Purpurogallin is a more powerful protector of kidney cells than Trolox and allopurinol.

Phase contrast and electron microscopic experiments demonstrated that oxyradicals generated with xanthine oxidase and hypoxanthine markedly damage rat kidney mesangial and porcine tubular epithelial cells. Purpurogallin, a phenol found in oak nutgalls, prolongs survival of the xanthine oxidase exposed renal cells three- to nine-fold longer than those without purpurogallin present. At levels equimolar to purpurogallin, either Trolox or allopurinol is less effective in delaying cell necrosis. Purpurogallin scavenges not only xanthine oxidase generated oxyradicals, but also non-enzymatically produced peroxyl radicals, more actively than equimolar levels of Trolox or allopurinol. Purpurogallin inhibits xanthine oxidase with severalfold higher potency than allopurinol and its more active metabolite oxypurinol. Therefore, purpurogallin is a stronger antioxidant than Trolox and a more potent inhibitor of xanthine oxidase than allopurinol as well as oxypurinol.     

Purpurogallin--a natural and effective hepatoprotector in vitro and in vivo.

Purpurogallin is a plant phenol that is sometimes added as an oxidation retardant to fats-oils or to certain fuels or lubricants. However, it was unknown if purpurogallin is cytoprotective. Here we examined this issue, both in isolated hepatocytes and in vivo. From 0.5 to 2.0 mM, purpurogallin prolongs survival of rat hepatocytes substantially against oxyradicals generated with xanthine oxidase and hypoxanthine. The protection was dose dependent and surpassed that given by such antioxidants as ascorbate, mannitol, superoxide dismutase, catalase, and Trolox, when each was examined at or near its optimal concentration in the same system. When 1.5, 3, and 6 mumol of purpurogallin in saline were infused into rats with postischemic livers shortly before reperfusion, the mean hepatic salvages were 42, 76, and 86%, respectively. Such salvage effects would rank purpurogallin highly among the hepatoprotectors known. Over the range of 31 to 500 microM, purpurogallin inhibited the rate of O2 consumption in the xanthine oxidase reaction by approximately 90%, which was 2- to several-fold higher than the inhibition elicited by allopurinol over the same concentrations. Thus, purpurogallin is an effective natural hepatoprotector that may operate partly or principally as an inhibitor of xanthine oxidase.     

The cytoprotective effect of Trolox demonstrated with three types of human cells

Trolox, a hydrophilic analogue of alpha-tocopherol, was reported to scavenge peroxyl radicals better than vitamin E in sodium dodecyl sulfate micelles and in liposomes. However, it was not known if Trolox protects human cells against oxyradical damage or if it acts as an antioxidant there. Here we demonstrate that Trolox prolonged substantially the survival of human ventricular myocytes and hepatocyte against oxyradicals generated with xanthine oxidase plus hypoxanthine, and prevented lysis of red cells exposed to an azo-initiator (2,2'-azo-bis(2-amidinopropane) HCl). Note that Trolox did not inhibit xanthine oxidase. In each cell type, the protection by Trolox was dose dependent and surpassed those given by such water-soluble antioxidants as ascorbic acid, superoxide dismutase, and (or) catalase, each examined at or near its optimal level in the same system. Using hepatocytes as a model, we further observed that Trolox reduced markedly the quantity of phospholipid conjugated dienes (a chemical imprint of oxyradical damage) in cells despite their exposure to oxyradicals. These data suggested that Trolox behaves as an antioxidant in cells as illustrated in hepatocytes.     

Albumin-bound bilirubins protect human ventricular myocytes against oxyradical damage.

Synthetic delta-bilirubin (i.e., bilirubin that is covalently bonded to albumin) and, to a lesser extent, bilirubin that is physically adsorbed to albumin substantially prolonged the survival of human ventricular myocytes against in situ generated oxyradicals. This cytoprotection, manifested at micromolar concentrations of either form of bilirubin, surpasses those given by unconjugated bilirubin alone, by equimolar levels of albumin (which has some cytoprotective activity), by known antioxidants ascorbate, mannitol, or Trolox, and by oxyradical scavenging enzymes superoxide dismutase (24,200 IU/L) plus catalase (92,000 IU/L) on the same cells. Our human cell-based data provide the first direct evidence that albumin-bound bilirubins, especially delta bilirubin, are potent circulating cytoprotectors. The latter may be important in defending, among others, the myocardium, which is much less equipped in antioxidant defences than for example the liver or intestines.     

