Albumin Transfer Research Articles

Indomethacin reduces ischemia-induced alteration of blood-brain barrier transport in piglets.

Cerebral ischemia-reperfusion (Isc-Rep) alters blood-brain barrier (BBB) transport properties in piglets. Pretreatment with superoxide dismutase and catalase partially attenuates these effects. Activated O2 species produced with Isc-Rep in piglets are generated via prostaglandin (PG) H synthase. This experiment determines if products of PGH synthase alter BBB transport of sodium and albumin. Piglets anesthetized with nitrous oxide and halothane were divided into four groups: 1) control, 2) indomethacin (5 mg/kg iv) with no Isc-Rep, 3) Isc-Rep alone, and 4) Isc-Rep after pretreatment with indomethacin (Indo). Regional transfer coefficients (Kin) and regional cerebral blood flow (microspheres) were measured at 2 h reperfusion after 20 min total global cerebral ischemia. Kin values are represented as absolute values and also relative to blood flow to account for any changes in perfusion caused by ischemia and/or Indo. Indo alone did not alter sodium or albumin transfer compared with control animals. Isc-Rep alone caused a significant increase in sodium and albumin transport compared with all other groups (control cerebral sodium Kin was 18.2 +/- 2.7 cm3.g-1.s-1.10(6) vs. 32.9 +/- 3.1 for Isc-Rep, P < 0.05). In contrast, there was no significant difference in sodium or albumin transfer with Isc-Rep after Indo pretreatment (e.g., cerebral sodium Kin was 22.0 +/- 2.0 for Isc-Rep after Indo) compared with the control or Indo alone groups. Indo pretreatment effectively attenuates increased BBB transport of both sodium and albumin following cerebral ischemia. We conclude that products of PGH synthase are involved in BBB alterations of protein and cation transport that follow the early stages of cerebral reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Endotoxin-induced arterial endothelial barrier dysfunction assessed by an in vitro model.

Using an in vitro model in which albumin transfer across monolayers of bovine aortic endothelial cells (BAEC) was measured, we have shown that lipopolysaccharide (LPS) induces a concentration-dependent increase in endothelial permeability. This increase was biphasic, having an early peak at 2 h and rising again by 24 h. Both peaks were abolished by polymixin B (PMB) but were unaffected by N omega-monomethyl-L-arginine, N omega-nitro-L-arginine methyl ester or dexamethasone. Furthermore, LPS did not stimulate nitric oxide production by BAEC following 24 h exposure. Thus, the LPS-induced increase in permeability may account for the vascular leakage of septic shock, but the L-arginine-nitric oxide system does not appear to be involved.

Proteoglycans and endothelial barrier function: effect of linoleic acid exposure to porcine pulmonary artery endothelial cells

Certain fatty acids induce changes in endothelial barrier function which may be mediated by alterations in normal proteoglycan synthesis/metabolism. To test this hypothesis, pulmonary artery derived endothelial cells were treated with media supplemented with linoleic acid (18:2), and/or a known proteoglycan synthesis inhibitor, β- d-xyloside. Independent exposure to 1 MM β- d-xyloside or 90 μM 18:2 increased albumin transfer, i.e., decreased barrier function, when compared with control cultures. 18:2 and β- d-xyloside increased albumin transfer additively, suggesting that the mechanisms by which 18:2 and β- d-xyloside alter the proteoglycan metabolism are different. Compared with the control group, treatment with 18:2 inhibited proteoglycan synthesis, decreased anionic properties of heparan sulfate proteoglycans in the cell monolayers and caused the release of a unique chondroitin sulfate proteoglycan into the culture media. Treatment with β- d-xyloside caused an increased incorporation of radioactive sulfate into glycosaminoglycans but inhibited proteoglycan synthesis. These results suggest that the fatty acid- and β- d-xyloside-induced impairment in endothelial barrier function may involve changes in the synthesis, release and physicochemical properties of proteoglycans.

