μM Vitamin Research Articles

Culture of in vitro-produced bovine embryos with vitamin E improves development in vitro and after transfer to recipients.

Detrimental effects of oxygen-derived free radicals on embryos during culture have been demonstrated in several species. Vitamin E occurs naturally in cell membranes and protects cells from oxidative stress. Under some conditions, vitamin C acts synergistically to enhance the antioxidant effects of vitamin E, a benefit that may be further enhanced by EDTA. The present experiments concerned culture of bovine embryos derived from in vitro-matured, fertilized oocytes with vitamin E, vitamin C, and EDTA in a chemically defined culture medium + 0.2% BSA at 5% O(2), 5% CO(2), and 90% N(2). In the first experiment, more zygotes developed to expanded blastocysts (17%, n = 224, P < 0.05) when culture medium contained 100 microM vitamin E than in control medium (11%, n = 234). Development to early, expanded, and hatched blastocysts was lower with vitamins E and C combined than with vitamin E alone (15%, 9%, and 2% vs. 24%, 17%, and 5%, respectively; P < 0.05), as was the mean number of cells per blastocyst (56 vs. 84, P < 0.05). Addition of EDTA (3 microM) failed to improve development over that in culture with vitamin E + vitamin C. In experiment 2, in vitro-produced embryos cultured 5.5 days in medium with or without 100 microM vitamin E were transferred nonsurgically to recipient cows and heifers and then collected nonsurgically 7 days later. Embryos cultured with vitamin E (n = 37) were approximately 63% larger in surface area than controls (1.16 mm(2) vs. 0.71 mm(2) surface area; n = 27, P < 0.04).

Growth inhibition of rat mesangial cells by high glucose: effect of vitamin E

Background. We aimed to examine both whether vitamin E prevented and whether it reversed the growth inhibitory effect of high glucose. Methods. For the preventive experiment, rat mesangial cells (RMC) were grown in control glucose medium with the addition of 100 μM of vitamin E. High glucose (27.5 mM) was added to the medium concurrent with the vitamin E addition. The 3-(4,5-di-methylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay was used to measure RMC proliferation. Our data confirmed the growth inhibitory effect of high glucose and showed that the growth inhibition was prevented by vitamin E. To examine whether vitamin E reversed the growth inhibitory effects of high glucose, RMC were grown in control and high glucose medium. Contrary to previous prevention type studies, vitamin E was not added to the medium until growth inhibition of the RMC by the high glucose was established. Results. Our data show that it took 5 days of vitamin E administration to reverse the growth inhibitory effect of high glucose. Conclusion. This is the first time that vitamin E has been shown to reverse this high-glucose-induced inhibition of RMC, suggesting that vitamin E reverses a potentially important pathogenetic process.

Effect of Hydroxytyrosol Found in Extra Virgin Olive Oil on Oxidative DNA Damage and on Low-Density Lipoprotein Oxidation

Hydroxytyrosol found in extra virgin olive oil strongly inhibited low-density lipoprotein oxidation stimulated by 2,2‘-azobis(2-amidinopropane) hydrochloride (AAPH), suggesting the ability to scavenge the AAPH-derived peroxyl radicals. Hydroxytyrosol inhibited iron-dependent phospholipid liposome peroxidation at low concentrations (IC50 = 50 ± 1.3 μM). In similar experiments, the calculated, IC50 values for other antioxidants compared are 1.5 ± 0.05 μM (carnosol), 2.25 ± 0.08 μM (carnosic acid), 65 ± 2.6 μM (Trolox C), and 250 ± 10 μM (vitamin E). Hydroxytyrosol and ascorbate reduced copper(II) ions to their copper(I) prooxidant form, but this was not reflected by their abilities to induce oxidative DNA damage in the complex copper−phenanthroline. Only high, nonphysiological, millimolar concentrations of pure hydroxytyrosol weakly stimulated copper-dependent chemical modification to DNA bases. The prooxidant (redox actions on metal ions) concentrations in vitro may never be achieved in vivo (following con...

