Principles of General Physiology

In this connection we may call to mind what Parker (1913, 1) points out, namely, that man}' apparent adaptations are not really such. That a person who faints falls with muscles limp is appropriate for recovery, and it is also the safest way to fall, but these conditions are the direct consequence of the faint, and that they are advantageous is purely incidental ; they might, in fact, have been the opposite, but they would happen, notwithstanding. Parker holds that the majority of animal reactions are, probably, neither of advantage nor disadvantage, in any notable degree, to the life of the individual, but dependent on the con-

struction, physical and chemical, of the given organism. At the same time, he points out that there are real adaptations. The capacity of an individual to react appropriately to his environment has been brought about by the elimination of myriads of individuals who failed to do so. Adaptation has been regarded as a sort of transcendental property of organisms, an entelechy, allied to intelligence. But, as Parker remarks, what do we really mean by intelligence other than "that aggregate of nervous states and actions which is our chief means of adaptation"? so that the proper understanding of adaptive reactions implies that of intelligence, and conversely. The introduction of such notions as entelechies consists, practically, in argument in a circle and is rather calculated to retard progress by apparent explanation, when what is really wanted is research into the very questions which they pretend to answer. "The details of animal reactions are then, in the main, free from adaptive restraint and tlu-ir diversity is dependent chiefly upon the fluctuating momentary condition of the animal iMidy : further, the main outlines of animal reactions are adaptive, but are not to lie explained by the assumption of something like intelligence."

The effect of Rise of Temperature on hydrogen ion concentration is of some importance. In a previous page the large temperature coefficient of electrolytic dissociation of water was referred to in another connection, as being of the order of those regarded as characteristic of chemical reactions. That of sodium salts, on the other hand, is the very low one of salts in general, which do not obey the Ostwald " dilution law." On mixtures of bicarbonate and CO., the net effect of a rise of temperature will be to increase the alkalinity, since the dissociation of water will be increased more than that of the bicarbonate. Thus water at 18° has a dissociation constant of O64 x 10~14, i.e.,

A solution of sodium bicarbonate and CO.,, which has, at 18°, a hydrogen ion concentration of 0*30 x 10~", has accordingly a hydroxyl ion concentration of since the product of (C)OH. and (C)H. is constant in all solutions in water at the same temperature. At 42°, the hydrogen ion concentration of the bicarbonate mixture has risen only to 0-42 x 10~", owing to its low temperature coefficient, whereas the dissociation constant of water has become 3-76 x 10~14 (Kohlrausch and Heydweiller, 1894, p. 209). Hence the hydroxyl ion concentration has risen to

The proteins present in the blood and tissues play some part in the maintenance of neutrality, owing to their amphoteric nature. They form salts with both strong acids and strong bases, just as amino-acids do. But they do not readily form salts with weak acids or bases and, as present in the organism, they themselves are but slightly dissociated. L. J. Henderson (1909, p. 289) dialysed serum, in order to remove the bicarbonates and phosphates, adding sodium chloride at intervals to keep the globulins in solution. It was then found that a fairly large amount of sodium hydroxide required to be added in order to change the colour of rosolic acid in the serum. The proteins are active therefore in the same way as the bicarbonates and phosphates, but their part seems to be a subordinate one and dependent on their high concentration rather than on their special properties as acids or alkalies. They are, however, able to drive off carbon dioxide from bicarbonates, owing to the fact that it is eliminated by the lungs as gaa, as it is formed. By this removal from the reacting system, a very weak acid is enabled to decompose the bicarbonate. Suppose that there is produced even a very small increase of the HCO3' in the solution ; this increase involves that of the pressure of the dissolved COa gas and escape of a part of it. A further amount of HCO3' is then formed by the weak acid, in order to hrini: nack equilibrium, and thus the process continues.

