estimates were made of the normal rate of increase in plasma urea over periods in skin and in plasma, hypertonic sodium chloride solution was

Transcription

1 482 J. Physiol. (I95I) II5, THE STTE OF BODY WTER IN THE CT BY M. GRCE EGGLETON From the Department of Physiology, University College, London (Received 5 July 1951) In the course of an investigation into the relationship between chloride concentration in skin and in plasma, hypertonic sodium chloride solution was injected. The resulting increase in chloride concentration in the plasma water enabled calculation to be made of the volume of water in the body in which the added chloride appeared to become distributed. It proved to be significantly less than the generally accepted figure for total body water. This, consequently, was measured in addition by determining the volume of water into which urea would diffuse. METHODS The experiments were made on cats, anaesthetized with nembutal (sodium ethyl (1-methylbutyl) barbiturate) by intraperitoneal injection of 40 mg./kg. body weight. In order to avoid loss of the injected solutions in the urine, or their concentration in the kidneys, the renal pedicles were tied. The solutions were injected into one external jugular vein and blood samples removed from the opposite carotid artery. fter collection of the first blood sample, a 20% solution of urea in 0-9% NaCl was injected intravenously in a dosage of 1 g./kg. body weight. In a few experiments the saline was replaced by isotonic NaCNS (0.25 g./kg.). fter min. a second blood sample was collected, preceded by removal of a sample of muscle (gracilis). 20% solution of NaCl was next infused in a dosage of 2 g./kg., and after j-5 hr. a further sample of muscle and blood. In one experiment, the order of injection of the two solutions was reversed. The water content of the tissues was determined by drying for hr. at C. Chloride was determined by electrometric titration with N/50-gNO3 in a deproteinized trichloroacetic acid filtrate, and thiocyanate colorimetrically, after reaction with ferric nitrate reagent. The colour was not completely stable, but satisfactory results were obtained by reading a set of standards both before and after the unknown solutions. Plasma urea was determined by the method of Conway & O'Malley (1942). RESULTS The resting plasma urea concentration was found to be considerably higher than in man. The lowest value encountered was 57 mg./100 ml. plasma water, and the average of ten animals was 82 mg./100 ml.; the eleventh had a value of 230 mg./100 ml., and this plasma itself was yellow-brown in colour. Eight estimates were made of the normal rate of increase in plasma urea over periods

2 BODY WTER IN THE CT 48-Q3 of 3-4 hr., and these varied from 1 to 5 mg./100 ml. plasma water/hr. Since this value is small in relation to the increase caused by the urea injection, no allowance has been made for it in calculating the volume of water into which the injected urea has penetrated. Had such allowance been made, the calculated body water would be higher than the values shown below. In Table 1 are given details of a typical experiment. Fifty-five minutes elapsed between the collection of plasma 1 and plasma 2, the latter being taken 20 min. after the end of the urea injection. Four hours and 20 min. elapsed between the collection of plasma 2 and plasma 3, the hypertonic NaCl solution being injected during the 20 min. immediately after collection of sample 2. The increase in plasma urea concentration indicates that the amount injected had been distributed in an amount of water which was 63% of the body weight. The increase in NaCl concentration, however, indicates that the water in which it had apparently been distributed amounted to only 49% of the body weight. TBLE 1. Measurement of body water by injection of urea and of hypertonic NaCl solution Cat 2-26 kg g. urea injected between plasmas 1 and 2; 4-15 g. NaCl (20% solution) between plasmas 2 and 3. NaCl Urea r -r, mg./100 ml. mg./l00 ml.h0 m./100 ml. mg./100 ml. Plasma * Plasma Plasma Urea: Increase of mg./100 ml..., g. in = 63% body weight. NaCl: Increase of 375 mg./100 ml g. in =49% body weight. It may be well to emphasize at this point that it is generally supposed that the chloride concentration of muscle cells is small and not far from zero. It may also be relatively small in the cells of other organs and tissues. The added NaCl, therefore, must not be supposed to penetrate, quantitatively, into the cells; but the increased concentration in the extracellular fluid will have the effect of drawing water out of the cells. If the cell contents behaved, osmotically, as an 'ideal' solution, the amount of water withdrawn would be such that the final concentration of chloride in the extracellular fluid would be the same as that which would have obtained if the chloride had penetrated freely into the cell contents. That there is undoubtedly considerable movement of water from intra- to extracellular fluid is shown by the fact that the concentration of total solids in skeletal muscle increases and that the ratio of chloride concentration in muscle water/chloride concentration in plasma water also increases, indicating a shrinkage of cells with increase in extracellular volume. This was consistently observed in all experiments, confirming the results of Eichelberger & Hastings (1937). The observation was amplified in four experiments by determination PH. CXV. 32

