Solvent Drag Reflection Coefficient Research Articles

Microvascular permeability transients due to histamine in cat limb

We measured the protein solvent drag reflection coefficient (sigma f) and the capillary filtration coefficient (CFC) before and after adding 1 or 10 microM histamine to the recirculating fluid (20% plasma, remainder albumin and electrolytes, hematocrit of 1-2%) perfusing the isolated cat hindlimb preparation. Transient sigma f measurements were made at 3- to 15-min intervals after histamine using a modification of the steady-state integral-mass balance method. CFC measurements were made at approximately 10-min intervals after histamine in separate experiments. A 1 microM dose of histamine caused sigma f to fall from approximately 0.8 to approximately 0.3 in 2-3 min; sigma f then returned to control in approximately 20 min. CFC response to the 1 microM histamine was a peak increase approximately 2 times control and a return to control in approximately 40 min. A 10 microM dose caused sigma f to fall rapidly to near zero. In general, recovery was much slower than for the 1 microM dose, most of the limbs not returning to control by 40 min after histamine. CFC measurements after 10 microM histamine increased only approximately 5 times control even though sigma f was near zero at the same time. CFC remained above control for approximately 60 min. The combined sigma f and CFC data could be described quantitatively if histamine simultaneously opened both short-lived large gaps (approximately 1,000 A) and a longer-lived pathway that sieved protein like the normal pathway and if the numbers of channels of each pathway closed exponentially with 4- and 15-min time constants, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Activated neutrophils increase microvascular permeability in skeletal muscle: role of xanthine oxidase

To determine the role of xanthine oxidase in the microvascular dysfunction produced by activated granulocytes, we examined the effect of xanthine oxidase depletion or inhibition on the increase in microvascular permeability produced by infusion of the neutrophil activator phorbol myristate acetate (PMA). Changes in vascular permeability were assessed by measurement of the solvent drag reflection coefficient for total plasma proteins (sigma) in rat hindquarters subjected to PMA infusion in xanthine oxidase-replete and -depleted animals, in animals pretreated with the xanthine oxidase inhibitor oxypurinol, and in animals depleted of circulating neutrophils by pretreatment with antineutrophil serum (ANS). Xanthine oxidase depletion was accomplished by administration of a tungsten-supplemented (0.7 g/kg diet) molybdenum-deficient diet. In animals fed the tungsten diet, muscle total xanthine dehydrogenase plus xanthine oxidase activity was decreased to less than 10% of control values. Estimates of sigma averaged 0.84 +/- 0.04 in control hindquarters, whereas PMA infusion was associated with a marked increase in microvascular permeability (decrease in sigma to 0.68 +/- 0.03). PMA infusion also caused an increase in the amount of the radical-producing oxidase form of xanthine oxidase (from 3.9 +/- 0.05 to 5.6 +/- 0.4 mU/g wet wt). ANS pretreatment attenuated this permeability increase (sigma = 0.77 +/- 0.04) and diminished the rise in xanthine oxidase activity (4.9 +/- 0.5 mU/g wet wt). Xanthine oxidase depletion with the tungsten diet or pretreatment with oxypurinol had no effect on this neutrophil-mediated microvascular injury (sigma = 0.69 +/- 0.06 and 0.67 +/- 0.03, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidant-mediated, CD18-dependent microvascular dysfunction induced by complement-activated granulocytes.

