decomposition carbonic acid, which is produced by every cell, comes first in importance. Lactic acid, oxalic acid, and sulphuric acid are important. The cholesterins also are to be regarded at least as derivatives of proteids ; they seem to occur in all living substance, but appear in great quantity only under certain circumstances in the form of iridescent scales, as upon the surface of the skin and the beak of birds, and in pathological conditions as gall-stones in the bile. Chemically the cholesterins are univalent alcohols, which with fatty acids can form fat-like compounds. Finally, there appear as decomposition-products of proteids certain carbohydrates, particularly grape-sugar and glycogen, and fats, which must be considered somewhat in detail in connection with allied substances.

In contrast to its presence in the proteids, nitrogen is wanting in the carbohydrates. The latter contain only the three elements, carbon, hydrogen and oxygen ; in the natural carbohydrates the number of carbon atoms within the molecule is always six or a multiple of six, while the number of hydrogen atoms is always double that of the atoms of oxygen ; hence hydrogen and oxygen are present in the same relative proportions as in water — a foct which led to the designation " carbohydrates." The carbohydrates are very wide-spread and are of great importance, especially in the manufacture of living substance in plant-cells; but there are varieties of living substance in which they cannot be demonstrated ; in other words, they are not general constituents of such substance. They present far simpler chemical relations than the proteids, and a brief glance will show their most essential features.

The natural carbohydrates may be divided into monosaccharids, disaccharids and polysaccharids, of which the two latter groups are different anhydride forms of the first group. The monosaccharids all have the formula C6H12Og, and are, therefore, isomeric ; but they are not all stereo-isomeric, that is, their individual atoms are not grouped alike in all. To the monosaccharids belong chiefly grape-sugar (dextrose or glucose) andfruitsugar (Isevulose), both of which are wide-spread in plant juices, the former in great quantity also in animal tissues. One of the most remarkable characteristics of the monosaccharids is that they readily take up oxygen from their surroundings and thus reduce bodies that are rich in oxygen, a peculiarity upon which depend the most important tests for their recognition. The most re-

liable of these reductiontests are Trommer's test and Bottger's test. They may be performed very simply in a test-tube. The former consists in the reduction of cupric hydroxide to cuprous oxide by an alkaline solution of grape-sugar. If a few drops of a very dilute solution of cupric sulphate be added to a sugar solution, made alkaline by caustic potash or soda, until a blue flocculent precipitate of cupric hydroxide appears, on boiling the latter is reduced to red cuprous oxide or yellow cuprous hydroxide. In Bottger's test a few drops of a solution of basic nitrate of bismuth is added to the alkaline solution of grape-sugar ; the former is then reduced to black metallic bismuth. A further very characteristic property of the monosaccharids is their power of fermentation. They become decomposed by the action of yeast-cells (Saccharomyces) into alcohol and carbonic acid —

Such an experiment can be carried on best in a fermentation - glass (Fig. 39), by introducing into it a solution of grape-sugar FIG. 39.— Fermentation-tube— a, newly filled ; b, with carbonic acid developing. At the top of the straight limb a quantity of carbonic acid has already accumulated. mixed with fresh yeast, so that the liquid fills completely the long closed limb of the glass. At a temperature of c. 30° — 40° C. there appears a fairly energetic cleavage of the grape-sugar, small bubbles of carbonic acid rising continually as in a glass of champagne, and accumulating at the upper end. The more carbonic acid accumulates above, the more the liquid is forced out of the long limb into the spherical part of the vessel, until finally the former may be entirely filled with the gas. The presence of alcohol may be recognized at once by the odour of the liquid. One more characteristic of the monosaccharids may be mentioned,

which they share with all soluble carbohydrates — viz., the power of rotating the plane of polarised light. As their names indicate, dextrose rotates it to the right ; Isevulose, to the left. The disaccharids may be regarded as having arisen from the monosaccharids by the combination of two molecules of the latter and the loss of a molecule of water ; this would yield the formula — Among the disaccharids are to be noted especially cane-sugar (saccharose), which is contained in large quantities in the cell-sap of the sugar-cane ; and milk-swgar (lactose), the carbohydrate of milk. By certain methods, as by boiling with dilute inorganic acids or by the action of certain bacteria, the disaccharids can be made to undergo hydrolytic cleavage, so that they pass over into the monosaccharids. This change is termed inversion. In contact with certain fermentation-agents, especially Bacterium lacticum, the disaccharids are induced not to ferment directly, but to pass over into monosaccharids, which are themselves subject to the fermentative action of these organisms. If Bacterium lacticum be employed as the fermentation-agent, lactic acid results —