Influence of vitamin E succinate on retinal cell survival

In this study, we analyzed the influence of vitamin E succinate (5–80 μM), supplemented in the culture medium, on the survival of cultured retinal cells. The release of lactate dehydrogenase (LDH) was decreased in the presence of low concentrations (10–20 μM) of vitamin E succinate, whereas high concentrations (80 μM) induced a significant increase (about 2-fold) in the release of LDH, indicating a reduction of plasma membrane integrity. Supplementing with vitamin E succinate (80 μM) greatly enhanced its cellular content, as compared to vitamin E acetate (80 μM), and the membrane order of the retinal cells, as evaluated by the fluorescence anisotropy ( r) of TMA-DPH (1-(4-(trimethylammonium)-phenyl)-6-phenylhexa-1,3,5-triene), was not altered. Furthermore, vitamin E succinate was more potent than vitamin E acetate in reducing thiobarbituric acid reactive substances (TBARS) formation upon ascorbate–Fe 2+-induced oxidative stress (TBARS formation after cell oxidation decreased by about 15-fold or 1.6 fold, respectively, in the presence of 20 μM vitamin E succinate or 20 μM vitamin E acetate). A decrease in MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction induced by supplementing with vitamin E succinate (80 μM), to 35.99±1.96% as compared to the control, but not by vitamin E acetate (80 μM), suggests that vitamin E succinate may affect the mitochondrial activity. Vitamin E succinate also reduced significantly the ATP:ADP ratio in a dose-dependent manner, indicating that vitamin E succinate-mediated cytotoxic effects involve a decrement of mitochondrial function.

Comparative in vitro oxygen radical scavenging ability of zinc methionine and selected zinc salts and antioxidants

1. 1. The concentration-dependent scavenging abilities of zinc dl-methionine, zinc sulfate, zinc gluconate, zinc picolinate and selected free radical scavengers, including superoxide dismutase (SOD), catalase, mannitol, allopurinol and dl-methionine, were examined against biochemically generated superoxide anion, hydroxyl radical and hypochlorite radical plus hypochlorous acid, by chemiluminescence and cytochrome c reduction. 2. 2. Zinc methionine was the most effective of the zinc compounds that were tested. Following incubations with superoxide anion, hydroxyl radical, and hypochlorite radical-generating systems, in the presence of 50 μM zinc dl-methionine approximately 38%, 47% and 28% inhibition in reactive oxygen species generation was observed, respectively, compared to control groups. 3. 3. The protective abilities of various zinc salts, as well as selected free radical scavengers and antioxidants were also assessed on phorbol ester (TPA)-induced lactate dehydrogenase (LDH) release from cultured PC-12 cells. Preincubation showed better protection than coincubation. Approximately 45% and 50% inhibition in TPA-induced LDH leakage was observed following preincubation with 50 μM zinc dl-methionine and 50 μM vitamin E succinate, respectively. Zinc dl-methionine exhibited better protection against LDH leakage than any other zinc salt tested 4. 4. The results indicate that zinc dl-methionine can attenuate the biochemical consequences of oxygen free radicals, and is comparable to other well-known antioxidants and free radical scavengers in the in vitro system that was employed.