To determine the mechanisms whereby complement-activated granulocytes induce microvascular dysfunction in skeletal muscle, we examined the effect of antineutrophil serum (ANS), IB4 (a monoclonal antibody that inhibits CD18-dependent neutrophil adherence), xanthine oxidase inhibition or inactivation, deferoxamine, and catalase on the increase in canine gracilis muscle microvascular permeability induced by intravascular administration of zymosan-activated plasma (ZAP). Changes in vascular permeability were assessed by measurement of the solvent drag reflection coefficient (sigma) for total plasma proteins, and the extent of neutrophil infiltration was estimated by assessing muscle myeloperoxidase activity. ZAP infusion was associated with a marked increase in vascular permeability compared with control muscles that received no treatment or to muscles treated with zymosan heat-inactivated plasma (ZIP) (sigma = 0.51 +/- 0.04, 0.89 +/- 0.02, and 0.90 +/- 0.01, respectively). Estimates of sigma in animals rendered neutropenic with ANS, or treated with IB4, deferoxamine, or catalase before ZAP infusion were not significantly different from values obtained in control or ZIP-treated muscles (sigma = 0.96 +/- 0.02, 0.88 +/- 0.03, 0.85 +/- 0.02, and 0.79 +/- 0.01, respectively). However, xanthine oxidase inactivation or inhibition provided no protection from this ZAP-induced microvascular dysfunction (sigma = 0.58 +/- 0.02 and 0.58 +/- 0.01, respectively). In addition, neutropenia and inhibition of neutrophil adherence also prevented ZAP-induced increases in vascular resistance and tissue neutrophil infiltration.(ABSTRACT TRUNCATED AT 250 WORDS)

Pichinde Virus Infection in Strain 13 Guinea Pigs Reduces Intestinal Protein Reflection Coefficient with Compensation

Pichinde virus inoculation into strain 13 guinea pigs is a model with features reputed to be similar to hemorrhagic fever in humans. Although the infection is lethal by day 13-19, guinea pigs of approximately 600 g do not show edema or effusions. This raises the questions of whether capillary damage is present in such infected animals and, if it is, why edema is absent. The effects of Pichinide virus on protein transport across jejunal capillaries were examined in 38 normal and 7 infected strain 13 guinea pigs 12 days after inoculation. The latter lost 20.3% body weight but maintained normal blood pressure, serum protein concentration, and jejunal lymph flow. However, their protein solvent drag reflection coefficient (sigma) was reduced to .52 +/- .03 (mean +/- SE) from .73 +/- .02 (2P less than .001), while permeability-surface area product was not changed. In the absence of gross edema or effusions, Pichinde virus-infected guinea pigs demonstrated a leaky gut capillary wall to protein compatible with an increase in pore size or large pore number less than sufficient to change permeability-surface area product. Compensatory mechanisms that prevent edema at this stage are efficient and may include reduced capillary pressure or some degree of capillary flow stasis.

Polyethylene glycol 400 penetration of the colonic epithelial barrier of the rat

Permeability changes of polyethylene glycol 400 have been seen in patients with inflammatory bowel diseases. Because the colon can be involved in inflammatory bowel disease, the mechanisms, kinetics, and influence of intraluminal factors on polyethylene glycol 400 permeation of perfused colonic segments of rats were studied. The absorption rate of polyethylene glycol 400 was linearly related to its luminal concentration (r = 0.94), suggesting that passive diffusion is a significant mechanism involved in polyethylene glycol 400 absorption. Changing the perfusate pH from 6.0 to 7.5 did not affect water absorption or polyethylene glycol 400 permeation. Increasing luminal osmolarity significantly decreased water and polyethylene glycol 400 absorption (P < 0.01). The relationship between polyethylene glycol 400 and water absorption at different luminal osmolarities was linear (r = 0.97). At luminal osmolarity of 0.3 osm/L, 14.3% of polyethylene glycol 400 absorption was mediated by passive diffusion and 85.7% was mediated by convection. The solvent drag reflection coefficient for polyethylene glycol 400 in the colon was 0.03. Taurocholic acid (10 mmol/L) and chenodeoxycholic acid (5 mmol/L) decreased polyethylene glycol 400 and water absorption (P < 0.01). Addition of 1 μg/mL of 16,16,-dimethyl prostaglandin E2, 2 mmol/L of dibutyryladenosine-3′,5′-cyclic monophosphate, or 10 mmol/L of aminophylline significantly decreased water and polyethylene glycol 400 absorption (P < 0.01). These studies demonstrate that polyethylene glycol 400 permeation of the colon is mediated by both passive diffusion and solvent drag. Convective absorption is the major mechanism of polyethylene glycol 400 permeation of the colon. Polyethylene glycol 400 permeation is modified by bile acids, prostaglandins, and cyclic nucleotides through changes in water flux.