The Reaction of Blood. — By the most sensitive methods available, the hydrogen ion concentration of blood at 38° is found to be 0'4 x 10"" and the corresponding OH' ion concentration, 7 '2 x 10"" molar. So that it is just on the alkaline side of neutrality. At room temperature, the alkalinity would be somewhat less, owing to the increase of OH' ion concentration in CO2 — bicarbonate systems with rise of temperature, as described above. Direct measurements of the effect of temperature on such systems, in moderately concentrated form, have shown about four times as great an alkalinity at 38° as at 18°. This is dependent on

the fact that the electrolytic dissociation of water rises more quickly with temperature than that of sodium bicarbonate does. Compared with mixtures of the alkaline and acid phosphates, the H* ion concentration in blood varies between relative proportions of the two phosphates between 6 to 4 and 1 to 0. Since it is to be presumed that variations of this magnitude are innocuous, it will be seen that in the presence of phosphates, protoplasm possesses an efficient mechanism for avoiding any considerable change in reaction. All the phosphate must be converted into the acid salt before the hydrogen ion concentration can rise beyond that due to this salt, or into the alkaline salt before the alkalinity can become greater than that of solutions of Na2HPO4.

"Buffers." — The effect of such substances as bicarbonates, phosphates, aminoacids, etc., in " soaking up," as it were, excess of hydrogen or hydroxyl ions was compared by Fernbach and Hubert (1900, p. 295) to that of "tampons." Sorensen (1909) adopted the word and, in the translation of his paper into German, it was rendered " Puffer " and thence into English as " Buffer." This latter word does not seem to me to be a very descriptive one nor to convey correctly the meaning of the original " tampon." A railway buffer does not absorb the engine itself, as the substances referred to absorb ions. A word more suggestive of a sponge would probably be better, but is not easy to find.

The Practical Use of Phosphate Mixtures. — In certain cases it is of much importance to be able to obtain a solution of a definite but very small concentration in hydrogen ions, as also to possess the means of maintaining constant this value in a system in which chemical changes, sensitive to change of reaction, are going on. Such cases are the action of enzymes, or the solutions used for perfusion of living organs. The bicarbonates are the most appropriate for the latter purpose. For the making of standard solutions as well as for use with enzymes, the phosphate systems are most valuable.

These phosphate mixtures are most readily prepared by the addition of standard sodium hydroxide in different proportions to standard phosphoric acid solution. The following table, from the paper by Prideaux (1911) with additions, will be found useful : — The mixtures are diluted to 100 c.c. since the hydrogen ion concentration refers to solutions which are decimolar in PO4. The equation by which any other required hydrogen ion concentration can be

obtained will be found in the paper by Prideaux on p. 125. The values may also be read on the curves of Fig. 58, copied from this paper. The abscissse give the number of c.c. of molar sodium hydroxide to be added to 10 c.c. of molar phosphoric acid to make 100 c.c. of solu tion, in order that we may have a hydrogen ion concentration of the ordinate selected. The second part of the figure on the succeeding page is the steeper parts of the complete curve drawn on a larger scale, so that greater accuracy may be attained by preparing a larger volume of the solution, say a litre.

To illustrate the use of the curve : — Suppose that a solution of the optimal acidity for emulsin is required. This is, according to Vulquin (1911), ox 10-6 in H- ions. The exponent of 10 which we require is log. 5 minus 6 times log. 10=-5'39. Corresponding to this or-' dinate in the table we find ID'S; we must, therefore, add 10'8 c.c. of molar NaOH to 10 c.c. of molar phosphoric acid and dilute to 100 c.c. In the case of organisms whose cells are unprotected by a resistant envelope, it has been already pointed out that the solutions which bathe them must have the same osmotic pressure as the cell contents. Otherwise the cell will contract or expand, by the loss or gain of water, until its osmotic

pressure is equal to that of the surrounding solution. If this is impossible, the cell will be destroyed. In any case, the concentration or dilution of the contents will seriously impair their functional activity. These remarks apply especially to animal cells and it is in the investigation of these that the necessity for solutions of equal osmotic pressure to themselves is met with. It is often required to replace the blood or other solution with which the cells are normally in contact by some artificial

