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Colloid osmotic pressure of subcutaneous wick fluid in rats
MICROVASCULAR
RESEARCH
35, 139-142 (1988)
BRIEF COMMUNICATION Colloid
Osmotic J. A.
Drpurtment
Pressure JOLES,
c?fNephrology
H. A.
of Subcutaneous KOOMANS,
and Hypertension, Received
AND
University
Decrmher
R.J.
Wick Fluid in Rats BERCKMANS
Hospital,
Utrecht,
The Netherlcmds
1, I986
INTRODUCTION Interstitial fluid colloid osmotic pressure (COP) is an important force in the plasma-to-interstitial fluid balance (Renkin, 1986). Subcutaneously placed nylon wicks are used to collect interstitial fluid and enable us to measure this variable in the skin. This method is easy to perform and practicable for use in human study. In a recent methodological study in rats, however, Kramer et al. (1986) found that saline-soaked wicks, which are most commonly used (Fadnes, 1975: Fauchald et al., 1984, 1985; Koomans et al., 1985, 1986; Noddeland et al., 1982; Wiig and Reed, 1985, 1986; Fadnes rt al., 1986), yielded about 25% lower concentrations of total protein than dry wicks, serum-soaked wicks, or wicks placed immediately postmortem. Measurement of COP was included in part of their study only. Again, saline-soaked wicks in live animals tended to give lower values than serum-primed wicks placed in freshly killed rats, although the difference was not significant (Kramer et al., 1986). They concluded that saline-soaked wicks probably underestimate protein concentration of skin interstitial fluid. However, they used l.O-mm-diameter wicks, whereas in a number of the abovementioned studies, 0.5 mm-diameter wicks have been used (Fadnes, 1975; Koomans et al., 1985, 1986). To analyze whether saline-primed wicks indeed yield lower COP-values than the other wick techniques, we performed paired implantations of saline-soaked and dry 0.5-mm-diameter wicks in live rats. MATERIALS
AND METHODS
Eighteen male Wistar rats were anesthetized by intraperitoneal injection of sodium pentobarbital, and two saline-soaked and two dry double-stranded multifilamentous nylon wicks 0.5 mm in diameter (Enkalon 3 x 3, Enka BV, Arnhem) were implanted simultaneously on the shaved back of each rat. After 1 hr the wicks were removed and stored under mineral oil. Then a blood sample was drawn. Colloid osmotic pressure was measured using a strain gauge oncometer (Aukland and Johnsen, 1974) in plasma and in wick fluid recovered as described 139 (X)26-2862iXX $3.00 CopyrIght !Z 198X by Academx Press. Inc. All right\ of rrproductmn in any form reserved. Printed in U.S.A
140
BRIEF COMMUNICATION
previously (Johnsen, 1974). In nine of these rats the wick fluid collected was sufficient to allow paired measurements of total protein (Bradford, 1976)in addition to COP. RESULTS Plasma COP was 19.0 ? 1.3 mm Hg (mean + SD, N = 18) and total protein 64.7 ? 8.7 g/liter (N = 9). No systematic differences in either COP (P = 0.35, paired t test; Fig. 1) or total protein concentration (P = 0.76; Fig. 2) were found between the two groups of wicks. COP in the dry and saline-soaked wicks was 10.5 2 2.0 and 10.0 + 2.7 mm Hg, respectively. Total protein in dry and salinesoaked wicks was 39.7 + 7.8 and 39.1 & 7.7 g/liter respectively. The transcapillary COP gradient (plasma COP -interstitial COP) was 8.5 + 3.4 and 8.9 + 3.3 mm Hg for dry and saline-soaked wicks, respectively. DISCUSSION We found equal values of COP and total protein in fluid sampled with salinesoaked and dry nylon wicks inserted in live rat subcutaneous tissue, which is collold osmotic prossuro(mmHg) 21-
A j
18-
* -.*
15-
12-
9-
6-
3-
L-1-I plasm.3
-Idry
wicks
saLno-soaked wicks
FIG. 1. Colloid osmotic pressure determined from dry and saline-soaked wicks implanted simultaneously in 18 rats, Plasma values are plotted for comparison. The mean + SD is indicated.
BRIEF COMMUNICATION
141
-.-ie . : -7 -.5. .