Cellular Antioxidant Properties of Human Natural Killer Enhancing Factor B

The protein, NKEF (natural killer enhancing factor), has been identified as a member of an antioxidant family of proteins capable of protecting against protein oxidation in cell-free assay systems. The mechanism of action for this family of proteins appears to involve scavenging or suppressing formation of protein thiyl radicals. In the present study we investigated the antioxidant protective properties of the NKEF-B protein overexpressed in an endothelial cell line (ECV304). Nkef-B-transfected cells displayed significantly lower levels of reactive oxygen species (ROS) compared with control or vector-transfected cells. Tert-Butylhydroperoxide-induced ROS was 15% lower in nkef-8-transfected cells and cytotoxicity was slightly, though not significantly, lower. NKEF-B had no effect on ROS induced by menadione or xanthine plus xanthine oxidase. NKEF-B overexpression resulted in slightly (≈ 10%) lower levels of cellular glutathione (GSH) and had no effect on rate or extent of GSH depletion following either diethylmaleate (DEM) or buthionine sulfoximine (BSO) treatment. Lipid peroxidation, assessed as thiobarbituric acid-reactive substances, was 40% lower in nkef-B-transfected cells compared with vector-only-transfected cells. DEM-induced lipid peroxidation was suppressed by NKEF-B at DEM concentrations of 20 μM to 1 mM. At 10 mM DEM, lipid peroxidation was unaffected by NKEF-B. NKEF-B expression also protected cells against menadione-induced inhibition of [³H]-thymidine uptake. The NKEF-B protein appears most effective in suppressing basal low-level oxidative injury such as that produced during normal metabolism. These results indicate that overexpression of the NKEF-B protein promotes resistance to oxidative stress in this endothelial cell line.     

Endothelium-dependent effect of bradykinin and ATP on rabbit ventricular myocytes.

We have investigated the effects of bradykinin (BK) and ATP on Ca2+ transient induced by electrical-field stimulation in freshly isolated rabbit ventricular myocytes, in the presence or absence of rabbit aortic endothelial cells. BK and ATP induced an increase in intracellular Ca2+ concentration ([Ca2+]i) in the endothelial cells, but had no significant effect on Ca2+ transient in electrical-field stimulated ventricular myocytes. In the presence of cultured endothelial cells, the amplitude of Ca2+ transient induced by electrical stimulation in ventricular myocytes was decreased. BK and ATP further reduced the amplitude of Ca2+ transient induced by electrical stimulation in ventricular myocytes. These data show that BK and ATP have endothelium-dependent depressing effects on ventricular myocytes and indicate that substances released from endothelial cells in response to BK and ATP stimulation can modulate ventricular myocytes excitation-contraction coupling. However, identification of the cardioactive mediators produced by the endothelial cells requires further study.     

Effect of variable glutathione peroxidase activity on H2O2-related cytotoxicity in cultured aortic endothelial cells

Primary cultures of porcine aortic endothelial cells were used to assess the effects of O2 intermediates produced by 10-40 mU/ml xanthine oxidase (XO; +2 mM hypoxanthine) or 25-100 mU/ml glucose oxidase (GO; +5 mM glucose). A 60-min incubation in the presence of the enzyme systems resulted in a dose-dependent toxic effect with evidence of cytolysis (increased LDH release) and cell loss (decrease in DNA and protein content), when these indexes were measured 24 hr after completion of the enzyme reaction. Decreased [3H]thymidine incorporation into DNA was the most sensitive index of cell dysfunction for both enzyme systems. The effects of various scavengers and enzymes indicated that H2O2 was the main O2 intermediate involved in the cytotoxicity resulting from the XO-hypoxanthine reaction. Increased glutathione peroxidase activity associated with the addition of 2 X 10(-7) M selenomethionine to culture medium had a partial protective effect which could be related to an increased rate of H2O2 degradation. Evidence for increased DNA synthesis after injury was found in cells previously exposed to XO-hypoxanthine, the degree of increase in [3H]thymidine incorporation being dependent on the intensity of the initial cytotoxicity. Cultured endothelial cells provide a useful tool to evaluate the role of O2 intermediates in endothelial cell injury, to test the effects of protective agents, and to study the repair process.     

