Principles of General Physiology

An interesting fact is the occurrence of the colourless chromogen (reduced pigment) in the flower, together with both an oxidising and a reducing enzyme. The former is active in water, inactive in alcohol. The latter is inactive in water, active in alcohol. Or perhaps this reducing enzyme is overpowered in water by the opposite action of the oxidase. The colour thus disappears in alcohol, returns in water. In our discussion of the synthetic action of enzymes we saw reason to believe that there are mechanisms in the cell capable of reducing the effective concentration of water, and we see here that the removal of water retards oxidation, and thus facilitates the action of reducing enzymes.

There are some substances which are oxidised by the oxygen of the air. Others, and these are the food-stuffs of most physiological importance, require the oxygen to be made "active," as it is often called. In other words, the system which causes the oxidation of such substances as sugar must have a higher oxidation potential than molecular oxygen has. It seems to be most in accordance with experimental facts to suppose that it is when in the process of changing its valency, or electric charge, that oxygen is "active." This conception is practically the same as that of the nascent state. In the course of another reaction oxidative effects are frequently obtained, such as oxygen alone is unable to bring about. Chemical energy is thus made use of before it has become degraded to heat.

When a substance undergoes " autoxidation " by molecular oxygen, there are two oxides formed in equivalent proportion. In the production of the lower oxide, energy is given out, and this energy is utilised to build up the other oxide, which is a peroxide, and has a higher oxidation potential than the original system. The reaction is what is known as a " coupled reaction." There is reason to believe that reactions between three or more different molecules at the same time rarely, if ever, take place. Probably all reactions occur in stages between two molecules at a time.

Those peroxides produced in the autoxidation of phosphorus and in some other cases, have a considerably higher oxidation potential than such peroxides as that of hydrogen. In the presence of water, as in the living cell, peroxides of the former type, if produced, react with water to form hydrogen peroxide. Now such a peroxide as that of hydrogen has not sufficient oxidation power to bring about the combustion of glucose, for example. But there are inorganic catalysts, such as iron, and also enzymes, called "peroxidases," which decompose hydrogen peroxide with the liberation of " active " oxygen. These latter enzymes are found in the living cell.

Since the oxidations brought about by cells do not occur in the absence of oxygen although a peroxidase is present, we must conclude that the peroxide is absent. Hence, the peroxide is formed by the action of molecular oxygen on some autoxidisable substance in the cell. In the actual process of autoxidation, another substance, itself difficult of oxidation, may be drawn into the reaction, as it were, and become oxidised. But the peroxides also formed in the process are acted on by the peroxidase of the cell, with formation of additional " active " oxygen.

Artificial oxidation systems, similar to the natural ones, "oxidases," can be made by the association of colloidal hydroxides of iron or manganese with an emulsoid colloid. The function of the latter appears to be that of maintaining the active constituent in a state of high dispersion and protecting it from aggregation by electrolytes. Living tissues also produce reducing systems, which require the presence of substances such as aldehydes in order to show their activity.

In these reduction processes, the reactions called " hydrolytic-oxidativereducing " have to be taken into account. The explanation of these reactions. MS given by Bach, will be found in the text. They seem to consist in the decomposition of water and the formation of " unstable complexes " of hydrogen and hydroxyl ions with water molecules. The former acts -as oxygen perhydride, the latter is the hydrate of hydrogen peroxide. The perhydride is apparently decomposed by an enzyme, "perhydridase," analogous to peroxidase, with the activation of hydrogen. An enzyme of this nature has been prepared from liver.

In the living cell, the presence of autoxidisable substances, together with peroxidase, is not in itself sufficient to bring about the oxidations which actually occur. Disintegration of the cell, as by rubbing with sand, nearly puts an end to the consumption of oxygen by it, although the cell constituents are all present as chemical compounds. These constituents must be organised into some kind of a mechanism or structure. This is not the ordinary structure visible under the microscope, since the latter may be unaltered, but the power of consuming oxygen absent.

There are some facts which show that a certain degree of oxygen consumption may remain after disintegration of the morphological structures by rubbing with sand. The properties of the cell membrane are of importance for the oxidative processes in the cell. The importance of " structure," no doubt, consists partly in the provision of surfaces for adsorption and activation by concentration of the catalysts concerned in the cell processes. Probably the maintenance of ultra -microscopic " reactionchambers," by provision of senuperrneable membranes, also plays a part.

