David G. Cornwell

Role of hydroxyl radical scavengers dimethyl sulfoxide, alcohols and methional in the inhibition of prostaglandin biosynthesis.

Prostaglandin biosynthesis from eicosa-8, 11, 14-trienoic acid in microsomes from bovine seminal vesicles is inhibited by relatively high concentrations of hydroxyl radical scavengers: dimethyl sulfoxide, n- and t-butanol, and methional. Methional is a more effective scavenger than t-butanol and dimethyl sulfoxide, two compounds which are more miscible with water than methional. The synthesis of both PGE and PGF is inhibited with incubation systems that promote PGE formation and with incubation systems that promote PGF formation. Furthermore, dimethyl sulfoxide and methional inhibit arachidonic acid-induced platelet aggregation, a reaction involving endoperoxide biosynthesis. The water soluble alcohol, ethanol, stimulates PGF biosynthesis when it is added in the same concentration range as t-butanol. Thus hydroxyl radical scavengers inhibit biosynthesis when their effective concentrations are high and stimulate biosynthesis when their effective concentrations are low. The results of this study and other studies where low concentrations of hydroxyl radical scavengers stimulate both prostaglandin biosynthesis and lipid peroxidation are consistent with a mechanism involving the hydroxyl radical both in the generation of singlet oxygen and the elimination of hydrogen peroxide.

Differential inhibitory effects of vitamin E and other antioxidants on prostaglandin synthetase, platelet aggregation and lipoxidase

Prostaglandin biosynthesis from eicosa-8,11,14-trienoic acid in microsomes from the bovine vesicular gland is inhibited by the antioxidants alpha-naphthol. guaiacol, NDGA and propyl gallate. Prostaglandin biosynthesis in this system is not inhibited by the antioxidants BHT, DL-alpha-tocopherol and Trolox C. Arachidonic acid induced platelet aggregation is inhibited by specifically by alpha-naphthol. guaiacol, NDGA and propyl gallate. Both arachidonic acid induced platelet aggregation and ADP induced platelet aggregation are inhibited non-specifically by the antioxidants BHT, DL-alpha-tocopherol and Trolox C. All antioxidants tested in this study inhibit soybean lipoxidase. Thus alpha-naphthol, NDGA and propyl gallate are non-specific inhibitors of both prostaglandin synthetase and soybean lipoxidase while BHT, DL-alpha-tocopherol and Trolox C are specific inhibitors of soybean lipoxidase alone.

Modulation of platelet thromboxane A2 and arterial prostacyclin by dietary vitamin E

Platelets from vitamin E-deficient and vitamin E-supplemented rats generate the same amount of thromboxane A2 (TxA2) when they are incubated with unesterified arachidonic acid. Platelets from vitamin E-deficient rats produced more TxA2 than platelets from vitamin E-supplemented rats when the platelets are challenged with collagen. Arterial tissue from vitamin E-deficient rats generates less prostacyclin (PGI2) than arterial tissue from vitamin E- supplemented rats. The vitamin E effect with arterial tissue is observed when the tissue is incubated with and without added unesterified arachidonic acid. These data show that arterial prostacyclin synthesis is diminished in vitamin E-deficient rats. Vitamin E, in vivo, inhibits platelet aggregation both by lowering platelet TxA2 and by raising arterial PGI2.

Restoration of Prostacyclin/Thromboxane A2 Balance in the Diabetic Rat: Influence of Dietary Vitamin E

The effect of dietary vitamin E on the synthesis of platelet thromboxane A2 (TxA2) and vascular prostacyclin (PGI2) in streptozotocin-induced diabetic rats was investigated. Thrombin-induced platelet TxA2 synthesis in diabetic rats was significantly elevated, whereas no significant difference was found with collagen-induced synthesis. Arterial PGI2 production was significantly decreased in diabetic rats. Serum lipid peroxide levels were elevated, and platelet vitamin E levels decreased in diabetic rats. When the diabetic rats were maintained on a high vitamin E diet for 2-3 mo, thrombin-induced platelet TxA2, vascular PGI2, and lipid peroxides were restored to levels found in nondiabetic rats. Platelets from diabetic rats maintained on a high vitamin E diet synthesized significantly less collagen-induced TxA2 than platelets from nondietary vitamin E-supplemented diabetic rats or nondiabetic controls.