3 484 M. GRCE EGGLETON also of thiocyanate spaces. The resting value obtained in nine cats was O8 (S.E. of mean) percentage of body weight. It increased to a slight extent with time; increases of 0 5, 4-5 and 1F5 in 3-4 hr. were observed in three experiments. fter injection of hypertonic NaCl solution, however, the resting values had increased by 9, 9 5, 13-5 and 13-5 respectively in a similar time. The magnitude of this water shift from intra- to extracellular compartment can be calculated for muscle if it be assumed that the interstitial fluid contains 1-5 % protein (Maurer, 1938) and that the muscle cells contain no chloride. typical example of such a calculation is given in Table 2. TBLE 2. The effect of intravenous hypertonic NaCl solution on the intra- and extracellular volumes of skeletal muscle. Cat 1-96 kg. 3-5 g. NaCl (20% solution) given 3-7 hr. before second sampling. Muscle Plasma r Cl concentration Cl concentration (g./loo g.) Solids (g./loo g.) mg./loog. mg./100 g. (g./10 g.) mg./100g. mg./100 g. Before fter Before: Extracellular water of muscle = 159/778 x 98-5 =20-1 g./100 g. muscle. Intracellular water of muscle = =54-2 g./100 g. muscle =54.2/25-7 =2-11 g./g. muscle solid. fter: Extracellular water of muscle =354/1123 x 98-5 =31-0 g./100 g. muscle. Intracellular water of muscle = =40-6 g./100 g. muscle =40-6/28-4 = 1-42 g./g. muscle solid. Thus, 0 69 g. muscle. intracellular water is released per g. muscle solid or 0 69 x 25*7 = 17-8 g./100 g. Nevertheless, it would seem that not all the body water behaves as if all the solutions were 'ideal' osmotically. The difference between 63% of the body weight (into which urea can diffuse), and 49% (into which chloride can apparently diffuse) is too large to be accounted for by experimental error; moreover, a difference in the same direction, though of varying magnitude, was observed in all experiments, as may be seen in Table 3. One possible cause of the large individual variation may be age. The smallest difference of 5 was obtained in a cat which was noted at the time of experiment as very old, and the two succeeding ones of 13-6 and 18 in cats which were noted as young. The animals were obtained in the usual way and their past history not known, so this factor of age can be accepted only as an impression rather than as a fact. In three further experiments urea was injected but not NaCl, and the average of the total eleven urea experiments gives a value of % body weight for the total body water; in a further nine experiments the reverse was done, and the average value of body water (derived from changes in chloride concentration) in seventeen experiments was %.

4 BODY WTER IN THE CT 485 It is clear also from Table 3 that the individual variation is not due to lack of equilibrium. This appears to be complete within half an hour of the end of the injection, for a value of 54% was obtained on the one occasion when so short a period was allowed. On another, in which again only hypertonic NaCl had been injected, 5 hr. was allowed before sampling, and this yielded one of the lowest values, 43'5 %. TBLE 3. comparison of the total body water as measured (a) by the amount into which urea will diffuse and (b) by the amount in which hypertonic NaCl solution appears to be distributed. Body water as % body weight Time for Body weight equilibrium of (kg.) Urea method NaCl method Difference NaCl (hr.) * * * verage 61-5± ± ± 1-6 DISCUSSION The value of body water given by the urea method, 63% body weight, is almost identical with that given by Skelton (1927) obtained by direct analysis. The value obtained by the hypertonic NaCl method, 49.5% body weight, is appreciably less, and a difference between the two was observed consistently in each experiment in which both methods were used. It would appear, therefore, that the quantity of water expelled from the cells into which NaCl cannot penetrate freely is less than the quantity which would be expected if the cells behaved as simple osmometers containing 'ideal' solutions. This behaviour could be accounted for in one of several ways: (1) The cell contents are in osmotic equilibrium with the interstitial fluid, in which case either (a) some water in the tissues might be free to dissolve urea but sufficiently 'bound' to be unable to partake in osmotic exchanges; or (b) some water might be 'bound' or 'non-solvent' in the strict sense of the word and, in addition, some urea adsorbed; or (c) when the osmotic pressure outside is raised, additional solutes either penetrate into or are formed within the cells. (2) The cell contents are not in osmotic equilibrium with the interstitial fluid, but a steady state is maintained at the expense of metabolic activity. For the first of these possibilities (la) there is little to be said except that, though possible, it is extremely improbable. gainst the second (1 b) there is already negative evidence, for Hill (1930) found no 'bound' water in frog muscle and strongly criticized the technique of earlier workers who had appeared to obtain a positive result. In addition, it is rather improbable that urea should be adsorbed at all, still more so that it should be adsorbed in just

