Olga Rounova

Wines and grape juices as modulators of platelet aggregation in healthy human subjects

To test the hypothesis that red wine, by virtue of its relatively high concentration of polyphenols, is more protective against atherosclerosis and coronary heart disease (CHD) than white wine, and that grape juice enriched in one of these, trans-resveratrol, may share some of these properties, studies were performed on 24 healthy males aged 26-45 years. Each consumed the following beverages for periods of 4 weeks: red wine, white wine, commercial grape juice and the same grape juice enriched with trans-resveratrol. Apart from the last beverage, 2 weeks abstinence was maintained before commencing the schedule. Blood was taken at the beginning and end of each schedule to determine plasma thromboxane B2 (TxB2) concentration and the IC50 (concentration required for 50% aggregation) for ADP and thrombin-induced platelet aggregation. White wine (P < 0.05) but not red wine increased the IC50 for ADP. Both wines increased the IC50 for thrombin (P < 0.02 and P < 0.001, respectively) and also lowered plasma TxB2 concentrations (P < 0.01 and P < 0.025, respectively). Neither grape juice altered ADP-induced aggregation or TxB2 concentrations, but the commercial juice lowered the IC50 for thrombin (P < 0.001) whereas the resveratrol-enriched juice caused a dramatic increase (P < 0.001). In vitro experiments demonstrated that the aggregation of fresh washed human platelets by ADP and thrombin was moderately reduced by both grape juices, strongly by red wine and not at all by white wine. The synthesis of TxB2 by platelets from labelled arachidonate was stimulated by commercial grape juice, slightly enhanced by resveratrol-enriched juice and strongly inhibited by red wine with white wine having little effect. Platelets from subjects consuming the commercial juice had a higher ratio of cyclo-oxygenase to lipoxygenase product formation and those consuming the resveratrol-enriched juice a lower ratio than during the control period. We conclude that trans-resveratrol can be absorbed from grape juice in biologically active quantities and in amounts that are likely to cause reduction in the risk of atherosclerosis. The failure of red wines (which have a 20-fold excess of polyphenols over white wines) to show any advantage suggests that, in vivo, ethanol is the dominant anti-aggregatory component in these beverages which are more potent than grape juices in preventing platelet aggregation in humans.

Hepoxilin A3 is oxidized by human neutrophils into its omega-hydroxy metabolite by an activity independent of LTB4 omega-hydroxylase.

Hepoxilin A3-methyl ester is taken up by intact human neutrophils where it is first hydrolyzed into the free acid which is subsequently converted into a single major metabolite. The structure of this metabolite was determined through mass spectral analysis of several derivatives, and through identity with an authentic compound prepared by chemical synthesis. The metabolite was identified as omega-hydroxy-hepoxilin A3 showing that the epoxide functionality of the parent hepoxilin is not opened during incubation with human neutrophils. All attempts to investigate hepoxilin metabolism in broken cells, despite the presence of protease inhibitors (Aproteinin, PMSF, DFP) and supplementation with NADPH were unsuccessful. Metabolism of hepoxilin A3 required the intact cell, while parallel experiments with LTB4 as substrate demonstrated that this eicosanoid was metabolized into its omega-hydroxy metabolite regardless of whether intact or broken cell preparations were used provided that NADPH was present in the latter. Hepoxilin metabolism in intact cells was inhibited dose-dependently by CCCP (0.01-100 microM), a mitochondrial uncoupler, whereas LTB4 metabolism was unaffected by CCCP. This data suggests that metabolism of hepoxilin A3 occurs in intact human neutrophils through omega-oxidation, is likely located in the mitochondrial compartment of the cell (inhibition by CCCP) and is carried out by an activity that is independent of the well characterized, relatively stable microsomal LTB4 omega-hydroxylase.

Hepoxilin A3 is Metabolized Into its ω-Hydroxy Metabolite by Human Neutrophils

Hepoxilins (Hx) elicit a variety of biological actions based on their ability to affect ion movements in the cell [Pace-Asciak 1994; Pace-Asciak et al., 1995a; Pace-Asciak et al., 1995b]. The most notable of these actions includes the release of insulin from pancreatic islets [Pace-Asciak and Martin 1984] as well as the regulation of cell volume in platelets [Margalit et al., 1993]. In the human neutrophil, HxA3 releases calcium from intracellular stores and this release is Hx-receptor mediated [Reynaud et al., 1996; Reynaud et al., 1995]. During the course of our investigations in neutrophils, we noted that radiolabeled HxA3 was metabolized into a single product which was identified as the corresponding ω-hydroxy product retaining the basic hydroxy-epoxide functional group of the parent hepoxilin intact [Reynaud et al., 1997]. Additional studies reported herein also indicate that the hepoxilin ω-hydroxylase is different from that which metabolizes LTB4.