Vitamin E Attenuates Peroxideinduced Vasoconstriction in the Human Placenta

Objective: Placental lipid peroxides and thromboxane are abnormally increased in preeclampsia. Peroxides stimulate thromboxane to increase placental vasoconstriction. Vitamin E is an antioxidant that inhibits lipid peroxidation. We, therefore, evaluated whether vitamin E would attenuate peroxideinduced vasoconstriction in the human placenta.Methods: Isolated human placental cotyledons (n = 6) were perfused for 20-min periods with control Krebs-Ringer-bicarbonate (KRB) buffer, 200 μM t-butyl hydroperoxide, control KRB buffer, 50 μM vitamin E, t-butyl hydroperoxide plus vitamin E, control KRB buffer, and t-butyl hydroperoxide alone.Main Outcome Measures: Fetal perfusion pressure was continually recorded and maternal (M) and fetal (F) effluent samples were collected for each treatment. Samples were analyzed for thromboxane and prostacyclin by RIA of their stable metabolites TXB2 and 6-keto PGF1α, respectively.Results: Compared to control KRB buffer, peroxide perfusion significantly increased perfusion pressure, vascular resistance, TXB2 secretion and 6-keto-PGF1α. After 20 min of perfusion with vitamin E, the ability of peroxide to increase perfusion pressure, vascular resistance, and TXB2, secretion was significantly attenuated and there was even further attenuation after 40 min of perfusion with vitamin E. Vitamin E did not affect the ability of peroxide to increase the secretion of 6-keto-PGF1α.Conclusions: (i) Vitamin E attenuates the ability of peroxide to increase placental vasoconstriction and thromboxane secretion, but not its ability to increase prostacyclin secretion, (ii) The attenuating effects of vitamin E increase with increased duration of exposure and its effects persist after it is discontinued, most likely because it is incorporated into the cell membranes.

Vitamin E Slows the Rate of Free Radical-Mediated Lipid Peroxidation in Cells

Much of what is known about the antioxidant mechanism of vitamin E has been learned from studies of lipid dispersions, solutions, or subcellular organelles. We have investigated the effect of vitamin E supplementation on intact live eucaryotic cells. L1210 murine leukemia cells were exposed to an oxidative stress induced by 20 μMFe2+and 100 μMascorbic acid introduced immediately before oxidative measurements were begun, and the kinetics of the generation of lipid-derived free radicals, as measured by EPR spin trapping (a product) and O2consumption (a reactant) were measured. Cells grown for 24 h with supplemental (5–100 μM) vitamin E in their media had a slower rate of lipid radical generation compared to cells grown without vitamin E supplementation; this inhibition in the rate of oxidation was generally dependent upon the amount of vitamin E supplementation. In complementary studies measuring O2consumption, 5–100 μMvitamin E slowed the rate of oxidation (10-fold with 100 μMsupplemental vitamin E) consistent with the EPR studies. The membrane active drug edelfosine accentuated the vitamin E effects; vitamin E introduced a discernible lag phase (time delay) in both lipid radical generation and O2consumption that was not seen in the absence of edelfosine. Vitamin E supplementation of cells also altered the kinetics of ascorbate free radical formation. We conclude that vitamin E inhibits lipid peroxidation in cells by slowing the rate of lipid peroxidation; but with iron/ascorbate as the initiating system, vitamin E does not delay the onset of peroxidation. Of special interest is that these free radical peroxidation events parallel cell membrane damage as detected using trypan blue exclusion. These observations are consistent with the free radical events preceding and causing the observed membrane damage.

Arachidonic acid supplementation enhances hydrogen peroxide induced oxidative injury of neonatal rat cardiac myocytes