Vitamin E protects porcine but not bovine cultured aortic endothelial cells from oxygen-derived free radical injury due to hydrogen peroxide

The antioxidative effects of vitamin E (VE) are well known and have been demonstrated in in vitro studies. Since we previously observed that dextran sulfate was markedly more protective of porcine versus bovine aortic endothelial cells when damaged by hydrogen peroxide (H2O2), our objectives were to determine if a similar species difference could be observed with VE. The effects of VE or Trolox (a more water-soluble VE) against oxygen-derived free radical (OFR) injury produced by H2O2 was studied in porcine aortic endothelium (PAE) vs. bovine aortic endothelium (BAE) and bovine brain microvessel endothelium (BBME). VE or Trolox was added to culture medium for at least 24 h prior or immediately prior to H2O2 addition. In PAE, pretreatment with VE dissolved in either ethanol (VE-EtOH) or Tween 20 (VE-Tween 20), or Trolox dissolved in DMSO (Trolox-DMSO) was protective, shown by increased percent viable cells and reduced lactate dehydrogenase (LDH) release. EtOH, Tween 20 or DMSO alone was protective in PAE although DMSO or Tween 20 alone was less effective than when added with VE. VE-Tween 20 or Trolox-DMSO protected PAE when added just prior to H2O2 injury, but protection was significantly less than with pretreatment. DMSO immediately prior to H2O2 injury had no protective effect. Tween 20 immediately prior resulted in complete cell death. In BAE and BBME, pretreatment with VE-EtOH, EtOH, Trolox-DMSO, or DMSO alone had little or no protective effect. Pretreatment with VE-Tween 20 or Tween-20 alone was protective of BAE with Tween 20 being more effective than VE-Tween 20 suggesting that Tween 20 was the protective agent. These studies show that the protective effects of VE and Trolox as well as DMSO, EtOH, and Tween-20 are species dependent.     

Purine metabolism in cultured aortic and coronary endothelial cells

Purine salvage pathways in cultured endothelial cells of macrovascular (pig aorta) and microvascular (guinea pig coronary system) origin were investigated by measuring the incorporation of radioactive purine bases (adenine or hypoxanthine) or nucleosides (adenosine or inosine) into purine nucleotides. These precursors were used at initial extracellular concentrations of 0.1, 5, and 500 microM. In both types of endothelial cells, purine nucleotide synthesis occurred with all four substrates. Aortic endothelial cells salvaged adenine best among purines and nucleosides when applied at 0.1 microM. At 5 and 500 microM, adenosine was the best precursor. In contrast, microvascular endothelial cells from the coronary system used adenosine most efficiently at all concentrations studied. The synthetic capacity of salvage pathways was greater than that of the de novo pathway. As measured using radioactive formate or glycine, de novo synthesis of purine nucleotides was barely detectable in aortic endothelial cells, whereas it readily occurred in coronary endothelial cells. Purine de novo synthesis in coronary endothelial cells was inhibited by physiological concentrations of purine bases and nucleosides, and by ribose or isoproterenol. The isoproterenol-induced inhibition was prevented by the beta-adrenergic receptor antagonist propranolol. The end product of purine catabolism in aortic endothelial cells was found to be hypoxanthine, whereas coronary endothelial cells degraded hypoxanthine further to xanthine and uric acid, a reaction catalyzed by the enzyme xanthine dehydrogenase.     