Fatty acid specificity in the inhibition of cell proliferation and its relationship to lipid peroxidation and prostaglandin biosynthesis

Primary cultures of smooth muscle cells were established from the medial layer of guinea pig aorta. Cells at passage level 4 were treated with different series of fatty acids belonging to the n-9, n-6 and n-3 families. Lipid peroxidation was measured by the thiobarbituric acid assay and prostaglandin biosynthesis was measured by the radioimmunoassay of PGE and 6-keto-PGF1α. Cell proliferation was estimated from the total cell number of cultures seeded at low density. 18∶1(n-9) did not form lipid peroxides and this fatty acid stimulated cell proliferation. All fatty acids which generated lipid peroxides inhibited cell proliferation, but inhibition was correlated with the degree of lipid peroxidation only in the n-9 fatty acid family. 22∶4(n-6) and 22∶6(n-3) inhibited prostaglandin biosynthesis. 18∶2(n-6), 18∶2(n-9), 18∶3(n-3), 20∶2(n-9), 20∶3(n-3) and 20∶5(n-3) had no effect on prostaglandin biosynthesis. 18∶3(n-6), 20∶3(n-6) and 20∶4(n-6) generated prostaglandins. 20∶3(n-9) generated metabolites with prostaglandin immunoreactivity. The inhibition of cell proliferation did not correlate with enhanced or inhibited prostaglandin synthesis. The inhibition of cell proliferation was related to the structures of the different polyunsaturated fatty acid families decreasing in the order n-9>n-6>n-3. Eicosatrienoic acids were the most effective inhibitors of cell proliferation in each fatty acid family and 20∶3(n-9) was the most potent eicosatrienoic acid. These data show that specific as yet unrecognized products of fatty acid metabolism are responsible for the inhibition of cell proliferation.

Surface Area of Human Erythrocyte Lipids: Reinvestigation of Experiments on Plasma Membrane

Ratios of the lipid monolayer area to the erythrocyte surface area are 2:1 at low surface pressures and approach 1: 1 at collapse pressures. Un saturated phospholipids in cholesterol-phospholipid complexes of membrane ex tracts resemble their saturated derivatives at collapse pressures. Area ratio and phospholipid area data are related by an equation that tests hypothetical values for molecular areas used in membrane models.

Polyunsaturated fatty acids, vitamine E, and the proliferation of aortic smooth muscle cells

Smooth muscle cell cultures were obtained from the aortas of prepubertal guinea pigs. Cell proliferation in these cultures was inhibited by 8,11,14-eicosatrienoic acid, 5,8,11,14-eicosatetraenoic acid, and their prostaglandin E derivatives, PGE1 and PGE2. Prostaglandin F derivatives, PGF1α and PGF2α, stimulated cell proliferation. Cell proliferation was also inhibited by 5,8,11-eicosatrienoic acid and 11,14,17-eicosatrienoic acid. The monoene and diene precursors of the triene acids, 9-octadecenoic acid and 9,12-octadecadienoic acid, did not inhibit cell, proliferation. Indomethacin alone had no effect on cell proliferation, and indomethacin did not suppress the inhibition of cell proliferation with a triene acid. The antioxidant α-naphthol alone stimulated cell proliferation and suppressed prostaglandin E formation. α-Naphthol in the presence of either triene or tetraene acids also stimulated cell proliferation and suppressed prostaglandin E formation. The antioxidants butylated hydroxy toluene and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid either alone or in the presence of triene and tetraene acids stimulated cell proliferation and had no effect on prostaglandin E formation. Vitamin E either alone or in the presence of triene or tetraene acids stimulated cell proliferation and had no effect on prostaglandin E formation. More prostaglandin E was formed from 8,11,14-eicosatrienoic acid than from 5,8,11,14-eicosatetraenoic acid in the presence of antioxidants. Vitamin E suppressed the inhibitory effects of both PGE2 and palmitic acid on cell proliferation. The cyclic nucleotide phosphodiesterase inhibitors, caffeine and papaverine, suppressed the stimulatory effect of vitamin E on cell proliferation and enhanced the inhibitory effect of a triene acid on cell proliferation. Substrate and inhibitor specificities are consistent with the oxidative regulation of cell proliferation through the formation of hydroperoxy fatty acids. We propose that hydroperoxy fatty acids may regulate both cyclase and cyclic nucleotide phosphodiesterase enzymes through sulfhydryl-disulfide interconversions. We suggest that this regulatory mechanism may help to explain the acculation of 5,8,11-eicosatrienoic acid in essential fatty acid deficiency, the effects of antioxidants on cell proliferation, and one of the several effects of polyunsaturated fatty acids in proliferative disorders such as cancer and atherosclerosis.