Selective fatty acid supplementation of non-myocardial cells has been reported to enhance oxidative injury. The purpose of this study was to extend this research by determining if supplementation of isolated cardiac myocytes with arachidonic acid increases myocyte sensitivity to H2O2 induced lipid peroxidation and cytotoxicity. Myocytes were supplemented with arachidonic acid by complexing it with calf serum and adding it to myocytes at a final concentration of 0, 50, or 100 microM for 36 h. Myocytes were then exposed to 50 or 100 microM H2O2 for 1 h and lipid peroxidation and cytotoxicity were assessed by measuring thiobarbituric acid reactive substances and lactate dehydrogenase release into culture medium. To determine if unesterified arachidonic acid contributed to oxidative injury, 100 microM arachidonic acid was added directly to cultured myocytes in serum-free buffer. Vitamin E and desferrioxamine were assessed for their ability to protect against oxidative injury induced by unesterified arachidonic acid or H2O2. Supplementation with 100 microM arachidonic acid for 36 h increased the arachidonic acid content of phospholipids from 0.58(SD 0.06) to 0.90(0.19) nmol.nmol-1 lipid phosphorus. A 1 h exposure to H2O2 induced lipid peroxidation and cytotoxicity in a concentration dependent manner. Pretreatment of myocytes with 10 microM vitamin E or 1 mM desferrioxamine prevented H2O2 induced effects. Arachidonic acid supplementation at 50 or 100 microM significantly enhanced lipid peroxidation induced by 100 microM H2O2, whereas cytotoxicity was increased only in myocytes supplemented with 100 microM arachidonic acid. Addition of unesterified arachidonic acid directly to cultured myocytes caused marked cytotoxicity and lipid peroxidation which were prevented by vitamin E or desferrioxamine. Arachidonic acid supplementation of cardiac myocytes modifies fatty acid content of phospholipids and enhances the susceptibility of myocytes to H2O2 induced oxidative injury.

Vitamin e succinate potentiates the inhibitory effect of prostaglandins on oral squamous carcinoma cell proliferation

Previous studies have shown that prostaglandin E 2 (PGE 2) and vitamin E succinate can act in an additive manner to inhibit the proliferation of human oral squamous carcinoma cells (SCC-25). The initial studies on the additive anticancer activity of PGE 2 and vitamin E succinate have been extended to include antineoplastic PGs, Δ 12-PGJ 2 and PGJ 2. Treatment of oral squamous carcinoma cells (SCC-15) with Δ 12-PGJ 2, PGJ 2, and vitamin E succinate, individually, caused significant concentration-dependent inhibition of cell proliferation to various degrees. PGJ 2 was most potent and caused an inhibition that corresponded to 85.55% at 10 −5 M. Addition of 1 μM of vitamin E succinate to Δ 12-PGJ 2 or PGJ 2 resulted in a significant increase in the inhibitory potency of the lower concentrations of the two PGs. These results suggest a novel role for a mixture of PGs and vitamin E as potent antitumor proliferative agents.

Very-Low-Density and Low-Density Lipoproteins Induce Nitric Oxide Synthesis in Macrophages

The effect of lipoproteins on rat peritoneal macrophage nitric oxide (NO) production was studied. Very low density (VLDL) and low density (LDL) but not high density lipoprotein (HDL) stimulated NO production at 12, 24 and 48 hr of incubation with macrophages. Forty-eight hour incubation of macrophages with VLDL or LDL increased NO production from 10.8 nmoles/l06 cells in control to 249 and 60 nmoles/l06 cells, respectively. VLDL-induced nitric oxide production was 825 nmoles/106 after 12 days of incubation. VLDL by itself or in the presence of LDL and HDL induced similar levels of NO in 24 hrs. Lipoproteins were without effect on NO, when incubated with macrophages in the absence of L-arginine and in the presence of nitric oxide synthase inhibitor monomethyl-L-arginine (L-NMMA). Oxidized VLDL was as effective as native form in the induction of macrophage NO, whereas oxidized LDL did not induce NO production. Further, the presence of 100 μM vitamin E in the incubation had no effect on VLDL- and LDL-induced NO production. Preincubation of macrophages with VLDL and LDL for 3hr or 6hrs activated macrophages to generate NO. These effects of lipoproteins could be of interest in the pathophysiology of lipoproteins and NO mediated diseases.