Microvascular injury after ischemia and reperfusion in skeletal muscle of exercise-trained rats

Ischemia and reperfusion in skeletal muscle is associated with increases in total vascular resistance (Rt) and the microvascular permeability to plasma proteins. To determine whether exercise training can attenuate ischemia and reperfusion-induced microvascular injury in skeletal muscle, intact (with skin) and skinned, maximally vasodilated (papaverine), isolated hindquarters of control (C) and exercise-trained (ET) rats were subjected to ischemia (intact 120 min; skinned 60 min) followed by 60 min of reperfusion. ET rats ran on a motorized treadmill at 32 m/min (8% grade), 2 h/day for 12 wk, whereas the C rats were cage confined. Before ischemia, ET hindquarters had higher isogravimetric flow, lower Rt, and similar solvent drag reflection coefficients (sigma f) compared with C. During reperfusion in intact hindquarters, flow was higher (P less than 0.05) and Rt tended to be lower (15 +/- 2 vs. 25 +/- 5 mmHg.ml-1.min.100 g; P less than 0.1) in ET compared with C; however, in skinned hindquarters flow and Rt (14 +/- 2 vs. 13 +/- 2 mmHg.ml-1.min.100 g) were not different between C and ET. During reperfusion, sigma f was reduced (P less than 0.05) in both intact (C 0.68 +/- 0.03; ET 0.68 +/- 0.02) and skinned (C 0.66 +/- 0.03; ET 0.68 +/- 0.03) hindquarters, indicative of an increased microvascular permeability to plasma proteins. These results indicate that exercise training did not attenuate the microvascular injury (increased Rt and decreased sigma f) associated with ischemia and reperfusion in rat skeletal muscle.

Neutrophil-mediated microvascular dysfunction in postischemic canine skeletal muscle. Role of granulocyte adherence.

Recent studies implicate a role for granulocytes in the genesis of the microvascular and parenchymal cell dysfunction, which occurs upon reperfusion of ischemic tissues. Although the molecular mechanisms underlying this neutrophil-mediated injury are not completely understood, it is clear that an essential first step in granulocyte migration from the vascular lumen to the interstitial space is adherence to vascular endothelium. The purpose of this study was to determine whether prevention of neutrophil adherence with monoclonal antibody IB4 directed against the neutrophil CD11/CD18 glycoprotein adherence complex or neutrophil depletion with a specific polyclonal antineutrophil serum would attenuate the microvascular dysfunction seen in postischemic skeletal muscle. Changes in vascular permeability were assessed by measurement of the solvent drag reflection coefficient for total plasma proteins (sigma) in isolated canine gracilis muscle subjected to ischemia/reperfusion, ischemia/reperfusion plus antineutrophil serum, or ischemia/reperfusion plus IB4. Estimates of sigma averaged 0.83 +/- 0.04 in nonischemic, control gracilis muscles, while ischemia/reperfusion was associated with a marked increase in vascular permeability (decrease in sigma to 0.54 +/- 0.04) and vascular resistance (increased by 135 +/- 41% over the control value). Prevention of neutrophil adherence or neutrophil depletion prevented this increase in vascular permeability (sigma = 0.80 +/- 0.03 and 1.01 +/- 0.06, respectively) and resistance (decrease of 16.51 +/- 8.0% and increase of 2.4 +/- 4.6% over control values, respectively). The results of this study suggest that neutrophils play a critical role in the genesis of microvascular dysfunction in postischemic skeletal muscle. Furthermore, neutrophil adherence to vascular endothelium appears to be a prerequisite for the production of this injury.

Effects of elevated venous pressure on capillary permeability in cat hindlimbs.