Selective Disruption of Endothelial Barrier Function in Culture by Pure Fatty Acids and Fatty Acids Derived from Animal and Plant Fats

Endothelial cell integrity has been suggested to play a role in the development of atherosclerosis. The effects of fatty acids on endothelial barrier function were tested by measuring albumin transport across endothelial monolayers cultured on polycarbonate filters. Compared with control cultures, a 24-h exposure to 90 mumol/L lauric (12:0) and linoleic acid (18:2) but not to butyric (4:0), hexanoic (6:0), octanoic (8:0), decanoic (10:0), myristic (14:0), palmitic (16:0) or stearic acid (18:0) caused an increase in albumin transfer across endothelial monolayers. Selective enrichment of a "physiological" serum fatty acid mixture (FA-Mix; 90 mumol/L) with 90 mumol/L of 12:0 or 18:2 significantly increased albumin transfer, whereas enrichment with 90 mumol/L of 4:0, 16:0 or 18:0 significantly decreased albumin transfer relative to 180 mumol/L FA-Mix. Only 12:0- or 18:2-treated cultures showed increased Ca(++)-ATPase activity and the presence of lipid droplets. Fatty acids (60 mumol/L) extracted from butter fat and beef tallow had no effect on albumin transfer, whereas fatty acids extracted from chicken fat and corn oil consistently disrupted endothelial barrier function. This fat-induced disruption of endothelial barrier function seems to be related to the amount of 18:2 present in each fat source. These data indicate that unsaturated fats cause cellular perturbations that result in a decrease in endothelial barrier function in this model system, and that high dietary levels of unsaturated fats may be detrimental to cell integrity.

Age-related changes in barrier function in mouse brain I. Accelerated age-related increase of brain transfer of serum albumin in accelerated senescence prone SAM-P/8 mice with deficits in learning and memory

The time course of brain accumulation of radiolabelled human serum albumin ( 125I-HSA) injected intravenously and the transfer of 125I-HSA from blood to brain were evaluated in DDD mice using a double isotope technique. The brain accumulation of 125I-HSA at 3 and 9 h but not at 24 h postinjection and the brain transfer rates were significantly higher in 22-month-old DDD mice than in 4-month-old ones. The brain transfer rates of 125I-HSA were measured also in senescence accelerated prone mice (SAM-P/8) with age-related deficits in learning and memory, and in senescence accelerated resistant mice (SAM-R/I) without these deficits. The brain transfer rates were significantly higher in 13-month-old SAM-P/8 and 22-month-old SAM-R/1 than in 3-month-old mice of the same strains, respectively. The mean brain transfer rates in five regions observed in 22-month-old DDD mice, 22-month-old SAM-R/1 and 13-month-old SAM-P/8 increased by 31%, 41% and 51% compared with corresponding values in 3- or 4-month-old mice of the same strains. DDD mice and SAM-R/1 mice with normal characteristics of aging showed similar age-related significant changes in brain transfer rates. Age-related increase in the brain transfer rate was manifested at the youngest age in SAM-P/8 among the three strains examined. These findings show that the transfer of human serum albumin into the mouse brain increases with aging and suggest that the barrier function in the mouse brain against macromolecules changes with aging.

Arterial endothelial barrier dysfunction: actions of homocysteine and the hypoxanthine‐xanthine oxidase free radical generating system

1. Endothelial barrier function was assessed by use of an in vitro model in which transfer of trypan blue-labelled albumin was measured across monolayers of bovine aortic endothelial cells grown on polycarbonate membranes. 2. Addition of either hypoxanthine (0.2 mM) or xanthine oxidase (20 mu ml-1) alone during a 90 min incubation did not affect albumin transfer across endothelial cell monolayers, but a combination of both increased transfer. 3. The increase in albumin transfer induced by hypoxanthine and xanthine oxidase was abolished by catalase (3 u ml-1), reduced by allopurinol (4 mM), but unaffected by superoxide dismutase (6000 u ml-1), the hydroxyl radical scavengers, mannitol (15 mM), dimethylthiourea (10 mM) and N-(2-mercaptopropionyl)-glycine (1 mM), the iron chelator, deferoxamine (0.5 mM), ferric chloride (50 microM), an inhibitor of nitric oxide synthase, NG-nitro-L-arginine (30 microM), or the antioxidant, dithiothreitol (3 mM). 4. Hydrogen peroxide (0.1-30 mM) itself increased albumin transfer across endothelial cell monolayers, exhibiting a biphasic concentration-response curve. The increase induced by 0.1 mM hydrogen peroxide was abolished in the presence of 0.3 u ml-1 catalase whilst that induced by 10 mM hydrogen peroxide was abolished by 3000 u ml-1 catalase. 5. Homocysteine (0.5-1.5 mM) did not affect albumin transfer across endothelial monolayers when added alone, but when added in combination with copper sulphate (50 microM), which catalyses its oxidation, a significant increase in albumin transfer was observed. 6. The increase in albumin transfer induced by the combination of homocysteine (1.5 mM) and copper sulphate was abolished by catalase (1 u ml-1), but was unaffected by superoxide dismutase (6000 u ml-1), mannitol (15 mM), dimethylthiourea (1 mM) or deferoxamine (0.5 mM).7. The data suggest that the endothelial barrier dysfunction induced by the combination of hypoxanthine and xanthine oxidase is mediated solely by the action of hydrogen peroxide and not by superoxide anion, hydroxyl radical, peroxynitrite anion or hypochlorous acid. The copper-catalysed oxidation of homocysteine also induces endothelial barrier dysfunction through the generation of hydrogen peroxide.These findings may have relevance to the endothelial barrier dysfunction associated with ischaemia reperfusion injury and the atherogenic actions of homocysteine.

