6I2.I22:547a being used. The subjects of these experiments are described in the text.

Transcription

1 443 6I2.I22:547a472.3 BLOOD LACTIC ACID IN MAN DURING REST. BY LESLIE COLIN COOK AND RICHARD HENRY HURST1. (From the Central Pathological Laboratory of the London County Mental Hospitals.) THE problems connected with lactic acid production in the animal body have been extensively investigated by previous authors, but the obvious difficulties encountered in working with the intact organism have rendered the solution to some of these still a matter of speculation. When, for instance, the organism is subjected to a severe stress, the presence of lactic acid in the circulating blood can be readily understood, but its constant presence in small concentration, even during complete bodily rest, is less easy of interpretation. No satisfactory explanation has, in fact, yet been given either for the origin of blood lactic acid in the resting state or for the wide variations in its concentration. The existence of some muscular activity during bodily rest has led most authors to attribute the blood lactic acid to this source. In this paper, however, an attempt will be made to show that muscular activity can have little influence in this connection and that conversion of blood sugar (glycolysis) is the most probable source of this lactic acid. The wide variations in the resting levels of blood lactic acid will then be explained as one of the factors through which the organism endeavours to maintain a constant blood ph. EXPERIMENTAL. In some of the experiments blood was taken from one of the superficial veins of the arm after initial compression, either manual or by tourniquet, which was released as soon as the needle was in the vein. The subjects were normal young adult males. In other experiments the femoral artery, femoral vein and jugular bulb were punctured, no compression being used. The subjects of these experiments are described in the text. The blood was collected into a small amount (approx. 003 g. per 15 c.c. 1 Maudsley Fellow.

2 444 L. C. COOK AND R. H. HURST. blood) of Evans's 9: 1 mixture of potassium oxalate and sodium fluoride, part of which was placed in the receiver and a smaller part on the barrel of the syringe. After thorough mixing the specimens were placed in a refrigerator or a portable ice-box. Deproteinization was carried out by the Folin-Wu tungstatesulphuric acid method. Actually 2 c.c. of whole blood were pipetted into a large centrifuge tube containing 14 c.c. of distilled water and 2 c.c. of 2/3N sulphuric acid; 2 c.c. of sodium tungstate were then added and the contents well shaken before being centrifuged. The customary method is then to remove sugar by treatment with copper sulphate and lime. It was found, however, that this was an unnecessary procedure, since by the Friedemann method of lactic acid determination the amount of bisulphite binding material formed from the small amount of glucose present is negligible. On the other hand, when trichloroacetic acid was used as deproteinizing agent, it was usually necessary to treat the filtrate with copper sulphate and lime, not, presumably, to remove sugar, but to remove bisulphite-binding material other than lactic acid which remained in solution or suspension. The results recorded in Table I illustrate these points. TABLE I. Lactic acid in mg./100 c.c. of whole blood Deproteinizing Filtrate treated with Filtrate used agent Material copper sulphate and lime directly Sodium tungstate Sheep's blood *0 and sulphuric acid,,,, Human blood ,,,,,, ,,- VP,, Human blood after exercise Trichloroacetic acid Human blood , 11.1 t p ,,.0 Human blood after exercise Lactic acid was determined in 10 c.c. of the filtrate by the method of Friedemann, Cotonio and Shaffer, modified by Friedemann and Kendall [1929]. The apparatus used was that designed by Davenport and Cotonio [1927], except that the inlet tube of the boiling flask was connected by means of rubber tubing to the air outside the building. In these circumstances the "blank" value obtained was constant and amounted to 0.15 c.c. of 0-002N iodine solution. In all experiments at least two estimations were performed, the specimens being separated before deproteinization. In the great majority of cases, duplicate results agreed to within 0.2 c.c. of 0*002N iodine solution, i.e. approximately