We investigated the effects of elevated venous pressure, Pv, (up to 140 mmHg) on the solvent drag reflection coefficient, sigma f, for protein and on the capillary filtration coefficient, CFC, in the isolated cat hindlimb perfused at constant flow. The perfusate contained 30% cat plasma and the remainder was a dialyzed albumin-electrolyte mixture. Cat red cells were added to a hematocrit of approximately 2%. sigma f was measured from the changes in hematocrit and plasma protein concentration (Integral-Mass Balance method) resulting from the fluid filtration caused by the Pv elevation. CFC was measured from the slope of the limb weight recording 2-4 min after the Pv elevation. sigma f decreased linearly from 0.807 (Pv less than 50 mmHg) to approximately 0.2 at 140 mmHg. CFC increased linearly from 0.0086 ml.min-1.mmHg-1.100 g-1 to about 0.04 over the same pressure range. A weight-independent filtration coefficient calculated from the change in hematocrit and a measurement of the initial perfusate volume gave comparable results, except at the very highest of pressures, where this coefficient was sometimes 20-40% less than CFC. Successive sigma f determinations at Pv at about 40 mmHg did not return to control after an initial measurement in which Pv was approximately 110 mmHg. Pore-theory analysis of the data suggests that the elevated Pv causes large pores to open as opposed to the stretching of small pores. Also, these large pores may remain open for a period of hours.

Role of xanthine oxidase in postischemic microvascular injury in skeletal muscle

Previous reports indicate that allopurinol, a xanthine oxidase inhibitor, attenuates the microvascular injury produced by reperfusion of ischemic skeletal muscle. To further assess the role of xanthine oxidase in ischemia/reperfusion (I/R) injury, we examined the effect of xanthine oxidase depletion or inhibition on the increase in microvascular permeability produced by I/R. Changes in vascular permeability were assessed by measurement of the solvent drag reflection coefficient for total plasma proteins (sigma) in rat hindquarters subjected to 2 h of ischemia and 30 min of reperfusion in xanthine oxidase-replete and -depleted animals and in animals pretreated with the xanthine oxidase inhibitor oxypurinol. Xanthine oxidase depletion was accomplished by administration of a tungsten-supplemented (0.7 g/kg diet), molybdenum-deficient diet. In animals fed the tungsten diet, muscle total xanthine dehydrogenase plus xanthine oxidase activity was decreased to less than 10% of control values. Estimates of sigma averaged 0.85 +/- 0.04 in nonischemic (continuous perfusion for 2.5 h) hindquarters, whereas muscle xanthine oxidase activity averaged 3.3 +/- 0.4 mU/g wet wt. I/R was associated with a marked decrease in sigma (0.54 +/- 0.02), whereas xanthine oxidase activity was increased to 5.8 +/- 0.5 mU/g wet wt. These results indicate that I/R produced a dramatic increase in vascular permeability coincident with an increase in muscle xanthine oxidase activity. Xanthine oxidase depletion with the tungsten diet or pretreatment with oxypurinol attenuated this permeability increase (sigma = 0.72 +/- 0.03 and 0.77 +/- 0.7, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of polyethylene glycol 400 permeability of perfused rat intestine

Abnormal permeability to polyethylene glycol 400 (PEG 400) has been demonstrated in various disorders with defective intestinal barrier functions. To understan the basic mechanisms of PEG 400 permeability, we compared PEG 400 permeation in different segments of the intestine and studied the kinetics and influence of intraluminal factors on PEG 400 absorption in vivo in perfused intestinal segments of the rat. The permeation rate of PEG 400 was dependent on the luminal concentration (y = 12.99x + 3.5; r = 0.97), indicating that passive movement is the mechanism involved in PEG 400 absorption. Changing the perfusate pH from 6 to 7.4 or modifying the unstirred water layer resistance by changing luminal flow rate did not affect PEG 400 absorption. When luminal osmolarity was varied from 0.225 to 0.6 osmol/L, higher osmolarity decreased both water and PEG 400 absorption (p > 0.01). The relationship between PEG 400 and water absorption at different osmolarities was linear (y = 0.9x + 5.7; r = 0.98). At a luminal osmolarity of 0.3 osmol/L 43% of PEG 400 permeation was mediated by passive diffusion and 57% was mediated by solvent drag. Increasing water absorption by decreasing luminal osmolarity resulted in proportional increase of PEG 400 permeation through solvent drag or convection. The solvent drag reflection coefficient (σf) for PEG 400 permeation of the jejunum was 0.1. Taurocholic acid (10 mM) alone or with oleic acid (2.5 mM) did not affect PEG 400 absorption. Permeabilities of 1 mM PEG 400 and water were similar in jejunum and ileum but were markedly increased in the colon (p > 0.01). These studies demonstrate that PEG 400 is absorbed by both passive diffusion and by solvent drag, with the latter accounting for a greater fraction of the absorptive drive under normal conditions. Polyethylene glycol 400 uses aqueous pathways for its permeation across the intestinal epithelium.

