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A modified colloidal osmotic transducer for the determination of transcapillary fluid movement
MICROVASCULAR
RESEARCH
5,
222-221 (1973)
TECHNICAL
REPORT
A Modified Colloidal Osmotic Determination of Transcapillary JULIUS
Transducer for the Fluid Movement’
J. FRIEDMAN
Department of Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202 Received September 27,1972 A modification of the Prather et al. (3,4) osmometer has been effected to enable its application to the study of transcapillary fluid movement. The basic change in the unit concerns the filter mounting and the gasketing in order to minimize the compliance of the assembly to allow measurement of changes in colloidal osmotic pressure as low as 0.02 mmHg. The necessity for low-noise high amplification and rigorous temperature regulation should be self-evident.
INTRODUCTION Prather et al. (3) describedthe development of a modification of the Hansen (l)-type colloidal osmotic transducer which possesseda greater frequency response, was insensitive to the red cell effect, and was applicable to the continuous determination of blood colloidal osmotic pressure. Whereas this transducer was entirely suitable for macrodeterminations of colloidal osmotic pressure, attempts to apply it to the dynamic study of transcapillary fluid movement proved ineffective due primarily to its relatively high compliance as well as the great temperature sensitivity of the transducer system. More recently Prather et al. (4) reported modifications of the original design which improved its performance in macro applications. The suitability of the system for microdeterminations has not been specified. This report representsa description of an adaptation of the osmometerwhich permits its application to dynamic studies of transcapillary fluid movement associated with changesin microvascular function. MATERIALS AND METHODS OsmometerAssembly As specifiedin the earlier reports (3,4) the fundamental principle of this transducer is to separatea sample chamber from a transducer chamber by means of a molecular selective filter-one which restricts molecules of plasma protein dimensions. The filter selected for this purpose is the Amicon PM-30. It possessesan effective molecular restriction at about 30,000 MW which provides a retention of bovine albumin to an r This work was supported by PHS Grant HE 04684-13. Copyright All rights
0 1973 by Academic Press, Inc. of reproduction in any form reserved.
222
TECHNICAL REPORT
223
effect greater than 95 ‘A when subjected to 55 psi for periods of 10-30 min of continuous ultrafiltration in a stirred cell. When utilized in a physiological study the unit is operated close to atmospheric pressure so that retention is undoubtedly greater than 95 %. The unit assembly is similar to that of Prather et al. (3, 4) with a couple of notable exceptions (Fig. 1). First, the O-rings used to seal the transducer and sample chamber to the base were replaced. The transducer seal is provided by Teflon gaskets (0.003 in. thick) which are compressed by screw tension. The number of gaskets used vary with
FIG. 1. (Left) schematic representation of the osmotic transducers assembly. (Right) cross-sectional diagram of the osmometer base indicating gaskets and filter locations.
the dimensions ofthe particular transducer employed. Usually two or three are adequate. The sample chamber seal is created with the filter itself. When adequately compressed the filter margins outside the sample chamber form water-tight seals. Second, the base upon which the filter sits is a flat stainless-steel surface provided with two rows of small holes (No. 80 drill) to allow for communication between the back of the filter and the transducer chamber. To provide the gain needed in this application the Statham P23De Pressure Transducer output was fed to a high-gain, low-noise amplifier (Keithley Model 155 Microvoltmeter) and then to a Beckman Type R Recorder. In operation blood is continuously pumped from the appropriate source to the transducer .unit at constant flow. The outflow port leading from the sample chamber
224
TECHNICAL
REPORT
is situated at or close to atmospheric pressure.The entire assemblyis housed in a water bath maintained at 37°C by means of a temperature-controlled water circulator. In isolated organ studies it has been found to be necessaryto defibrinate as well as heparinize the blood usedfor perfusion to prevent clot formation in the samplechamber which modifies transducer behavior. RESULTS AND DISCUSSION The time-response characteristic of the unit used in the flow-through mode of operation to various concentrations of human serum albumin is shown in Fig. 2. The time constant of the responsesis about 20-30 sec.Much of this time is due to the flow replacement of the previous solution in the tubing and samplechamber. Although not examined routinely, syringe presentation of samplesdirectly to the filter provides time constants of about 5 sec. ALBUMIN
4%
CONCENTRATION 6%
2%
(pm%) 7%
3%
-
5%
SALINE
-
111 min
TIME
FIG. 2. The time response of the osmometer to step inputs of albumin at concentrations of 2 to 8 g/100 ml.