3 BLOOD LACTIC ACID IN MAN DURING REST. 2 mg./100 c.c. of lactic acid. Any experiment in which duplicate results showed a difference in excess of this figure was ignored. Similarly, in comparing the lactic acid levels of blood taken under varying conditions, differences below 2 mg./100 c.c. were regarded as insignificant. The percentage recovery of added lactic acid (as zinc lactate) was between 95 and 98. On some of the blood samples ph measurements were made with the glass electrode apparatus designed by Kerridge, the precise technique employed being that previously described by one of us [Hurst, 1932]. For this purpose the blood was collected under paraffin. Blood lactic acid at rest. The introduction of the Clausen method in 1922 gave a fresh impetus to work on this subject, since previously no thoroughly reliable and convenient technique had been known. Using this method or one of its more recent modifications, numerous investigators have found that the resting levels of the lactic acid of whole blood of man vary within wide limits. Clausen [1922] gave results varying from 15 to 32 mg./100 c.c., whilst Owles [1930], with more refined technique, obtained results varying from 7 to 18 mg./100 c.c. The results of authors who used other methods for lactic acid determination are in close agreement and it seems clear that the normal resting levels differ by as much as 100 p.c. In the present investigation 26 estimations on 9 normal subjects showed variations between 8-4 and 16-6 mg./100 c.c. (Table II). In each experiment the subject was in the post-absorptive state and was either in bed or had rested in an arm-chair for at least half an hour. TABLE II. Lactic acid in mg./100 c.c. blood Subject A M. M. M *7 I. H. 16* *0 13*5 E. I A. M. T. 10* F. J. S * P.C. 14*5 9*0 J.C.B F. P *6 K.C.H The values are lower than those of previous authors except Owles; a fact which suggests that they were obtained by an improved technique and afford a truer index of the lactic acid content of blood. Table II shows also that even with the same subject under apparently identical conditions the variations are considerable. It seems probable that these wide variations are due neither to experimental error nor to changes in the blood during or after shedding.

4 446 L. C. COOK AND R. H. HURST. POSSIBLE FACTORS INFLUENCING BLOOD LACTIC ACID PRODUCTION AT REST. (1) Muscular contraction. It is known that lactic acid is constantly being produced in the muscles even during complete bodily repose. This was first shown in the case of frogs by Fletcher and Hopkins [1907] and amply confirmed by Meyerhof [1924] in the cases of amphibians and of mammals. It has usually been held that small amounts of the lactic acid so formed escape into the blood stream and that this supply constitutes the "resting" blood lactic acid. Against this hypothesis much evidence is accumulating. According to Hill, Long and Lupton [1924] the process of recovery after muscular exertion may be divided into two phases, the first being "nothing more than the oxidative removal of lactic acid in the muscles where it was formed before it has had time to escape into the blood and still further afield." This would appear to be the only phase necessary in very mild exercise, where lactic acid removal can keep pace with its formation. The second phase occurs when lactic acid formation is so rapid that some of it escapes into the blood stream, to be resynthesized in the liver and in other muscles. Until recently the view that such moderate exercise as walking at rates of about 31 m.p.h. produces a definite rise in blood lactic acid had not been questioned. This view depended mainly upon the work of Hill, Long and Lupton [1924], who found that a man walking at 3-5 m.p.h. for 28 min. raised his blood lactic acid from 20-9 mg. to 36-6 mg./100 c.c., whilst the same man walking at 4-1 m.p.h. for 33 min. raised it from 21>4 to 58-9 mg./100 c.c. Several other subjects were found by Long [1926] to give similar figures. Recent work by Owles [1930], however, has opened up the whole question as to the threshold at which lactic acid overflows into the blood after muscular exercise. Owles found in the case of two subjects in good training that walking for approximately 30 min. at speeds up to 4k m.p.h. produced no rise in lactic acid in the blood taken from a superficial arm vein. At first sight these results suggested that, although no rise in lactic acid could be demonstrated in the blood from an antecubital vein, there might nevertheless be an escape of lactic acid from the leg muscles, but that the amount was insufficient to show a significant rise by the time it had been diluted in the general circulation and had passed through the inactive muscles of the arm. In order to test the validity of Owl e s's results the following experiments were performed. A man of 48 (W. P. W.), who was accustomed to walking for about an hour every morning at a brisk pace (about 41 m.p.h.), was made to walk at 4 m.p.h. for 30 min. Specimens of blood were taken from one of the antecubital veins at rest before exercise and from an antecubital vein and the femoral vein immediately after the exercise. The experiment was done on two separate occasions with identical results, which were corroborated in the case of another subject (B. T.), aged 55, who was accustomed to walking exercise and who walked for 30 min. at 4j m.p.h. In neither case was there any rise in the lactic acid content of blood from the femoral vein, which demonstrates clearly that there was no escape of lactic acid into the veins immediately draining the muscles mainly involved in the exercise. Further experiments with