Influence of saline infusion on blood-tissue albumin transport.

Anesthetized rats were infused with lactated Ringer solution (LR) at constant rate for 30 or 60 min; delivered volume loads ranged from 0.03 to 0.08 ml/g body wt. Controls were given only a sustaining infusion of saline at 0.002 ml.g-1.h-1. Only 7-14% of the LR remained in the plasma at the end of the infusion; 76-88% entered the interstitial compartment, and 7-17% was excreted. The amount of plasma protein lost from the circulation with the extravasated fluid was studied simultaneously by two methods: 1) material balance in the whole animal and 2) changes in 131I-labeled albumin uptake (VA) and water content (VW) in individual tissues. The extravasation of 0.03-0.06 ml fluid/g body wt (75-160% initial plasma volume) did not significantly increase plasma protein extravasation in the whole rat. Nearly all of the sampled tissues of LR-infused rats had higher VW than controls. Tissue VA tended to increase with VW, but the regression slopes (delta VA/delta VW), a measure of the tracer albumin concentration of capillary filtrate relative to plasma, were low; skin, 0.006; paw, 0.018; skeletal muscles, 0.007; heart, 0.057; jejunum, 0.095; ileum, 0.045; cecum, 0.026; and colon, 0.027. These ratios are consistent with the very small loss of total plasma protein observed and attest to high solvent-drag reflection coefficients (sigma approximately equal to 1 - delta VA/delta VW): greater than 0.98 in capillaries of skeletal muscles, skin, and paw and 0.91-0.97 in heart and intestine.

Role of iron in postischemic microvascular injury.

Iron-catalyzed formation of hydroxyl radicals has been postulated to occur during reperfusion of ischemic tissues. To assess the role of iron-catalyzed oxidant production in ischemia/reperfusion (I/R) injury to skeletal muscle, we examined the effects of deferoxamine (an iron chelator) and apotransferrin (an iron-binding protein) on the increased vascular permeability produced by I/R in isolated, pump-perfused rat hindquarters. Solvent drag reflection coefficients (sigma) were measured in hindquarters subjected to 2 h of ischemia and 30 min of reperfusion with either no pretreatment, pretreatment with 50 mg/kg deferoxamine, 200 mg/kg apotransferrin, or iron-loaded deferoxamine (50 mg/kg). I/R alone was associated with an increase in vascular permeability as indicated by the significantly lower estimates of sigma obtained after I/R (0.68 +/- 0.03) compared with those obtained in nonischemic preparations (0.82 +/- 0.02). Pretreatment with deferoxamine or apotransferrin attenuated this permeability increase (sigma = 0.83 +/- 0.03 and 0.86 +/- 0.02, respectively), whereas pretreatment with iron-loaded deferoxamine afforded no protection (sigma = 0.71 +/- 0.02). These findings are consistent with the hypothesis that iron-catalyzed oxidant production is important in the genesis of microvascular injury following I/R. Since the enzyme xanthine oxidase has been implicated as a major source of oxidants generated during reperfusion, we also measured tissue levels of xanthine oxidase and xanthine dehydrogenase in muscle samples obtained from the same hindquarters in which we measured permeability changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypoxic reperfusion attenuates postischemic microvascular injury.