Vitamin K 3 Inhibits Growth of Human Hepatoma HepG2 Cells by Decreasing Activities of Both p34 cdc2 Kinase and Phosphatase

Synthetic vitamin K 3 is known to exhibit in vitro and in vivo antitumor activity against rodent and human cancer cells. To determine vitamin K 3′s effect on the cell cycle, we treated both synchronized and asynchronized human hepatoma HepG2 cells with 100 μM vitamin K 3 for 1 h and followed cells to recover for various times, then monitored cell cycle progression by flow cytometry and by phosphorylation status of p34 cdc2 kinase. Vitamin K 3 induced a delay at the S/G2 phase of the cell cycle. This was accompanied by constant hyperphosphorylation status and decreased activity of p34 cdc2 kinase. Protein-tyrosine phosphatase activity also decreased 2- to 3-fold after vitamin K 3 treatment. Our results suggest that vitamin K 3 inhibits the growth of HepG2 cells via a cell cycle progression delay and altered phosphorylation patterns and activities of both p34 cdc2 kinase and protein-tyrosine phosphatase.

Toxicity of oxidised low density lipoprotein towards mouse peritoneal macrophages in vitro

The addition of cupric sulphate-oxidised low density lipoprotein (oxLDL) to mouse peritoneal macrophage (MPM) cultures caused toxicity towards the cells, measured by tritiated adenine release. The degree of toxicity increased with increasing concentrations of oxLDL up to 18 h incubation with MPM, after which the toxicity appeared to be independent of the concentration of oxLDL used. Toxicity was significantly reduced when vitamin E in the form of d,l-α-tocopherol was added to the LDL before the artificial oxidation, but vitamin E had no effect on the toxicity when added alongside the oxLDL in the culture medium. However, if the MPM were pre-incubated for 24 h with 80 μM vitamin E, there was a significant reduction in the level of toxicity up to 6 h in culture with oxLDL. There was a strong correlation ( r = 0.81) between the degree of oxidation of the LDL, measured as thiobarbituric-reactive substances, and the corresponding toxicity. This indicated that the more oxidised the LDL was, the more toxic it was to the macrophages. Native LDL also led to toxicity, but only after a time-lag of 20 h. Again, this toxicity was decreased by pre-incubation of the MPM with vitamin E, but not by addition of vitamin E at the same time as LDL. The findings suggest that oxLDL is capable of contributing to the onset of necrosis during atherogenesis and that the oxidative capacity of the macrophage foam cells themselves might contribute to the process.

Potentiation of sodium chromate(VI)-induced chromosomal aberrations and mutation by vitamin B 2 in Chinese hamster V79 cells

The effect of vitamin B 2, which is capable of reducing chromium(VI) to chromium(V), on chromosomal aberrations and mutation caused by Na 2CrO 4 was investigated in Chinese hamster V79 cells. Pretreatment with 200 μM vitamin B 2 (riboflavin) for 24 h prior to exposure to Na 2CrO 4 (2.5–5 μM) resulted in an increase of metal-induced chromosomal aberrations and mutation at the HGPRT locus. These and other previous studies suggest that vitamin B 2 enhances the clastogenic and mutagenic action of chromate compounds, through its ability to directly reduce chromium(VI) in cells.

Induction of the FMS proto-oncogene product in HL-60 cells by vitamin D: A flow cytometric analysis

Agents which induce monocytic characteristics in HL-60 human acute promyelocytic leukemia cells induce mRNA for the fins proto-oncogene, which encodes the receptor for M-CSF. Previous studies of fms expression in HL-60 cells have characterized chiefly induction by phorbol esters of fms mRNA. Our studies of fms expression in HI-60 cells have characterized induction by vitamin D 3 of the fms protein. We have used flow cytometry to correlate fms antigen with a monocyte-specific differentiation antigen recognized by antibody MO2 (CD14), with DNA content, and with the nuclear antigen Ki-67, a marker of cell cycling. HL-60 cells were cultured with or without 1 μM vitamin D for 7 days. fms antigen was found on 42±5.8% of the cells cultured without vitamin D, but on 63±4.3% of the cells cultured with vitamin D. MO2 binding was detected on only 2±0.5% of the cells without vitamin D, but on 59±9% with vitamin D. Cells cultured with vitamin D that were fms-positive were also predominantly (83%) MO2-positive. Analysis of DNA content, measured by propidium iodide staining, showed that 57±1.5% of cells cultured without vitamin D, but 93±0.5% of cells cultured with vitamin D, were in the G 0/G 1 cell cycle phase. Analysis of nuclear antigen Ki-67 revealed that, of the vitamin D-treated cells that were fms-positive, a significant proportion (37%) were still cycling. We conclude that (1) fms is demonstrable on some uninduced HL-60 cells, (2) when HL-60 cells are induced to develop monocytic characteristics by vitamin D, fms induction is part of the program for monocytic differentiation that includes MO2 expression, yet (3) some induced cells expressing fms are still cycling.

