Principles of General Physiology

The experiments of Falloise (1901) and of Durig (1903) are regarded by Zuntz as affording definite proof of the absence of any kind of storage of oxygen on the part of the cell. Falloise showed that, if an animal were caused to breathe for a considera hittime a mixture rich in oxygen, and then the supply cut off, symptoms of asphyxia appeared only forty-five seconds later than they did if ordinary air had been breathed. If the oxygen inhalation only lasted for one minute, the same effect resulted, and, if air were breathed for one minute after the oxygen inhalation, its effect was removed. Hence the only effect produced is that of the residual air in the lungs and the extra oxygen dissolved in the plasma.

Durig's experiments were made in Zuntz' laboratory by the most accurate methods and they resulted in confirming the work of Falloise. Dogs were given mixtures of air with various percentages of oxygen, and the intake per minute was determined. During the first two to three minutes after changing the mixture, the oxygen intake was increased or decreased in proportion to the oxygen content of the air breathed. Special experiments were then made to determine the amount present in the air of the lungs and that dissolved in the tissues and blood. It was found that the whole of that taken in or given out beyond the normal amount was required for these purposes, so that none at all was left over for storage in any other form.

We saw above (page 343) that the results of the experiments of Barcroft and Brodie (1905, p. 65) are opposed to the view of intra-molecular oxygen in this case. Those of Evans and Ogawa (1914) are also of interest in this connection, as well as with regard to the mechanism of tissue respiration. They found that in the heart, under the action of adrenaline, great increase both in oxygen consumption and in carbon dioxide output occurred, but that the two processes do not coincide in time. The oxygen intake reaches its maximum during the first few minutes after the drug is given, while the output of carbon dioxide reaches its maximum some time later, after the oxygen intake has begun to diminish again. The respiratory quotient is thus first lowered and then raised before returning to normal, but the mean value is unaltered. The explanation suggested is that a definite time is required for the chemical reactions which occur in the intermediate stages of oxidation, so that, if there is an increase in the rate of oxidation generally, the amount of oxygen consumed alters at once, while that of the carbon dioxide output attains its new level more slowly. This view is confirmed by the fact that, if adrenaline is continually added, the mean respiratory quotient during the administration becomes constant, but at a lower level than before the adrenaline was given. This can be seen in detail in the consideration given on p. 456 of the paper. It is merely necessary to remember that the carbon dioxide given out in a particular period does not correspond to the oxygen used in that period, but to that of an earlier period.

Certain interesting experiments on the growth of yeast by Horace Brown (1914) appear, at first sight, to show that there is, in this case, a storage of oxygen. If yeast be placed into a culture solution, which has been saturated with oxygen at its tension in air by shaking with air, the oxygen is removed rapidly and serves for subsequent combustion purposes by the yeast cells which have taken it up. In interpreting this result, it should be remembered that the amount of oxygen present in the solution was only 0'559 c.c. per cent., and also that (p. 212) it was found impossible to increase the "oxygen charge" of normal yeast, which had been washed in contact with air, by submitting it to more extensive aeration. The possibility of peroxides may be taken into account here (see the footnote on p. 212 of the paper), and also that of adsorption of oxygen on surfaces in the cell, since the amount taken up was so small.

The Relation of Oxygen Tension to its Consumption. — We find almost invariably that the supply of oxygen is sufficient to meet the requirements of the cell, so that increase of its pressure ( = concentration) does not lead, by mass action, to increased consumption. In the case of the slug, the earthworm, and the mealworm, Thunberg (1905, 2) found, on the contrary, that the consumption of oxygen was, within fairly wide limits, in proportion to its tension.

Some phenomena and theories of narcosis have been discussed previously (pages 138-140). That of Verworn (1912), according to which the process consists in the inhibition of oxidation, was left until the present chapter. Allied to this view is that of Mansfeld (1909), which attributes the process to an effect on the cell membrane by which access of oxygen is prevented. This blockage is supposed to be due to the diminution of the solubility of oxygen in the lipoid membrane, owing to the presence of the narcotic there ; but, if ordinary solution be meant, it is difficult to reconcile the view with the ordinary laws of solubility.