Relationship between blood flow and blood-brain barrier permeability of sodium and albumin in focal ischaemia of rats: a triple tracer autoradiographic study.

Local cerebral blood flow, the permeability of the blood-brain barrier to sodium and serum albumin, and the content of electrolytes were investigated in rats before and at 4 h and 24 h following permanent occlusion of the middle cerebral artery (MCA). Measurements were carried out by triple tracer autoradiography, using 131I-iodoantipyrin, 22NaCl and 125I-iodinated bovine serum albumin, respectively. Regional sodium and albumin transfer coefficients were calculated by multiple time point analysis, and correlated with the corresponding flow and tissue electrolyte values. In sham operated controls regional sodium and albumin transfer coefficients ranged between 2.16-2.30 x 10(-3) and 0.22-0.48 x 10(-3) ml/min per g, respectively. Four hours after MCA occlusion sodium and albumin transfer coefficients were unchanged although tissue sodium content was already increased. After 24 h the sodium-but not albumin-transfer coefficient increased 2-3 fold but the rise in tissue sodium content was slower than after 4 h. At both ischaemia times the unidirectional sodium influx was substantially higher than the actual changes of tissue sodium content. The development of stroke oedema is, therefore, not limited by the alterations of barrier permeability.

Modulation of barrier function of bovine aortic and pulmonary artery endothelial cells: dissociation from cytosolic calcium content.

1. Barrier function and cytosolic free calcium content [Ca2+]i was measured in monolayers of bovine pulmonary artery endothelial cells (BPAEC) and bovine aortic endothelial cells (BAEC). 2. Thrombin (1 u ml-1) increased albumin transfer across monolayers of BPAEC but not BAEC, yet induced biphasic increases in [Ca2+]i in both endothelial cell types, consisting of a rapid, initial phasic component which decayed to a lower, more sustained plateau phase. 3. 4 beta-Phorbol 12-myristate 13-acetate (PMA; 0.3-3000 nM) increased albumin transfer across monolayers of BPAEC and BAEC, but had no effect on basal levels of [Ca2+]i in either endothelial cell type. 4. Treatment of BPAEC and BAEC with forskolin (30 microM), an activator of adenylate cyclase, had no effect on resting transfer of albumin, but inhibited that stimulated by PMA (600 nM). It also inhibited the thrombin (1 u ml-1)-induced increase in albumin transfer across monolayers of BPAEC, but enhanced the plateau phase of the associated increase in [Ca2+]i. 5. Treatment of BPAEC and BAEC with either atriopeptin II (100 nM), an activator of particulate guanylate cyclase, or 8 bromo cyclic GMP (30 microM) had no effect on resting or PMA (600 nM)-stimulated transfer of albumin. Both agents did, however, inhibit the thrombin (1 u ml-1)-induced increase in albumin transfer across monolayers of BPAEC, but had no effect on the associated increase in [Ca2+]i. 6. These data suggest a dissociation between the ability of agents that increase or decrease albumin transfer and their effects on [Ca2+]i. Consequently, activation of protein kinase C may be the major stimulus for trans-endothelial transfer of macromolecular solutes. Endothelial barrier function is enhanced by elevation of either cyclic AMP or cyclic GMP content. Cyclic AMP appears to act by inhibiting the actions of protein kinase C, while cyclic GMP may act to inhibit a key step proximal to activation of this enzyme.