The results of several recent studies have demonstrated that reactive oxygen metabolites are responsible for a major portion of ischemia/reperfusion (I/R) injury in skeletal muscle. Presumably, the cytotoxic oxidants are produced during reperfusion when molecular oxygen (the source of the reactive oxygen metabolites) is reintroduced to the tissues. The purpose of this study was to test the hypothesis that molecular oxygen must be provided at reperfusion to produce I/R injury in skeletal muscle. Isolated, maximally vasodilated (papaverine) canine gracilis muscles were reperfused, after 4 h of inflow occlusion, from reservoirs containing autologous blood equilibrated with either 95% O2-5% CO2 or 95% N2-5% CO2 gas mixtures. Arterial PO2 fell from approximately 120 mmHg to less than 3-5 mmHg, during the use of nitrogen. The solvent drag reflection coefficient for total plasma proteins (sigma f) and total vascular resistance was determined for the following conditions: control (no ischemia), reperfusion with oxygenated blood after 4 h ischemia; and reperfusion (after 4 h ischemia), first with anoxic blood and then oxygenated blood. Reperfusion with oxygenated blood, after 4 h of ischemia, significantly reduced solvent drag reflexion coefficient (sigma f) from 0.93 +/- 0.02 to 0.63 +/- 0.02, indicating a dramatic increase in vascular permeability. Total vascular resistance increased from 6.1 +/- 1.1 mmHg.ml-1.min.100 g during the preischemic period to 12.9 +/- 3.0 mmHg.ml-1.min.100 g during normoxic reperfusion. In muscles reperfused with anoxic blood, sigma f averaged 0.82 +/- 0.06, whereas vascular resistance increased by 56 +/- 13%.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary embolism: analysis of endothelial pore sizes in canine lung

The effect of glass-bead microemboli (diameter 100 micron, range 77-125 micron) in the absence of fibrinolysis inhibition on pulmonary hemodynamics and microvascular permeability was determined in anesthetized, microfilaria-free dogs acutely prepared for the collection of lung lymph. Pulmonary vascular resistance, pulmonary capillary pressure (Pc), lymph flow (QL), and the ratio of lymph (CL) to plasma (Cp) protein concentrations were measured after 0.2 (n = 4), 0.4 (n = 6), or 0.6 (n = 3) g/kg beads. In all cases, emboli increased resistance and QL severalfold (P less than 0.05), while CL/Cp remained unchanged. In part, the increase in QL could be attributed to an increase in Pc compared with control (12.4 +/- 2.2 vs. 6.7 +/- 0.6 mmHg, P less than 0.05). Furthermore, since the solvent-drag reflection coefficient (sigma f) for total proteins approaches the osmotic reflection coefficient (sigma d) at high QL, sigma d was estimated under these conditions with sigma f approximately equal to sigma d approximately equal to 1 - (CL/Cp)min. The sigma d was decreased (P less than 0.05) after 0.4 and 0.6 g/kg beads to 0.55 +/- 0.03 and 0.50 +/- 0.07, respectively, when compared with that in control lungs (sigma d = 0.62 +/- 0.02; Parker et al., Circ. Res. 48: 549-561, 1981). A pore-stripping analysis demonstrated that after emboli the pulmonary endothelial barrier could be described by a population of small (80 A) and large (350 A) pores. However, the number of large to small pores was 1:1,195, compared with 1:195 in control lungs, suggesting an increased contribution of extra-alveolar vessels upstream of the emboli.(ABSTRACT TRUNCATED AT 250 WORDS)

Calculation of the reflection coefficient from measurements of endogenous vascular indicators.