5 BLOOD LACTIC ACID IN MAN DURING REST. the subject B. T. showed that as soon as the rate of walking was accelerated to a speed incompatible with comfort, a rise in blood lactic acid occurred. Thus, after walking for 30 min. at 5j m.p.h., it rose from 10 mg. to 23*5 mg./100 c.c. In the case of men not used to strenuous walking, an increase of blood lactic acid was obtained after walking at lower speeds. The figures obtained in the above experiments are shown in Table III. Blood lactic acid in mg./100 c.c. TABLE III. Arm Arm vein, Femoral vein, after vein, after Rate and period Subject Age resting exercise exercise of walking Remarks W.P.W min. at 4 m.p.h. Good walker 15* ,1,... B. T min. at 4i m.p.h * min. at 5k m.p.h. Considerable effort needed to maintain this pace M. S *5 20*5 20 min. at 4j m.p.h. Fair walker for age D. T. J *5 21X0 24 min. at *k m.p.h. Very poor training 447 It appears from these experiments that the threshold of exercise beyond which an increase of lactic acid appears in the blood depends on the "fitness" or "training" of the individual, a condition which is determined partly by the constitution of the individual, but mainly by accustoming the organism to exercise. Training, according to Briggs [1920], develops the efficiency of the mechanism which supplies oxygen to the tissues. As Owle s suggests, the power of the muscles to eliminate in situ the lactic acid formed during exercise probably depends upon the adequacy of the blood supply to them. In the light of the above results it is unlikely that the muscles are unable to eliminate in situ the relatively small amounts of lactic acid they produce during bodily rest. It is even more unlikely that the amounts produced in the same person under apparently identical conditions of rest should vary so much as to cause the large fluctuations actually found. Further evidence against the probability of the lactic acid content of the blood at rest being due to muscular activity was supplied by taking specimens from the femoral artery and the femoral vein at the same time. In no case was there any appreciable difference between the venous and

6 448 L. C. COOK AND R. H. HURST. arterial sample (Table IV). This indicates clearly that the passage of blood through a large group of muscles during rest causes no significant change in its lactic acid content. TABLE IV. Blood lactic acid in mg./100 c.c. Subject No. Femoral artery Femoral vein * *9 6 9* *7 The value of these observations, however, would be considerably diminished unless a definite rise in the lactic acid content of the femoral vein over that of the femoral artery could be shown in conditions where the leg muscles were knows 1,'be producing large amounts of lactic acid, for example, after vigorou, exercise. At first sight this might appear to be an easy matter, but when it is remembered how rapidly the blood comes into equilibrium and how great an increase of the circulatory rate occurs after severe exercise, it becomes clear that at best no more than a small increase can be expected. Recovery curves after severe exercise of short duration, such as standing-running "all out" for 1 min., show that the blood lactic acid rises rapidly for a few minutes after the exercise and then falls very much more slowly, regaining approximately its resting level in about an hour. The more severe the exercise, the longer is the period of ascent of the curve, the higher the maximum point and the longer the recovery period. The maximum, however, is always reached within 5 or 6 mi. At this point the arterial and venous blood are in approximate equilibrium, and in order to show any excess lactic acid in the blood of the femoral vein, simultaneous arterial and venous punctures must be made immediately after the exercise. In order to demonstrate this, standingrunning "all out" for 1 min. was carried out in three cases. The subject was first stripped and the femoral area cleaned up in order to save time. As soon as the exercise was completed the subject lay upon a couch, and the left femoral artery and right femoral vein were punctured simultaneously by two operators. Withdrawal of the blood was delayed until both operators were ready, and was then accomplished at the same rate and to the same amount. In two cases the blood had been transferred from the syringe to the receiving vessel within 3 min. In the third experiment a delay occurred owing to the femoral artery being entered instead