— a process which, in contrast to alcoholic fermentation by the yeast, plant, is termed lactic acid fermentation ; to it is due the souring of milk exposed to the air. Finally, under the influence of another fermentation agent, Bacillus butyricus, lactic acid can be still further decomposed into butyric acid, carbonic acid and hydrogen — The polysaccharids are anhydride stages of the monosaccharids. still further removed ; in them several monosaccharid molecules combine with the loss of a molecule of water, so that their formula is a multiple of C6H10O5. Among the polysaccharids occurs a series of bodies that play an important role and are widespread, some in the life of the plant-cell, others in many animal-cells. They are, first, starch, which occurs in all green cells of plants in the form of granules, in which the layers are arranged concentrically (Fig. 40) ; secondly, glycogen, which occurs as flakes and irregular particles, especially in the cells of the liver, but in smaller quantities in many other tissue-cells; thirdly, cellulose, which constitutes the cell-membranes of all plant-cells, and has been demonstrated also in the leathery mantle of the Tunicates. These members of the group of polysaccharids may be distinguished from one another in a very characteristic manner by their behaviour towards solutions of , iodine : by iodine starch is coloured an intense blue, glycogen a mahogany brown, and cellulose

not at all ; the latter, however, becomes blue in the presence of iodine and sulphuric acid. In addition to the free carbohydrates, combinations of carbohydrates exist in living substance — e.g., combinations with proteids, as an example of which mucin has already been mentioned. The most important decomposition-products of carbohydrates have also been mentioned, such as lactic acid, butyric acid, carbonic acid, etc., all of which are met with in living substance.

The fats likewise do not belong to the general constituents of living substance, but they are wide-spread, chiefly in animal cells. Like the carbohydrates, the fats are non-nitrogenous, and contain only the elements carbon, hydrogen and oxygen. But chemically they differ fundamentally from the carbohydrates. For example, they represent the so-called compound ethers, or esters — i.e. compounds in which an acid has combined with alcohol with the loss of water. The alcohol that is the basis of all fats is glycerine, C3H5(OH)3, and the acids that are combined with glycerine belong to the series of fatty acids, whose general formula is CUH211O2. Since the glycerine represents a trivalent alcohol, in the neutral fats three atoms of fatty acid are always combined with one atom of glycerine into tri-glycerides. The general formula of the fats is, therefore—

As examples of the fatty acids there may here be mentioned palmitic acid, stearic acid, butyric acid, valeric acid and capronic acid. In addition to these, oleic acid, which does not belong to the normal series of fatty acids, occurs in the various oils combined with glycerine. In correspondence with their composition, the neutral fats may by certain methods be split up by hydrolysis into their constituents — i.e., into glycerine and free fatty acids ; this process takes place in the organism as the result of the action of the digestive juices. It

FIG. 40. — /. Plant-cell containing starch-grains. II. Starch-grains isolated — a, from the potato ; b, from the corn ; c, from the pea. occurs also when neutral fats are boiled with alkaline liquids, such as caustic potash or soda. The fatty acids thus set free combine with the alkali to form the so-called soaps, which may be distinguished as potash soaps, sodium soaps, calcium soaps, etc. The fats are all lighter than water and do not dissolve in water, but they are easily soluble in ether. A characteristic property, which is important for the microscopic recognition of the fat-droplets in cells, is their power of reducing perosmic acid to metallic osmium, the latter forming a black coating to the fat-droplet. This osmic acid reaction is not to be employed alone as a sure test in the diagnosis of fat ; for doubtless other reducing substances exist, which, under certain circumstances, can be blackened by osmium; hence it should be used only in conjunction with other tests, solubility in ether, strong refracting power, etc.

The fact that fats, like carbohydrates, can appear as cleavageproducts of proteids has already been mentioned. In the case of the organic compounds of the cell the general constituents (proteids) and the special constituents (carbohydrates and fats) can be contrasted ; the same distinction can be made with the inorganic compounds. Here, also, the greater interest is associated with the general inorganic constitiients, among which there are distinguished water, salts, and gases.