Vitamin E Binds to Specific Binding Sites and Enhances Prostacyclin Production by Cultured Aortic Endothelial Cells

We evaluated the effect of d-alpha-tocopherol (vitamin E) on the production of prostacyclin (PGI2) by cultured bovine aortic endothelial cells. Vitamin E at physiological doses significantly enhanced the production of PGI2 by aortic endothelial cells when added to the culture simultaneously with histamine, the Ca2+ ionophore A23187 (A23187), plasma-derived serum (PDS), or arachidonic acid. This effect was found to occur in a time- and dose-dependent manner, and the maximal enhancement was produced by 9.28 microM of vitamin E for 1 h incubations. Significantly lower amounts of lipid peroxides were measured in endothelial cells stimulated by 10% PDS with 9.28 microM of vitamin E than in those stimulated without vitamin E for over 24 h, although the stimulation during the initial 1 to 12 h period did not have a significant effect on lipid peroxide formation in cultured aortic endothelial cells. We also demonstrated that bovine aortic endothelial cells have specific binding sites for [3H]vitamin E that exhibited time- and temperature-dependent saturability. At 4 degrees C, the nonspecific binding was 8-12% of the total binding, and the specific binding reached equilibrium by 2 h. Specific binding increased with the concentration of [3H]vitamin E and became saturated at concentrations between 1.5 microM and 2.0 microM per 2.0 x 10(5) cells. Raising the unlabeled vitamin E concentration from 97.7 nM to 1,000 microM reduced the specific binding of 2.0 microM [3H]vitamin E.(ABSTRACT TRUNCATED AT 250 WORDS)

Circumvention of Adriamycin resistance: effect of 2-methyl-1,4-naphthoquinone (vitamin K 3) on drug cytotoxicity in sensitive and MDR P388 leukemia cells

The effect of vitamin K 3 (2-methyl-1,4-naphthoquinone) on Adriamycin (ADR) induced growth inhibition of drug sensitive and multidrug resistant P388 leukemia cells was evaluated. Exposure to ADR concentrations of 100–5000 ng simultaneously with 1 μM vitamin K 3 elicited an enhanced inhibition of tumor cell survival. The effect of treatment with ADR alone, or in combination with vitamin K 3 on DNA and RNA biosynthesis in the sensitive and resistant tumor cells, was also assessed. DNA and RNA biosynthesis inhibition was increased in P388/S (the parental cell line) and P388/ADR cells (the ADR resistant cell line which exhibits the multidrug resistant (MDR) phenotype) exposed to ADR after pretreatment for 3 h with vitamin K 3. Concurrent administration in vivo of vitamin K 3 and ADR illustrated a therapeutically significant increase (P < 0.05) in the life span of sensitivie and resistant tumor cell bearing animals. Vitamin K 3 caused a depletion of the intracellular glutathione (GSH) levels in P388/S and P388/ADR leukemia cells but at concentrations greater than those that enhanced ADR cytotoxicity. Pretreatment of the tumor cells with 1 μM vitamin K 3 induced a 35–50% (P < 0.001) elevation in the intracellular ADR accumulation in MDR P388 leukemia cells, while such an effect was absent in P388/S tumor cells. DNA binding studies performed utilizing calf thymus DNA, indicated that vitamin K 3 enhanced the intercalation potential of ADR and also altered the equilibrium between the free and bound form of ADR in a cell free system. These factors and their possible effects on the potentiation of ADR cytotoxicity and the therapeutic significance of utilizing vitamin K 3 as an adjuvant in the chemotherapy of MDR tumors is discussed.