The relationship between colloidal osmotic pressure of human serum albumin and protein concentration determined by refractometry is shown in Fig. 3. Included are data reported by Landis and Pappenheimer (2). The data generated in this study are situated above those presented by Landis and Pappenheimer. This discrepancy is attributed to the fact that about 20 % of the total protein in the human serum albumin generally available at the pharmacy consists of protein fragments as revealed by Sephadex column and electrophoretic separation. These fragments contribute to the colloidal osmotic pressure in excessof their protein concentration. Human serum albumin is used, nevertheless,becauseof convenient availability. Figure 4 revealsthat the determination of colloidal osmotic pressureby this technique is relatively insensitive to changesin hematocrit over the range of 0.35-0.70. When used in studies of transcapillary fluid movement, a conversion factor for calculating changesin fluid movement from changes in colloidal osmotic pressure is
TECHNICAL
225
REPORT
obtained from the data of Landis and Pappenheimer (2) at the appropriate level of plasma protein concentration. This procedure is considered feasible provided the blood possesses a reasonably normal albumin/globulin ratio and the selective filter is intact and functioning properly. The range of plasma protein concentration which encompasses the changes associated with variations in transcapillary fluid movement is 5.5-6.5 g/l00 ml. The integrity of the filter is assessedusing human serum albumin over this range of concentration to verify the slope of the relationship shown in Fig. 3. The Landis and 46
G X
l
;r
36
32
!3 B 26
4
0 0
1
2 ALBUMIN
3
4
5
6
CONCENTRATION
7
8
(@II%)
FIG. 3. The colloidal osmotic pressure response/albumin concentration relationship. Open circles represent data taken from Landis and Pappenheimer (2).
Pappenheimer slope at the protein concentration of 6.0 g/l00 ml is 5 mmHg per 1 g/l00 ml albumin or 0.2 g/l00 ml/mmHg. At 6.0 g/100 ml, 0.2 g/l00 ml represents 3.3 % change in plasma fluid of the sample. Thus, the conversion factor is 0.033 ml of plasma fluid per ml of plasma per mmHg colloidal osmotic pressure. This conversion factor is susceptible to variations of the A/G ratio to the extent that a 100% albumin sample would provide a factor 21% lower and a 100 % globulin sample a factor 27 % higher than that of the plasma. Extremes of this order are entirely unlikely; rather it is expected that physiological variations in the A/G ratio would alter the conversion factor by no more than 10 %. Transcapillary fluid movement is calculated as : &FM = idn,
(mmHg) x
ml plasma fluid ml plasma. mmHg
X
ml blood x ml plasma ml blood ’ min
226
TECHNICAL
REPORT
HEMATOCRIT
L 0
I
I
I
I
2
4
6
6
ALBUMIN
CONCENTRATION
(pm%)
FIG. 4. The effect of hematocrit on the colloidal osmotic pressure/albumin concentration relationship.
%21.2 g i 20.6 ” rl
20.4
8
20.0
i 8
16.6
Ill min
_._
I
TIME
FIG. 5. Theyeffect of venous pressure elevation on tissue volume and venous blood colloidal osmotic pressure.
TECHNICAL
REPORT
227
Figure 5 shows the result of applying this system to the dynamic assessmentof transcapillary fluid movement after the elevation of venous pressure. The capillary filtration coefficient calculated by this method is about 0.002 ml per minute per 100 g of muscle (gracilis) per mmHg change in capillary pressure. The change in capillary pressure was taken to be 80% that of venous pressure. The discrepancy between this and the volumetric estimate of 0.006 ml per minute per 100 g of muscle per mmHg change in capillary pressure is the subject of a study in progress. Since transcapillary fluid movement is largely dependent on the ratio of pre- to postcapillar:y resistance, when combined with a method to assessthe status of the precapillary sphincters, such as 86Rb extraction, the method presented here permits the assessmentof the status of postcapillary resistanceelements. REFERENCES 1. HANSEN, A. T. (1961). A self-recording electronic osmometer for quick, direct measurements of
colloidal osmotic pressure in small samples. Actu Physiol. Stand. 53, 197-213. 2. LANDIS, E. M., AND PAPPENHEIMER, J. R. (1963). Exchange of substances through the capillary
walls. In “Handbook of Physiology,” (W. F. Hamilton, ed.), Vol. II, p. 972. Amer. Physiol. Sot. Washington, DC. 3. PRATHER, J. W., GAAR, K. A., JR., AND GUYTON, A. C. (1968). Direct continuous recording of plasma colloidal osmotic pressure of whole blood. J. Appl. Physiol. 24,602-605. 4. PRATHER, J. W., BROWN, W. H., AND ZWEIFACH, B. W. (1972). An improved osmometer for measurement of plasma colloid osmotic pressure. Microuusc. Res. 4,300-305.