Direct evidence exists, moreover, which shows that there is no connection between narcosis and oxidation. Thus Warburg (1910, 2) found that, although the segmentation of the sea urchin's egg was stopped by phenyl-urethane, the consumption of oxygen was not ; a greater concentration of the narcotic, however, stopped the latter also, as would be expected. Winterstein (1913) shows that there is no relation between the narcotic action of various substances, and their effect on oxidation. Further, anaerobic worms cau be anaesthetised. In a further paper (1914), it is shown that the spinal cord of the frog, when narcotised by urethane, shows diminished oxidation, but when narcotised by alcohol, the oxidation is increased. The two processes are independent. The fact that nerve centres, after asphyxia, cannot be recovered by oxygen, if alcohol be present, shows that there is some intermediate process between oxidation and excitability, which process is attacked by the narcotic.

As to what the process actually consists in, certain facts have been given previously, and we may remember that Claude Bernard (1875, p. 143) suggested that the various forms of anaesthesia, as by drugs, heat, asphyxia, and so on, are essentially the same physico-chemical process. He also clearly pointed out that the ordinary phenomena of asphyxia have nothing to do with those of narcosis (p. 96). Loewe (1913), as the result of detailed investigations of the relation of narcotics to lipoids, came to the conclusion that the cell membrane consists of a complex colloidal system of hydrophile colloids together with lipoids, and that the narcotics are adsorbed by the latter, with the result that their hydrophile nature is changed into a hydrophobe nature, or one that behaves as such, although no water is lost. Hence the decrease of permeability found experimentally as the accompaniment of typical narcosis, as opposed to the increase associated with lethal action. There may also be a diminution of "elective" permeability, resulting in diminution of potential difference and injury to "specific" functions of the membrane. But it is difficult to attach very definite meaning to the last statements.

We have seen that certain organisms, both animal and vegetable, such as some bacteria and nucleated red blood corpuscles, are not killed by deprivation of oxygen, although no oxidation proceeds and cell activities are suspended. Recovery takes place on admission of oxygen. In other cases, such as intestinal worms, the leech, and yeast cells, chemical activities of a special kind proceed, together with certain manifestations of life, in absence of oxygen. A further condition is that of certain bacteria, which are killed by oxygen and are capable of existence only in its absence. Thus we have facultative and obligatory anaerobiosis.

The manifestations of life require the supply of free energy. This is usually obtained from oxidative reactions, and the interesting problem arises, How is it obtained in absence of oxygen ? Perhaps the best example to start with is that of the mould, Mucor racemosus, which, as shown by Pasteur (1876, pp. 130-132), in the presence of oxygen burns up glucose to carbon dioxide and water, but when submerged and deprived of oxygen, certain morphological changes occur and it now forms alcohol and carbon dioxide from glucose.

Yeast. — We have seen that no growth takes place in absence of oxygen, but that the fermentation proceeds. In this fermentation, in which sugar is split into alcohol and carbon dioxide, there is production of heat ; so that we may put it in this way, the combination of part of the carbon with oxygen to form carbon dioxide sets free more energy than is required to make up the difference between the heats of combustion of alcohol and of sugar. There is, then, energy at the disposal of the organism for what activities it is capable of, if this energy can be made use of. The reaction from glucose to alcohol probably passes through several stages, similar to those given on page 273.

Putrefactive Organisms. — Pasteur (1861) showed that certain organisms, responsible for butyric acid formation in putrefaction, were actually killed by oxygen, although, presumably, their spores are able to withstand its presence. In a protein undergoing putrefaction, it was shown by Hoppe-Seyler (1887) that the chemical products of putrefaction are different when the process proceeds with or without air. In the presence of air, aerobic organisms develop at the surface, while anaerobic ones grow in the depths. In presence of oxygen, carbon dioxide, water, and ammonia are formed ; in its absence, hydrogen, marsh gas, leucine, and tyrosine. Nencki (1904, 1, p. 376) showed that certain aromatic derivatives, phenyl-propionic acid, parahydroxyphenylpropionic acid, and skatol-acetic acid, together with lower fatty acids, butyric, caproic, etc., were formed in anaerobic putrefaction. It is chiefly to these lower fatty acids, together with indol and skatol, that putrefactions owe their objectionable smell. Methyl-mercaptan is also sometimes present. Decarboxylation of amino-acids occurs, giving rise to various amines, and, from diamino-acids, putrescine and cadaverine. (tetra- and penta-methylene-diamines).