The solvent drag reflection coefficient (sigma) for total proteins can be estimated by comparing the relative degrees of concentration of erythrocytes and plasma proteins that occur during fluid filtration in an isolated perfused organ. In this analysis, we evaluated the accuracy of equations proposed by Pilati and Maron [Am. J. Physiol. 247 (Heart Circ. Physiol. 16): H1-H7, 1984] and Wolf et al. [Am. J. Physiol. 253 (Heart Circ. Physiol. 22): H194-H204, 1987] to calculate sigma from these concentration changes. We calculated sigma with each equation using data generated from a mathematical model of fluid and solute flux in membranes with known sigma's. We found that the equation of Wolf et al. provided the closest approximation to the true sigma over the entire range of filtration fractions tested (0.1-0.6), with the differences between the two equations increasing with filtration fraction. At low filtration fractions, the difference in sigma obtained using either approach was found to be inconsequential. At larger filtration fractions, a closer approximation of the true sigma can be obtained using the equation of Wolf et al.

Integral-mass balance method for determination of solvent drag reflection coefficient.

We have developed the integral-mass balance (IMB) method to measure the solvent drag reflection coefficient (sigma f) for transcapillary macromolecular transport in skeletal muscle and other organs. Of course, sigma f is calculated from the cumulative amounts of water and macromolecule that move convectively across the microvascular membrane as determined from changes in hematocrit and plasma macromolecule concentration over a period of fluid filtration. We have investigated the effects of both theoretical and experimental factors that affect the validity and accuracy of the method. The effect of the following factors on sigma f determination by the IMB method were explored: low Peclet number; random-measurement errors; and systematic errors due to vascular leakage, hemolysis of red blood cells, evaporation, and osmolality changes. We found that all of these factors produced overestimations of sigma f, but their effects could be corrected. Also, appropriate experimental design could minimize these effects. Experiments using the IMB method in the isolated, perfused cat hindlimb preparation to determine sigma f for albumin and plasma proteins resulted in mean values of 0.82 +/- 0.08 (SD) (n = 7) and 0.83 +/- 0.02 (n = 4), respectively.

Quantitative fluorescence microscopy on single capillaries: alpha-lactalbumin transport.

We have extended the use of a microscope densitometric technique [Am. J. Physiol. 245 (Heart Circ. Physiol. 14): H495-H505, 1983] to measure the solute permeability coefficients (Pa) of fluorescently labeled solutes in single perfused capillaries of frog mesentery. The method enables the transcapillary flux of solutes larger than 10,000 mol wt to be measured under conditions where the forces that determine both solute and water flows across the capillary wall are known. The Pa for alpha-lactalbumin (mol wt 14,176, Stokes radius 2.02 nm) increased from a mean value of 2.1 X 10(-6) cm/s when capillary pressure was 3.0 cmH2O (no net filtration) to greater than 4.0 X 10(-6) cm/s when capillary pressure was 15 cmH2O. Taking a value of 0.35 for the solvent drag reflection coefficient for alpha-lactalbumin, we conclude that the increased solute flux represents solvent drag through a water pathway with a hydraulic conductivity of 3.6 X 10(-7) cm X s-1 X cmH2O-1. Our data conforms to the hypothesis that alpha-lactalbumin is transported across the capillary wall by restricted diffusion and solvent drag in a pathway that carries 90% of the transcapillary water flow (the principle water pathway). In vitro and in vivo calibration experiments have been carried out to test the assumption that the measured fluorescent light intensity is proportional to the number of fluorescent molecules in the measuring window of the photometer.

Preparation and high-performance size-exclusion chromatographic (HPSEC) analysis of fluorescein isothiocyanate-hydroxyethyl starch: Macromolecular probes of the blood-lymph barrier