Certain fine particles have been separated fi'om liver cells, which absorb oxygen and give off carbon dioxide to the extent of one-fifth of that of the intact cells. The consumption of energy by cells for the purpose of opposing the mixing of their constituents by diffusion, maintaining intact the properties, electrical or otherwise, of the semipermeable membranes and so on, must be remembered in the interpretation of the oxygen consumption by tissues which perform no external work.

There is some evidence that, in the cases of yeast and bacteria, energy is used up for the purpose of growth. The reducing power of tissues depends to a large extent on their degree of activity, or, in other words, of the relative rate at which oxygen is consumed and supplied. The experiments of Ehrlich on the question are described briefly in the text. When organisms emit light, it is by a process of chemi-luminescence, in which the wave length of the light is much shorter than that corresponding to the temperature of the source. It is an oxidative process in which the chemical energy is used directly for conversion to light energy, without passing through the stage of heat. The system concerned, although secreted by cells, is active apart from living protoplasm.

The nature of certain flower pigments is described in the text and their relation to oxidising and reducing enzymes indicated. WE have seen in the previous chapters how the essential energy changes in cells are of the nature of oxidation, and we have discussed the nature of the mechanism by which ordinary molecular oxygen, arriving at the cell, is rendered active in order to burn up substances, not otherwise easily oxidised. Oxygen must, theiefore, be supplied to the cells and, in warm-blooded animals, at a considerable rate. In unicellular organisms, no special mechanism is necessary, but in larger organisms, it is clearly of importance that oxygen should be conveyed directly to the active cells, without having to diffuse through thick layers of cells, themselves consuming oxygen. At the same time, provision must be made for the escape of the carbon dioxide formed in combustion. The mechanisms concerned in this process are known as those of respiration.

In the majority of organisms, oxygen is conveyed to, and carbon dioxide removed from, the tissues in a state of solution of some kind in a liquid circulating through a system of tubes. This liquid is the blood. In insects, there is a system of ramifying tubes containing air. These are known as tracheae, and the air contained in them is periodically changed by muscular movements as well as by diffusion. In organisms provided with circulating blood, there is usually a means by which free gaseous interchange of blood with the external medium containing oxygen, be it water or air, is enabled to take place. In water animals we have gills, in land animals, lungs. Arrangements are also present by which the water or air, with which the interchange of gases takes place, is periodically replaced by a fresh supply. This is done by the aid of muscular movements. The external surface of the organism not being of a sufficiently large area for gaseous exchange, special organs are developed for the purpose of affording a larger surface. The mechanisms here referred to are usually known as those of external respiration, as contrasted with the oxidation process in the tissues, called internal respiration. Intervening between the two, we have to consider the process by which oxygen is carried in the blood. The question of internal respiration is closely connected with that discussed in the preceding chapter, although there are some aspects of it more appropriately described here.

That a continued supply of air is necessary to life, at all events in the higher animals, was shown clearly by Robert Hook (or Hooke) (1667, p. 539) in experiments made before the Royal Society at some of their early meetings. He showed at one meeting a dog, which was kept alive, after removal of the ribs and the diaphragm, by blowing air into the windpipe with bellows. The absence of convulsions was noted. These made their appearance when the supply of air was stopped, but were put an end to by renewing the blowing in of air. He showed also that the actual mechanical movement of the lungs had nothing to do with the recovery, as had been supposed, since he caused a continuous current of air to be blown through, and allowed to escape by means of holes pricked in the lungs. Hook himself points out that " it was not the subsiding or movelessness of the lungs that was the immediate cause of death, or the stopping of the circulation of the blood through the lungs, but the want of a sufficient supply of fresh air." (The italics are in the original.) .

The actual constituent of the air that is required was not recognised until the experiments of John Mayow (1674), who showed that it is what we now call "oxygen," but which he called " spiritus nitro-aereus," identifying it with that constituent on which ordinary combustion depends. Mayow was also aware that this substance entered into the blood, and was indispensable for vital activity (see Domian's reprint, pp. 27 and 38). The reader will probably be interested to have before him the actual words used in one or