7 BLOOD LACTIC ACID IN MAN DURING REST. of the vein. This necessitated a change of syringe and the blood was ultimately withdrawn about 5 mi. after the exercise. The results of these experiments, although too few to be convincing, were satisfactory (Table V). TABLE V. Simultaneous punctures of femoral artery and femoral vein after severe exercise. Blood lactic acid in mg./100 c.c. Subject No. Femoral artery Femoral vein Remarks Within 3 min.* Within 3 min.* About 5 min.* * After completion of exercise. In Cases 8 and 9 the blood was taken within the period of lactic acid increase and a slight but definite excess in the venous specimen appeared. In Case 10 the blood was withdrawn approximately at the lactic acid maximum and showed no significant difference between the arterial and venous figures. The subjects of these experiments (Nos. 1-10) were chronic mental defectives or psychotics in good physical condition. It has now been shown that no increase in blood lactic acid is caused by the passage of blood through a large group of resting muscles nor by the taking of a moderate amount of exercise, as long as it is not severe enough to produce dyspncea or discomfort. This evidence clearly indicates the error of assuming that muscular activity is responsible for the blood lactic acid during bodily rest. It has also been shown that the blood draining a group of muscles, which have been vigorously exercised, contains slightly more lactic acid than that of their arterial supply, provided that the lactic acid maximum has not been reached. This indicates the possibility of demonstrating changes of lactic acid in the blood occurring during its passage through muscle, but only when the formation of lactic acid is very rapid and before its removal has begun to keep pace with its formation. (2) Stimulation of the sympathetic nervous system. The effect of administration of adrenaline upon carbohydrate metabolism has been investigated in great detail, a review of the work being given by Cori [1931]. It has been demonstrated conclusively that injection of this substance leads to the breaking down of muscle glycogen without any associated muscular contraction, and that this results in the escape into the blood of large amounts of lactic acid. The effects of subcutaneous injections on the blood lactic acid of man are shown in Table VI. It is known that excitement, fright or any emotion which powerfully stimulates the sympathetic nervous system 449

8 450 L. C. COOK AND R. H. HURST. TABLE VI. Effect of 17 minims of 1/1000 solution of adrenaline hydrochloride given subcutaneously. Blood lactic acid in mg./100 c.c. ph Pulse rate Sub- Before 20 mm. 20 min. 10 min. 20 min. 30 min. ject adrenaline after Before after Before after after after F. J. S * A. C No significant change increases the secretion of adrenaline with consequent rise of blood lactic acid. For this reason it might be suggested that the mood and emotional state of the individual, through their influence upon adrenaline secretion, are the regulators of the lactic acid content of the blood during rest. The present work, however, supplies little evidence in support of this view, since the blood lactic acid levels in the same subject appear to be entirely independent of mood. Furthermore, in experiments, not included in this paper, upon neurotic patients some of the highest levels were found in the most sluggish and phlegmatic of cases, whilst subjects showing sympatheticonia or suffering from anxiety states showed levels not appreciably above the average normal. (3) Lactic acid production in the brain. The production of lactic acid in>1relatively large amount by the braintissue has been shown to occur both in vitro [Warburg, Posener and Negelein, 1924] and in vivo [Holmes and Sherif, 1932]. The possibility of some of this lactic acid overflowing into the blood and thus constituting a part of the resting blood lactic acid had therefore to be considered. Specimens of blood were accordingly collected from the jugular bulb and from the femoral artery. It is obvious that the blood in the main arteries does not vary significantly, and for this reason the femoral artery, being the most convenient to enter without delay, was chosen in preference to the carotid artery. As not more than 2 mi. elapsed between the removal of the venous and arterial blood, the samples may be taken as simultaneous for comparative purposes. The subjects were in the resting state and were of the same type as those used for other arterial punctures. Table VII shows that the lactic acid level of the blood directly issuing from the brain is no higher than that of the arterial blood, that is to say, that all the lactic acid formed in the brain is dealt with in situ and plays no part in forming the resting blood lactic acid. The free passage of CO2 into the blood, on the other hand, is demonstrated by the differences between arterial and venous ph values.