RESEARCH
5,
222-221 (1973)
TECHNICAL
REPORT
A Modified Colloidal Osmotic Determination of Transcapillary JULIUS
Transducer for the Fluid Movement’
J. FRIEDMAN
Department of Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202 Received September 27,1972 A modification of the Prather et al. (3,4) osmometer has been effected to enable its application to the study of transcapillary fluid movement. The basic change in the unit concerns the filter mounting and the gasketing in order to minimize the compliance of the assembly to allow measurement of changes in colloidal osmotic pressure as low as 0.02 mmHg. The necessity for low-noise high amplification and rigorous temperature regulation should be self-evident.
INTRODUCTION Prather et al. (3) describedthe development of a modification of the Hansen (l)-type colloidal osmotic transducer which possesseda greater frequency response, was insensitive to the red cell effect, and was applicable to the continuous determination of blood colloidal osmotic pressure. Whereas this transducer was entirely suitable for macrodeterminations of colloidal osmotic pressure, attempts to apply it to the dynamic study of transcapillary fluid movement proved ineffective due primarily to its relatively high compliance as well as the great temperature sensitivity of the transducer system. More recently Prather et al. (4) reported modifications of the original design which improved its performance in macro applications. The suitability of the system for microdeterminations has not been specified. This report representsa description of an adaptation of the osmometerwhich permits its application to dynamic studies of transcapillary fluid movement associated with changesin microvascular function. MATERIALS AND METHODS OsmometerAssembly As specifiedin the earlier reports (3,4) the fundamental principle of this transducer is to separatea sample chamber from a transducer chamber by means of a molecular selective filter-one which restricts molecules of plasma protein dimensions. The filter selected for this purpose is the Amicon PM-30. It possessesan effective molecular restriction at about 30,000 MW which provides a retention of bovine albumin to an r This work was supported by PHS Grant HE 04684-13. Copyright All rights
0 1973 by Academic Press, Inc. of reproduction in any form reserved.
222
TECHNICAL REPORT
223
effect greater than 95 ‘A when subjected to 55 psi for periods of 10-30 min of continuous ultrafiltration in a stirred cell. When utilized in a physiological study the unit is operated close to atmospheric pressure so that retention is undoubtedly greater than 95 %. The unit assembly is similar to that of Prather et al. (3, 4) with a couple of notable exceptions (Fig. 1). First, the O-rings used to seal the transducer and sample chamber to the base were replaced. The transducer seal is provided by Teflon gaskets (0.003 in. thick) which are compressed by screw tension. The number of gaskets used vary with
FIG. 1. (Left) schematic representation of the osmotic transducers assembly. (Right) cross-sectional diagram of the osmometer base indicating gaskets and filter locations.
the dimensions ofthe particular transducer employed. Usually two or three are adequate. The sample chamber seal is created with the filter itself. When adequately compressed the filter margins outside the sample chamber form water-tight seals. Second, the base upon which the filter sits is a flat stainless-steel surface provided with two rows of small holes (No. 80 drill) to allow for communication between the back of the filter and the transducer chamber. To provide the gain needed in this application the Statham P23De Pressure Transducer output was fed to a high-gain, low-noise amplifier (Keithley Model 155 Microvoltmeter) and then to a Beckman Type R Recorder. In operation blood is continuously pumped from the appropriate source to the transducer .unit at constant flow. The outflow port leading from the sample chamber
224
TECHNICAL
REPORT
is situated at or close to atmospheric pressure.The entire assemblyis housed in a water bath maintained at 37°C by means of a temperature-controlled water circulator. In isolated organ studies it has been found to be necessaryto defibrinate as well as heparinize the blood usedfor perfusion to prevent clot formation in the samplechamber which modifies transducer behavior. RESULTS AND DISCUSSION The time-response characteristic of the unit used in the flow-through mode of operation to various concentrations of human serum albumin is shown in Fig. 2. The time constant of the responsesis about 20-30 sec.Much of this time is due to the flow replacement of the previous solution in the tubing and samplechamber. Although not examined routinely, syringe presentation of samplesdirectly to the filter provides time constants of about 5 sec. ALBUMIN
4%
CONCENTRATION 6%
2%
(pm%) 7%
3%
-
5%
SALINE
-
111 min
TIME
FIG. 2. The time response of the osmometer to step inputs of albumin at concentrations of 2 to 8 g/100 ml.