Modulation of thiol pools by vitamin K3 and its effect on survival of sensitive and resistant murine tumor cells

Cytotoxic effects of vitamin K3 were evaluated utilizing the P388/S, L1210, EAT, S-180 and a multidrug-resistant variant of the P388 leukemia cells (P388/ADR). Antitumorigenic potential of vitamin K3 was assessed by MTT and DNA and RNA biosynthesis inhibition assay. A dose-dependent inhibition of P388/S and P388/ADR cell survival and [3H]thymidine and [3H]uridine incorporation (as a function of DNA and RNA biosynthesis) was observed in tumor cell types exposed to vitamin K3 concentrations ranging from 1 to 100 microM. One hundred mg/kg vitamin K3 caused a 32 and 52% increase in life span of the sensitive and resistant P388 leukemia tumor-bearing mice. Induction of DNA strand breaks at 100 microM vitamin K3 was greater in P388/S than in P388/ADR cells. In vitro treatment with vitamin K3 (100 microM) reduced the intracellular levels of GSH by 40, 47, 6, 15 and 14% in P388/S, P388/ADR, EAT, S-180 and L1210 tumor cells, respectively. In vivo treatment with 100 mg/kg vitamin K3 reduced the GSH content by 18 and 38% and increased the activity of the enzyme GSH-S-transferase and gamma-glutamyl transpeptidase. Effects of free radical scavengers and of compounds that modulate the GSH metabolism on the cytotoxicity of vitamin K3 were also investigated. Results indicate that vitamin K3 interacts with the tumor cell thiol pools while eliciting its antitumor effects and suggest the utility of vitamin K3 in dealing with the growing problem of multidrug resistance.

Inhibition of cyclooxygenase-independent platelet aggregation by low vitamin E concentration

Platelet aggregation induced by threshold concentrations of agonists such as collagen, PAF or epinephrine was inhibited in vitro by 100 μM aspirin but was restored by stimulating platelets with high concentrations of collagen, PAF or by a combination of epinephrine and PAF. Incubating aspirin-treated platelets with 50-100 μM vitamin E or vitamin E acetate inhibited platelet aggregation by high concentrations of collagen and PAF and by the combination of epinephrine and PAF; platelet thromboxane A 2 formation was less than 10% in samples incubated with 100 μM aspirin. Apyrase, added to aspirin-treated platelet, did not influence platelet aggregation induced by epinephrine and PAF. The present study suggests that concentrations of vitamin E as low as 50-100 μM inhibit cyclooxygenase-independent platelet aggregation when combined with an inhibitor of the arachidonate pathway.

The cooperative action of vitamins E and C in the protection against peroxidation of parinaric acid in human erythrocyte membranes