9 BLOOD LACTIC ACID IN MAN DURING REST. 451 TABLE VII. Lactic acid in mg./100 c.c. blood - I A Subject No. Jugular bulb Femoral artery Jugular bulb Femoral artery (4) Threshold for tactic aaid resynthesis. The possibility of a threshold below which lactic acid is not resynthesized by the muscles would afford an attractive hypothesis, in that it would solve in an easy manner the problem of the source of blood lactic acid at rest. The existence of such a threshold value would also account for the fact that the resting levels of the blood supplying and leaving a group of muscles on the one hand and the liver on the other are all approximately the same [Himwich, Koskoff and Nahum, 1930]. It would not, however, account for the wide variations obtained and there is at present no practical evidence to justify the assumption of such a threshold. (5) Glycolysis. The conversion of blood sugar to lactic acid in vitro has long been an established fact. The disappearance of glucose in shed blood was noted by Claude Bernard [1877], and has since been studied by many workers. Lovatt Evans [1922] has shown that large amounts of lactic acid are produced by glycolysis in shed blood and that the reaction is accelerated by alkali. It appears that the phenomenon is due to some extent to leucocytic activity and that it cannot occur in the plasma or serum alone. It is known to be accelerated by moderate increases of temperature or by raising the blood ph, to be retarded by decrease in ph and to be stopped completely by the addition of sodium fluoride in suitable concentration. The existence of glycolytic activity in the blood in vivo is more difficult to prove, but considerable evidence is accumulating in its support. Eggleton and Evans [1930] have demonstrated in a lung-limb preparation of a dog a fall of blood sugar coincident with a rise of lactic acid, caused by raising the ph of the blood by means of sodium bicarbonate. Still more convincing is the work of Anrep and Cannan [1923] who found that in a heart-lung preparation an increase in ph led to an increase in blood lactic acid. In this case there could be little production of blood lactic acid by muscular activity, since it has been shown by Himwich, Koskoff and Nahum [1928] that the heart is more concerned with removing than with forming lactic acid. Anrep and Cannan considered the increase in lactic acid to be due more to interference with its oxidative removal than to its formation from sugar. Against this, however, it has been shown by Hartree and Hill [1924] that in the frog a fall in ph tends to retard lactic acid removal and that a rise in ph has no such effect. In the present investigation, the effect of alkali ingestion upon man was determined, 20 g. of sodium bicarbonate dissolved in water being given by the mouth. The results recorded in Table VIII show that 30 mi. after ingestion a small but definite increase in both blood ph ph

10 452 L. C. COOK AND R. H. HURST. TABLE VIII. Ingestion of 20 g. sodium bicarbonate. Blood lactic acid in ph mg./100 c.c. Subject Before 30 mnm. after Before 30 min. after J. C W. S A. M M. H and blood lactic acid could be demonstrated. On the hypothesis that glycolysis is the source of the "resting" blood lactic acid, these results supply one at least of the reasons for the variations of blood lactic acid found in the same resting individual at different times. It is known that slight changes in ph are constantly taking place in the circulating blood, and it is to be expected from the above evidence that these are accompanied by changes not only in C02 tension but also in lactic acid concentration. Thus glycolytic activity, stimulated by a tendency to increased blood ph and depressed by a tendency to decreased blood ph, offers a material basis for the suggestion of Anrep and Cannan [1923] that blood lactic acid changes serve as a third line of buffer resistance, and for the statement of Peters and van Slyke [1931] that (in states of alkalosis) the "lactic acid increase must be looked upon as an adaptive reaction, as it tends to mitigate the alkalosis to which it is a response." SUMMARY. 1. The technique of lactic acid estimation by means of one of the more recent modifications of Claus en's method is described. It is shown that the removal of sugar is unnecessary as long as tungstate-sulphuric acid deproteinization is used. 2. An attempt is made to trace the source of blood lactic acid in man during bodily rest. Resting blood lactic acid values observed by previous authors are reviewed and are compared with the figures obtained in the present studies. The results show that any satisfactory explanation of the existence of this lactic acid must also account for the wide variations observed in the same individual at different times, but under apparently identical conditions of bodily rest. 3. Possible sources of blood lactic acid at rest are discussed and experimental evidence is presented in support of the foliowing conclusions: (a) During bodily rest the muscles supply no lactic acid to the blood. In the case of men in good training, walking at speeds up to 4i m.p.h.

