STUDIES ON TISSUE WATER

STUDIES ON TISSUE WATER I. THE DETERMINATION OF BLOOD WATER BY THE DISTILLRTIOS METHOD BY A. T. MILLER, (From the Department of Physiology, School of Medicine, University 0.1 Vorth Carolina, Chapel Hill) JR. (Received for publication, December 15, 1911) The standard method for determining the water content of biological materials is drying to constant weight in an oven, usually at, 105. The loss in weight is assumed to represent the original water content of the sample. The absolute accuracy of this procedure depends on the validity of t.wo assumptions, (a) that the attainment of constant weight implies complotc removal of water, and (b) that the entire weight loss is due to the volatilization of water. The first assumption is subject to the criticism that material dried to constant weight at one temperature loses weight when exposed to a higher temperature (1). Neuhausen and Patrick (2) heated a silica gel to 300 in vacua for 6 hours without reducing the water content below 4.8 per cent and Bartell and Almy (3) state that water persists within silica gels at temperatures well above the critical temperature of water. Rimington (4) states that 2 to 7 per cent of water adheres to protein after it has been dried to constant weight preliminary to elementary analysis. His evidence for this statement is the fact that the values for hydrogen and oxygen in proteins are too high to agree with what is known of their amino acid composition. Benedict and Manning (5) demonstrated that proteins dried under a high vacuum for several weeks actually gain weight in an oven at 110 but lose it when again dried in a high vacuum. The validity of the second assumption, that all the weight loss in oven drying represents volatilization of water, is equally questionable. Benedict and Manning (6) demonstrated experimentally the volatilization of both fatty materials and nitrogen from proteins heated in an oven at 100-105. This would increase the weight loss and hence yield high values for water. The magnitude of the error is (in all except adipose tissue, at least) much smaller than the opposing error due to incomplete removal of water (6), so that the net result is a value lower than the actual water content. 65

66 BLOOD WATER DETERMINATION A further source of error in the determination of water by oven drying is gain in weight due to absorption of oxygen (oxidation), likewise demonstrated by Benedict and Manning (6). The errors due to volatilization of substances other than water and to oxidation are minimized by drying in vacua at 20, but several weeks may be required for the attainment of constant weight. The error due to incomplete removal of water is, of course, not diminished by this procedure; in fact it probably is increased. Aside from the errors inherent in the drying method, the length of time required and the numerous weighings involved make it inconvenient as a routine procedure. The determination of water by distillation with liquids immiscible with water has certain advantages which should make it a method of choice for biological work. The apparatus is inexpensive and is easily constructed by any competent glass-blower. The method is rapid, requiring 30 to 40 minutes for blood plasma or serum and 1 hour for whole blood. The removal of water is complete (see the analyses on protein solutions in this paper), there is no contact with oxygen, and, if substances other than water are volatilized, they are probably much more soluble in the organic distillation fluid than in water, so that water recovery is not measurably increased. Marcusson (7) apparently was the first to determine moisture by distilling the sample with a liquid immiscible with water (he used xylene). Rogers (8) recommended the use of toluene. Dean and Stark (9) improved the method by providing the receiving tube with a side arm which connected to the distillation flask, allowing the reflux distillation of the toluene, while the water was trapped by dropping to the bottom of the receiving tube. The method presented in this paper is a modification of the reflux distillation method of Dean and Stark. EXPERIMENTAL Apparatus-The apparatus is shown in Fig. 1. It is constructed of Pyrex and all joints are No-lub standard taper 24/40. The 250 ml. distilling flask is flat bottomed and has ashort neck formed byan outer standard taper. The condenser is the West type (jacket length 300 mm.) with a sealed-on drip-tip, which must be long enough to dip about 10 mm. beneath the surface of the toluene in the receiving tube when the apparatus is assembled. This prevents the return of water droplets to the distillation flask, decreasing the distillation time. The open portion of the tip faces the side arm. The receiving tube is made from 25 mm. tubing; to the tapered bottom is sealed a 1.00 ml. Mohr pipette, with graduation intervals of 0.01 ml. The receiving tube has a side arm, 10 mm. in diameter, which leads to the distillation flask. A mercury leveling bottle is attached by