(From the painting \>y Fuseli in 1783. Copied from the print in the Yates Album in the possession of the Royal Society. P»y permission of the Council. The signature is from the Charter Book of the Royal Society.) two passages. I have added the translation given in the "Alembic Club" reprint. On p. 96 of Mayow's book we read : " Nempe imprimis pro concesso habeo, Aerem particulas quasdam, quas alibi Nitro-aereas nuncupavimus, ad Ignem conflandum omnino necessarias continere ; atque eas per flamma; deflagrationem ab acre exhauriri, et absumi ; ita ut idem particulis istis deprivatus, in futurum ad igriem sustinendum prorsus inidoneus evadat, uti supra ostendum

est." "In the first place, then, I take it for granted that the air contains certain particles termed by us elsewhere nitro-aerial which are absolutely indispensable for the production of fire, and that these in the burning of flame are drawn from the air and removed, so that the latter when deprived of these particles ceases to be fit for supporting fire, as has been shown above" (p. 67 of "Alembic Club" translation). On p. 299: "Circa respirationis ergo usum affirmare fas sit, nonnihil, quicquid sit, Aereum ad vitam sustinendam necessarium, in sanguinis massam transire. Hinc aer e pulmonibus egestus, e quo particulae istae vitales exhauriuntur, non amplius ad respirationem

idoneus eat." " With respect, then, to the use of respiration, it may be affirmed that an aerial something, whatever it may be, essential to life, passes into the mass of the blood. And thus air driven out of the lungs, these \jtal particles having been drained from it, is no longer fit for breathing again " (p. '204). On p. 107 "Ex quibus manifestum est, aerem per animalium respirationem, baud multo secus, ac per flammie deflagrationem, vi sua elastica deprivari ; et utique credendum est, Animalia, tgnemque particxilas e jusdem generis ex acre exhaurire ; id quod sequenti experimento majua adhuc confirmatur," "Hence it is manifest that air is deprived of its elastic

force " (that is, diminished in volume) " by the breathing of animals very much in the same way as by the burning of flame. And indeed we must believe that animals and fire draw particles of the same kind from the air, as is further confirmed by the following experiment" (p. 75). On p. 151 : " Quemadmodum sanguinis fermentationem, ita etiam illius incalescentiam a particulis nitro aereis cum particulis cruoris salino-sulphureis exsestuantibus, oriri existimo." "Just as the fermentation of the blood, so also its heat arises I think from the effervescence of nitro-aerial particles with salino-sulphureous particles of the blood " (p. 104).

On p. 152: " Quanquam calor iste in animalibus, per exercitia violenta excitatus, etiam ab effervescentia particularum nitro-aerearum et salino-sulphurearum in partibus motricibus orta, partim provenit, ut alibi ostendetur." "Nevertheless, the heat excited in animals by violent exercise is in part also due to the effervescence, originating in the motor parts themselves, between the nitro-aerial particles and the salino-sulphureous particles, as will be pointed out elsewhere. "

With regard to the two last passages, we must remember that, as is evident from other parts of the book, "salino-sulphureous particles" are what we now call combustible substances. It appears from the last passage that Mayow rightly held that combustion went on in the muscles themselves, although he was incorrect in his statement that it took place in the blood also. I have ventured to put this passage into slightly different words from those used by the translator of the " Alembic Club." It is clear that " effervescentia " agrees with " orta," that is, it is the "effervescence" (combustion) that arises in the muscle, not merely the salinosulphureous particles, which would escape into the blood and be burnt there. This is a point of some importance, since it was held even by Lavoisier that the combustions take place in the lungs, so that Mayow was in advance of his successors. A portrait of Mayow is given in Fig. 184, being that placed at the front of his book.

The importance of Mayow's discovery was lost sight of in the rapid development of the phlogiston doctrine and oxygen was rediscovered by Priestley (1774), who called it " dephlogisticated air." As we have seen, Priestley also showed that air, which had been "spoilt" by animal respiration, was restored by green plants. Priestley's portrait is given in Fig. 185 and a copy of the frontispiece to his book in Fig. 186. As is well known, the doctrine of phlogiston was overthrown by Lavoisier (1770, etc.), who showed the true nature of oxidation and gave the name "oxygen" to Priestley's "dephlogisticated air." With Lavoisier, modern chemistry with its use of the balance commences. It has been stated that his discovery of oxygen was suggested by the account given to him by Priestley. However this may be, there is no doubt that he was the first, after Mayow, who saw the phenomena in their real aspect. A portrait of Lavoisier with his wife will be found in Fig. 187. The product of combustion in living beings was not known to Mayow. It was shown by Black (1755) to be something quite different from common air, and was called by him, " fixed air," but its true nature as an oxide of carbon was discovered by Lavoisier. A sketch by Madame Lavoisier of an experiment on respiratory exchange in work, performed in Lavoisier's laboratory, is reproduced in Fig. 188. Madame Lavoisier is taking notes.

As we commenced the study of the subject with the oxidation processes in the cell, it will be most appropriate to take in order the stages backwards from the cell to the lungs and the outer atmosphere. We saw in the preceding chapter how the processes of oxidation and reduction in the cell are under the control of enzymes and the part played by peroxides therein. Thus, at a given moment, there will be a certain amount of available oxygen present in the cell as peroxide. But this must be extremely small.