solution, whose composition is known and can be modified at will. The blood plasma of the same species of animal has been supposed indispensable for the growth of excised tissues in the work of Ross Harrison, and others. (But see Thomson, 1914.) But if an efficient substitute can be found for other purposes, the advantages are obvious. It might be supposed that a solution of any substance, so long as it is not actually toxic, would suffice, provided that the cell membrane is impermeable to the solute and it is present in the correct concentration. Sodium chloride, as one of the salts present in all animal fluids, was selected at an early date and was found to serve well for the histological examination of fresh tissues or for the dilution of blood without causing changes in volume in the corpuscles. But when used by Ringer (1880-82, 1882-83, 1 and 2) for continuous perfusion of the heart of the frog, it was found unable to maintain the normal beat. The work of Ringer on this question is fundamental and enabled a satisfactory perfusion fluid to be made. Although this solution is used everywhere and known as " Ringer's Solution," its origin is apt to be forgotten, so that it is necessary to give a brief account of the re-

1, B, Tracing obtained eight minutes after replacing blood by pure sodium chloride, 075 per cent. 1, C, Six minutes later. 1, />, After another four minutes' action. searches which led to its composition being established. A portrait of Sydney Ringer himself will be found in Fig. 59. When the heart was perfused with a solution of sodium chloride in distilled water, isotonic with the blood, that is, Og75 per cent., the beats gradually diminished in extent and ultimately ceased (1882-83, 1, p. 31), as shown in Fig. 60. The excitability to electrical stimuli also disappeared.

We may note here that subsequent work has shown that this action of pure sodium chloride is not only due to the want of some essential salt, but also to a toxic action of the Na- ions, similar to, but less marked than that of potassium ions to be described presently. Clark (1913, 2, p. 77) finds indeed that the ordinary Ringer solution is improved when a part of the sodium chloride is replaced by isotonic cane-sugar, and Abel (1914), to avoid oedema in his " vivi-diffusion" experiments, found it advisable to reduce the sodium chloride to 0'6 per cent.

FIG. 61. The effect of adding 5 c.c. of 0"25 per cent, calcium chloride solution to 100 c.c. of the pure sodium chloride solution. The heart-beats, which had ceased under the pure sodium chloride, became spontaneous after one artificial stimulus, but the diastole was prolonged so that the beats fused. (Ringer, 1882-83, 1, p. 33.) To proceed with the experiments of Ringer, it was found that, if calcium chloride were added to the pure sodium chloride solution when the heart had ceased to beat, the excitability to stimuli returned and was scon followed by spontaneous beats, but that the relaxation was imperfect and delayed, so that there was a tendency to a tonic, systolic state (Fig. 61).

This condition is seen, although less markedly, in the figures of Plate 2 of the first paper (1880-82), where saline solutions made with tap water, containing calcium, were used. It was next discovered (1882-83, 1, p. 35) that a trace of a potassium salt (1 c.c. of 1 per cent. KC1 to 100 c.c. of the solution of sodium chloride in tap water) abolished this tonic action of calcium, without depriving it of the power of neutralising the injurious effect of the pure sodium chloride (Fig. 62). A solution capable of maintaining the heart beat at a satisfactory height for a considerable time was thus obtained, but, from what has been said in the previous pages of the present chapter, it is not surprising to find, as Ringer himself did, that the addition of a small amount of sodium bicarbonate was beneficial. The investigator himself pointed out that this addition had the effect of producing a slight alkalinity similar to that of the blood, and of neutralising acid produced in the contractions of the heart muscle (see 1882-83, 2, p. 223). The amount used was 5 c.c. of a 1 per cent, solution to 100 c.c. of the circulating fluid.