A. T. MILLER, JR. 67 rubber tubing to the tip of the pipette. The rubber tubing is pretreated with dilute acid and alkali to remove the bloom, and the mercury should be redistilled; both must be cleaned and dried if the mercury becomes dirty. An L-shaped glass tube, connected to the top of the condenser by a standard taper joint, dips about 10 mm. beneath the surface of toluene in an open flask. This allows free vapor expansion during distillation, while preventing the condensation of atmospheric moisture in the condenser tube. The position of the flask is adjusted so that the vertical distance between the top of the condenser and the level of toluene in the flask is about 300 mm. This permits adequate but not too rapid siphoning of toluene for rinsing the condenser at the end of an analysis (described below). D F- e I FIG. 1. Receiving tube with condenser tip in place. A, No-lub joint between condenser and receiving tube; B, drip-tip sealedto bottom of condenserjoint; C, toluene level during distillation; D, Mohr pipette (1.00 ml. with graduation intervals of 0.01 ml.); E, side arm of receiving tube; P, joint to which distillation flask is attached. Procedure The receiving tube is attached, without lubrication, to the condenser. The mercury leveling bottle is connected to the receiving pipette, the mercury level is raised to the junction of the pipette and the receiving tube, and the rubber tubing clamped with a screw clamp. A 1.00 ml. sample of blood or plasma is delivered into a distillation flask containing 100 ml. of 4 per cent n-amyl alcohol in toluene. The amyl alcohol reduces the tendency toward the formation of adherent water films in the condenser and receiving tube. The joint between the distillation flask and the side arm of the receiving tube is lubricated lightly with rubber cement, which may be obtained from any shoe repair shop, and the flask is secured in place.

68 BLOOD WATER DETERMINATION Toluene is delivered through the top of the condenser until it reaches the level of the side arm of the receiving tube. The glass tube connecting the top of the condenser to the open flask of toluene is adjusted as described above. With the water jacket of the condenser empty, the distillation flask is heated until the first appearance of boiling, and then the water current through the condenser jacket is turned on. This preliminary heating frees the system of all atmospheric moisture. Distillation is allowed to proceed at a brisk rate for 1 hour for whole blood or 40 minutes for plasma or serum. At the end of this time the drop of water which usually adheres to the condenser tip is dislodged by raising the mercury cautiously until the water in the receiving tube makes contact with the drop. The mercury is now lowered until the water-toluene junction is about 10 mm. below the junction of the receiving tube and the pipette, and the heating is discontinued. As cooling proceeds, contrac,tion of the air in the system results in the siphoning of sufficient toluene from the open flask to rinse the condenser thoroughly, dislodging any water droplets which may adhere to the walls. After the recovered water has cooled to room temperature, it is drawn into the pipette and measured. Water droplets occasionally adhere to the walls of the receiving tube. They are easily dislodged by disconnecting the condenser and scraping the walls of the receiving tube with a rubber-tipped stirring rod. After each analysis the receiving tube is cleaned by rinsings in the following order: alcohol, water, warm chromic-sulfuric acid cleaning fluid, water, alcohol. The tube is dried in a stream of dry compressed air or in an oven. The condenser is cleaned in like manner after three or four analyses. Calibration and Correction Factors-The pipette of the receiving tube is calibrated with mercury in the usual manner before it is sealed to the tube, or it may be calibrated after sealing by measuring the mercury delivered into it from a calibrated burette. The Mohr pipettes examined have been found to contain from 0.995 to ml. In addition, there is a constant volume correction characteristic of each assembly and independent of the volume of water recovered. This factor, which amounts to 0.03 ml. for an assembly with the dimensions given above, is determined by blank analyses on distilled water; this volume is added to themeasuredvolume of water. If a subst.ance is added to the toluene to reduce the glass-wetting tendency of water, a correction must be applied for the solubility of this substance in water. The use of 4 per cent n-amyl alcohol in toluene increases the apparent recovery of water by 1.0 per cent. Theoretically this factor should vary with the volume of water in the sample, since the greater the relative proportion of water, the lower the equilibrium concentration of amyl alcohol in toluene. Actually the low solubility of amyl alcohol in water and the great excess of toluene result in a very constant factor (see Table I).