' Water is that constituent of living substance that gives to it its liquid nature and thus renders possible the easy shifting of its particles, which is so necessary for the occurrence of vital phenomena. It is contained in the cell, in part chemically combined as water of constitution, and in part free, as the solvent medium of all sorts of substances. Accordingly, water is present in abundant quantity, constituting upon the average more than 50 per cent, by weight of living substance. If, e.g., the whole water contents of the human body be investigated, which with the great variety of the forms of tissue affords a good average, approximately 59 per cent, of water is found; this is shown especially by the detailed investigations of Bezold. The different tissues vary very greatly in this respect. Thus, bones contain only about 22 per cent, of water, the liver 69 per cent., muscles 75 per cent., and the kidneys 82 per cent. Hence it is not strange that the water contents of living substance varies much more in different species of animals, and that all intermediate stages in percentage composition are met with between the slight traces of water contained in a rotifer when dried but still capable of life, and the water-contents, amounting to more than 99 per cent., of certain pelagic Ctenophora.

Many salts occur dissolved in water, and they are present in all living substance. The compounds of chlorine appear to be especially important, as well as the carbonates, sulphates, and phosphates of the alkalies and alkaline earths, particularly sodium chloride (common salt), potassium chloride, ammonium chloride, and sodium, potassium, magnesium, ammonium, and calcium carbonates, sulphates and phosphates. Finally, as regards gases, there occur in all living substance oxygen and carbonic acid. When not in chemical combination, they are usually absorbed in water, and rarely, as in many unicellular organisms, e.g., Rhizopoda, in the form of bubbles of gas.

The special inorganic constituents of cells comprise a great variety of substances, but for present purposes it is unnecessary to discuss them. It is remarkable that in certain cells even free mineral acids appear, such as hydrochloric acid, which is produced by certain cells of the gastric glands in vertebrates, and sulphuric acid, which in many marine snails is secreted by the cells of the salivary glands. Although during the last few years our knowledge of cellmorphology has increased greatly, and microscopic investigation of the cell has revealed its finest structural relations, comparatively little is known of the chemical nature of its individual morphological constituents. Here is the point where physiological microchemistry must institute its work. The combination of microscopic observation and chemical reaction alone is able to bridge the gap between that which has become known morphologically as groundsubstance and solid constituents in the protoplasm and the nucleus, and that which gross chemical analysis has shown to be the constituents of living substance. The building of the bridge between the morphology and the chemistry of the cell is a difficult undertaking, since the majority of reactions that can be employed conveniently and easily in the test-tube, under the microscope on account of the minuteness of the objects either give very indistinct results, or are entire failures. Hence, first of all, delicate and reliable micro-chemical methods need to be devised. The first steps in this direction have already been taken, and we have begun to obtain here and there an insight into the distribution of the chemically known substances within the cell-contents.

It has been shown that the bodies that have been found as morphological differentiations in the cellcontents, differ also chemically. Especially the investigations of Miescher, Schwarz, Zacharias, Altmann, Kossel, Lowitt, Malfatti and others have proved that characteristic chemical differences exist between the constituents of the two essential cell-elements, the protoplasm and the nucleus. The proteids, which are the sole general chemical constituents of the cell, occur in both the protoplasm and the nucleus, but a very remarkable difference between them has been discovered. It has been found that the compounds of proteids, containing phosphoric acid, the so-called nucleins, preponderate greatly in the nucleus,1 while in the protoplasm they seem to be wanting entirely, or at least to appear only in combination with other proteids as nucleo-albumins ; the protoplasm, on the other hand, is constructed chiefly of simple proteids and proteid compounds that lack phosphorus. The employment of a simple chemical method confirms this fact. As Miescher ('74) has shown, the nucleins, in contrast to all other proteids, resist the digestive action of the gastric juice. If, therefore, cells of very different kinds be brought under the influence of artificial gastric juice, all other proteids are digested, while the nucleins remain. It is then found that the whole protoplasmic body is digested, while the nuclei are left with an inconsiderable decrease in volume and a somewhat ragged contour. If, now, the remaining substance of the nuclei be tested with the known nuclear stains, it is shown that what is wanting is the nuclear sap,2 and perhaps the achromatic substance also, for the whole remaining mass takes up the nuclear stain more or less strongly. It follows, therefore, that the chromatic substance and the nucleoli consist of nucleins, while the protoplasm of the cell is composed of other proteids. Lilienfeld and Monti ('93), in Kossel's laboratory, have endeavoured to prove by means of a micro-chemical reaction that phosphorus is localised especially in the nucleus. If ammonium molybdate be added to a substance containing phosphoric acid, a compound is formed, phospho-molybdic acid, which with pyrogallol takes on a dark brownish-black colour.