Higher Fungi. — Kostytschev (1910) showed that mushrooms in absence of oxygen do not form alcohol. In their press juice an interesting phenomenon was observed. Carbon dioxide is formed and can be driven off by boiling. It arises only in small part from carbonates and chiefly from some substance which splits off carbon dioxide by hydrolysis. The carbamino-acids of Siegfried were excluded by the observation that the phenomenon could be observed in the absence of proteins or amino-acids. The substance in question seems to be some intermediate stage of oxidation, formed by previous exposure to oxygen. It was found that mannite disappears, if added to the press juice, without giving off carbon dioxide until the solution is heated, and it is thought probable that this substance is the source of the interesting compound in question.

Higher Plants. — Considerable evidence exists that, in absence of oxygen, higher plants attack sugar as yeast does, forming alcohol and carbon dioxide. Further details may be found in the essay by Lesser (1909). In Animals. — The behaviour of frog's muscle in absence of oxygen has.been described above (page 444). We saw that there is no evidence of any chemical process going on in rest, apart from the action of micro-organisms ; -when stimulated, the substance of high potential energy content gives off lactic acid and, when the store of the substance is exhausted, the muscle ceases to contract. When the muscle is placed in oxygen, lactic acid is replaced in the system by aid of an oxidation process, which forms carbon dioxide from some other substance. In a certain sense, we may say that lactic acid is the product of anaerobic change in muscle, carbon dioxide that of aerobic change. But it must not be forgotten that they do not arise from the same source.

Intestinal Worms. — These are the only multicellular animals known which normally exist in absence of oxygen. Although they have no need to produce heat, they require energy for other purposes, muscular movement, growth, and so on. They are, therefore, very instructive for investigation. The most recent work is that of Weinland (1901-1906). These worms were found to contain large quantities of glycogen, which was consumed in starvation, giving as products, in absence of oxygen, carbon dioxide as the only gas. In -the liquid around the animals, valerianic acid was found, in amount corresponding to 0'3 g. per 100 g. of Ascaris in twenty-four hours, together with a nitrogenous substance containing 0'015 g. nitrogen for the same time and weight of animals. The carbon dioxide was 0'4 g. The process is represented as follows : —

The hydrogen is supposed to be used up at once for reduction processes. If this be so, we have a true fermentation process. The Leech. — Putter (1907) has investigated the metabolism of the leech, which can live ten days without oxygen. He states that hydrogen is formed in these conditions. When first placed in water deprived of oxygen, the carbon dioxide production goes up considerably for a time, but afterwards falls again. Energetics of Anaerobiosis. — It appears from the preceding paragraphs that a larger amount of carbon dioxide has to be given off by a fermentation process than by an oxidation in order to give the amount of energy required by an organism. Indeed, Warburg (1914, p. 262) calculates that the same quantity of glucose when decomposed to alcohol and carbon dioxide only gives 3 to 5 per cent, of the energy which it gives when completely burnt to carbon dioxide and water. But the general conclusion seems to be justified that the cell mechanisms are such as to be able to use chemical energy whether it comes from oxidation or otherwise, and that they are independent of the particular chemical reaction which affords it.

It would naturally be supposed that the anaerobic changes of a food-stuff would pass through the same stages as those which the same substance undergoes in the presence of oxygen, but stop short. Thus, glucose might be thought in all cases to pass through the stages of alcohol and carbon dioxide, but that, in the presence of oxygen, the alcohol is further oxidised to carbon dioxide and water. This, in fact, was the view suggested by Pfeffer (1881-1885, p. 664), and it appears to be the case in some instances. Yeast, however, does not ferment

any more -sugar to alcohol in the absence of oxygen than in its presence (Buchner and Rapp, 1899). It may be held, nevertheless, that yeast is an abnormal organism, produced under the process of repeated selection for a special purpose. In the case of the animal cell, we have already (page 276) seen reason to hold that alcohol is not a normal stage of sugar metabolism. Further, the valerianic acid produced by Ascaris must be a special form of anaerobic metabolism. It is difficult to make any statement as to the characteristic putrefaction products, since the organisms are inactive in the presence of oxygen and we do not know what their metabolism might be in such circumstances.