The relationship between colloidal osmotic pressure of human serum albumin and protein concentration determined by refractometry is shown in Fig. 3. Included are data reported by Landis and Pappenheimer (2). The data generated in this study are situated above those presented by Landis and Pappenheimer. This discrepancy is attributed to the fact that about 20 % of the total protein in the human serum albumin generally available at the pharmacy consists of protein fragments as revealed by Sephadex column and electrophoretic separation. These fragments contribute to the colloidal osmotic pressure in excessof their protein concentration. Human serum albumin is used, nevertheless,becauseof convenient availability. Figure 4 revealsthat the determination of colloidal osmotic pressureby this technique is relatively insensitive to changesin hematocrit over the range of 0.35-0.70. When used in studies of transcapillary fluid movement, a conversion factor for calculating changesin fluid movement from changes in colloidal osmotic pressure is
TECHNICAL
225
REPORT
obtained from the data of Landis and Pappenheimer (2) at the appropriate level of plasma protein concentration. This procedure is considered feasible provided the blood possesses a reasonably normal albumin/globulin ratio and the selective filter is intact and functioning properly. The range of plasma protein concentration which encompasses the changes associated with variations in transcapillary fluid movement is 5.5-6.5 g/l00 ml. The integrity of the filter is assessedusing human serum albumin over this range of concentration to verify the slope of the relationship shown in Fig. 3. The Landis and 46
G X
l
;r
36
32
!3 B 26
4
0 0
1
2 ALBUMIN
3
4
5
6
CONCENTRATION
7
8
(@II%)
FIG. 3. The colloidal osmotic pressure response/albumin concentration relationship. Open circles represent data taken from Landis and Pappenheimer (2).
Pappenheimer slope at the protein concentration of 6.0 g/l00 ml is 5 mmHg per 1 g/l00 ml albumin or 0.2 g/l00 ml/mmHg. At 6.0 g/100 ml, 0.2 g/l00 ml represents 3.3 % change in plasma fluid of the sample. Thus, the conversion factor is 0.033 ml of plasma fluid per ml of plasma per mmHg colloidal osmotic pressure. This conversion factor is susceptible to variations of the A/G ratio to the extent that a 100% albumin sample would provide a factor 21% lower and a 100 % globulin sample a factor 27 % higher than that of the plasma. Extremes of this order are entirely unlikely; rather it is expected that physiological variations in the A/G ratio would alter the conversion factor by no more than 10 %. Transcapillary fluid movement is calculated as : &FM = idn,
(mmHg) x
ml plasma fluid ml plasma. mmHg
X
ml blood x ml plasma ml blood ’ min
226
TECHNICAL
REPORT
HEMATOCRIT
L 0
I
I
I
I
2
4
6
6
ALBUMIN
CONCENTRATION
(pm%)
FIG. 4. The effect of hematocrit on the colloidal osmotic pressure/albumin concentration relationship.
%21.2 g i 20.6 ” rl
20.4
8
20.0
i 8
16.6
Ill min
_._
I
TIME
FIG. 5. Theyeffect of venous pressure elevation on tissue volume and venous blood colloidal osmotic pressure.
TECHNICAL
REPORT
227
Figure 5 shows the result of applying this system to the dynamic assessmentof transcapillary fluid movement after the elevation of venous pressure. The capillary filtration coefficient calculated by this method is about 0.002 ml per minute per 100 g of muscle (gracilis) per mmHg change in capillary pressure. The change in capillary pressure was taken to be 80% that of venous pressure. The discrepancy between this and the volumetric estimate of 0.006 ml per minute per 100 g of muscle per mmHg change in capillary pressure is the subject of a study in progress. Since transcapillary fluid movement is largely dependent on the ratio of pre- to postcapillar:y resistance, when combined with a method to assessthe status of the precapillary sphincters, such as 86Rb extraction, the method presented here permits the assessmentof the status of postcapillary resistanceelements. REFERENCES 1. HANSEN, A. T. (1961). A self-recording electronic osmometer for quick, direct measurements of
colloidal osmotic pressure in small samples. Actu Physiol. Stand. 53, 197-213. 2. LANDIS, E. M., AND PAPPENHEIMER, J. R. (1963). Exchange of substances through the capillary
walls. In “Handbook of Physiology,” (W. F. Hamilton, ed.), Vol. II, p. 972. Amer. Physiol. Sot. Washington, DC. 3. PRATHER, J. W., GAAR, K. A., JR., AND GUYTON, A. C. (1968). Direct continuous recording of plasma colloidal osmotic pressure of whole blood. J. Appl. Physiol. 24,602-605. 4. PRATHER, J. W., BROWN, W. H., AND ZWEIFACH, B. W. (1972). An improved osmometer for measurement of plasma colloid osmotic pressure. Microuusc. Res. 4,300-305.