A procedure is described for the preparation of fluorescein isothiocyanate-labeled hydroxyethyl starch (FITC-HES). Chromatographic techniques for the purification and analysis of FITC-HES that include the development of a high-performance size-exclusion chromatographic (HPSEC) technique with a fluorescence spectrophotometer-computer detection system are described. FITC-HES macromolecules have a wide range of hydrodynamic radii (a e > 120− < 10 Å) and the substitution ratio of FITC to the size-selected HES macromolecules remains constant throughout the chromatographic range. The predominant isoelectric point (p I) of the multiple acidic isomers of FITC-HES is 4.6. In vitro, the very large FITC-HES macromolecules (>100 Å) are rapidly degraded (15 sec) by α-amylase in control dog plasma. While most of the large molecules (100-20 Å) remain intact for >24 hr, this degradation is not associated with the loss of FITC from HES. In vivo, the rate of this reaction appeared to be accelerated and the degradation of FITC-HES between 0.1 and 12 hr was small. Illustration of the HPSEC quantitation of the spectrum of FITC-HES macromolecules in “near steady-state” lymph (L) and plasma (P) samples and the L P ratio show that this technique can be used to describe the size selectivity of the blood-lymph barrier under conditions of unaltered capillary pressure. We propose that the size-selected solvent-drag reflection coefficient ( σ f) curves for the anionic FITC-HES under conditions of elevated capillary pressure is a measure of macromolecular convective permeability of the blood-lymph barrier.

Effects of hypoproteinemia on lung microvascular protein sieving and lung lymph flow.

Experiments were conducted on five chronically instrumented unanesthetized sheep to determine the effects of sustained hypoproteinemia on lung fluid balance. Plasma total protein concentration was decreased from a control value of 6.17 +/- 0.019 to 3.97 +/- 0.17 g/dl (mean +/- SE) by acute plasmapheresis and maintained at this level by chronic thoracic lymph duct drainage. We measured pulmonary arterial pressure, left atrial pressure, aortic pressure, central venous pressure, cardiac output, oncotic pressures of both plasma and lung lymph, lung lymph flow rate, and lung lymph-to-plasma ratio of total proteins and six protein fractions for both control base-line conditions and hypoproteinemia base-line conditions. Moreover, we estimated the average osmotic reflection coefficient for total proteins and the solvent drag reflection coefficients for the six protein fractions during hypoproteinemia. Hypoproteinemia caused significant decreases in lung lymph total protein concentration, lung lymph-to-plasma total protein concentration ratio, and oncotic pressures of plasma and lung lymph. There were no significant alterations in the vascular pressures, lung lymph flow rate, cardiac output, or oncotic pressure gradient. The osmotic reflection coefficient for total proteins was found to be 0.900 +/- 0.004 for hypoproteinemia conditions, which is equal to that found in a previous investigation for sheep with a normal plasma protein concentration. Our results suggest that hypoproteinemia does not alter the lung filtration coefficient nor the reflection coefficients for plasma proteins. Possible explanations for the reported increase in the lung filtration coefficient during hypoproteinemia by other investigators are also made.

Fluid filtration and protein clearances through large and small pore populations in dog lung capillaries

Solvent drag reflection coefficients ( σ f) for six protein fractions with hydrodynamic radii ranging from 37 to 120 Å were determined using dog lung lymph C L C P data over a range of lung lymph flows (Q̊ L). Two equivalent pore populations with effective radii of 75–85 Å and 200–325 Å were determined for the pulmonary capillaries over a range of mean lymph flows for 1.8 to 6.3 times control. Fractional fluid clearance through the large pore population was observed to decrease from 0.28 to 0.16 as Q̊ L increased. These data indicate that lymph flow (filtration) increased primarily by an increased filtration through small pores with relatively little increase in filtration through the large pore population. The shift to small pore filtration at high filtration rates accounts for the dependence of the homoporous estimate of total protein σ f on lymph flow previously observed using lung lymph (Parker et al., 1981). If capillary membrane heteroporosity is present then there should be a net convective clearance of protein across the capillary caused by a volume circulation between large and small pore populations. Minimal estimates of 6–10% of protein clearance are attributed to this mechanism even in the absence of net fluid movement across the capillary. The optimal solute radius for clearance due to volume circulation is about 60 Å, based on the membrane pore sizes determined for lung capillaries. Although convective transport of macromolecules is generally acknowledged to account for most transcapillary protein clearance at high filtration rates, the inclusion of a volume circulation component suggests that convection may also be the dominant mode of macromolecular transport at low filtration rates.