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A simple colloid osmometer and determination of some factors influencing the measurement of colloid osmotic pressure in rat serum
MICROVASCULAR
RESEARCH
36, 181-186 (1988)
BRIEF COMMUNICATIONS A Simple Colloid Osmometer and Determination Factors Influencing the Measurement of Colloid Pressure in Rat Serum
of Some Osmotic
M. CHIBA AND K. KOMATSU Department
of Pharmacology,
School of Dental Medicine, Tsurumi University, Tsurumi-ku, Yokohama, Japan, 230
2-l-3 Tsurumi,
INTRODUCTION There have been many reports on the measurement of colloid osmotic pressure (COP) of plasma and/or serum in human and experimental animals by using various types of osmometers with different characteristics (e.g., Hansen, 1961; Prather et al., 1968; Intaglietta and Zweifach, 1971; Zweifach and Intaglietta, 1971; Worning and Steven, 1973; Navar and Navar, 1977; Kakiuchi et al., 1979). The validity of the measurement of COP seems to depend partly upon the characteristics of the semipermeable membrane (Prather et al., 1972), and of the pressure transducer utilized to construct an osmometer. In addition, there seem to be various other factors which may cause fluctuations of COPS during the course of measurements (Marty and Intaglietta, 1970; Marty and Zweifach, 1970; Miki et al., 1983). The main purposes of the present study were to construct a simple colloid osmometer of our own, to examine the validity of the measurement by the apparatus, and also to examine the effect of some of the factors causing fluctuations on the estimation of COPS in the rat serum. MATERIALS
AND METHODS
The general construction of the colloid osmometer is shown in Fig. 1. The dome of the pressure transducer (Type MPU, Toyo Baldwin, Tokyo) was connected with the base of the osmometer. The Amicon PM-30 membrane (Amicon, Lexington, MA), 7.5 mm in diameter, was placed between the rubber rings, mounted between the base and top of the osmometer, and clamped tightly by screws. Since the diameter of the sample chamber was 4 mm, the free surface area of the membrane was about 12.6 mm’. Serum samples were obtained from five male rats of the Wistar strain weighing from 177 to 192 g. The blood was taken from the left carotid artery, sedimented at about 4” for 2 hr, and then centrifuged at 3000 rpm for 15 min. The supernatant was used to measure the COP. The ultrafiltrate was also obtained by using a 181 @X6-2862/88 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.
182
BRIEF COMMUNICATIONS SAMPLE -
-
CHAMBER
OSYOMETER
TOP
OSMOMETER
BASE
PRESSURE
TRANSDUCER
FIG. 1. Diagrammatic representation of the colloid osmometer.
microvolume stirred ultrafiltration cell with PM-30 membrane under a nitrogen gaseous pressure of 3 kg/cm*. In order to prevent evaporation of water in the serum, the top of the sample (50 ~1) was covered with a few drops of silicone oil. Changes of the COP were measured for about 2 hr. Observations were also made when the top of the sample was not covered with silicone oil. Ten measurements of two successive experiments were taken when the dome of the pressure chamber was filled either with saline or with deionized water. The serum sample (40 ~1) was put into the sample chamber for each measurement. The COP was recorded for 15 min. Then the sample chamber was washed by the same fluid as that in the dome. In order to determine the least amount of the sample required to obtain a reliable measurement, the COPS of various amounts of the serum sample, that is, 40, 20, 10, 5, and 3 ~1 were measured. Solutions containing 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100% of the serum were prepared. The serum was diluted by saline. The COPS of these solutions were measured successively from high to low and from low to high concentrations. The relationship between the COPS and the serial dilutions of the serum was determined by the method of least squares. RESULTS The shift of the baseline was very small, that is, 0.02 mm Hg at 17.2 - 21.2 for 12 hr, -0.23 mm Hg at 20.3 - 22.8” for 10 hr and -0.17 mm Hg at 20.0 22.0” for 3 hr. When the top of the serum sample was covered with silicone oil, changes in the COP were very small after an initial acute response. The equilibration time was about 8 min, the values varying from 15.5 to 15.7 mm Hg over a period of 1.8 hr following application of the sample. When the top of the sample was not
183
BRIEF COMMUNICATIONS
(a)
(b)
TIME (h)
TIME (h)
FIG. 2. Effects of fluids within the dome and those used to wash the sample chamber on the COP of rat serum. Ten measurements of two successive experiments are shown. The dome of the pressure chamber was filled either with saline (a) or with deionized water (b). The serum sample (40 ~1) was put into the sample chamber which was washed with the same fluid as that in the dome after the measurement. Arrows indicate the start of each measurement.