Lilienfeld and Monti were able to show that in a great variety of cells the nuclei stain black because of this reaction, while the protoplasm is left unstained ; but it should be mentioned that soon after the publication of their results Raciborski, Gilson and Heine raised the objection against the reaction that there was simply an accumulation of ammonium molybdate in the nucleus, which is analogous to the accumulation there of nuclear stains. Hence caution is still necessary in drawing conclusions from this reaction.

The carbohydrates appear to be limited to the protoplasm ; at least, thus far no carbohydrates have been found in the nucleus. In the protoplasm they appear not rarely as solid constituents, e.g., glycogen in the form of scales and irregular particles in the protoplasm of liver-cells, starch-grains in general in the protoplasm of all green plant-cells, and cellulose as a protoplasmic product upon the surface of cells. The fats also appear to be limited to the protoplasm. Without •exception they seem to be wanting in the nucleus, but are very wide-spread in the protoplasm as fat- and oil-droplets. They may always be recognised by their great refracting power, or, in dubio, by their blackening with perosmic acid and solubility in ether.

Concerning the distribution of the inorganic constituents of the cell almost nothing whatever is known. As to the potassium compounds, however, the investigations of Vahlen appear to show that they are to be found exclusively in the protoplasm, and not in the nucleus. These are the few facts thus far known. The chemical composition of the great mass of substances in the protoplasm that are termed granules, as well as that of the substances in solution, is thus far wholly unknown. Here an unbounded field is open to the physiological chemists of the future, and in a more distant future shall we have to look to the micro-chemical investigation of living substance for the solution of the final riddle of life.

The main points of the above examination of living substance may be summarised as follows : Living substance, as it now exists upon the surface of the earth, appears solely in the form of elementary organisms, the cells, some of which live separately, while some Are united together into coherent communities. Each cell is a bit of liquid substance, usually microscopic in size, in which various constituents, partly solid, partly in solution, are stored Only the liquid ground-mass, the protoplasm, and the somewhat more solid nucleus contained within the former can be regarded as general cell-constituents. A bit of protoplasm containing a nucleus is a complete cell, and, vice versa, there are no cells that do not possess nucleus and protoplasm. Just as very different morphological constituents may be distinguished in living substance, so very different chemical bodies are present. The elements of which they consist are only such as exist in the inanimate world also, but their number is small, and it is chiefly the elements having the lowest atomic weights that compose living substance. A special vital element does not exist, but the compounds in which these elements occur are characteristic of living substance, and in great part are absent from the inorganic world. They are, first of all, proteids, the most complex of all organic compounds, which consist of the elements C, H, O, N, and S, and are never wanting in living substance. Further, there occur other complex organic compounds, such as carbohydrates, fats, and simpler substances, all of which either are derived from the decomposition of proteids or are necessary to their construction ; and inorganic substances,

such as salts and water; the latter gives to living substance its requisite liquid consistency. In its main outlines the above is the picture that the anatomical, microscopic, physical, and chemical investigation of living substance has afforded. But the picture of living substance is still incomplete. In the above pages there have been presented the details of its composition as known at present, but the most essential point is still wanting. In what does the characteristic difference between living and lifeless substance consist ? This question is weighty, for it contains nothing less than the problem of all physiology — namely, the problem of life, which since the earliest times has had an irresistible fascination for inquiring minds.

As has already been seen, the conception of life has not been always the same. Since its origin among primitive peoples, it has become changed in diverse ways. We will now inquire whether it is possible to outline the conception scientifically by considering the differences between living and lifeless substance. Because of the sharp distinction between objects that never have lived, such as stones, and those that have lived and died, or corpses, this undertaking must be extended in two directions — first, to the differences between organisms and inorganic substances, and, secondly, to the differences between living and dead organisms.

In comparing organisms with inorganic substances, the mistake has been made of contrasting the organism with a crystal, instead of with a substance that has a consistency, and, in general, physical relations similar to those of living substance, i.e., with a semiliquid mass. Because of this mistaken comparison, a host of differences have been set up, the incorrectness of which is evident. Thus, it has been said that inorganic bodies have forms constructed according to simple mathematical laws and possessing perfectly definite angles and edges, while organisms have bodily shapes that cannot be represented mathematically. It is not necessary to cite in refutation the " crystallised human folk " which Mephistopheles claims to have seen in his years of travel ; the untenableness of this distinction becomes clear when it is recalled that, in the first place, mathematically simple body-forms do actually occur among organisms, as in the Kadiolaria, which are