Novel complexes of guanylate cyclase with heat shock protein 90 and nitric oxide synthase

Soluble guanylate cyclase (sGC) is an important downstream intracellular target of nitric oxide (NO) that is produced by endothelial NO synthase (eNOS) and inducible NO synthase (iNOS). In this study, we demonstrate that sGC exists in a complex with eNOS and heat shock protein 90 (HSP90) in aortic endothelial cells. In addition, we show that in aortic smooth muscle cells, sGC forms a complex with HSP90. Formation of the sGC/eNOS/HSP90 complex is increased in response to eNOS-activating agonists in a manner that depends on HSP90 activity. In vitro binding assays with glutathione S-transferase fusion proteins that contain the alpha- or beta-subunit of sGC show that the sGC beta-subunit interacts directly with HSP90 and indirectly with eNOS. Confocal immunofluorescent studies confirm the subcellular colocalization of sGC and HSP90 in both endothelial and smooth muscle cells. Complex formation of sGC with HSP90 facilitates responses to NO donors in cultured cells (cGMP accumulation) as well as in anesthetized rats (hypotension). These complexes likely function to stabilize sGC as well as to provide directed intracellular transfer of NO from NOS to sGC, thus preventing inactivation of NO by superoxide anion and formation of peroxynitrite, which is a toxic molecule that has been implicated in the pathology of several vascular diseases.     

A new member of the natriuretic peptide family is present in the venom of the green mamba (Dendroaspis angusticeps).

This paper describes the purification, sequence, and biological properties of a 38-amino acid residue peptide from the venom of Dendroaspis angusticeps which shared important sequence homologies with natriuretic peptides. Dendroaspis natriuretic peptide (DNP) relaxed aortic strips that had been contracted by 40 mM KCl with a potency (K0.5 = 20 nM) similar to that of atrial natriuretic peptide (ANP) and larger than that of C type natriuretic peptide (CNP). The relaxing actions of ANP and DNP (both at 100 nM) were mutually exclusive. Bovine aortic endothelial cells responded to ANP (K0.5 = 3 nM) and DNP (K0.5 = 3 nM) but not to CNP by a large activation of guanylate cyclase. Rat aortic myocytes showed larger cGMP responses to CNP (K0.5 = 10 nM) than to ANP or DNP (K0.5 = 100 nM). Finally, DNP completely prevented the specific 125I-ANP binding to clearance receptors in cultured aortic myocytes with a potency (Kd = 10 nM) that was less than that of ANP (Kd = 0.3 nM). It is concluded that DNP is a new member of the family of natriuretic peptides and that it recognizes ANPA receptors and clearance, ANPc receptors, but not CNP-specific ANPB receptors.     

Anti-inflammatory functions of purpurogallin in LPS-activated human endothelial cells

Enzymatic oxidation of commercially available pyrogallol was efficiently transformed to an oxidative product, purpurogallin. Purpurogallin plays an important role in inhibiting glutathione S-transferase, xanthine oxidase, catechol O-methyltransferase activities and is effective in the cell protection of several cell types. However, the anti-inflammatory functions of purpurogallin are not well studied. Here, we determined the effects of purpurogallin on lipopolysaccharide (LPS)-mediated proinflammatory responses. The results showed that purpurogallin inhibited LPS-mediated barrier hyper-permeability, monocyte adhesion and migration and such inhibitory effects were significantly correlated with the inhibitory functions of purpurogallin on LPS-mediated cell adhesion molecules (vascular cell adhesion molecules, intracellular cell adhesion molecule, E-selectin). Furthermore, LPS-mediated nuclear factor-κB (NF-κB) and tumor necrosis factor-α (TNF-α) releases from HUVECs were inhibited by purpurogallin. Given these results, purpurogallin showed its anti-inflammatory activities and could be a candidate as a therapeutic agent for various systemic inflammatory diseases. [BMB reports 2012; 45(3): 200-205].     

Responses of vascular endothelial oxidant metabolism to lipopolysaccharide and tumor necrosis factor-alpha.