covered with silicone oil, an initial acute response followed by a gradual increase of the COP was observed. The COPS commenced at 16.5 mm Hg and progressively increased to 19.9 mm Hg over the same period of time. During the experiment, the environmental temperature ranged from 21.0 to 22.7” and the humidity from 73 to 74%. Figure 2a shows the result from 10 successive measurements of the COP when the dome of the pressure chamber was filled with saline. The sample chamber was washed by saline after each measurement. The mean and the standard deviation of the COP were 16.7 and 0.2 mm Hg. Those of the shift of the zero point were - 0.04 and 0.13 mm Hg. Similar observations were made when deionized water was filled in the pressure chamber and the sample chamber was washed by deionized water after each measurement (Fig. 2b). The mean and standard deviation of the COP were 16.5 and 0.3 mm Hg. Those of the shift of the zero point were -0.04 and 0.24 mm Hg. A significant difference of the COPS was not found between the two experiments. However an overshoot was observed when the sample chamber was washed by deionized water in the latter experiment (Fig. 2b). The COP decreased when smaller sample size was measured. However, reproducible unvarying values were obtained when the sample volumes were greater than 10 ~1. In order to examine the relationship between the COPS and the ratios of the serum in diluted solution, the measured COPS were plotted against the ratios and a best-fit cubic equation was estimated by the method of least squares (Fig. 3). The formula was represented as y = 0.227 + 10.784x + 3.351~’ + 2.079x3, where y is the COP in mm Hg and x is the ratio of the serum in diluted solutions. It was found that a third degree polynominal is a satisfactory fit. Average COPS of the ultrafiltrate and the serum were 0.62 and 17.1 mm Hg,
184
BRIEF
COMMUNICATIONS
RATIO OF SERUM IN DILUTED SOLUTION FIG. 3. Relationship between the COP and the ratio of serum in the diluted solutions. Curve describes the best-fit cubic equation, y = 0.227 + 10.784.x + 3.351x2 + 2.079.x’, where y is the COP in mm Hg and x is the ratio of serum in diluted solutions.
respectively. It is possible that a small fraction of protein was forced to pass through the membrane into the ultrafiltrate and generated this COP. DISCUSSION It has been reported that the Amicon PM-30 ultrafiltration membrane represents the membrane of choice because of its optimal reproducibility and response time as well as its stability and long operational life (Prather et al., 1972). In the present experiments, it was found that the PM-30 membrane also gave satisfactory results as to its reproducibility and stability when measurements were performed several times a week for over a month. The response time or the equilibration time of the osmometer has been reported to vary from 4 set to tens of minutes (Hansen, 1961; Prather et al., 1968, 1972; Intaglietta and Zweifach, 1971; Wiederhielm et al., 1973; Kakiuchi et af., 1981; Marty and Intaglietta, 1970) whereas that of ours was approximately 8 min. It is supposed that the response time and/or the baseline recovery of our osmometer could be reduced if the membrane area was widened and the volume of fluid in the dome of the pressure chamber of the transducer was diminished. It is assumed that ordinary variations in room temperature and also in pH of the sample will not disturb the measurement materially as suggested by Hansen (1961). The concentration of the sample due to evaporation of water during the measurement could effectively be prevented by covering the top of the sample with silicone oil. The overshoot at washing (Fig. 2b) might be caused by salt concentration difference between the fluid in the sample chamber and that in the pressure chamber of the transducer as suggested by Aukland and Johnsen (1974). Such an overshoot could be reduced if we minimize the salt concentration difference across the membrane. In fact, the initial transient overshoot has been reduced
BRIEF
COMMUNICATIONS
185
when serum ultrafiltrate was applied repeatedly to the membrane until no pressure response was observed (Zweifach and Intaglietta, 1971; Aukland and Johnsen, 1974). It has been suggested that the exact size of the sample is immaterial, except that it must cover the whole effective part of the membrane; smaller samples did not always cover the membrane surface and gave too low pressures (Hansen, 1961). In addition it is recommended that the sample size should be the same and the baseline should be determined by adding the same amount of saline in the sample chamber because it was found that the measurement was affected by the amount of sample. Although we have not compared the values obtained from our apparatus with those obtained from another proven method, the COPS obtained in the present study (15.5 - 17.1 mm Hg) were within the range of values (15.7 - 25.4 mm Hg) reported by other investigators in the rat serum and plasma (Zweifach and Intaglietta, 1971; Worning and Steven, 1973; Navar and Navar, 1977). It seems necessary to use a sufficient number of rats in order to examine changes of COPS under various experimental conditions. Relationships between protein concentrations and COPS for human, dog, rat, and rabbit plasma and/or serum samples have been examined (Landis and Pappenheimer, 1963; Zweifach and Intaglietta, 1971; Worning and Steven, 1973; Navar and Navar, 1977). However, the relationship between the ratio of serum in a diluted solution and the COP was only examined in the present experiment. In order to obtain excellent linear correlations between COP and the total protein concentration, it seems important to standardize the protein determinations properly (Mullins et al., 1983). ACKNOWLEDGMENT The authors thank Professor R. W. Fearnhead for his valuable advice and kind help in preparing the manuscript.