Iron and transferrrin uptake by brain and cerebrospinal fluid in the rat

Iron and transferrin uptake into the brain, CSF and choroid plexus, and albumin uptake into the CSF and choroid plexus, were determined after the intravenous injection of [ 59Fe- 125]Itransferrin and [ 131I]albumin into control rats aged 15, 21 and 63 days and 21-day iron-deficient rats. Iron uptake by the brain was unidirectional, greatly exceeded that of transferrin and was equivalent to 39 and 36% of the plasma iron pool per day in the 15-day control and 21-day iron-deficient rats. The rate of transferrin catabolism in the rats was only about 20% of the plasma pool per day. Iron and transferrin uptake into the brain and CSF decreased with increasing age and was greater in the iron-deficient than in the control 21-day rats. The quantity of 125I-transferrin recovered in the CSF could account for only a small proportion of the iron taken up by the brain. Albumin transfer to the CSF also decreased with age but was lower than that of transferrin and was not affected by iron deficiency. Similarly, the plasma: CSF concentration ratios of transferrin and albumin, as determined immunologically, decreased with age and were greater for transferrin than albumin. It is concluded that iron uptake by the brain is dependent on iron release from transferrin at the cerebral capillary endothelial cells with recycling of transferrin to the plasma and transfer of the iron into the brain interstitium. Only a small fraction of the transferrin bound by brain capillaries is transcytosed into the brain and CSF, this being one source of CSF transferrin while other sources are local synthesis and transfer from the plasma by the choroid plexuses.

Oxysterol-induced endothelial cell dysfunction in culture.

Cholesterol oxidation products (oxysterols), such as cholestan-3 beta,5 alpha,6 beta-triol (Triol), may be atherogenic by altering the barrier function of the vascular endothelium. We have shown that incubation of endothelial cell monolayers with Triol increased transendothelial albumin transfer (i.e., decreased barrier function) in a concentration- and time-dependent manner. Such dysfunction of endothelium could result from alterations in membrane characteristics, including changes in membrane-associated enzyme activities. To test this hypothesis, endothelial monolayers were treated with 20 microM Triol and the activities of selected membrane enzymes were measured at 0, 2, 4, 6, 12 and 24 hours. Calcium-adenosine triphosphatase (Ca(++)-ATPase) and sodium, potassium, magnesium-adenosine triphosphatase (Na+, K+, Mg(++)-ATPase) activities were significantly increased after 4 or 2 hours incubation with 20 microM Triol, respectively. 5'-nucleotidase activity was significantly elevated only after a 24-hour exposure to Triol, whereas there was no change in angiotensin-converting enzyme (ACE) activity in response to 20 microM Triol treatment at any time studied. Compared with all concentrations tested 40 microM Triol increased Ca(++)-ATPase activity most markedly, with a significant increase already after a 2-hour exposure. No major morphological changes were noted until 12 hours of exposure to 20 microM Triol; obvious cellular damage was observed by 24 hours. Cultures treated with Triol for 24 hours showed significant signs of toxicity, measured by an elevated [3H]adenine release, compared with control cultures. These data demonstrate that Triol alters the activity of certain membrane-bound enzymes, particularly Na+, K+, Mg(++)-ATPase and Ca(++)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)

Atriopeptin-induced increases in endothelial cell permeability are associated with elevated cGMP levels.

To investigate the mechanism of nonrenal capillary hyperfiltration, we studied the effect of atriopeptin (AP) III and AP I on permeability and intracellular cyclic nucleotide levels in cultured bovine pulmonary artery endothelial cell monolayers. Permeability to albumin was assessed by the albumin transfer rate across endothelial cell monolayers, following a 4-h incubation with atriopeptins. AP III (0.01, 0.1, and 1 microM) caused a concentration-dependent increase in the albumin transfer rate. AP III induced a threefold increase in intracellular guanosine 3',5'-cyclic monophosphate (cGMP) levels during the incubation period. A phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), enhanced the AP III-induced increase in permeability and cGMP accumulation by 16-fold at maximum. 8-Bromoguanosine 3',5'-cyclic monophosphate, a hydrolysis-resistant cGMP analogue, caused a slight but significant increase in permeability. In contrast, AP I, a weak agonist of the cGMP-coupled ANP receptor, did not elicit an increase in permeability at concentrations of 0.1 and 1 microM. Although AP I (1 microM) caused a significant increase in cGMP by 33 and 60% in the absence and presence of IBMX, the increase was markedly less compared with AP III. AP III did not cause a change in intracellular cAMP levels during the incubation period. These observations suggest that in our system AP III increases the permeability of endothelial cell monolayers in association with an elevated cGMP level. Thus an increase in permeability might be involved in the mechanism of ANP-induced capillary hyperfiltration.