We have already referred, incidentally, to the requirements of certain tissues as regards oxygen supply, both in rest and in activity. For further data, the essay by Barcroft (1908), together with his book (1914), may be consulted. Xubmaxillary Gland. — In the experiments of Barcroft and Piper (1912), the oxygen used in the resting gland amounted to 0-027 c.c. per gram per minute. The results as regards activity have been referred to above (page 342). The consumption went up to 0*089 c.c. in a particular case and continued to be raised for a hundred seconds or more after the flow of saliva has ceased. For the production of 0-3 c.c. of saliva, 0-18 c.c. of oxygen was used over and above that of the resting condition.

The Kidney. — The most recent measurement is that of Neuman (1912). Under ordinary conditions, the oxygen consumption was found to be from 0'026 to 0*06 c.c. per gram per minute. Results under stimulation to secretory activity have been given above (page 358). The increase was about four to five times that in rest. The Liver. — Barcroft and Shore (1912) found that, in cats unfed for thirty six hours, the oxygen consumption amounted to from 0-005 to 0*018 c.c. per gram per minute. In animals fed eighteen hours previously, 0*024 to 0'05 c.c. For the viscera drained by the portal vein, chiefly intestine, the values were 0*008 to 0*013 c.c. for the unfed, and 0*011 to 0018 c.c. for fed animals. These facts indicate that the chief metabolism during late digestion is in the liver.

The Suprarenal Gland. — A striking fact about this organ is the rich supply of blood. Neuman (1912) found that a blood pressure of 130 mm. of mercury drives through it 6 to 7 c.c. of blood per gram per minute. This is higher than that of any other organ. Its oxygen consumption is 0*045 c.c. per gram per minute, and is increased threefold during a rise of blood pressure produced by adrenaline. The Heart. — The chief work on this organ has been done by Rohde (1910) and by Rohde and Nagasaki (1913) on the mammalian heart perfused with Ringer's solution and by Lovatt Evans (1912, 1, and 1914, 1) on the heart-lung preparation perfused with blood. The results will be considered in a later chapter, when dealing with tiltmechanism of the cardiac contraction. It may be stated here that the oxygen consumed in any one contraction varies directly with the maximal tension developed, in accordance with the results of A. V. Hill (page 443) on energy production in skeletal muscle. The oxygen used per minute depends directly on the number of beats ; so

that, as Rohde expresses it, -^, is a constant for normally beating hearts, where Q is the quantity of oxygen consumed per minute, N is the pulse rate, and T the maximum tension. The amount of oxygen consumed per gram weight, according to the results of Evans, is from 0*043 to 0*085 c.c. per minute. The Lungs. — In the course of the above work, Evans (1912, 1) determined the metabolism of the lung tissue. This is of some importance with regard to certain theories which supposed that a considerable degree of oxidation of metabolic products of tissues took place here. It amounts only to 0*015 c.c. per gram per minute, really a low figure.

The Nerve Centres. — Tfre metabolism of the nerve centres has been referred to previously (page 472). Further work is required on the question, especially in connection with the great sensibility of the higher centres to deprivation of oxygen, although it has been stated that the actual consumption of oxygen is not great. The Blood Itself. — We have already seen that the nucleated blood corpuscles have a fairly considerable oxidation metabolism. Morawitz (1909) showed that the blood of rabbits made anaemic by the injection of phenyl-hydrazine has also a fairly considerable metabolism, and that this is due to the young non-nucleated red cells which are present in such conditions in considerable numbers. In contrast with this, the actual metabolism in the normal blood is extraordinarily small.

Technique. — For the methods used in the various experiments referred to in the preceding paragraphs, the original papers must be consulted. There is a possible criticism to be brought against these methods, in which the rate of the blood flow is measured by the time taken to fill a certain volume of a graduated pipette inserted into the vein. This value is obviously of great importance in the determination of the oxygen consumed in a given time. When vascular dilatation occurs, as is usual in an active organ, the time taken to fill the tube is very short, and is only a small part of the total duration of an observation, so that the assumption must be made, that the rate of flow and consumption of oxygen continues to be the same as that during the small sample of the total effect of a stimulation which is actually measured. For this reason, it seems desirable that further observations should be made, in which the whole blood passing through an organ in a considerable time should be collected, and its oxygen and carbon dioxide contents compared with that of the arterial blood entering. This criticism is not intended to cast doubt on the results given above, but it seems to me that it may be quite easy to overestimate the oxygen consumption when vaso-dilatation occurs, since the measurement only applies to so short a period. The application of this consideration to the question of the nature of vaso-dilatation will be clear later, when we have to discuss the regulation of the blood supply in Chapter XXIII.