A suggested role for hydrogen peroxide in the biosynthesis of prostaglandins

Abstract Prostaglandin E and F biosynthesis from eicosa-8,11,14-trienoic acid in microsomes from bovine seminal vesicles is inhibited by catalase. Prostaglandin biosynthesis in the same system is also inhibited by 3-amino-1,2,4-triazole (AT) and NaN 3 , reagents which function as heme enzyme inhibitors. AT and NaN 3 inhibit arachidonic acid induced platelet aggregation. Chlorpromazine, a hydroxyl radical scavenger, inhibits platelet aggregation. 1,4-diazabicyclo[2.2.2.] octane, a singlet oxygen scavenger, does not inhibit either microsomal biosynthesis or platelet aggregation. The results are consistent with a mechanism requiring the enzymatic decomposition of H 2 O 2 to a radical, possibly ·OH, in the biosynthesis of prostaglandins.

Fatty acid metabolism and cell proliferation. V. Evaluation of pathways for the generation of lipid peroxides

Primary cultures of smooth muscle cells were established from the medial layer of guinea pig aorta. Confluent cells at passage level 4–6 were challenged with arachidonic acid and treated with a number of antioxidants and inhibitors of specific lipid peroxidation pathways. Lipid peroxidation was measured by the thiobarbituric acid test for malondialdehyde (MDA) and the isolation of hydroperoxy fatty acids (HPETE) by high performance liquid chromatography (HPLC). Prostanoids were measured by radioimmunoassay and the separation of labeled compounds by HPLC. MDA, 6-keto-PGF1α, and PGE2 were formed when cells were challenged with arachidonic acid and these cells synthesized small amounts of one HPETE isomer, 15-HPETE. The HPETE isomers characteristic of the lipoxygenase pathway, 12-HPETE and 5-HPETE, were not detected. Furthermore, the lipoxygenase inhibitors, eicosatetraynoic acid (ETYA) and 6,7-dihydroxycoumarin (Esculetin), did not block MDA formation. These data show that MDA is not generated in the cells by a lipoxygenase pathway. The cyclooxygenase inhibitors, indomethacin and ETYA, did not block MDA formation but these agents blocked the formation of 15-HPETE. These data show both that 15-HPETE is generated by a cooxidation pathway and that 15-HPETE and cooxidation are not involved in MDA formation. Three inhibitors of cytochrome P450 linked lipid peroxidation, 2-amino-3-ethoxycarbonyl-6-benzyl-4, 5,6,7-tetrahydrothieno-[2,3-C]-pyridine (Tinoridine), 3-methyl-1,2-di-3-pyridyl-1-propanone (Metyrapone) and phenobarbital, did not block MDA formation. These data support earlier studies that indicated that MDA is not generated by a P450 pathway. Cells contained a bound precursor that decomposed to MDA when cells were treated with Fe3+. The cells exhibited autofluorescence and concentric lamellae in lipid droplets that are characteristic of ceroid-lipofuscin. These observations are consistent with lipid peroxidation through increased peroxisomal activity leading to the generation of MDA and the accumulation of ceroid-lipofuscin. The natural antioxidants, vitamin E and vitamin E quinone (EQ), and the synthetic antioxidants, butylated hydroxytoluene and nordihydroguaiaretic acid (NDGA), α-naphthol (α-N) and propyl gallate (PrGa), all blocked MDA formation in confluent smooth muscle cells, showing that these antioxidants did not function solely as specific inhibitors of lipoxygenase, cooxidation or P450 pathways. Cell proliferation was measured in cells challenged with arachidonic acid and treated with antioxidants and other inhibitors. The least cytotoxic and most potent antioxidant, EQ, blocked MDA formation in confluent cells and promoted growth in proliferating cells when it was present in either system in the same concentration range. The synthetic antioxidants, NDGA, α-N and PrGa, blocked prostanoid synthesis and promoted growth in proliferating cells. The cyclooxygenase inhibitors, indomethacin, ETYA and Esculetin, did not enhance cell proliferation even though they were highly effective inhibitors of prostanoid synthesis. These data suggest, but do not prove, the hypothesis that cell proliferation is controlled in part by general peroxidation reactions rather than the specific peroxidation reaction involved in prostanoid synthesis.

Positions of double bonds in the monounsaturated alk-1-enyl groups from the plasmalogens of human heart and brain

Abstract The phospholipids of human heart were found to include 16.3 per cent alk-1-enyl acyl glycerophosphorylcholines and 14.9 per cent alk-1-enyl acyl glycerophosphorylethanolamines. A sample of human brain with both gray and white matter contained 0.9 per cent of the former and 22.4 per cent of the latter. Alkyl-1,3-dioxolane derivatives of the alk-1-enyl groups were prepared. The octadecenyl dioxolanes accounted for 18 per cent of the choline plasmalogens and 23 per cent of the ethanolamine plasmalogens from heart and 43 per cent of the ethanolamine plasmalogens from brain. The 18:1 dioxolanes from the heart plasmalogens had a 64:36 distribution of 9,10 and 11,12 double bonds whereas the corresponding value was 30:70 for the brain plasmalogens.