REFERENCES AUKLAND,K., ANDJOHNSEN,H. M. (1974). A colloid osmometer for small fluid samples. Acta Physiol. Stand.
90, 485-490.
HANSEN,A. T. (1961). A self-recording electronic osmometer for quick, direct measurement of colloid osmotic pressure in small samples. Acta Physiol. Stand. 53, 197-213. INTAGLIETTA,M., ANDZWEIFACH,B. W. (1971). Measurement of blood plasma colloid osmotic pressure. I. Technical aspects. Microvasc. Res. 3, 72-82. KAKILJCHI,Y., ARAI, T., HORIMOTO,M. KIKUCHI, Y., AND KOYAMA, T. (1979). A new needle-type colloid osmometer for continuous determination of blood oncotic pressure. Amer. J. Physiol. 236, F419-F422. KAKIUCHI, Y., HUGHES,G. M., ARAI, T., HORIMOTO,M., KIKUCHI, Y., AND KOYAMA,T. (1981). A microanalysis of colloid osmotic pressure. Experientiu 37, 376-377. LANDIS, E. M., ANDPAPPENHEIMER, J. R. (1963). Exchange of substances through the capillary walls. In “Handbook of Physiology. Circulation” (W. F. Hamilton and P. DOW, Eds.), Vol. II. pp. 9611034. Amer. Physiol. Sot., Washington, DC. MARTY, A. T., ANDINTAGLIETTA,M. (1970). Effect of anticoagulants on human plasma colloid osmotic pressure measurements. J. Appl. Physiol. 29, 740-741. MARTY, A. T., AND ZWEIFACH,B. W. (1970). The high oncotic pressure effects of dextrans. Arch. Surg. 101, 421-424.
186
BRIEF
COMMUNICATIONS
MIKI, K., SAGAWA,S., AND SHIRAKI, K. (1983). Factors influencing the determination of colloid osmotic pressure of plasma. J. Univ. Occup. Environ. Health 5, 405-412. MULLINS, R. E., PAPPAS,A. A., AND GADSDEN,R. H. (1983). Correlation of standardized serum protein determinations with calculated and measured colloid osmotic pressure. Amer. J. C/in. Pathol. 80, 170-175. NAVAR, P. D., AND NAVAR, L. G. (1977). Relationship between colloid osmotic pressure and plasma protein concentration in the dog. Amer. J. Physiol. 233, H295-H298. PRATHER, J. W., BROWN,W. H., ANDZWEIFACH, B. W. (1972).An improved osmometer for measurement of plasma colloid osmotic pressure. Microvasc. Res. 4, 300-305. PRATHER,J. W., GAAR, JR., K. A., ANDGUYTON,A. C. (1968). Direct continuous recording of plasma colloid osmotic pressure of whole blood. J. Appl. Physiol. 24, 602-605. WIEDERHIELM, C. A., LEE, D. R., AND STROMBERG, D. D. (1973). A membrane osmometer for microliter samples. J. Appl. Physiol. 35, 432-435. WORNING, C., AND STEVEN, K. (1973). Serum protein concentration and oncotic pressure relationship in the rat. PJliigers Arch. 340, 77-80. ZWEIFACH, B. W., ANDINTAGLIETTA, M. (1971). Measurement of blood plasma colloid osmotic pressure. II. Comparative study of different species. Microvasc. Res. 3, 83-88.