It is evident that the amount of oxygen required by active organs is far larger than the blood could carry merely in the ordinary state of solution in liquids. We have, therefore, to consider in the next place the extraordinary substance, haemoglobin, contained in the red blood corpuscles, by whose agency oxygen in adequate amount is conveyed to the tissues. As far as difficulty of understanding is concerned, this mechanism, as we shall see, is similar to that of its near relative, chlorophyll.

Although it is this substance which is contained in the red blood corpuscles of the vertebrates, and is responsible for the taking up of oxygen, and the giving it off again when required, it is not to be supposed that there are no other similar substances. In fact, in the blood of molluscs and Crustacea there is a pigment, haemocyanin, which serves the same purpose. This pigment contains copper, whereas, as we shall see, haemoglobin contains iron. As yet we know comparatively little about haemocyanin, especially with regard to its relation to oxygen. It is a matter which would well repay investigation to determine whether it has the remarkable properties which haemoglobin has in this respect, properties which are at present unique. The work of Alsberg and Clark (1914), to be given presently, indicates that haemocyanin has not the peculiar properties of haemoglobin.

Haemoglobin, as is well known, is a compound of a protein with a complex acid substance, containing iron and pyrrol derivatives, as we have seen (page 560). We will leave, for the present, further remarks as to its chemical constitution, merely stating what is necessary for the immediate question. This is, that it exists in two forms, oxyhaemoglobin, which is regarded as a compound of the other form, haemoglobin, or " reduced haemoglobin," with oxygen. This oxygen can be removed by exposure to a vacuum, so that it is stated to be " loosely combined," and haemoglobin remains.

Now, suppose that we expose blood, or a solution of hemoglobin, to oxygen at its pressure in the air and shake together until no more oxygen is taken up. We find that, even when exposed to oxygen at a higher pressure, no more is taken up. At least, this is what is usually held to be the case, but there are very few experimental determinations which show this fact directly. At all events, it is practically " saturated," as shown by the form of the curve which we shall learn to call the "dissociation-curve."

Barcroft has recently made some determinations of the amount of oxygen taken up by blood exposed to a gaseous mixture of 8«> per cent, oxygen and lo per cent, nitrogen, and finds the following percentage degrees of saturation as compared with that regarded as complete : 102, 99, 98, and 97 in four experiments. These values were corrected for the gas physically dissolved, and point to a true saturation point. They were kindly communicated to me by the experimenter.

Next, let us take haemoglobin, which has been saturated with oxygen at the pressure in which it exists in the atmosphere, and compare the amount of its content in iron with the oxygen contained. It has been satisfactorily proved by Peters (1912), in very careful and accurate work, that the amount of oxygen taken up corresponds to that required to convert the iron into FeO0. Of course, this does not mean that the oxygen is actually combined in this way, as sometimes appears to be thought. Such a peroxide does not seem to be known and the iron is united also in organic combination. A trivalent iron might be united to two atoms of oxygen in peroxide form and the third valency attached to the organic group, but such a combination does not agree with the formula given by Kilster (1912, p. 469). Too much stress must not be laid on this point, since it is difficult to see what is the function of the iron, except to combine with oxygen. It is to be remembered that the iron in hemoglobin is not in such a form as to be electrolytically dissociated, and that it gives none of the reactions of iron salts. All that we are really justified in saying is that, when saturated with oxygen, each molecule of hemoglobin contains two atoms of oxygen to each atom of iron, or, in other words, that each molecule of hemoglobin takes up the same definite amount of oxygen. The work of Laidlaw (1904), however, tends to show that the iron is in different combination in reduced hemoglobin to that in which it is in oxyhemoglobin, since the iron-free derivative, hematoporphyrin, is easily obtained by the action of acid on the former, while, under the same conditions, hematin is obtained from the latter ; that is, the iron is not split off.