-4. T. MILLER, JR. 69 Choice of Distillation Fluid-Several liquids immiscible with water have been tested. Xylene, benzene, mixtures of the two, and petroleum ether give inconstant results. Toluene, either alone or with the addition of amyl alcohol, gives constant results. Butyl alcohol is satisfactory in reducing wetting, but causes unpredictable variations in water recovery. Solid camphor has the advantage of requiring no correction, but is somewhat less efficient than amyl alcohol. Heptane (b.p. 90-100 ), the only aliphatic hydrocarbon used, gives constant results (see Table I) and is superior to Analysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 No. TABLE I Recovery of Known Volumes of Distilled Water Distillation Toluene alone I ( ( I fluid 1 $& camphor in toluene 1 % 1 % l cl 1 % 1 % ( I 4 % n-amyl alcohol in toluene 4 % Ii ( I I 4 % IL I I 4 % ( ( 4 70 I *( < Heptane I Volume of sample ml. 0.700 0.700 l.ooo l.ooo Volume of water recovered ml. 0.502 0.990 0.995 0.700 0.710 0.999 0.995 0.990 1.003 toluene alone from the standpoint of reduction of glass-water films, but has the disadvantage of requiring a more prolonged distillation. Its lower boiling point should make it desirable for the analysis of materials high in volatile components. Results Analyses on Distilled Water-Table I gives the results of twenty consecutive distillations of known volumes of distilled water. The average recovery is 99.95 per cent of the theoretical, with a mean deviation of 0.26 per cent.

70 BLOOD WATER DETERMINATION Analyses on Protein Xolutions-The data presented in Table I indicate that water in the free form is quantitatively distilled and measured. It remained to be demonstrated that water is completely extracted from protein solutions by distillation with toluene. Since it is impossible to obtain proteins in dry form (4) (samples of powdered egg albumin tested contained 12 to 15 per cent water), solutions of egg albumin (Merck s impalpable powder) were made in distilled water. The solutions were allowed to stand overnight in the cold, and then centrifuged four times (1 hour at 3000 R.P.M.). The protein content was determined by Kjeldahl analysis (N X 6.25)) and specific gravity was determined by the falling drop method of Barbour and Hamilton (10). For a given sample, the weight of water is equal to the total weight minus the weight of protein. No distinction is made between free and bound water in the analyses of protein solutions, blood, and plasma. The results of a typical experiment are as follows: protein content, 9.79 gm. per 100 ml.; specific gravity, 1.03374 at 27.0 ; water content (calculated), 90.5 gm. per 100 gm.; water content by distillation, 90.3 gm. per 100 gm.; water content by oven drying, 89.0 gm. per 100 gm. Analyses on Blood and Plasma--In order to eliminate individual variations, all analyses have been made on blood from a single dog, an adult, male mongrel, weighing 26.2 kilos. Blood samples were drawn into oiled syringes from the femoral artery and transferred to flasks containing sufficient ammonium and potassium oxalate to give a final concentration of 2 mg. of oxalate per ml. of blood. Since the water content of whole blood depends primarily on the relative proportion of cells and plasma, the water content of the plasma also was determined. The water content of the cells was calculated by the formula given below. Hematocrit cell volume was determined by centrifugation (3000 R.P.M. for 1 hour) under oil in Wintrobe tubes. All determinations were made in quadruplicate. Plasma was obtained by centrifugation under oil (to prevent evaporation). Hemolyzed samples were discarded. The specific gravity of whole blood and plasma was determined by the falling drop method (10). 1 ml. samples of blood and plasma were delivered into distillation flasks from an Ostwald- Van Slyke blood pipette which had been repeatedly calibrated by weighing samples of blood and plasma delivered. A delivery time of 45 to 60 seconds is essential for complete drainage of the pipette. Flat, glass-stoppered drying vessels were used for water determination by the oven drying method. At 105 plasma samples attained constant weight in 24 hours; whole blood required 36 to 48 hours. 1 Constant weight was attained after 5 days in the oven at 105. No further loss of weight occurred after 22 days in the oven.

A. T. MILLER, JR. 71 The specific gravity of the cells was calculated by the formula where SG, = specific gravity of cells, SGb = specific gravity of blood, SG, = specific gravity of plasma, V, = hematocrit cell volume, VP = plasma volume (100 - V,). The water content of the cells was calculated by the formula WC = loowb SGb - VP w, SG, Vc SG where W, = water content of cells (by weight) and Wa and W, = water content of whole blood and plasma respectively. TABLE Comparison of Results by Distillation and by Oven Drying The results are measured in gm. per 100 gm. II Sample No. Blood water Plasma water - I Xstillation O- W drying Difference I Xstilla. tion OWIl drying Difference I Cell water (calculated)* Xstilla. OVUl Differtion drying ence 79.5 76.7 2.8 93.2 91.1 2.1 81.3 79.2 2.1 92.6 91.5 1.1 79.5 77.3 2.2 92.9 91.1 1.8 82.5 80.1 2.4 94.1 92.3 1.8 82.1 80.1 2.0 93.5 92.1 1.4 74.0t 73.5t 0.5t 93.0 91.6 1.4 83.7t 82.67 1.1t 93.0 91.6 1.4 Averages. 81.0 78.7 2.3 93.q 91.6 1.6 67.3 68.9 66.8 68.1 68.2 67.6 68.9 68.0 -_ 63.6 3.7 65.5 3.4 64.3 2.5 65.0 3.1 64.4 3.8 67.0t 0.61 66.lt 2.8t 64.6 3.3 * See the text. t Omitted from the averages for the reasons given in the text. The results of duplicate determinations on five samples of blood obtained on different days from the same animal are shown in Table II. Samples 6 and 7 were prepared from Sample 5 by centrifuging and resuspending the cells in plasma. Since the relative proportion of cells and plasma in Samples 6 and 7 was purposely altered, the values for the water content of whole blood are not included in the averages. The differences in Samples 6 and 7 between values for whole blood water by the drying method and by distillation are excluded from the averages because, due to a faulty thermostat, the oven temperature reached 150. The apparent increased water yield in these two samples (as compared to the distillation values) may be due to actual volatilization of more water at the higher temperature, or to partial pyrolysis with liberation of volatile components.

