fluid in the muscles of the rat and the frog following violent

612.766.1: 612.014.461.3 THE CHANGES IN PLASMA AND TISSUE FLUID VOLUME FOLLOWING EXERCISE. By H. CULLUMBINE and A. C. E. KoCH. From the Department of Physiology and Pharmacology, University of Ceylon, Colombo. (Received for publication 4th February 1949.) THERE is ample suggestive evidence that muscular exercise may produce marked changes in the distribution of water in the body. Barcroft and Kato [1915] demonstrated that there was an increase in the weight of skeletal muscle during activity. Fenn et al. [see Fenn, 1936] have shown that there is an increase in the volume of extracellular fluid in the muscles of the rat and the frog following violent stimulation. Muscular exercise in man produces an increased concentration of the blood [Dill et al., 1930]; while Gregersen [1941] has reported an experiment on one subject in whom the plasma volume (measured by means of the dye, T-1824) decreased by 16 per cent. following exercise. To obtain a more precise estimate of the magnitude of these changes, simultaneous determinations of plasma volume and tissue fluid volume before and after exercise have been made. METHODS. Thirteen Ceylonese adult males were used as subjects. They rested for two hours before the commencement of the experiment and, except when exercising, they remained seated throughout the experiment. No fluid was taken by the subjects for 3 hours before the exercise and for at least 2 hours after the exercise. The exercise consisted of stepping up and stepping down from a stool 20 inches high and at a rate of 30 "step-up and step-down" cycles per minute. The exercise rate was maintained by means of a metronome and was performed for five and, in some cases, ten minutes. Such exercise can be considered to be moderate in severity, and Cogswell et al. [1946] have shown that it produces a "steady state" in circulatory and respiratory responses. In the first experiment, for which 6 subjects were used, the procedure was as follows:- After two hours' rest a sample of venous blood was withdrawn, and then about 10 c.c. (the actual volume varied a little from subject to subject but was carefully measured in each case) of a solution containing 50 mg. Evans Blue dye and 500 mg. sodium thiocyanate per 10 c.c. was injected intravenously. Venous blood samples were withdrawn 39

40 Cullumbine and Koch 5, 10, 15, 20, 40, 60, 90 and 120 minutes later. To minimise haemolysis the blood samples were collected with paraffined syringes and needles and delivered into paraffin-coated centrifuge tubes containing 500 units Heparin (0.5 ml.). The subjects exercised for five minutes immediately before the 40-minute blood sample was collected. Haematocrit readings were taken on the pre-injection, 20-, 40-, 60- and 90-minute samples. Blood and plasma specific gravities were estimated by the copper sulphate technique of Phillips et al. [1945] on all samples. From these specific gravities the plasma protein concentration was determined. The concentrations of Evans Blue dye and of sodium thiocyanate in the plasma were determined for each sample. The Evans Blue was estimated after extraction by the method of Crooke and Morris [1942] and the sodium thiocyanate by the method of Bowler [1944]. Readings were made in a Spekker photoelectric colorimeter. The air temperature was 82.40 F. and the relative humidity 78 per cent. RESULTS. The "available fluid" volume was calculated by dividing the milligrams of thiocyanate injected by the milligrams of thiocyanate per litre of plasma [Gregersen and Stewart, 1939]. This volume corresponds to " Space A " of Stewart and Rourke [1941] and Kaltreider et al. [1941]. "Interstitial fluid" volumes were calculated by subtracting the plasma volume and 70 per cent. of the red cell volume from the available fluid volume. The relevant results are given diagrammatically in fig. 1, and the magnitude of the volume changes is indicated in Table I. TABLE I.--VARIATIONS IN THE VOLUME OF THE BODY FLUIDS AFTER MODERATE EXERCISE. Volume in litres of Subject. Plasma. Available fluid. Interstitial fluid. Before After Before After Before After exercise. exercise. exercise. exercise. exercise. exercise. 1 2.75 2.25 14-78 16-97 11-04 13-77 2 2-83 2*40 11-54 13X49 7-78 10*20 3 2.20 1-60 13-59 14-82 10-75 12-63 4 2.83 2-03 15-32 17-61 11*59 14-47 5 2.05 1*60 10-20 11-81 7-52 9.48 6 2-28 2-05 14-78 16-95 11-78 14*16 The "plasma volumes " given here are calculated direct from the observed plasma concentrations of the Evans Blue dye, the assumption being made that the whole of the injected dye is still in the circulation.