Quantification of intracellular and extracellular levels and production rates of reactive oxygen species is crucial to understanding their contribution to tissue pathophysiology. We measured basal rates of oxidant production and the activity of xanthine oxidase, proposed to be a key source of O2- and H2O2, in endothelial cells. Then we examined the influence of tumor necrosis factor-alpha and lipopolysaccharide on endothelial cell oxidant metabolism, in response to the proposal that these inflammatory mediators initiate vascular injury in part by stimulating endothelial xanthine oxidase-mediated production of O2- and H2O2. We determined a basal intracellular H2O2 concentration of 32.8 +/- 10.7 pM in cultured bovine aortic endothelial cells by kinetic analysis of aminotriazole-mediated inactivation of endogenous catalase. Catalase activity was 5.72 +/- 1.61 U/mg cell protein and glutathione peroxidase activity was much lower, 8.13 +/- 3.79 mU/mg protein. Only 0.48 +/- 0.18% of total glucose metabolism occurred via the pentose phosphate pathway. The rate of extracellular H2O2 release was 75 +/- 12 pmol.min-1.mg cell protein-1. Intracellular xanthine dehydrogenase/oxidase activity determined by pterin oxidation was 2.32 +/- 0.75 microU/mg with 47.1 +/- 11.7% in the oxidase form. Intracellular purine levels of 1.19 +/- 1.04 nmol hypoxanthine/mg protein, 0.13 +/- 0.17 nmol xanthine/mg protein, and undetectable uric acid were consistent with a low activity of xanthine dehydrogenase/oxidase. Exposure of endothelial cells to 1000 U/ml tumor necrosis factor (TNF) or 1 microgram/ml lipopolysaccharide (LPS) for 1-12 h did not alter basal endothelial cell oxidant production or xanthine dehydrogenase/oxidase activity. These results do not support a casual role for H2O2 in the direct endothelial toxicity of TNF and LPS.     

Expression of intercellular adhesion molecule-1 induced by high glucose concentrations in human aortic endothelial cells

We examined the effects of high glucose concentrations on the expression of adhesion molecules in human aortic endothelial cells. Expression levels of both mRNA and protein of intercellular adhesion molecule-1 (ICAM-1) were increased after incubation of endothelial cells with 30 mM glucose for 24 h. The effect of glucose on ICAM-1 was concentration dependent, partially attributable to osmolarity, and enhanced by glycated-collagen. Staurosporine (10 nM), epalrestat (10 microM) suppressed the expression of ICAM-1 mRNA and protein induced by high glucose to variable extents. Aminoguanidine (50 mM) suppressed the expression of ICAM-1 protein. It is thought that soluble ICAM-1 protein is produced by shedding in human aortic endothelial cells because RNA for the soluble form of ICAM-1 formed by variant splicing has not been detected. These results show that glucose is an important determinant of ICAM-1 expression in endothelial cells, and suggest that ICAM-1 molecules induced by hyperglycemia may contribute to the development of atherosclerosis in diabetes mellitus.     

Differential effects of diabetes on the expression of the gp91phox homologues nox1 and nox4

The nox2-dependent NADPH oxidase was shown to be a major superoxide source in vascular disease, including diabetes. Smooth muscle cells of large arteries lack the phagocytic gp91phox subunit of the enzyme; however, two homologues have been identified in these cells, nox1 and nox4. It remained to be established whether also increases in protein levels of the nonphagocytic NADPH oxidase contribute to increased superoxide formation in diabetic vessels. To investigate changes in the expression of these homologues, we measured their expression in aortic vessels of type I diabetic rats. Eight weeks after streptozotocin treatment, we found a doubling in nox1 protein expression, while the expression of nox4 remained unchanged. This was associated with a significant increase in the NADPH oxidase activity in membrane fractions of diabetic heart and aortic tissue. Furthermore, we observed a decreased sensitivity of diabetic vessels to acetylcholine and nitroglycerin and a decrease in both acetylcholine-stimulated NO production and phosphorylation of VASP, despite an increase in endothelial NO synthase (NOSIII) expression. In addition, xanthine oxidase activity was markedly increased in plasma and 100,000 g supernatant of cardiac tissue of diabetic rats, while myocardial mitochondrial superoxide formation was only weakly enhanced. We conclude that in addition to phagocytic NADPH oxidase, also nonphagocytic, vascular NADPH oxidase subunit nox1, uncoupled NOSIII, and plasma xanthine oxidase contribute to endothelial dysfunction in the setting of diabetes mellitus.     