RESEARCH
36, 181-186 (1988)
BRIEF COMMUNICATIONS A Simple Colloid Osmometer and Determination Factors Influencing the Measurement of Colloid Pressure in Rat Serum
of Some Osmotic
M. CHIBA AND K. KOMATSU Department
of Pharmacology,
School of Dental Medicine, Tsurumi University, Tsurumi-ku, Yokohama, Japan, 230
2-l-3 Tsurumi,
INTRODUCTION There have been many reports on the measurement of colloid osmotic pressure (COP) of plasma and/or serum in human and experimental animals by using various types of osmometers with different characteristics (e.g., Hansen, 1961; Prather et al., 1968; Intaglietta and Zweifach, 1971; Zweifach and Intaglietta, 1971; Worning and Steven, 1973; Navar and Navar, 1977; Kakiuchi et al., 1979). The validity of the measurement of COP seems to depend partly upon the characteristics of the semipermeable membrane (Prather et al., 1972), and of the pressure transducer utilized to construct an osmometer. In addition, there seem to be various other factors which may cause fluctuations of COPS during the course of measurements (Marty and Intaglietta, 1970; Marty and Zweifach, 1970; Miki et al., 1983). The main purposes of the present study were to construct a simple colloid osmometer of our own, to examine the validity of the measurement by the apparatus, and also to examine the effect of some of the factors causing fluctuations on the estimation of COPS in the rat serum. MATERIALS
AND METHODS
The general construction of the colloid osmometer is shown in Fig. 1. The dome of the pressure transducer (Type MPU, Toyo Baldwin, Tokyo) was connected with the base of the osmometer. The Amicon PM-30 membrane (Amicon, Lexington, MA), 7.5 mm in diameter, was placed between the rubber rings, mounted between the base and top of the osmometer, and clamped tightly by screws. Since the diameter of the sample chamber was 4 mm, the free surface area of the membrane was about 12.6 mm’. Serum samples were obtained from five male rats of the Wistar strain weighing from 177 to 192 g. The blood was taken from the left carotid artery, sedimented at about 4” for 2 hr, and then centrifuged at 3000 rpm for 15 min. The supernatant was used to measure the COP. The ultrafiltrate was also obtained by using a 181 @X6-2862/88 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.
182
BRIEF COMMUNICATIONS SAMPLE -
-
CHAMBER
OSYOMETER
TOP
OSMOMETER
BASE
PRESSURE
TRANSDUCER
FIG. 1. Diagrammatic representation of the colloid osmometer.
microvolume stirred ultrafiltration cell with PM-30 membrane under a nitrogen gaseous pressure of 3 kg/cm*. In order to prevent evaporation of water in the serum, the top of the sample (50 ~1) was covered with a few drops of silicone oil. Changes of the COP were measured for about 2 hr. Observations were also made when the top of the sample was not covered with silicone oil. Ten measurements of two successive experiments were taken when the dome of the pressure chamber was filled either with saline or with deionized water. The serum sample (40 ~1) was put into the sample chamber for each measurement. The COP was recorded for 15 min. Then the sample chamber was washed by the same fluid as that in the dome. In order to determine the least amount of the sample required to obtain a reliable measurement, the COPS of various amounts of the serum sample, that is, 40, 20, 10, 5, and 3 ~1 were measured. Solutions containing 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100% of the serum were prepared. The serum was diluted by saline. The COPS of these solutions were measured successively from high to low and from low to high concentrations. The relationship between the COPS and the serial dilutions of the serum was determined by the method of least squares. RESULTS The shift of the baseline was very small, that is, 0.02 mm Hg at 17.2 - 21.2 for 12 hr, -0.23 mm Hg at 20.3 - 22.8” for 10 hr and -0.17 mm Hg at 20.0 22.0” for 3 hr. When the top of the serum sample was covered with silicone oil, changes in the COP were very small after an initial acute response. The equilibration time was about 8 min, the values varying from 15.5 to 15.7 mm Hg over a period of 1.8 hr following application of the sample. When the top of the sample was not
183
BRIEF COMMUNICATIONS
(a)
(b)
TIME (h)
TIME (h)
FIG. 2. Effects of fluids within the dome and those used to wash the sample chamber on the COP of rat serum. Ten measurements of two successive experiments are shown. The dome of the pressure chamber was filled either with saline (a) or with deionized water (b). The serum sample (40 ~1) was put into the sample chamber which was washed with the same fluid as that in the dome after the measurement. Arrows indicate the start of each measurement.