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42 Cullumbine and Koch It will be seen that moderate exercise of the nature of a 5-minute Harvard Step Test produces a haemoconcentration, as evidenced by the rise in plasma protein content and the haematocrit readings. This hsemoconcentration is reflected in the fall in plasma volume, which is indicated by the changes in the plasma dilution of the Evans Blue dye. This fall in plasma volume varies between 225 to 800 c.c. in magnitude in the different subjects. The plasma volume does not return to normal for 40 to 60 minutes after the exercise. There is also a fall in the concentration of plasma thiocyanate following the exercise. In each subject the plasma thiocyanate had reached a constant level before the exercise (i.e. the thiocyanate was uniformly distributed in the "available fluid" of the body), and the concentration returned to its constant level about 1 hour after the exercise. This fall in plasma thiocyanate concentration indicates an increase in the available fluid- volume, and it will be seen from the table that the interstitial fluid volume also increases. Some increase in interstitial fluid volume would be expected, since the plasma lost from the circulation would have passed into the interstitial spaces. However, the increase in interstitial fluid volume is, in all cases, greater than can be accounted for by mere plasma diffusion. Presumably the water produced by the increased metabolism during muscular exercise has aided in this rise in interstitial fluid volume. In most cases too the maximum rise in available fluid volume occurs about 20 minutes later than the maximum haemoconcentration. To ensure that the variations in the plasma sodium thiocyanate and Evans Blue dye concentrations were caused by the exercise, the experiment was repeated on 4 of the 6 subjects, but this time no exercise was performed. In these instances no compa-rable increase in the concentration of Evans Blue dye and no fall in the concentration of sodium thiocyanate were found for three hours subsequent to the injection (see fig. 2). The procedure adopted to demonstrate the change in plasma volume after exercise cannot give a measure of the absolute plasma volume after exercise, and, in addition, the return of the plasma volume to its normal pre-exercise level may be masked by the loss of dye from the circulation. To obtain a more accurate measure of the plasma volume immediately after exercise and to ascertain more precisely the time for return to normal, a different procedure was adopted. Seven fresh subjects were used. A control sample of blood was obtained, and then the Evans Blue dye and the sodium thiocyanate injected intravenously. Blood samples were obtained 10, 20, 40, 60 and 90 minutes after this injection. When the 90-minute sample had been taken, the subjects at once performed the Harvard Test for 5 minutes. Immediately on completion of the exercise, 5 c.c. of 1 per cent. Evans Blue dye solution was injected intravenously and further

Changes in Plasma and Tissue Fluid Volume following Exercise 43 venous blood samples obtained 10, 20, 40, 60 and 90 minutes after the exercise. All the blood samples were analysed as before and, in this way, an estimation of the plasma volume before and after exercise was Subject 1 Subject 2 Subject 3 Subject 4 3000,-,. 2900 2800.0 2700 2600 a2500 Pq 2400 CO 2300 Pq 2200 2100 2000 6.0 5*0 Cs C> 4-0 3-0 9 1 20 0 1 0 20 40 60 90 120 10 20 40 60 90 120 10 20 40 60 90 120 Time in minutes after injections of dye and thiocyanate. FIG.2.-Variations in body fluid volumes-resting subjects. obtained [Overbey et al., 1947; Cruickshank and Whitfield, 1945]. The air temperature was 84.30 F. and the relative humidity 83 per cent. The results are summarised in Table II. TABLE II.-ITARIATIONS IN THE VOLUME OF PLASMA AND AVAILABLE FLUID AFTERE.XERCISE. Plasma volume Available fluid Time after in C.C. Time to volume in litres. exercise Time to Subject. return to for return to Subject. normal in maximum normal in Before After minutes. Before After volume in minutes. exercise. exercise. exercise. exercise. minutes. 7 2130 1840 25 11-446 12-05 10 40 8 2250 1900 25 13-163 13-900 30 50 9 2450 2050 25 13-163 14-040 40 60 10 2850 2300 30 13,320 14*450 40 60 11 3100 2600 75 14.70 15-85 60 90 12 2950 2700 75 12-40 13-16 60 90 13 2450 1900 60 10-88 11-88 60 90