Although the electrolytic dissociation theory was unknown at the time these experiments were made, it was clearly recognised by Ringer that the effects of calcium and potassium salts were due to the calcium and potassium components of the salts added. He himself used indifferently carbonate, sulphate, phosphate and chloride of calcium. In view of the fundamental importance of these facts, the simplest way of demonstrating !::./>'. Effect of adding calcium. The first three beats show the prolongation of the systole.

At the arrow, 3 minims of 1 per cent, potassium chloride were added to the solution. The calcium effect is partially abolished. 13.C, Addition of a further 2 minims of potassium chloride solution. The beat becomes quite normal. them may be described. The heart of the frog or the tortoise is tied on to a cannula inserted into the ventricle through the auricle by the method of Symes (1911). The way in which the effect of different electrolytes can be shown will best be understood from the description of an actual experiment. A tortoise heart was used and a tracing taken, by a lever attached to the apex of the ventricle, before any perfusion fluid was introduced (Fig. 63). The beats were small, as frequently happens, a. Perfusion was then commenced with a solution containing 0'75 per cent, sodium chloride and OK)1 per cent, sodium bicarbonate, b. The beats were not improved, and would probably have ceased, if the perfusion with this solution had been continued. Portions of the tracing are omitted for want of space. At c, a solution consisting of 100 c.c. of the previous one, to which 3 c.c. of decimolar calcium chloride had been added, was perfused. This contained a sb'ght excess of calcium above the normal one. An immediate improvement is to be noticed, but relaxation is incomplete, as shown by the gradual risiin the level of the diastolic position. 6 c.c. of decimolar potassium chloride were then added to the solution already containing sodium and calcium. The tonic action of the calcium was removed, and, after a minute or two, the tracing d was obtained, showing a regular, powerful beat, which would have continued for a long time. At e, the solution containing sodium alone is returned to ; the small irregular beat reappears. At /, potassium chloride, in the same proportion as before, is added. No improvement in the beat results, but the characteristic relaxing effect of potassium in the fall of the diastolic position is observed.

At y, calcium chloride is added to the mixture in the same proportion as before, and we see the powerful regular beat produced by the normal Ringer solution, containing sodium, potassium, and calcium. In order to observe the effect of calcium in a more marked way, at h, another 3 c.c. of the calcium chloride solution were added, and a further 3 c.c. before each step in the tracing. We note the increase of tonic contraction brought about by each addition. At k potassium chloride was added, but, although it diminished the systolic condition, the size of the beat was rather decreased ; in fact, the antagonism is not complete when there is excess of either calcium or potassium much beyond the normal proportion. Finally at I normal Ringer solution was perfused.

It is a remarkable fact that the proportion of sodium, potassium, and calcium ions in sea water is almost identical with that found by Ringer to be the best for maintaining the beat of the heart, although the total concentration in sea water is higher. Magnesium salts, however, are present in sea water, in addition to those mentioned. The presence of magnesium does not appear to be necessary in an artificial physiological saline solution, although Neu lurch

FIG. 63. ACTION OF ELECTROLYTES ON THE HEART OF THE TORTOISE. b, Perfusion with sodium chloride, 075 per cent., sodium bicarbonate, O'Ol per cent. c, Addition of 3 c.c. of O'l molar calcium chloride to 100 c.c. d, Potassium chloride added, 6 c.c. of O'l molar to 100 c.c. of the mixture containing sodium and calcium salts. h, 3 c.c. calcium chloride added before each step in the tracing. k, More potassium chloride added, in amount'corresponding to the calcium chloride present. The tonic action of

the calcium is partly abolished, but the beat does not return to its normal height. /. Normal Ringer's solution. (1912) found that the contractions of the excised intestine of the rabbit were more regular when magnesium was present. If we look at the i-elative proportions of these ions in blood serum and in sea water, viz. : — we notice that the proportion of calcium to sodium is very similar and that of potassium to sodium is not very far different in the two solutions, but there is a great excess of magnesium in sea water. The probable reasons for the divergences will be seen presently. Bunge (1894, p. 120) made the suggestion that the high content in sodium chloride of the blood of land vertebrates in comparison with that of their surroundings is an inheritance from marine