72 BLOOD WATER DETERMINATION The values for water content of whole blood, plasma, and cells by the oven drying method are in good agreement with those in the literature (11). The values obtained by the distillation method are uniformly higher. In the case of plasma, the average difference is almost identical with that observed in the analyses on egg albumin solutions. The discrepancy is greater in the case of cells, as might be expected from their higher protein content and greater structural complexity. In order to determine the reproducibility of results with the distillation method, ten analyses were performed on a single sample of blood (Sample 4 of Table II). The results, in order of determination, were 82.4, 82.6, 82.5, 82.3, 82.3, 82.5, 82.4, 82.5, 82.3, 82.5 gm. per 100 gm. These results, together with those on distilled water and on protein solutions, indicate an average maximum deviation between duplicate determinations of 0.2 per cent. Preliminary investigations have been made into the cause of the low results obtained with the oven method. Two 10 gm. samples of blood were dried to constant weight in the oven. The dried residue from one sample was transferred, with minimal exposure to the atmosphere, to a distillation flask and distilled for 2 hours. The water recovered accounted for 20 per cent of the difference between the results by oven drying and by distillation. The dried residue from the other sample was ground under toluene in a mortar and then transferred to a distillation flask and distilled for 2 hours. The water recovered in this case accounted for 65 per cent of the difference between oven drying and distillation values. These results are best interpreted on the assumption that small amounts of water are trapped in pockets in the heat-denatured protein during oven drying. Mechanical grinding of the dried residue breaks down the walls of these pockets and exposes the residual water to the action of the toluene. When fresh samples of blood are distilled, the extraction of water is complete before heat denaturation occurs. Toluene denaturation apparently does not result in the trapping of water, since blood samples preserved under toluene for 24 hours yield the same results as samples freshly distilled. SUMMARY A method is described for the determination of water in blood by distillation with toluene. The analysis requires 1 hour and has a reproducibility of 0.2 per cent. The results are uniformly higher than by the oven drying method (2.3 per cent for whole blood, 1.6 per cent for plasma, and 3.3 per cent for cells (calculated)). The sources of error in the oven drying method are discussed, and reasons are given for believing that lower results by this method are due to incomplete extraction of water.

A. T. MILLER, JR. 73 BIBLIOGRAPHY 1. Nelson, 0. A., and Hulett, G. A., J. Ind. and Eng. Chem., 12, 40 (1920). 2. Neuhausen, B. S., and Patrick, W. A., J. Am. Chem. Sot., 43, 1844 (1921). 3. Bartell, F. E., and Almy, E. G., J. Physic. Chem., 36, 475 (1932). 4. Rimington, C., Tr. Faraday Sot., 26, 699 (1930). 5. Benedict, F. G., and Manning, C. R., Am. J. PhysioZ., 18, 213 (1907). 6. Benedict, F. G., and Manning, C. R., Am. J. Physiol., 13, 309 (1905). 7. Marcusson, J., Mitt. k. Materialpr@fungsamt, 23, 58 (1905). 8. Rogers, J. S., U. S. Dept. Agric., Bur. Chem., Bull. 137, 172 (1910). 9. Dean, E. W., and Stark, D. D., J. Ind. and Eng. Chem., 12, 486 (1920). 10. Barbour, H. G., and Hamilton, W. F., J. Biol. Chem., 69, 625 (1926). 11. Bodansky, M., Introduction to physiological chemistry, New York, 4th edition, 222 (1938).