Principles of General Physiology

Effect of Increase of Structural Differentiation. — The comparison of the oxygen consumption of the fertilised egg of the sea urchin with that of the unfertilised egg gave Warburg (1908 and 1910) opportunity to study this factor. The increase is considerable, but not directly proportional to the number of new nuclei formed. The change from one nucleus to a thousand, for example, only causes a threefold increase in oxygen consumption. This fact indicates that the " structure " in question is not the visible one of nuclei, and so on. A remarkable fact, however, is that, if the eggs are cytolysed by placing in distilled water, and shaking, the resulting suspension of apparently structureless debris consumed as much oxygen as the normal cells in the case of the unfertilised . eggs ; but it was reduced to one-tenth in the fertilised, dividing eggs, although the structure did not appear to be so completely destroyed as in the former case. A significant fact, which shows that the oxidation process, even in the unfertilised eggs, was not normal after cytolysis, is that the carbon dioxide production ceased.

Effect of the Cell Membrane. — We have already referred to the fact that changes of permeability occur in the act of fertilisation, and Warburg (1908) has shown that, coincidently with this, the rate of oxygen consumption rises considerably. Further, it was shown, as already mentioned (page 142), that alkali, even when it does not enter the cell, causes a large rise in the oxygen consumption. It is evident, then, that changes in the cell surface alone produce profound effects on the cell mechanism, and further evidence is afforded that the " structures " are of very minute character, since no obvious change takes place in the cell. The minute nature of the protoplasmic elements was pointed out above (page 19). The machinery can be put out of work, although no visible change may have occurred. As if, in a petrol motor, the accumulator cells used for ignition were discharged.

Lillie states (1913) that the formation of indo-phenol blue by oxidation of a-naphthol and dimethyl-paradiamino-benzene takes place most rapidly at the nuclear and cell membranes of the frog's blood corpuscles. The passage of induction shocks is said to accelerate this reaction, so that electrical polarisation of these surfaces is held to play a part. Effect of Cyanide. — The action of potassium cyanide in extremely small concentration is to stop all oxidation processes in cells, without doing any permanent damage. Recovery can be obtained by washing away the cyanide. This paralysis of oxidation has been referred to above (page 448) in relation to the analysis of the muscle processes. The work of Weksacker (1912) on the heart of the frog gives some interesting facts. We find, to take an example from his table on p. 140, that a particular heart, in absence of cyanide, performed 1,180 g.-cm. of work with a consumption of oxygen corresponding to 23 mm. of

the scale of Barcroft's apparatus, and an evolution of 25 mm. of carbon dioxide. In the presence of m/6,000 potassium cyanide, the same heart performed more work (1,380 g.-cm.) with the consumption of only 8 mm. of oxygen, and gave off 9 mm. of carbon dioxide. On p. 143, it is shown that the excitability of the heart to electrical stimuli, even when the consumption of oxygen has been completely abolished by m/2,000 cyanide, is unchanged. The explanation given by Warburg of the action of cyanide will be found immediately.

Relation to Catalysts. — Warburg and Meyerhof (see Warburg, 1914, p. 334) have obtained results with unfertilised, cytolysed sea urchin eggs which show the importance of iron. The ash of the eggs was found to contain considerable amounts of iron. The addition of iron salts to the egg substance caused very considerable increase in the oxygen consumption, an effect not produced by other metallic salts, not even by manganese. Alcohol extracts were also made, evaporated to dryness, and the residue extracted with ether. A part remained undissolved, and this part consumed no oxygen, even on the addition of iron salt. The ether extract was evaporated, and the residue suspended in water. This suspension, by itself, consumed no oxygen, but, on addition of iron, the oxidation amounted to as much as that of the original egg substance. Substances of a "lipoid" nature are, therefore, responsible for the phenomenon.