covered with silicone oil, an initial acute response followed by a gradual increase of the COP was observed. The COPS commenced at 16.5 mm Hg and progressively increased to 19.9 mm Hg over the same period of time. During the experiment, the environmental temperature ranged from 21.0 to 22.7” and the humidity from 73 to 74%. Figure 2a shows the result from 10 successive measurements of the COP when the dome of the pressure chamber was filled with saline. The sample chamber was washed by saline after each measurement. The mean and the standard deviation of the COP were 16.7 and 0.2 mm Hg. Those of the shift of the zero point were - 0.04 and 0.13 mm Hg. Similar observations were made when deionized water was filled in the pressure chamber and the sample chamber was washed by deionized water after each measurement (Fig. 2b). The mean and standard deviation of the COP were 16.5 and 0.3 mm Hg. Those of the shift of the zero point were -0.04 and 0.24 mm Hg. A significant difference of the COPS was not found between the two experiments. However an overshoot was observed when the sample chamber was washed by deionized water in the latter experiment (Fig. 2b). The COP decreased when smaller sample size was measured. However, reproducible unvarying values were obtained when the sample volumes were greater than 10 ~1. In order to examine the relationship between the COPS and the ratios of the serum in diluted solution, the measured COPS were plotted against the ratios and a best-fit cubic equation was estimated by the method of least squares (Fig. 3). The formula was represented as y = 0.227 + 10.784x + 3.351~’ + 2.079x3, where y is the COP in mm Hg and x is the ratio of the serum in diluted solutions. It was found that a third degree polynominal is a satisfactory fit. Average COPS of the ultrafiltrate and the serum were 0.62 and 17.1 mm Hg,
184
BRIEF
COMMUNICATIONS
RATIO OF SERUM IN DILUTED SOLUTION FIG. 3. Relationship between the COP and the ratio of serum in the diluted solutions. Curve describes the best-fit cubic equation, y = 0.227 + 10.784.x + 3.351x2 + 2.079.x’, where y is the COP in mm Hg and x is the ratio of serum in diluted solutions.
respectively. It is possible that a small fraction of protein was forced to pass through the membrane into the ultrafiltrate and generated this COP. DISCUSSION It has been reported that the Amicon PM-30 ultrafiltration membrane represents the membrane of choice because of its optimal reproducibility and response time as well as its stability and long operational life (Prather et al., 1972). In the present experiments, it was found that the PM-30 membrane also gave satisfactory results as to its reproducibility and stability when measurements were performed several times a week for over a month. The response time or the equilibration time of the osmometer has been reported to vary from 4 set to tens of minutes (Hansen, 1961; Prather et al., 1968, 1972; Intaglietta and Zweifach, 1971; Wiederhielm et al., 1973; Kakiuchi et af., 1981; Marty and Intaglietta, 1970) whereas that of ours was approximately 8 min. It is supposed that the response time and/or the baseline recovery of our osmometer could be reduced if the membrane area was widened and the volume of fluid in the dome of the pressure chamber of the transducer was diminished. It is assumed that ordinary variations in room temperature and also in pH of the sample will not disturb the measurement materially as suggested by Hansen (1961). The concentration of the sample due to evaporation of water during the measurement could effectively be prevented by covering the top of the sample with silicone oil. The overshoot at washing (Fig. 2b) might be caused by salt concentration difference between the fluid in the sample chamber and that in the pressure chamber of the transducer as suggested by Aukland and Johnsen (1974). Such an overshoot could be reduced if we minimize the salt concentration difference across the membrane. In fact, the initial transient overshoot has been reduced
BRIEF
COMMUNICATIONS
185
when serum ultrafiltrate was applied repeatedly to the membrane until no pressure response was observed (Zweifach and Intaglietta, 1971; Aukland and Johnsen, 1974). It has been suggested that the exact size of the sample is immaterial, except that it must cover the whole effective part of the membrane; smaller samples did not always cover the membrane surface and gave too low pressures (Hansen, 1961). In addition it is recommended that the sample size should be the same and the baseline should be determined by adding the same amount of saline in the sample chamber because it was found that the measurement was affected by the amount of sample. Although we have not compared the values obtained from our apparatus with those obtained from another proven method, the COPS obtained in the present study (15.5 - 17.1 mm Hg) were within the range of values (15.7 - 25.4 mm Hg) reported by other investigators in the rat serum and plasma (Zweifach and Intaglietta, 1971; Worning and Steven, 1973; Navar and Navar, 1977). It seems necessary to use a sufficient number of rats in order to examine changes of COPS under various experimental conditions. Relationships between protein concentrations and COPS for human, dog, rat, and rabbit plasma and/or serum samples have been examined (Landis and Pappenheimer, 1963; Zweifach and Intaglietta, 1971; Worning and Steven, 1973; Navar and Navar, 1977). However, the relationship between the ratio of serum in a diluted solution and the COP was only examined in the present experiment. In order to obtain excellent linear correlations between COP and the total protein concentration, it seems important to standardize the protein determinations properly (Mullins et al., 1983). ACKNOWLEDGMENT The authors thank Professor R. W. Fearnhead for his valuable advice and kind help in preparing the manuscript.