5 486 M. GRCE EGGLETON the right amount to yield the same value for body water as that found by direct determination. gainst the third possibility (1 c), there is the evidence of Conway (1946) who found that isolated frog's muscle behaved as might be expected whenthe concentration of sodium ions in the external solution was changed, the total cation concentration remaining constant. It may be argued that the present experiments are not comparable with those of Conway, since the total cation concentration was not constant. Indeed, it is possible that the hypothetical solute which penetrates the cells may be sodium. It is known that in both muscle cells and in red blood cells the sodium concentration is maintained at a low value by the performance of osmotic work; no prediction, from physicochemical considerations, can be made as to what will happen when the sodiumion concentration in the external fluid is changed. But if sodium ions do enter the cells they must be accompanied by some anion other than chloride. If chloride ions penetrated into the cells, in the amount required theoretically, there should be no discrepancy to be accounted for in the present series of observations. On the other hand, the results obtained by Hetherington (1931) on cats may possibly be accepted as evidence in favour of this possibility (1 c), though her technique was not altogether satisfactory. She determined the increase in vapour pressure of the blood following intravenous injection of hypertonic NaCl solution and calculated from it the body water; but considerable quantities of urine were secreted and its total osmotic pressure regarded as due entirely to NaCl. This may account for the fairly wide variations found even in the same animal at different times after the injection. Her recorded value for six cats was 59% of the body weight, but if that from the seventh is included, the average drops to 55 %. Thus the value yielded by this method is higher than that now obtained by measurement of chloride changes. Comparison of Smirk's (1933) values for dilution of blood chloride following ingestion of water by human subjects, however, with those later obtained by Baldes & Smirk (1934) by the vapour-pressure method, indicates a similar degree of change in the two: a 4% dilution of blood chloride after 1500 ml. water and a 2-7% reduction in total osmotic pressure after 1000 ml. respectively. It would seem, therefore, that the possibility of movement of solutes into or out of cells when the osmotic composition of the surrounding fluid is altered, must remain an open question. For the last possibility (2), however, there is a certain amount of positive evidence. Robinson (1951) has recently concluded from the results of various experiments on isolated kidney slices that the osmotic pressure within the cells is % greater than that in the surrounding fluid. Moreover, when such slices are exposed to hypotonic solutions, the change in weight of the tissue is less than that to be expected on osmotic considerations. One further piece of

6 BODY WTER IN THE CT 487 evidence suggests that, if these results are applicable also to other tissues, the discrepancy between expected and observed change in volume is greater when the tissues are exposed to hypertonic than when they are exposed to hypotonic surroundings. From an earlier series of experiments (Eggleton, 1937), in which water was given to cats, either injected into the intestine or intravenously, calculation has now been made of the body water from the observed dilution of plasma chloride. value of % is obtained, from sixteen cats. This is just significantly less than the value of 63% yielded by urea and significantly greater than the 49-5 % yielded by hypertonic NaCl solution. If the osmotic pressure within the cells is indeed greater than that of the surrounding fluid, these results might be interpreted as indicating that the hypothetical 'pump' which maintains the steady state becomes less effective as the concentration within the cells becomes greater. In these earlier experiments, the potassium-ion concentration in the extracellular fluid was lowered as well as the sodium-ion concentration; there would thus be a loss of potassium ions from the muscle cells and perhaps also from other cells, as Boyle & Conway (1940) have shown for the isolated muscles of the frog. This might be sufficient to account for the fact that the discrepancy is less when the body fluids are diluted than it is when NaCl is added. SUMMRY 1. When urea is injected intravenously into cats, its concentration in the plasma indicates that it has diffused into % of the body weight. 2. When hypertonic NaCl solution is so injected, its concentration in the plasma indicates that the NaCl appears to become distributed in only P08% of the body weight. 3. The origin of the difference between the two values is discussed. No satisfactory explanation has been found, and it would appear that some, at least, of the cells are not in simple osmotic equilibrium with the extracellular fluid. REFERENCES Baldes, E. J. & Smirk, F. H. (1934). J. Phy8iol. 82, 62. Boyle, P. J. & Conway, E. J. (1940). J. PhysioZ. 100, 1. Conway, E. J. (1946). Nature, Lond., 157, 715. Conway, E. J. & O'Malley, E. (1942). Bsochem. J. 36, 655. Eggleton, M. G. (1937). J. Phy8iol. 90, 465. Eichelberger, L. & Hastings,. B. (1937). J. biol. Chem. 1i8, 205. Hetherington, M. (1931). J. Phy8iol. 73, 184. Hill,. V. (1930). Proc. Roy. Soc. B, 106, 477. Maurer, F. W. (1938). mer. J. Phy8iol. 124, 546. Robinson, J. R. (1951). Proc. Roy. Soc. B, 137, 378. Skelton, H. (1927). rch. intern. Med. 40, 140. Smirk, F. H. (1933). J. Phy8iol. 78, 127.