Disruption of endothelial barrier function by lipolytic remnants of triglyceride-rich lipoproteins

Remnants, resulting from the lipolysis of triglyceride-rich lipoproteins, injured cultured endothelial cells and resulted in decreased barrier function of the vascular endothelium. Endothelial cells were cultured on micropore filters. Albumin transfer across endothelial cell monolayers was measured after a 24-h exposure to media enriched with control or in vitro-lipolyzed samples of various hypertriglyceridemic (HTG) sera and its isolated lipoprotein (VLDL, LDL and HDL) and serum free protein ( d > 1.21 g/ml) fractions. Compared with control cultures, neither control HTG serum nor its isolated lipoprotein and serum-free protein fractions had any effect on albumin transfer. In contrast, lipolyzed HTG (L-HTG) serum and all of its isolated lipoprotein fractions (L-VLDL, L-IDL, L-LDL and L-HDL) caused a marked decrease in endothelial barrier function, evidenced by a significant increase in albumin transfer across endothelial monolayers. The L-IDL and L-HDL fractions were more effective in increasing albumin transfer than the L-VLDL and L-LDL fractions. The extent of the L-IDL and L-HDL mediated increases in albumin transfer was concentration dependent. An exposure of 12 h was required for L-HDL to increase albumin transfer. The L-HDL mediated increase in albumin transfer was reversible only after a 12-h exposure at low concentrations. The free protein fraction from L-HTG serum had no significant effect on the barrier function of endothelial cells. The presence of normolipidemic HDL in culture medium prevented disruption of the endothelial barrier induced by L-IDL but not by L-HDL. The decrease in endothelial barrier function induced by lipolyzed samples of HTG serum or lipoproteins appeared to be correlated with the level of free fatty acids contained in lipolytic remnants. Enrichment of LDL, and in particular HDL, with fatty acid significantly increased albumin transfer. Compared with lipolyzed samples, sera/lipoproteins oxidized in vitro by Cu 2+, ions had little effect on endothelial barrier function, which did not correlate with their respective thiobarbituric acid-reacting substance (TBARS) values. TBARS remained within normal range after L-HDL incubation with endothelial cells for up to 48 h. At most concentrations tested, exposure to lipolyzed but not oxidized lipoproteins resulted in morphological perturbations of cell monolayers. These data suggest that lipolytic remnants of triglyceride-rich lipoproteins may play an important role in the development of atherosclerosis by decreasing the barrier function of the vascular endothelium. The remnant-induced injury of the arterial wall may permit the entry of cholesterol-rich lipolytic remnants as well as LDL into the arterial wall.

Enhanced permeabilities of cationized-bovine serum albumins at the blood-nerve and blood-brain barriers in awake rats.

Permeability-surface area products (PAs) of the blood-nerve barrier (BNB) and blood-brain barrier (BBB) to 125I-labeled native bovine serum albumin (nBSA, pI approximately 4), and to 2 cationized albumins (cBSA) of differing pI (pI approximately 8 and 11), were quantitatively determined in awake rats, using an i.v. bolus injection technique. Mean PAs of the BNB and BBB to 125I-nBSA, after a circulation time of up to 120 min, were (0.17 +/- 0.23) and (0.09 +/- 0.05) x 10(-5) ml/s.g. wet wt, respectively (n = 12 rats), and were not significantly different from 0 (P greater than 0.05). Mean PAs of the BNB and BBB to 125I-cBSA (pI approximately 8), after circulation time of 12 min, were (1.9 +/- 0.1) and (1.7 +/- 0.1) x 10(-5) ml/s.g wet wt, respectively (n = 8). Significant greater PAs, at both the BNB and BBB to 125I-cBSA (pI approximately 11) [(8.2 +/- 1.8) and (3.0 +/- 0.6) x 10(-5) ml/s.g wet wt, respectively (n = 12)], than both PA's of nBSA and cBSA (pI approximately 8) were found. The accumulation of 125I-cBSA in epi-perineurial tissues also was higher than that of 125I-nBSA, and was related to the degree of cationization. Our results indicate that, as at the BBB, the transfer of cationized serum albumin is enhanced over that of native albumin at the BNB of the mammalian peripheral nerve.

