ascorbic-acid and 15-hydroperoxy-5-8-11-13-eicosatetraenoic-acid

Reactive oxygen species convert the omega-6 polyunsaturated fatty acid arachidonic acid into 15-hydroperoxy-5,8,11,13-(Z,Z,ZE)-eicosatetraenoic acid (15-HPETE). Cyclooxygenases and lipoxygenases can also convert arachidonic acid into 15-HPETE. Vitamin C mediated decomposition of 15(S)-HPETE to protein- and DNA-reactive bifunctional electrophiles was examined by normal-phase liquid chromatography/atmospheric pressure chemical ionization/mass spectrometry (LC/APCI-MS). The individual bifunctional electrophiles, trans-4,5-epoxy-2(E)-decenal (t-EDE), cis-4,5-epoxy-2(E)-decenal (c-EDE), 4-oxo-2(E)-nonenal (ONE), and 4-hydroxy-2(E)-nonenal (HNE), exhibited protonated molecules at m/z 169, 169, 155, and 157, respectively. The MH+ ion at m/z 173 for 4-hydroperoxy-2(E)-nonenal (HPNE) was very weak with an ion corresponding to the loss of OH at m/z 156 as the major ion in the APCI mass spectrum. The bifunctional electrophiles were all separated under normal-phase LC conditions. All five bifunctional electrophiles were formed when 15-HPETE was treated with vitamin C. The LC/MS-based methodology showed that t-EDE was the major bifunctional electrophile formed during vitamin C mediated 15(S)-HPETE decomposition. Stable isotope dilution LC/MS studies revealed that this did not result in the formation of increased levels of unsubstituted etheno-dGuo adducts in calf thymus DNA when compared with 13(S)-hydroperoxy-9,10-(Z,E)-octadecadienoic acid [13(S)-HPODE], a lipid hydroperoxide derived from linoleic acid. However, the formation of heptanone-etheno-dGuo adducts in calf thymus DNA was reduced when compared with the 13(S)-HPODE. This was attributed to the reduced formation of ONE from 15-HPETE when compared with its formation from 13-HPODE. In contrast to reactions with dGuo or DNA conducted using 13(S)-HPODE, no carboxy-containing adducts were observed with 15(S)-HPETE.

Topics: Ascorbic Acid; Chromatography, High Pressure Liquid; DNA Adducts; Electrons; Leukotrienes; Lipid Peroxides; Oxidation-Reduction; Spectrometry, Mass, Electrospray Ionization

Flavonols (quercetin and rutin) and flavanes (cyanidol and meciadonol) were studied for their effect on non-enzymatic lipid peroxidation, lipoxygenase and cyclo-oxygenase activities, binding to albumin and platelet membranes. These biochemical properties of four flavonoids were compared with respect to their antithrombotic action in vivo and their efficacy at influencing the platelet-endothelium interaction in vitro. All four flavonoids inhibited the ascorbate-stimulated formation of malondialdehyde by boiled rat liver microsomes (quercetin greater than rutin approximately cyanidol approximately meciadonol) and inhibited platelet lipoxygenase activity (quercetin greater than cyanidol greater than meciadonol greater than rutin) whereas only flavonols, but not flavanes, stimulated cyclo-oxygenase and were bound to platelet membranes. The same two flavonols dispersed platelet thrombi which were adhering to the rabbit aortic endothelium in vitro (EC50 for quercetin was 80 nM and for rutin 500 nM) and prevented platelets from aggregation over blood-superfused collagen strip in vivo (ED50 for quercetin was 5 nmol/kg and for rutin 33 nmol/kg i.v.). Cyanidol and meciadonol were not effective as anti-thrombotic agents. It is concluded that activated platelets adhering to vascular endothelium generate lipid peroxides and oxygen-free radicals which inhibit endothelial biosynthesis of prostacyclin and destroy endothelium-derived relaxing factor (EDRF). Flavonols are anti-thrombotic because they are selectively bound to mural platelet thrombi and owing to their free radical scavenging properties resuscitate biosynthesis and action of endothelial prostacyclin and EDRF. Thus, flavonols release the thrombolytic and vasoprotective endothelial mediators only in these vascular segments which are covered by a carpet of aggregating platelets.

Topics: Albumins; Animals; Anthocyanins; Antithrombins; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Arachidonic Acids; Ascorbic Acid; Benzopyrans; Blood Platelets; Catechin; Flavonoids; Leukotrienes; Lipid Peroxides; Malondialdehyde; Microsomes, Liver; Prostaglandin-Endoperoxide Synthases; Quercetin; Rats; Rutin

The enhancing effect of vitamin C on the conversion of arachidonic acid, endoperoxide G2 and endoperoxide H2 to 6-keto-PGF 1 alpha, the stable metabolite of prostacyclin, by ram seminal vesicle microsomes was further investigated. From the incubations of these substrates with 1-tryptophan, catalase, superoxide dismutase and 15-HPETE it became clear that vitamin C apparently acts mainly through neutralization of the oxidative species formed during the reduction of endoperoxide G2 to endoperoxide H2. Although it has also a more direct stimulating activity on the prostacyclin synthase, a possible interference with hydroperoxy derivatives of arachidonic acid cannot be completely ruled out.

Topics: Animals; Arachidonic Acids; Ascorbic Acid; Catalase; Epoprostenol; In Vitro Techniques; Leukotrienes; Lipid Peroxides; Male; Microsomes; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandins G; Prostaglandins H; Seminal Vesicles; Sheep; Superoxide Dismutase; Tryptophan

Aortic rings from rats, rabbits and guinea-pigs produce different amounts of 6-oxo-prostaglandin F1 alpha (6-oxo-PGF1 alpha), the stable breakdown product of prostacyclin, i.e. 2760 +/- 195, 160 +/- 10 and 87 +/- 17 pg 6-oxo-PGF1 alpha per mg wet weight in 30 min. Vitamin C enhances the production of 6-oxo-PGF1 alpha by the aortic tissue of these three species, independent of their basal release. This increase was only significant if vitamin C was present in the preincubation as well as in the incubation fluid. 15-Hydroperoxy-arachidonic acid inhibits the production of 6-oxo-PGF1 alpha (IC50:6 microM) and this inhibitory effect was completely neutralized by vitamin C. The increased production of 6-oxo-PGF1 alpha is not due to an increased release of the substrate arachidonic acid. It is suggested that vitamin C enhances the formation of 6-oxo-PGF1 alpha by protecting the cyclo-oxygenase and PGI-synthase.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Aorta; Arachidonic Acids; Ascorbic Acid; Blood Vessels; Dose-Response Relationship, Drug; Epoprostenol; Guinea Pigs; Leukotrienes; Lipid Peroxides; Male; Rabbits; Rats