Now, Thunberg (1911) has observed that lecithin, in the presence of iron, consumes oxygen at a considerable rate; and lecithin is present in the eggs. Further, we have seen reason, in preceding pages of this chapter, to hold that the activity of the peroxidases of the cell depends on their content in iron (or manganese). But, since lecithin is not, to any perceptible degree, autoxidisable, the origin of the cell peroxides remains, as yet, unexplained. A further difficulty is the absence of carbon dioxide production, both in the cytolysed eggs and in the action of iron salts on lecithin.

According to information given me by Dr Weizsiicker, Warburg has recently found that the amount of potassium cyanide required to stop oxidation in the egg cells of the sea urchin is precisely, equal to that required to combine with the iron which they contain. This fact distinctly points to the iron as the catalyst concerned in oxidation. It is difficult, however, to see exactly what compound of iron, containing cyanide ion, could be reversible under the conditions of the life of the cell. It must be a complex ion of the nature of the ferrocyanic ion ; but it is not a simple process to recover the iron from such compounds under conditions which would be possible in a protoplasmic system.

Vernon (1914, p. 220) points out that the indophenol oxidase is inhibited by narcotics in a series closely corresponding to their anaesthetic action on tadpoles, and draws the conclusion that the oxidation process is connected with lipoids. He shows that nucleo-proteins have nothing to do with it. Mode of Action of "Structure." — Warburg (1914, p. 337) suggests that the essential importance of structure consists in the presence of surfaces for condensation of the catalysts active in the cell processes ; in other words, for adsorption. The action of narcotics of the alcohol series is much greater on the fermentative power of yeast cells than on that of the press juice, apparently because these substances are highly adsorbed and drive off the enzymes from their state of condensation on the surfaces (see the paper by Warburg, 1913, 1, p. 20).

This observer does not think that the view of separate " reaction-chambers," although it may apply to other chemical reactions in the cell, is applicable to the case of oxidation, because the fluid contents presumably contained in these spaces can be changed by diffusion in certain cases, without affecting the oxidation processes. It does not appear to me, however, that the evidence is sufficiently convincing to show that the essential contents were actually changed in these instances. The active contents of the hypothetical, ultra-microscopic vacuoles might be indiffusible, or held by adsorption. Probably both surface condensation and microscopic reaction-chambers play a part.

There are some further experiments by Warburg (1913, 2), which require mention here. Mammalian liver was rubbed with sand, water added, and the mixture centrifuged for ten minutes. A suspension of fine particles was obtained, which absorbed oxygen and gave off carbon dioxide, thus confirming certain statements of Battelli and Stern with respect to " water-soluble respiration." The amount of oxygen consumed was about one-fifth of that consumed by the intact liver in an equal time. The remaining four-fifths are what Battelli and Stern call the "chief respiration" (1909). The particles were small enough to show Brownian movements, but were removed by filtration through a Berkefeld filter. No doubt they would be removed by the Buchner method in Harden and Maclean's work. The Berkefeld filtrate certainly showed a slight oxygen consumption, about onetwenty-fifth of that of the entire liver, but there might have been a few ultramicroscopic particles present. .

As frequently pointed out already, the energy required for the various purposes of the organism is derived, except in very special cases, entirely from oxidation. The necessity of considerable combustion in muscle cells doing external work is clear, as also where gland cells are performing osmotic work. But, as Warburg points out (1914, pp. 256-258), the high oxygen consumption of nucleated blood corpuscles, of the central nervous system and of the developing egg, is difficult to understand, since there is no apparent work done, with the exception of a minimal amount. This is especially noticeable in the last case referred to, since the rate of oxidation has no relation even to the morphological changes taking place. What then becomes of the energy ? It would seem wasteful if it were merely degraded to heat. Warburg, therefore, makes the suggestion already referred to, that there may be work done in a way that is invisible, but yet indispensable. It may be required to maintain the " structure " of the cell, in the sense of preventing the mixing of constituents by diffusion, in maintaining intact certain properties of the semipermeable membranes, such as electric charge, or possibly irreciprocal permeability and other states of which we have, at present, little knowledge.