Superoxide potently induces ceramide formation in glomerular endothelial cells

Recent evidence suggests that the sphingolipid-derived second messenger ceramide and oxidative stress are intimately involved in apoptosis induction. Here we report that exposure of microcapillary glomerular endothelial cells to superoxide-generating substances, including hypoxanthine/xanthine oxidase and the redox cyclers DMNQ and menadione results in a dose-dependent and delayed increase in the lipid signaling molecule ceramide. Long-term incubation of endothelial cells for 2-30 h with either DMNQ or hypoxanthine/xanthine oxidase leads to a continuous increase in ceramide levels. In contrast, short-term stimulation for 1 min up to 1 h had no effect on ceramide formation. The DMNQ-induced delayed ceramide formation is dose-dependently inhibited by reduced glutathione, whereas oxidized glutathione was without effect. Furthermore, N-acetylcysteine completely blocks DMNQ-induced ceramide formation. All superoxide-generating substances were found to dose-dependently trigger endothelial cell apoptosis. In addition, glutathione and N-acetylcysteine also prevented superoxide-induced apoptosis and implied that ceramide represents an important mediator of superoxide-triggered cell responses like apoptosis.     

Trolox and ascorbate: are they synergistic in protecting liver cells in vitro and in vivo?

From in vitro studies involving multilamellar liposomes or other artificial systems, several groups of workers have deduced that Trolox (a water-soluble analogue of vitamin E) and ascorbate are synergistic antioxidants. Here, we demonstrate that while Trolox and ascorbate individually protect cultured hepatocytes against oxyradicals generated either with xanthine oxidase plus hypoxanthine or with hydrogen peroxide, the two antioxidants do not appear to be synergistic when used in equimolar combinations. Also, in a rat model of hepatic ischemia-reperfusion, we observed that infusion of Trolox or ascorbate (7.5-10 mumol/kg body weight) into the postischemic liver reduced the reperfusion injury by 76 or 67%, respectively. However, when both compounds were used together (each at the same dose as used separately), the organ salvage amounted to only 79%. Therefore, there is no evidence of synergism between Trolox and ascorbate in our in vitro and especially in vivo systems.     

Link between free radicals and protein kinase C in glucose-induced alteration of vascular dilation

Development of vascular complications in diabetes has been linked to the quality of glucose regulation and characterized by endothelial dysfunction. The exact mechanism behind vascular complications in diabetes is poorly understood. However, alteration of nitric oxide (NO) biosynthesis or bioactivity is strongly implicated and the mechanism behind such alterations is still a subject for research investigations. In the present study, we tested the hypothesis that glucose-induced attenuation of vascular relaxation involves protein kinase C (PKC)-linked generation of free radicals. Vascular relaxation to acetylcholine (ACh; 10(-9)-10(-5) M), isoproterenol (10(-9)-10(-5) M), or NO donor, sodium nitropruside (SNP; 10(-9)-10(-6) M) was determined in phenylephrine (PE, 10(-7) M) pre-constricted aortic rings from Sprague-Dawley rats in the presence or absence of 30 mM glucose (30 min), L-nitro-arginine methyl ester (L-NAME; 10(-4) M for 15 min), a NO synthase inhibitor, or xanthine (10(-5) M), a free radical generator. ACh dose-dependently caused relaxation that was attenuated by L-NAME, glucose, or xanthine. Pre-incubation (15 min) of the rings with vitamin C (10(-4) M), an antioxidant or calphostin C (10(-6) M), a PKC inhibitor, restored the ACh responses. However, high glucose had no significant effects on SNP or isoproterenol-induced relaxation. ACh-induced NO production by aortic ring was significantly reduced by glucose or xanthine. The reduced NO production was restored by pretreatment with vitamin C or calphostin C in the presence of glucose, but not xanthine. These data demonstrate that oxidants or PKC contribute to glucose-induced attenuation of vasorelaxation which could be mediated via impaired endothelial NO production and bioavailability. Thus, pathogenesis of glucose-induced vasculopathy involves PKC-coupled generation of oxygen free radicals which inhibit NO production and selectively inhibit NO-dependent relaxation.     