11 BLOOD LACTIC ACID IN MAN DURING REST. 453 for 30 min. produces no increase in the lactic acid concentration of blood drawn from the femoral vein. (b) Activity of the sympathetic nervous system with consequent secretion of adrenaline is an unlikely source. (c) Comparison of blood samples taken in immediate succession from the jugular bulb and from an artery during rest shows that the blood receives no demonstrable amount of lactic acid from the brain. (d) The existence of a threshold value below which lactic acid is not resynthesized by the muscles is an hypothesis which lacks supporting evidence of a practical nature and which would still leave unexplained the wide variations in lactic acid concentration. (e) Conversion of blood sugar (glycolysis) is the most probable source of the blood lactic acid at rest. The evidence of glycolytic activity, shown by previous workers to take place in vitro and in heart-lung preparations, is supported by experiments in vivo, in which an increase in blood ph by means of alkali ingestion is shown to increase the blood lactic acid concentration. These experiments also suggest that the variations in lactic acid coneentration during bodily rest are due to stimulation or depression of glycolytic activity and assist the organism in its endeavour to maintain a constant blood reaction. Our acknowledgments are due to Dr F. L. Golla, Director of the Central Pathological Laboratory of the London County Mental Hospitals, for his unfailing interest, and to Drs R. A. McCance and S. A. Mann for valuable advice and criticism. We would also thank Drs G. Clarke, D. Ogilvy, A. A. W. Petrie and N. Roberts for allowing us access to cases in their hospitals. A special word of thanks is due to Prof. F. R. Fraser for his advice concerning the technique of jugular bulb puncture. REFERENCES. Anrep, G. V. and Cannan, R. K. (1923). J. Physiol. 58, 244. Bernard, Claude (1877). LeQon8 &ur le diabete. Paris. Briggs, H. (1920). J. Physiol. 54, 292. Clausen, S. W. (1922). J. biol. Chem. 52, 263. Cori, C. F. (1931). Phy8iol. Rev. 11, 143. Davenport, H. A. and Cotonio, M. (1927). J. biol. Chem. 73, 359. Eggleton, M. G. and Evans, C. L. (1930). J. Phy8iol. 70, 261. Evans, C. L. (1922). Ibid. 56, 146. Fletcher, W. M. and Hopkins, F. G. (1907). Ibid. 35, 247. Friedemann, T. E. and Kendall, A. I. (1929). J. biol. Chem. 82, 23. Hartree, W. and Hill, A. V. (1924). J. Phy8iol. 58, 470.

12 454 L. C. COOK AND R. H. HURST. Hill, A. V., Long, C. N. H. and Lupton, H. (1924). Proc. Roy. Soc. B, 97, 96. Himwich, H. E., Koskoff, Y. D. and Nahum, L. H. (1928). Proc. Soc. exp. Biol., N.Y. 25, 347. Himwich, H. E., Koskoff, Y. D. and Nahum, L. H. (1930). J. biol. Chem. 85, 571. Holmes, E. G. and Sherif, M. A. F. (1932). Biochem. J. 26, 381. Hurst, R. H. (1932). Ibid. 26, Long, C. N. H. (1926). Proc. Roy. Soc. B, 99, 167. Meyerhof, 0. (1924). Chemical Dynamic.s of Life Phenomena. Lippincott. Owles, W. H. (1930). J. Phy8iol. 69, 214. Peters, J. P. and van Slyke, D. D. (1931). Quantitative Clinical Chemi8try. London, 1,476. Warburg, O., Posener, K. and Negelein, E. (1924). Biochem. Z. 152, 309.