REFERENCES AUKLAND,K., ANDJOHNSEN,H. M. (1974). A colloid osmometer for small fluid samples. Acta Physiol. Stand.
90, 485-490.
HANSEN,A. T. (1961). A self-recording electronic osmometer for quick, direct measurement of colloid osmotic pressure in small samples. Acta Physiol. Stand. 53, 197-213. INTAGLIETTA,M., ANDZWEIFACH,B. W. (1971). Measurement of blood plasma colloid osmotic pressure. I. Technical aspects. Microvasc. Res. 3, 72-82. KAKILJCHI,Y., ARAI, T., HORIMOTO,M. KIKUCHI, Y., AND KOYAMA, T. (1979). A new needle-type colloid osmometer for continuous determination of blood oncotic pressure. Amer. J. Physiol. 236, F419-F422. KAKIUCHI, Y., HUGHES,G. M., ARAI, T., HORIMOTO,M., KIKUCHI, Y., AND KOYAMA,T. (1981). A microanalysis of colloid osmotic pressure. Experientiu 37, 376-377. LANDIS, E. M., ANDPAPPENHEIMER, J. R. (1963). Exchange of substances through the capillary walls. In “Handbook of Physiology. Circulation” (W. F. Hamilton and P. DOW, Eds.), Vol. II. pp. 9611034. Amer. Physiol. Sot., Washington, DC. MARTY, A. T., ANDINTAGLIETTA,M. (1970). Effect of anticoagulants on human plasma colloid osmotic pressure measurements. J. Appl. Physiol. 29, 740-741. MARTY, A. T., AND ZWEIFACH,B. W. (1970). The high oncotic pressure effects of dextrans. Arch. Surg. 101, 421-424.
186
BRIEF
COMMUNICATIONS
MIKI, K., SAGAWA,S., AND SHIRAKI, K. (1983). Factors influencing the determination of colloid osmotic pressure of plasma. J. Univ. Occup. Environ. Health 5, 405-412. MULLINS, R. E., PAPPAS,A. A., AND GADSDEN,R. H. (1983). Correlation of standardized serum protein determinations with calculated and measured colloid osmotic pressure. Amer. J. C/in. Pathol. 80, 170-175. NAVAR, P. D., AND NAVAR, L. G. (1977). Relationship between colloid osmotic pressure and plasma protein concentration in the dog. Amer. J. Physiol. 233, H295-H298. PRATHER, J. W., BROWN,W. H., ANDZWEIFACH, B. W. (1972).An improved osmometer for measurement of plasma colloid osmotic pressure. Microvasc. Res. 4, 300-305. PRATHER,J. W., GAAR, JR., K. A., ANDGUYTON,A. C. (1968). Direct continuous recording of plasma colloid osmotic pressure of whole blood. J. Appl. Physiol. 24, 602-605. WIEDERHIELM, C. A., LEE, D. R., AND STROMBERG, D. D. (1973). A membrane osmometer for microliter samples. J. Appl. Physiol. 35, 432-435. WORNING, C., AND STEVEN, K. (1973). Serum protein concentration and oncotic pressure relationship in the rat. PJliigers Arch. 340, 77-80. ZWEIFACH, B. W., ANDINTAGLIETTA, M. (1971). Measurement of blood plasma colloid osmotic pressure. II. Comparative study of different species. Microvasc. Res. 3, 83-88.