Interleukin-2 directly increases albumin permeability of bovine and human vascular endothelium in vitro.

The direct effects of interleukin-2 (IL-2) on albumin permeability of cultured bovine pulmonary artery endothelial cell (BPAEC) and human arterial endothelial cell (HAEC) monolayers were studied. BPAEC were exposed to IL-2 (500 to 25,000 U/ml) for 4 h. The steady-state transfer rate of [125I]albumin across the BPAEC monolayer was 3.3 +/- 0.4%/h (n = 10) in control BPAEC (diluent alone), was significantly increased in BPAEC exposed to 500 U/ml of IL-2 (72 +/- 3% above control values, n = 6, P less than 0.02), and further increased in BPAEC exposed to 5,000 U/ml (60 +/- 2% increase above 500 U/ml values, n = 5, P less than 0.02). No further increase was noted after exposure to 25,000 U/ml of IL-2. Additionally, no further increase in [125I]albumin transfer rates was noted in BPAEC exposed to 5,000 U/ml of IL-2 for 24 versus 4 h. Similar changes were found using HAEC. Preincubation of HAEC with an anti-IL-2 low-affinity receptor antibody (anti-IL-2R alpha) inhibited the IL-2-induced permeability increase. Expression of IL-2R alpha receptors in HAEC incubated with 5,000 U/ml of IL-2 for 4 h was also found. Thus, IL-2 appears to have a direct effect on cultural arterial endothelial monolayers not requiring the presence of other cell types or serum proteins. IL-2-induced increases in endothelial macromolecular permeability may play an important role in the pathogenesis of the IL-2-induced vascular leak syndrome seen in vivo.

Interleukin-1 alpha and -beta augment pulmonary artery transendothelial albumin flux in vitro.

Human recombinant interleukin-1 alpha (rIL-1 alpha) and -beta were studied to determine whether either could alter the permeability of bovine pulmonary artery endothelial cell monolayers. Endothelial cells were grown to confluence on filters mounted in chemotaxis chambers placed in wells. Barrier function of the monolayers was assessed by placing 14C-labeled bovine serum albumin ([14C]BSA) in the upper chamber and sampling the lower well for [14C]BSA. rIL-1 alpha induced a significant (P less than 0.01) dose- and time-dependent increase in transendothelial [14C]BSA flux. rIL-1 alpha exposures as brief as 30 min increased permeability, but the increased albumin transfer could not be demonstrated before 4 h after exposure. Exposures up to 6 h were reversible at 24 h. rIL-1 alpha induced significantly (P less than 0.01) greater increments in [14C]BSA flux than did equivalent exposures to rIL-1 beta. No important differences between bovine and human rIL-1 beta were demonstrated. Increased transendothelial flux could not be ascribed to either endothelial cytotoxicity or growth inhibition. There was no additive or synergistic relationship between rIL-1 alpha and human recombinant tumor necrosis factor-alpha. Our studies suggest that IL-1 alpha and -beta may play a role in the pathogenesis of pulmonary vascular leak.