ancestors who lived in a solution fairly rich in this salt. Macallum (1903, p. 234), struck by the similarity between the proportion of potassium and calcium to sodium in the blood plasma of vertebrates and that in sea water, was led, independently, to advocate the same view. The ocean, ever since the first condensation of water on the earth's surface, has been continually receiving salts by dissolving them from its bed and from the contents of the rivers flowing into it. Since the salts are left behind on evaporation, while water vapour is continually rising to form new rivers, which wash away more constituents of the land, it is easy to understand why the total concentration of salts in sea water is, at the present time, so much higher than that which it was at the time when the ancestors of the land vertebrates left it.

It is generally believed that life began in the ocean and continued in it alone until the close of the Cambrian period. When vertebrates with a closed circulatory system took to the land, they took with them a blood of the same composition, as regards salt, as the sea water which they left behind. The Cambrian period was an extremely long one, judging by the thickness of the deposits, amounting to 40,000 feet in British Columbia, and 12,000 feet in Wales, although it varies in different places. It is to be expected, therefore, that the protoplasm would have become adjusted to the salts of the sea during this long period, and that mechanisms would have been produced to maintain the concentration in the blood at the same value. These mechanisms still continue to act since life on land began.

If this view is correct, the salt composition of the blood represents that of the ocean in the early Cambrian period. As regards the proportion of calcium and potassium in sea water, Macallum points out that, at the present time, the concentrations of these two salts is scarcely changing at all. Calcium is being continually removed by living animals for the formation of bones, coral, shells, etc., as fast as it is supplied by the rivers. Potassium, since the great development on land of plant life, with its comparatively large content in this element, is supplied by the rivers in much less quantity than it was in early geologic times. The chief difficulty is the magnesium,1 which is present now in so much greater ratio to sodium in sea water than it is in blood plasma. According to Macallum, the reason is that the magnesium content of sea water is still slowly increasing, so that " in the pre-Cambrian oceans it must have been very small, not perhaps as low as it is in blood plasma, for in the latter the magnesium would only represent the proportion of an earlier period than that in which the circulation became closed, as the tissues would only reproduce the proportion which had by long accommodation become fixed in them. Even the organisms which live in the sea to-day, whose ancestral forms have lived in the sea since the Cambrian, do not take up the magnesium from the sea water in the full proportion which it has in the latter" (1904, p. 8 of reprint). Chemical changes by which magnesium chloride in the primeval ocean became precipitated as magnesia must also be taken into account (1904, p. 12).

A further interesting question concerns the salts of the cells themselves, a more difficult problem; but, as Macallum puts it (1904, p. 9 of i-eprint), " If the blood plasma of vertebrates, because of the forces of heredity, reproduce the proportions which obtained in pre-Cambrian oceans, why should not the cells of the tissues, because of the same forces, reproduce in themselves the proportions which obtained in sea water of a much earlier geological period ? "

There are different questions involved in the discussion of this problem, the consideration of which would lead us too far. The reader interested may refer to the paper quoted, and to a further one on the salts of the blood (1910). Whatever may be the final decision on the question, the fact remains that sea water, diluted to the same osmotic pressure as the blood, is a very effective physiological solution, although the amount of magnesium is unnecessarily great.

Returning to the preparation of such solutions for experimental use, it is found that Ringer's solution of the following composition is the most satisfactory for the heart of the frog : — The osmotic pressure of the blood of warm-blooded vertebrates is higher than that of the frog, so that the concentration of the salts must be slightly raised. For the isolated mammalian heart, Locke (1900) found the following composition to serve well : — The addition of glucose is of advantage as a source of energy, unless there is objection to its presence for other reasons. For mammals, this solution must be thoroughly oxygenated, conveniently by blowing oxygen from a cylinder of compressed gas through a Berkefeld filter immersed in the solution, as suggested by Keith Lucas. The solution used by Locke should be called Ringer- Locke's solution.