FIG. 2. Total protein concentration determined from dry and saline-soaked wicks implanted simultaneously in 9 rats. Plasma values are plotted for comparison. The mean + SD is indicated.
at variance with data reported by Kramer et al. (1986). Using dry wicks, these workers found similar total protein concentrations as we did (37.5 ? 4.0 g/liter), but much lower values (26.4 2 4.9 and 29.5 ? 5.2 g/liter in two groups of rats) with saline-soaked wicks. The difference between Kramer et al.‘s (1986) and the present findings must originate from the method used. The theory underlying the wick method is that for a short period after wick insertion, 30 min or less, plasma fluid and proteins leak to the interstitium and fill the wick or mix with the fluid in the wick (Aukland et al., 1981). After this “priming” of the wick, the fluid within the wick will equilibrate osmotically with the surrounding interstitial fluid. The chance that equilibration is complete when the wick is extracted depends upon this priming of the wick that occurs in the subcutis by plasma leakage, and the implantation time. When leakage is impaired by pretreatment with indomethacin, or when relatively thick saline-soaked wicks are used, this priming may not be enough to allow osmotic equilibration within the generally used implantation time of 1 hr (Aukland et ul., 1981). This may result in too low values of COP and protein concentration, such as found by Kramer et al. (1986), or in dehydration of the wicks (Aukland et al., 1981). Because we have used thin wicks (0.5 mm) in the present (and former) studies, whereas wicks of 1.0 mm in diameter were used by Kramer et al. (1986), we conclude that the problem of underestimation of interstitial protein and COP by saline-soaked wicks implanted subcutaneously for 1 hr does not concern thin wicks.
142
BRIEF
COMMUNICATION
REFERENCES AKJKLAND, K.. AND JOHNSEN. H. M. (1974). A colloid osmometer or small fluid samples. Acta Physiol. Stand. 90, 485-490. AUKLAND, K., FADNES, H. O., NODDELAND. H., AND REED, R. K. (1981). Edema-preventing mechanisms in subcutis and skeletal muscle. In “Tissue fluid Pressure and composition” (A. R. Hargens, Ed.), pp. 87-93. Williams & Wilkins, Baltimore. BRADFORD, M. M. (1976). A rapid and sensitive method for the quantitation of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-253. FADNES, H. 0. (1975). Protein concentration and hydrostatic pressure in subcutaneous tissue of rats in hypoproteinemia. Stand. J. C/in. Lab. Invest. 35, 441-446. FADNES, H. O., PAPE, J. F., AND SUNDSFJORD,J. A. (1986). A study on oedema mechanism in nephrotic syndrome. Stand. J. C/in. Lab. Invest. 46, 533-538. FAUCHALD, P.. NODDELAND H., AND NORSETH, J. (1984). Interstitial fluid volume, plasma volume and colloid osmotic pressure in patients with nephrotic syndrome. Scund. J. C/in. Lab. Invest. 44, 661-667. FAUCHALD, P., NORSETH, J., AND JERVELL, J. (1985). Transcapillary colloid osmotic gradient, plasma volume and interstitial fluid volume in long-term Type I (insulin-dependent) diabetes. Diabrtologia 28, 269-273. JOHNSEN, H. M. (1974). Measurement of colloid osmotic pressure of interstitial fluid. Acto Physiol. Stand. 91, 142-144. KOOMANS, H. A., GEERS, A. B., DORHOUT MEES. E. J., AND KORTLANDT, W. (1986). Lowered tissuefluid oncotic pressure protects the blood volume in the nephrotic syndrome. Nephron 42, 317322. KOOMANS, H. A., KORTLANDT, W., GEERS, A. B., AND DORHOU~ MEES. E. J. (1985). Lowered protein content of tissue fluid in patients with the nephrotic syndrome: Observations during disease and recovery. Nephron 40, 391-395. KRAMER, G. C., SIBLEY, L., AUKLAND, K., AND RENKIN, E. M. (1986). Wick sampling of interstitial fluid in rat skin: Further analysis and modifications of the method. Microvosc. Res. 32, 39-49. NODDELAND. H., RIISNESS, S. M., AND FADNES, H. 0. (1982). Interstitial fluid colloid osmotic and hydrostatic pressures in subcutaneous tissue of patients with nephrotic syndrome. Stand. J. C/in. Lab. Invest. 42, 139-146. RENKIN, E. M. (1986). Some consequences of capillary permeability to macromolecules: Starling’s hypothesis reconsidered. Amer. 1. Physiol. 250, H706-H7lO. Wnc. H., AND REED, R. K. (1985). Interstitial compliance and transcapillary Starling pressures in cat skin and skeletal muscle. Amer. J. Physiol. 248, H666-H673. Wnc, H.. AND REED, R. K. (1986). Interstitial compliance and transcapillary fluid balance in renal hypertensive rats. Acfa Physiol. &and. 127, 407-417.