The relation of oxygen to the work of the muscle cell has been given in some detail above (page 446), arid reference also made to the work of secretion and so on. In the present place, some interesting investigations, chiefly by Meyerhof, on the total energy changes of certain isolated cells, as indicated by heat production, may be referred to. Experiments on whole organisms, such as those done by Rubner, Benedict, Macdonald, etc., show that the heat production is practically identical with the loss of chemical energy of the food-stuffs. Bohr and Hasselbalch (1903) determined the heat production of the developing chick, comparing it with the respiratory exchange, and found it to be identical with that of fat, as indicated by the respiratory quotient. The fact is interesting as showing that the formation of the morphological structures which contain nitrogen uses up no measurable amount of energy.

The experiments, of Meyerhof (1911) were concerned with the developing eggs of the sea urchin. The "caloric quotient" was first determined. This is the number expressing the amount of heat formed, in gram-calories, per milligram of oxygen consumed. Previous workers, Zuntz, Pfliiger, Rubner, found this number to be, neglecting the second decimal place, when protein is burnt, 3'2 ; when fat, 3 '3 ; when carbohydrate, 3*4 to 3-5. Now that of the developing egg is between 2'55 and 2 -9. This number is made slightly lower if the heat of solution of carbon dioxide and that of its combination to sodium bicarbonate is taken into account. This value, moreover, remains the same, whether fertilised or unfertilised eggs are taken, or if cell division is prevented by the presence of phenyl-urethane, as in Warburg's experiments. If work had been done in formation of coarse morphological structures, it is plain that the values could not be the same in these different cases.

It will be remembered that Warburg showed that ammonia, which enters the cells, stops cell division, but increases slightly the oxygen consumption. Now in such cases, Meyerhof found the caloric quotient raised to 3'3. If the heat of combination of ammonia with carbon dioxide be deducted, the value falls to 2 '95 ; but this is the maximum deduction permissible, so that the value is certainly raised above the normal one. As to the meaning of the low value of the caloric quotient, no evidence of the presence of carbohydrate was to be found, and there was no breakdown of protein. Fat, on the other hand, was found in sufficient amount to cover the heat produced.

Further experiments (191 '2, 1) were made with nucleated blood corpuscles of birds. In this case, a "normal" caloric quotient of a value between that of protein and fat was found. As Meyerhof remarks (1912, 2, p. 1), it can scarcely be an accidental coincidence that, when the normal caloric quotient was found, the cells were in a stationary condition, in the other case in active multiplication. In the case of aerobic bacteria, Meyerhof (1912, 2) finds that the caloric quotient is from 4 '5 to 4'7, whether growth is in progress or inhibited by deficiency of food material, being rather higher in the latter case. This high quotient seems to be due to reactions in the nutrient solution brought about by products of bacterial action.

Horace Brown (1914, p. 223) finds that the heat production of yeast, as grown in fermenting solutions, is, weight for weight, somewhere about seventy times as great as that of a man at rest. An explanation of this relatively enormous metabolism is suggested on the basis of the abnormal conditions under which yeast is cultivated for industrial purposes, as compared with its natural habitat on the outer skin of fruits. It must be remembered that we know now that there is no inverse proportion between fermentation and growth. In absence of oxygen, no growth takes place, but, as Pasteur showed, the fermentation process goes on with vigour. The cells remain constant in mass and in composition, so that no energy is needed for growth, yet, as Horace Brown puts it (p. 224), " there is an enormous activity in the metabolic mill, through which continues to pass an amount of substance which may amount to several times the mass of the cell in a few hours."