The influence of vitamins E and C on the initial stages of lipid peroxidation in human erythrocyte membranes was assessed with the fluorescent polyunsaturated fatty acid, parinaric acid, as probe molecule. Cumene hydroperoxide was used as initiator with either haemin-Fe 3+ or Cu 2+ as metal ion cofactor. The effect of vitamin C (pro- or antioxidant) appeared to be determined by the localisation of the metal ions, either in the water phase or in the membrane. Vitamin C is only able to reduce metal ions in the water phase, which results in acceleration of radical generation and subsequent enhancement of parinaric acid peroxidation. Thus, interaction of vitamin C with Cu 2+ in the water phase led to drastically enhanced peroxidation of parinaric acid. In contrast, when only membrane-associated haemin-Fe 3+ was present, vitamin C functioned as an antioxidant at all concentrations tested (0–10 μM). In a system with haemin-Fe 3+ equilibrated between the water phase and the membrane, <5 μM vitamin C produced an overall prooxidant, and >15 μM vitamin C an overall antioxidant effect. At vitamin C concentrations of 5–15 μM, continuous measurement of parinaric acid fluorescence revealed a shift in the vitamin C effect from antioxidant to prooxidant within the time-course of an assay. Vitamin E exhibited a protective effect on peroxidation initiated by cumene (per)oxyl radicals with haemin-Fe 3+ as cofactor, by inducing a concentration-dependent extension of the lag-phase in parinaric acid peroxidation. Vitamin E appeared to be much more effective compared with vitamin C in scavenging radicals in this system. This indicates that vitamin C has only a limited ability to react with cumene (per)oxyl radicals in the membrane. The combination of vitamins E and C produced a protective effect on parinaric acid peroxidation exceeding the sum of their individual contributions. Moreover, the rate of vitamin E consumption was drastically lowered in the presence of vitamin C, whereas the rate of vitamin C consumption hardly decreased in the presence of vitamin E. The results are discussed in terms of a reaction scheme where the relative contributions of a number of reactions are considered to determine the total effect of added vitamins C or E. Vitamin E radicals constitute an additional substrate for vitamin C, resulting in a more than additive shift in the overall effect to the antioxidant side.

Phosphatidylcholine metabolism in isolated rat heart: modulation by ethanol and vitamin E

Prolonged ethanol administration has been reported to cause defects in cardiac performance and abnormal cardiac lipid contents. However, little is known regarding the short-term administration of ethanol to the perfused heart and its effect on cardiac phospholipid metabolism. In this study, the isolated Langendorff heart perfusion was used as a model to study the effects of ethanol and a combination of ethanol and vitamin E ( dl-α-tocopherol) on phospholipid metabolism. When perfused with 1% ethanol for 4 h, the major cardiac phospholipids were not altered but a 60% increase in lysophosphatidylcholine level was observed. Studies on the lysophosphatidylcholine metabolic enzymes revealed that phospholipase A (both phospholipase A 1 and A 2) activity was enhanced in the ethanol-perfused heart, but lysophospholipase and acyltransferase activities were unaffected by ethanol treatment. When the heart was perfused with 1% ethanol in the presence of 50–100 μM vitamin E, the ethanol-induced lysophosphatidylcholine accumulation was completely abolished. This was largely attributed to the attenuation of phospholipase A activities by vitamin E. In order to delineate the opposing effects of ethanol and vitamin E on phospholipid metabolism in the heart, phospholipase A activities in the subcellular fractions were determined in the presence of 0.5–2.0% ethanol or a combination of 1% ethanol and 0–100 μM vitamin E. Ethanol alone exhibited a biphasic effect on phospholipase A activity with maximum stimulation of enzyme activities at 1% concentration. When phospholipase A was assayed in 1% ethanol and vitamin E (25–100 μM), its activity was inhibited by vitamin E in a dose-dependent manner. The mechanism by which ethanol enhanced phospholipase A activities was further investigated with a partially purified enzyme from the rat heart cytosol. Kinetic studies with different concentrations of phosphatidylcholine revealed that at low substrate concentrations, ethanol was inhibitory to the reaction, whereas at high substrate concentrations, the reaction was enhanced by ethanol. Vitamin E (50 μM) completely abolished the ethanol-induced enhancement of enzyme activity in a noncompetitive manner. Since lysophosphatidylcholine is cytolytic at high concentration and its accumulation in the heart has been postulated as a biochemical cause of cardiac dysfunction, the level of the lysolipid in the heart must be under rigid control. Our result suggest that the modulation of cardiac phospholipase A activity is an important mechanism for the the regulation of lysophosphatidylcholine levels in the rat heart.