RESEARCH
35, 139-142 (1988)
BRIEF COMMUNICATION Colloid
Osmotic J. A.
Drpurtment
Pressure JOLES,
c?fNephrology
H. A.
of Subcutaneous KOOMANS,
and Hypertension, Received
AND
University
Decrmher
R.J.
Wick Fluid in Rats BERCKMANS
Hospital,
Utrecht,
The Netherlcmds
1, I986
INTRODUCTION Interstitial fluid colloid osmotic pressure (COP) is an important force in the plasma-to-interstitial fluid balance (Renkin, 1986). Subcutaneously placed nylon wicks are used to collect interstitial fluid and enable us to measure this variable in the skin. This method is easy to perform and practicable for use in human study. In a recent methodological study in rats, however, Kramer et al. (1986) found that saline-soaked wicks, which are most commonly used (Fadnes, 1975: Fauchald et al., 1984, 1985; Koomans et al., 1985, 1986; Noddeland et al., 1982; Wiig and Reed, 1985, 1986; Fadnes rt al., 1986), yielded about 25% lower concentrations of total protein than dry wicks, serum-soaked wicks, or wicks placed immediately postmortem. Measurement of COP was included in part of their study only. Again, saline-soaked wicks in live animals tended to give lower values than serum-primed wicks placed in freshly killed rats, although the difference was not significant (Kramer et al., 1986). They concluded that saline-soaked wicks probably underestimate protein concentration of skin interstitial fluid. However, they used l.O-mm-diameter wicks, whereas in a number of the abovementioned studies, 0.5 mm-diameter wicks have been used (Fadnes, 1975; Koomans et al., 1985, 1986). To analyze whether saline-primed wicks indeed yield lower COP-values than the other wick techniques, we performed paired implantations of saline-soaked and dry 0.5-mm-diameter wicks in live rats. MATERIALS
AND METHODS
Eighteen male Wistar rats were anesthetized by intraperitoneal injection of sodium pentobarbital, and two saline-soaked and two dry double-stranded multifilamentous nylon wicks 0.5 mm in diameter (Enkalon 3 x 3, Enka BV, Arnhem) were implanted simultaneously on the shaved back of each rat. After 1 hr the wicks were removed and stored under mineral oil. Then a blood sample was drawn. Colloid osmotic pressure was measured using a strain gauge oncometer (Aukland and Johnsen, 1974) in plasma and in wick fluid recovered as described 139 (X)26-2862iXX $3.00 CopyrIght !Z 198X by Academx Press. Inc. All right\ of rrproductmn in any form reserved. Printed in U.S.A
140
BRIEF COMMUNICATION
previously (Johnsen, 1974). In nine of these rats the wick fluid collected was sufficient to allow paired measurements of total protein (Bradford, 1976)in addition to COP. RESULTS Plasma COP was 19.0 ? 1.3 mm Hg (mean + SD, N = 18) and total protein 64.7 ? 8.7 g/liter (N = 9). No systematic differences in either COP (P = 0.35, paired t test; Fig. 1) or total protein concentration (P = 0.76; Fig. 2) were found between the two groups of wicks. COP in the dry and saline-soaked wicks was 10.5 2 2.0 and 10.0 + 2.7 mm Hg, respectively. Total protein in dry and salinesoaked wicks was 39.7 + 7.8 and 39.1 & 7.7 g/liter respectively. The transcapillary COP gradient (plasma COP -interstitial COP) was 8.5 + 3.4 and 8.9 + 3.3 mm Hg for dry and saline-soaked wicks, respectively. DISCUSSION We found equal values of COP and total protein in fluid sampled with salinesoaked and dry nylon wicks inserted in live rat subcutaneous tissue, which is collold osmotic prossuro(mmHg) 21-
A j
18-
* -.*
15-
12-
9-
6-
3-
L-1-I plasm.3
-Idry
wicks
saLno-soaked wicks
FIG. 1. Colloid osmotic pressure determined from dry and saline-soaked wicks implanted simultaneously in 18 rats, Plasma values are plotted for comparison. The mean + SD is indicated.
BRIEF COMMUNICATION
141
-.-ie . : -7 -.5. .