Further considerations on the question of anaerobic existence will be found in the next chapter. In the paper referred to (p. 226), Horace Brown states that he has obtained evidence that less heat is evolved from the same amount of sugar fermented, when growth is taking place, than in its absence. Quantitative measurements of this kind would give, of course, what might be called the "heat of formation" of yeast. The experiments of Ehrlich (1885) are of much interest in this respect, although they are not, in all cases, easy to interpret. Two dyestuffs were used in intravenous injection, both capable of oxidation and reduction. One of them, alizarin blue, is reduced with difficulty ; it requires boiling with caustic alkali and glucose. The other, indophenol blue, is more easily reduced.

We saw in our first chapter that living protoplasm itself does not stain with soluble dyes ; the two dyes used in Ehrlich's experiments were, accordingly, introduced in the form of suspensions, or colloidal solutions, and the particles were then taken up by the cells of various organs and found therein subsequently, either as the reduced, colourless derivative, or the oxidised, blue one, according to the oxidation potential of the cell system. Alizarin Blue. — This dye does not become reduced in the blood, but is reduced in the liver, the renal cortex, the Harderian gland, and the lungs. The amount taken up depends, as would be expected, on the permeability of the cell membrane.

Indophenol Blue. — The heart and the brain, together with certain voluntary muscles, such as the diaphragm and the eye muscles, are blue. The renal cortex and some other secreting glands also do not reduce the dye. By all other organs it is reduced. In general, it may be said (Ehrlich, 1885, p. 109) that the reducing power of protoplasm lies between that required by alizarin blue and by indophenol blue. But it is to be remembered that "protoplasm," as used here, means the cell constituents as a whole.

1. Those of high "oxygen saturation," in which indophenol blue is not reduced. Such are the grey matter of the brain, the heart, and some other muscular organs. 2. Those which reduce indophenol blue, but not alizarin blue. Such are the greater number of the tissues, smooth muscle, most voluntary muscles, and secreting glands. 3. Those which reduce even alizarin blue. Lungs, liver, fatty tissue, Harderian gland. As to the meaning of the facts, Ehrlich points out that, in activity, as also in asphyxia, practically all cells become highly reducing, and that the state of a cell at any given moment depends on the rate at which it consumes oxygen in relation to that at which it is supplied. At the same time, it is necessary to assume that a series of substances of different reducing power make their appearance.' Thus, a substance which has less affinity for oxygen than alizarin blue has cannot reduce it, and a further stage of reduction must occur before this takes place.

The important question of the facility of access of oxygen belongs to those to be discussed in the next chapter. The fact that fat tissue has so high a reducing power, shows that oxygen avidity is not necessarily to be ascribed to great functional capacity. The chemical nature of certain permanent constituents of the cell must be taken into account. This should be kept in mind with regard to the paradoxical experimental fact of the reducing power of the lung tissue. Ehrlich ascribes the property to the stroma cells, not to the alveolar epithelium, and suggests that it may be due to an appropriate relative impermeability of these cells to oxygen, so that they shall not unnecessarily retard the aeration of the blood.

After what we have learnt in the present and preceding chapters in connection with the phenomena of autoxidation and their relation to catalysts, together with that of chemi-luminesceiice, further brief reference may profitably be made to the problem of the emission of light by living organisms. One of the most important practical questions at the present time is that of the improvement of the efficiency of our methods of artificial illumination. The majority of these, as used now, depend on the emission of light by substances when heated to a very high temperature. The higher they can be heated, the greater the proportion of light to heat rays. Hence the advantage of the metallic filament lamps over the old carbon filament, and especially that of the electric arc over other forms of illuminant.