Zinc Deficiency Alters Barrier Function of Cultured Porcine Endothelial Cells

Zinc is necessary for normal membrane function and stability. We postulated that Zn deficiency may disrupt the integrity of the vascular endothelium by decreasing its barrier function. To test this hypothesis, endothelial cells were cultured on polycarbonate filters and exposed to media enriched with either 1% fetal bovine serum (FBS) (low FBS; total Zn, 1.07 mumols/L medium) or 5% FBS (control; total Zn, 2.29 mumols/L) or low FBS plus two supplemental levels of Zn, 3.36 and 5.66 mumols total zinc/L. Endothelial cell barrier function, expressed as albumin transfer across cultured endothelial monolayers, was significantly lower in cultures exposed to low FBS compared with control medium. Supplementation with 5.66 mumols total Zn/L completely restored endothelial barrier function. A divalent cation chelator, 1,10-orthophenanthroline, was used to induce Zn deficiency in vitro. Compared with control cultures, the presence of 1,10-orthophenanthroline in the culture medium resulted in markedly lower endothelial barrier function that was increased by the addition of Zn but not calcium or magnesium. Activity of the membrane-bound zinc-dependent angiotensin-converting enzyme (ACE) was depressed by low zinc medium, whereas membrane-bound Ca(2+)-ATPase and total ATPase were not depressed. Furthermore, cells cultivated in low zinc medium did not have greater cytosolic release of adenine, indicating no increase in cell injury or death. These data suggest that Zn is vital to endothelial cell integrity and that Zn may play an important role in vascular endothelial barrier function.

Increased permeability across the blood-nerve barrier of albumin glycated in vitro and in vivo from patients with diabetic polyneuropathy.

The blood-nerve transfer of human plasma albumin glycated with D-glucose was investigated by measuring the permeability coefficient-surface area product (PS) of the blood-nerve barrier to radioiodinated albumin in normal adult rat sciatic nerve. Human albumin (ALB) from normal individuals, freshly isolated by CM-Affi-Gel Blue affinity chromatography, was glycated in vitro for 1, 3, 10, 19, and 30 weeks. Glycated ALB (gALB) was separated from the nonglycated form by boronate-affinity chromatography. The efficiency of this separation was assessed by chromatography of ALB glycated with [14C]glucose and by rechromatography of isolated ALB and gALB after radioiodination. The gALB was also shown to have a higher molecular weight and be completely separated from ALB after SDS/pore gradient electrophoresis in a Tris borate/EDTA buffer. After 1 week of glycation, the gALB PS was 2.2-fold greater than the ALB PS (0.724 +/- 0.063 x 10(-6) vs. 0.328 +/- 0.053 x 10(-6) ml.g-1.s-1; mean +/- SD; P less than 0.0001) and it increased with the time of glycation reaching a maximum value of 16.2-fold greater at 30 weeks (4.656 +/- 1.117 x 10(-6) vs. 0.288 +/- 0.042 x 10(-6) ml.g-1.s-1; mean +/- SD; P less than 0.0001). No change was observed in the residual endoneurial plasma volume. In addition, the PS of gALB isolated from patients with diabetic polyneuropathy was significantly increased (P less than 0.0001) compared to the PS for ALB isolated from the same patients. It is hypothesized that the increased permeability of gALB and presumably other glycated serum components across the blood-nerve barrier, as well as the observed quantitative increase in ALB, IgG, and IgM in sural nerve biopsies from patients with diabetic polyneuropathy contribute to the development of diabetic polyneuropathy over a prolonged period of time by mechanisms that might involve osmotic changes in the nerve microenvironment, direct toxic effects of glycated macromolecules on cells within the endoneurium, or nerve damage by classical immunological mechanisms due to trapping of glycated immunoglobulins within nerve.

Transfer of 125I-albumin from blood to brain in newborn rats and the effect of hyperbilirubinemia on the transfer.

Transfer of albumin from blood into cerebellum and cerebrum was examined in postnatal rats. 125I-Albumin was injected into rats on day 3, 7, 14, 28 or 70. The ratio of albumin concentration in the cerebellum or in the cerebrum to that in the plasma 2 h after the injection, hereinafter termed the relative albumin level (RA), was calculated and used as a criterion for the albumin transfer. The RA in both the cerebellum and the cerebrum decreased with age, but plateaued between days 28 and 70. The level was significantly higher in the cerebellum than in the cerebrum on days 3 and 7. This was not observed, however, on days 28 and 70. Significant correlations were indicated between 125I-albumin levels in the plasma and those in the cerebellum or cerebrum on days 7 and 14, but not on day 28. No significant difference in the RA in the cerebellum and the cerebrum was detected between jaundiced homozygous Gunn rats and control nonjaundiced rats at all ages examined. These results demonstrated that albumin could enter the brain tissue from the blood at least until postnatal day 14 and that the endogenous bilirubin had no effect on the albumin transfer into the brain. The relationship between the immaturity of brain and the transfer of albumin into the brain was discussed.

Species‐specific transfer of plasma albumin from blood into different cerebrospinal fluid compartments in the fetal sheep.