FIG. 2. Total protein concentration determined from dry and saline-soaked wicks implanted simultaneously in 9 rats. Plasma values are plotted for comparison. The mean + SD is indicated.
at variance with data reported by Kramer et al. (1986). Using dry wicks, these workers found similar total protein concentrations as we did (37.5 ? 4.0 g/liter), but much lower values (26.4 2 4.9 and 29.5 ? 5.2 g/liter in two groups of rats) with saline-soaked wicks. The difference between Kramer et al.‘s (1986) and the present findings must originate from the method used. The theory underlying the wick method is that for a short period after wick insertion, 30 min or less, plasma fluid and proteins leak to the interstitium and fill the wick or mix with the fluid in the wick (Aukland et al., 1981). After this “priming” of the wick, the fluid within the wick will equilibrate osmotically with the surrounding interstitial fluid. The chance that equilibration is complete when the wick is extracted depends upon this priming of the wick that occurs in the subcutis by plasma leakage, and the implantation time. When leakage is impaired by pretreatment with indomethacin, or when relatively thick saline-soaked wicks are used, this priming may not be enough to allow osmotic equilibration within the generally used implantation time of 1 hr (Aukland et ul., 1981). This may result in too low values of COP and protein concentration, such as found by Kramer et al. (1986), or in dehydration of the wicks (Aukland et al., 1981). Because we have used thin wicks (0.5 mm) in the present (and former) studies, whereas wicks of 1.0 mm in diameter were used by Kramer et al. (1986), we conclude that the problem of underestimation of interstitial protein and COP by saline-soaked wicks implanted subcutaneously for 1 hr does not concern thin wicks.
142
BRIEF
COMMUNICATION
REFERENCES AKJKLAND, K.. AND JOHNSEN. H. M. (1974). A colloid osmometer or small fluid samples. Acta Physiol. Stand. 90, 485-490. AUKLAND, K., FADNES, H. O., NODDELAND. H., AND REED, R. K. (1981). Edema-preventing mechanisms in subcutis and skeletal muscle. In “Tissue fluid Pressure and composition” (A. R. Hargens, Ed.), pp. 87-93. Williams & Wilkins, Baltimore. BRADFORD, M. M. (1976). A rapid and sensitive method for the quantitation of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-253. FADNES, H. 0. (1975). Protein concentration and hydrostatic pressure in subcutaneous tissue of rats in hypoproteinemia. Stand. J. C/in. Lab. Invest. 35, 441-446. FADNES, H. O., PAPE, J. F., AND SUNDSFJORD,J. A. (1986). A study on oedema mechanism in nephrotic syndrome. Stand. J. C/in. Lab. Invest. 46, 533-538. FAUCHALD, P.. NODDELAND H., AND NORSETH, J. (1984). Interstitial fluid volume, plasma volume and colloid osmotic pressure in patients with nephrotic syndrome. Scund. J. C/in. Lab. Invest. 44, 661-667. FAUCHALD, P., NORSETH, J., AND JERVELL, J. (1985). Transcapillary colloid osmotic gradient, plasma volume and interstitial fluid volume in long-term Type I (insulin-dependent) diabetes. Diabrtologia 28, 269-273. JOHNSEN, H. M. (1974). Measurement of colloid osmotic pressure of interstitial fluid. Acto Physiol. Stand. 91, 142-144. KOOMANS, H. A., GEERS, A. B., DORHOUT MEES. E. J., AND KORTLANDT, W. (1986). Lowered tissuefluid oncotic pressure protects the blood volume in the nephrotic syndrome. Nephron 42, 317322. KOOMANS, H. A., KORTLANDT, W., GEERS, A. B., AND DORHOU~ MEES. E. J. (1985). Lowered protein content of tissue fluid in patients with the nephrotic syndrome: Observations during disease and recovery. Nephron 40, 391-395. KRAMER, G. C., SIBLEY, L., AUKLAND, K., AND RENKIN, E. M. (1986). Wick sampling of interstitial fluid in rat skin: Further analysis and modifications of the method. Microvosc. Res. 32, 39-49. NODDELAND. H., RIISNESS, S. M., AND FADNES, H. 0. (1982). Interstitial fluid colloid osmotic and hydrostatic pressures in subcutaneous tissue of patients with nephrotic syndrome. Stand. J. C/in. Lab. Invest. 42, 139-146. RENKIN, E. M. (1986). Some consequences of capillary permeability to macromolecules: Starling’s hypothesis reconsidered. Amer. 1. Physiol. 250, H706-H7lO. Wnc. H., AND REED, R. K. (1985). Interstitial compliance and transcapillary Starling pressures in cat skin and skeletal muscle. Amer. J. Physiol. 248, H666-H673. Wnc, H.. AND REED, R. K. (1986). Interstitial compliance and transcapillary fluid balance in renal hypertensive rats. Acfa Physiol. &and. 127, 407-417.