We have seen, however (page 557), that, in the phenomena of chemiluminescence, we have light emitted at temperatures far below those to which it would be necessary to heat a metallic wire in order to obtain light of the same wave length. We may put it thus, chemical energy is transformed directly to light energy, without passing through the state of heat. Now the problem appears to have been solved by numerous organisms, although the quantity of light they emit is not great. Amongst these organisms we may mention fungi (including bacteria), protozoa, medusae, insects, molluscs, and fish. For individual details of these organisms, the reader is referred to the articles by Mangold (1910), Dubois (1903, 1913), Coblentz (1912), and for plants, Molisch

The fact that living cells can emit light shows at once that the fact is not due to their temperature, as in the case of the ordinary sources of light. Phosphorescence, in the true sense, requires previous exposure to light, and is easily excluded. We are left then with phenomena related to chemi-luminescence. Spectral examination shows, accordingly, that the light is limited to the middle region of the spectrum, usually having its maximum in the green (Langley

and Very, 1904). Fig. 183 gives photographs of the spectrum of the light of the fire-fly, taken by Coblentz. It will be seen that the spectrum extends considerably less, both towards the red and towards the violet, than that of the carbon filament lamp. It is, in fact, according to Coblentz, very like that of an ideal radiator at a temperature of 5,000°. Langley and Very (1890) showed that the heat radiation of Pyrophorus noctilucus (a South American beetle) is only one-four-hundredth of that given out by incandescent source^, such as candle light, reduced to the same luminosity.

In its chemical aspect, the phenomenon is evidently an oxidation. In many cases, the cessation of light in absence of oxygen and its reappearance on admission of oxygen have been demonstrated. The nature of the substance FIG. 183. LIGHT OF FIRE-FLY COMPARED WITH THAT OF ARTIFICIAL SOURCES. A, Photographs of spectrum of carbon {flow-lamp, for different exposures. 1, helium vacuum tube. 3, 5, and 6, fire-fly, Photinwi pyralix 2, carbon glow-lamp, four watts per candle power. | 4 and 7, fire-fly, Photinus pennxylvanica.

The colours of the bright lines in the spectrum at the" top are as follows : — jfote that the light emitted by the fire-fly is confined to the middle region of the spectrum and is most intense in the yellow-green, while that of the carbon glow-lamp is most intense in the orange (see the uppermost spectrum in A, below the bright line comparison spectrum). (Coblentz, 1912 ) oxidised is not yet known. The particular case of Pholas has been referred to above (page 362). According to Dubois (1913), the oxidation here is under the control of an enzyme, an " oxidase " ; the secretion is luminous after removal from the cells, as can readily be verified. In any case, the reaction seems to be independent of the "vital" activity of cells. Schultze (1864) finds a substance which reduces osmic acid in the luminous organs of the glow-worm, probably a derivative of oleic acid.

There is evidence that a certain small amount of heat is developed in the active glands of the fire-fly (Coblentz, 1912, p. 33), but this appears to be associated with the secretory process. Water is necessary. Dried material can, however, be brought to shine again by moistening. Although the production^ of light can occur by reaction with oxygen of substances formed in the cell, in many cases the process takes place already inside the cell itself. In other cases the luminosity does not make its appearance until the secretion is extruded.

The use of the production of light to the organisms themselves is somewhat problematical. It may serve to attract prey and, in some cases, it appears to assist the progression of the organism in the dark, like an ordinary lantern. In connection with chlorophyll and with oxidation enzymes, the nature of the coloured pigments of flowers is of interest. As Willstatter points out, it is a suggestive fact that the vitally important leaf pigment is the same in all plants, whereas those of the flower are of various chemical constitution. A large number, as shown by Willstatter and Everest (1913), are related to' flavones, but with the oxygen atom marked (A) replaced by hydrogen, while a varying number of the benzene hydrogens are oxidised or replaced by other groups. Flavone is : —

This quinonoid structure, as is well known, is associated with coloured substances. In the flower, the flavone derivatives are combined with glucose to form glucosides and are capable of oxidation under the influence of an oxidase. Thus Keeble, Armstrong, and Jones (1913) found that the yellow sap pigment of the wallflower is a mixture of hydroxy-flavone glucosides. These are readily hydrolysed by mineral acids, or, more slowly, by emulsin. The hydrolysed product, if reduced and again oxidised, gives a red pigment.