At one time, it was generally thought that the cell contained a store of " intramolecular" oxygen in some loosely combined form. In our first chapter we discussed the more modern form of this belief, in the guise of " biogen " molecules, supposed to contain loosely combined oxygen in one side chain, together with combustible substance in another, so that cell oxidations might proceed without immediate supply of fresh oxygen. Since this view is still held in some quarters, occasionally in a more or less modified form, it is important to give further evidence bearing upon it.

Of course, a very small amount of oxygen may exist in the cell fluids in ordinary solution, and the time taken to consume this would vary with the rate of oxidation in the cell. Further, a somewhat larger amount of carbon dioxide is dissolved or combined as bicarbonate. In our description of Fletcher's experiments (page 443), we have seen that the carbon dioxide given off by muscle in nitrogen is not greater than can be accounted for in the way mentioned. In absence of oxygen, therefore, no combustion process goes on in muscle ; in other words, there is no oxygen present in a form available for oxidation. Further experiments on the disengagement of carbon dioxide from muscle by heat, the results of which are incapable of explanation on the theory of intra-molecular oxygen, will be found in the paper by Fletcher and Brown (1914). Similar conclusions were drawn by Verzar (1912, 3, p. 47) with regard to the muscle of warm-blooded animals. He found that there is no oxygen tension in this tissue. If there is no oxygen tension, there can be no oxygen that it is possible to use for oxidation purposes in the

FIG. 189. WINTERSTEIN'S MICRO-RESPIRATION APPARATUS. — With tubes for introduction of various gases. To be used also for analysis of gases in small quantities of blood. cell. There is none that can be dissociated, either for combustion of another part of the " giant " molecule or for that of other molecules. If there is any store, it is in stable combination and does not concern us here. Peters (1913, p. 266) states that his experiments do not absolutely decide the question, but he could obtain no evidence in favour of a storage of oxygen.

Turning to other tissues, the experiments of Winterstein (1907) on the spinal cord of the frog may be referred to. The micro-respirometer of Thunberg (1905, 1) was used. This apparatus is similar to that represented in Fig. 189, which is the form given to the instrument by Winterstein in order to use it for the estimation of the gases in blood, as well as for respiratory experiments. Into this apparatus the isolated spinal cord of the frog, prepared by Baglioni's method (1904), was introduced. ' In oxygen it remains excitable for forty-eight hours, in nitrogen only for half an hour. The oxygen consumption at 16° to 18° is 268 to 300 c.mm. per gram. The respiratory quotient is less than unity, hence some substance other

than carbohydrate is oxidised. Now, supposing that, while in oxygen, the preparation has accumulated to itself a store of oxygen in disposable form, as " biogens " or otherwise, then, when nitrogen takes the place of oxygen, this store of oxygen will be used up. Therefore, the first thing that will happen, when the nitrogen is again replaced by oxygen, will be that the store is replenished and oxygen will disappear without the corresponding amount of carbon dioxide being given off; in other words, the respiratory quotient will not be the same as that after being some time in oxygen. In very carefully controlled experiments no indication was found of any change of this kind.

It appears, then, that, although inexcitable, the tissue remains alive in nitrogen, since its excitability can be restored in oxygen. Since the survival is not an 'oxidative process, why is oxygen necessary for restoration of excitability 1 Winterstein compares it to a clock which has stopped, not because the spring has run down, but because the movement of the pendulum is hindered. In our case the hindrance is the accumulation of asphyxial products, which require oxygen to remove them. The length of time necessary for recovery is not due to slowness of diffusion of oxygen, but to the rate of oxidation of these products. Whatever they may be, it seems clear from the non-alteration of the respiratory quotient that their chemical nature is similar to that of those oxidised under normal conditions. It is scarcely necessary to remark that, after asphyxia, the rate of consumption of oxygen was temporarily increased, but the point is that the respiratory quotient was unaltered, as it would have been if oxygen were being stored apart from simultaneous production of carbon dioxide.

In the researches of Battelli and Stern (1907), however they may be interpreted, there is no evidence of storage of oxygen. Meyerhof (1912, 1, p. 176), again, finds that in the absence of oxygen, there is no production of heat in the blood corpuscles of the goose, although it returns on admission of oxygen. Thunberg (1905, 3), in some experiments to be referred to again later, found that the oxygen consumption of the slug and the earthworm was increased by increase of oxygen pressure, but that the carbon dioxide production was always parallel to it, so that no storage of oxygen took place, even under increased pressure.