Altered gene and protein expression by Nm23-H1 in metastasis suppression

The aim of our work was to study (1) the antioxidant properties of lipoic acid (LA) and its reduced metabolite dihydrolipoic acid (DHLA) formed by reduction of LA and (2) the effects of treatment with LA and DHLA on (a) K(+) efflux from human red blood cells and (b) post-ischemic recovery and oxidative stress in isolated perfused rat hearts challenged with an ischemia-reperfusion (IR) sequence. In vitro, we used xanthine and xanthine oxidase to generate superoxide anion, which is not directly measurable by electron paramagnetic resonance (EPR), but specifically oxidizes the spin probe CPH into an EPR-detectable long lasting CP(*) nitroxide radical. While 5 mM of LA was ineffective in reducing the kinetics of CP(*) nitroxide formation, DHLA was shown to lessen this rate in a dose-dependent manner and at 30 mM was even more efficient than 300 UI/ml SOD. These results are in agreement with the fact that DHLA is able to directly scavenge superoxide anion. Red cells are a good model to investigate oxidative damage in biological membranes; hence, we used a suspension of erythrocytes incubated with 2,2(')-azobis(2-amidinopropane) hydrochloride (AAPH) which generates in vitro free radicals. DHLA provided more effective protection of red cells membranes than LA; DHLA was comparable to Trolox for its antioxidant potency. In vivo, treatment of rats (50 mg/kg/day i.p. for 7 days) with LA induced a slight increase in coronary flow (CF) in isolated perfused hearts, after 30 min of global total ischemia. This effect was not associated with an improvement in contractile function and reduction of myocardial oxidative stress. In conclusion, because of their ability to scavenge free radicals, LA and to an even greater degree DHLA were able to protect the membranes of red blood cells. This finding suggests that LA and DHLA might be useful in the treatment of diseases associated with oxidative stress such as diabetes.     

Protective mechanisms of Mg-gluconate against oxidative endothelial cytotoxicity.

The potential anti-radical properties and cytoprotective effects of Mg-gluconate were studied. When microsomal membranes were peroxidized by a *O2- driven, Fe-catalyzed oxy-radical system (R* = dihydroxyfumarate + Fe2+), Mg-gluconate inhibited lipid peroxidation (TBARS formation) in a concentration-dependent manner with IC50 being 2.3 mM. For the entire range of .25-2 mM, MgSO4 or MgCl2 were < or = 20% effective compared to Mg-gluconate. When cultured bovine aortic endothelial cells were incubated with the R* for 50 min. at 37 degrees C, 56% loss of total glutathione occurred. Pre-treatment (10 min.) of the cells with 0.25-4 mM Mg-gluconate before R* exposure significantly (p<0.05) prevented the GSH loss to varying degrees; the EC50 was 1.1 mM. In separate experiments, with 30 min. of free radical incubation of endothelial monolayers (approximately 65% confluent), cell survival/proliferation determined by the tetrazolium salt MTT assay, decreased to 38% of control at 24 hrs; Mg-gluconate concentration-dependently attenuated the lost cell survival with EC50 of approximately 1.3 mM. For comparison, the effects provided by MgSO4 or MgCl2 were significantly lower and were < or = 1/3 as potent as that produced by Mg-gluconate. In a Fenton-reaction system consisting of Fe(II)+ H2O2, Mg-gluconate but not other Mg-salts, significantly inhibited the formation of OH radicals as determined by the ESR DMPO-OH signal intensity. Mg-gluconate also dose-dependently inhibited the 'Fe-catalyzed' deoxyribose degradation suggesting that Mg-gluconate could displace Fe from 'catalytic sites' of oxidative damage. These data suggest that Mg-gluconate may serve as a more advantageous Mg-salt for clinical use due to its additional anti-radical and cytoprotective activities.