1. The blood-cerebrospinal fluid (CSF) transfer of endogenous sheep albumin and several exogenous species of albumin has been investigated in different CSF compartments of the immature fetal sheep brain, at an early stage of development (60 days gestation, term is 150 days) when the CSF concentration of total protein is high. 2. There were marked differences in the steady-state CSF/plasma ratios for all species of albumin (including endogenous sheep albumin) between different CSF compartments. Ratios measured in the cisterna magna were significantly higher than those in the dorsal subarachnoid space, which in turn were higher than those in the lateral ventricles. The ratios for endogenous sheep albumin were (%; mean +/- S.E.M.): lateral ventricle (LV), 4.0 +/- 0.03; dorsal subarachnoid (DSA), 6.1 +/- 1.0; cisterna magna (CM), 13.7 +/- 0.8. 3. Three hours after I.V. injection, the CSF/plasma ratios for bovine albumin (LV, 2.0 +/- 0.2; DSA, 2.4 +/- 0.1; CM, 7.2 +/- 0.7%) were significantly lower than the ratio for endogenous sheep albumin in all three compartments. The ratios for human albumin (LV, 0.7 +/- 0.2; DSA, 1.0 +/- 0.2; CM, 3.9 +/- 0.4%) were significantly lower than those for bovine albumin. 4. In all three CSF compartments, the endogenous sheep albumin ratios were higher than would be expected on the basis of transfer by passive mechanisms. Conversely, steady-state CSF/plasma ratios for [3H]sucrose and [14C]inulin were consistent with passive transfer, and there were no differences between the ratios for these markers measured in each of the three CSF regions. 5. Goat albumin and [35S]sheep albumin ratios were not significantly different, 5 h after injection, from the endogenous sheep albumin levels in each of the three CSF compartments. 6. It is concluded that in the 60-day-old fetal sheep, all of the endogenous albumin in CSF is derived from the plasma by a specific transfer mechanism that can distinguish between different species of the same protein. There is also some evidence of a small passive component of blood-CSF albumin transfer. 7. Immunocytochemical evidence suggests that the route of transfer from blood to CSF is transcellular, through the choroid plexus epithelial cells. 8. Regional variations in albumin ratios are probably due to differences in specific transfer into each CSF compartment. This is reflected in a differential immunocytochemical staining for albumin in choroid plexus epithelial cells from different regions of the brain. 9. The results are discussed in terms of differences in albumin amino acid sequences, structural homologies, and transfer by a specific transcellular mechanism.

Initial plasma disappearance and tissue uptake of 131I-albumin in normal rabbits

The simultaneous plasma disappearance curves of 131I-albumin and 125I-fibrinogen were recorded in normal rabbits for 1 hr. Using fibrinogen as a plasma reference, the disappearance curves of albumin were shown to contain two separate phases of efflux: one fast from zero to 10 min, comprising 8% of the total tracer; and one slow appearing in the interval of 10 to 60 min, containing another 9% of the tracer. Total albumin escape was analyzed to yield an initial slope of 0.024 ± 0.004 min −1, corresponding to a wholebody unidirectional albumin clearance (Cl(0)) of 0.090 ± 0.009 ml(min∗100 g) −1. The distribution of efflux was assessed by biopsy uptakes using the same tracers in spleen, kidney, heart, lung, liver, intestine, skin, muscle, and brain. The disappearance curve generally reflects a biphasic pattern of uptake in peripheral tissue, predominantly by muscle and lung. The rapid phase has contributions from the fast near equilibration of liver, and intestine and skin are significant codeterminants of the slow phase. Due to their low body masses highly perfused organs such as kidney, spleen, and heart have little influence on the plasma disappearance. In accordance, the Cl(0) determined for the wholebody was higher than initial clearances found in skin (0.053 ml(min∗100 g) −1) and muscle (0.054 ml(min∗100 g) −1), but much lower than those found in the highly perfused organs. The initial (unidirectional) rates of peripheral albumin transfer demonstrated, ranged from 10 to 30 times higher than estimates of lymphatic return, suggesting that transcapillary albumin exchange is mediated by high-rate bidirectional diffusion. The rapid decrease of net albumin exchange rates suggests a second, highly significant barrier located within the interstitial matrix, which restricts plasma escape and reduces plasma to lymph albumin transport.