44 Cullumbine and Koch Again it is obvious that the plasma volume decreases and the available fluid volume increases after exercise. Subjects 11, 12 and 13 performed the Harvard Test for 10 minutes. The fluid volume changes are not noticeably more with the longer exercise, though the individual variation is too great and the number of subjects too small to justify a precise statement. However, after the longer exercise the available fluid does seem to increase for a longer time than with the shorter exercise. In all cases the maximum available fluid volume occurred later than the maximum fall in plasma volume, and, indeed, in some cases the plasma volume had returned to normal while the total available fluid was still increasing in quantity. DIscUSSION. As suggested by the results of earlier workers [Gregersen, 1941; Dill et al., 1930], exercise results in a decrease in the plasma volume. In our experiments there were wide individual variations in the amount of fluid leaving the blood-stream (225 to 800 c.c.), but in all cases the loss of fluid occurred rapidly, and this loss was restored within 20 to 30 minutes for exercise of 5 minutes' duration and within about 75 minutes for exercise lasting 10 minutes. Even greater changes occurred in the volume of "available" and "interstitial" fluids. Here, too, wide individual variations in response occurred (e.g. 0-604 to 2-29 litres was the range of increase in available fluid volume). The maximum increase in the available and interstitial fluid volume in 11 of the 13 subjects occurred 20 to 60 minutes after the cessation of the exercise and the occurrence of maximum haemoconcentration. The longer the duration of exercise, the longer was the persistence of these increased available and interstitial fluid volumes. These fluid volumes were calculated after estimating the plasma concentration of sodium thiocyanate, and the changes in the concentration of this substance produced by exercise could be interpreted in other ways. Thus exercise may have induced a greater rate of excretion of sodium thiocyanate in the urine. If this had occurred, then we should not expect the plasma concentration to return, as it did, fairly rapidly to its pre-exercise level. Or it may be suggested that, during exercise, part of the sodium thiocyanate is removed from the body fluids and becomes an intracellular constituent. After exercise this intracellular sodium thiocyanate would have to be released to restore the plasma concentration. In the absence, at present, of any evidence to support this, we must assume that the changes in plasma sodium thiocyanate concentration following exercise reflect changes in the volumes of the body fluids.

Changes in Plasma and Tissue Fluid Volume following Exercise 45 Estimations of the total body water before and after exercise would help in the interpretation of our results. Painter [1940] has used urea and sulphanilamide for estimating the total body water in dogs. Their use in man would be complicated by the alteration in the rate of excretion of urea by the kidney during exercise, and the fact that sulphanilamide is conjugated in the liver of man but not in dogs. The Appendix gives details of age, height and weight of the 13 subjects. APPENDIX AGE, HEIGHT AND WEIGHT OF THE SUBJECTS EXAMINED. Subjects. Age, years. Height, cm. Weight, lb. 11 12 13 10 37 2 5 6 4 1 9 8 21 22 23 22 25 31 19 22 24 21 48 36 24 179 173 150 162 154 153 156 157 165 154 158 156 153 148 143 94 151 101 89 92 90 118 113 140 125 98 I- SUMMARY. Simultaneous determinations of plasma volume and tissue fluid volume before and after exercise have been made in 13 male Ceylonese. Exercise produces a prompt decrease in plasma volume, and a slower but greater increase in available fluid and interstitial fluid volume.

46 Changes in Plasma and Tissue Fluid Volume following Exercise REFERENCES. BARCROFT, J., and KATO, T. (1915). Proc. Roy. Soc., B, 88, 541. BOWLER, R. S. (1944). Biochem. J. 38, 385. COGSWELL, R. C., HENDERSON, C. R., and BERRYMAN, G. H. (1946). Amer. J. Physiol. 146, 422. CROOKE, A. C., and MORRIS, C. J. 0. (1942). J. Physiol. 101, 217. CRUICKSHEANK, E. W. H., and WHITFIELD, I. C. (1945). Ibid. 104, 52. DniT, D. B., TALBOTT, J. H., and EDWARDS, H. T. (1930). Ibid. 69, 267. FENN, W. 0. (1936). Phy8iol. Rev. 16, 478. GREGERSEN, M. I. (1941). Macleod's Physiology in Modern Medicine, 9th ed., p. 1087. London. GREGERSEN, M. I., and STEWART, J. D. (1939). Amer. J. Physiol. 125, 142. K.ALTREIDER, N. L., RENEELY, S. R., AuLTAN, J. R., and BALE, W. F. (1941). J. exp. Med. 74, 569. OVERBEY, D. T., MOORE, J. C., SHEDLE, 0. W., and LAWSON, H. C. (1947). Amer. J. Physiol. 151, 290. PAINTER, E. E. (1940). Ibid. 129, 744. PiHTTTTs, R. A., VAN SLYKE, D. D., DOLE, V. P., EMERSON, K., HAMILTON, P. B., and ARCHEALD, R. M. (1945). Copper Sulphate Method for Measuring Specific Gravities of WVhole Blood and Plasma. Macy Foundation, N.Y. STEWART, J. D., and ROURKE, S. M. (1941). J. Lab. clin. Med. 26, 1383.