Haloperidol and apomorphine differentially affect neuropeptidase activity

The effects of lipid peroxidation and the antioxidant vitamin E contained in LDL isolated from control plasma (LDL--) and from plasma preincubated with 0.5 mmol/ml alpha-tocopherol (LDL+) on the proliferation of estrogen-receptor positive (ER+ : ZR-75, T-47-D, MCF-7) and negative (ER--: HBL-100, MDA-MB-231) human breast cancer cells were studied. Human skin fibroblasts served as controls. Incubation of plasma with 0.5 mmol/ml alpha-tocopherol resulted in a 3-fold increase of its content and a significant reduction in lipid hydroperoxides and conjugated dienes in LDL. Incubation of fibroblasts or ER+ tumor cells with LDL- or LDL+ had an effect on neither cell proliferation nor on the cellular levels of peroxidation products as compared to control incubations in the absence of LDL. In ER- cells, however, LDL+ stimulated the proliferation, whereas LDL- yielded a cytotoxic effect. Moreover, LDL- supplementation resulted in an increase in the content of hydroperoxides and conjugated dienes. LDL+ supplemented cells exhibited hydroperoxide levels in these tumor cells comparable to the basal levels measured in the absence of LDL. Our data suggested that peroxidation products in LDL are cytotoxic to estrogen-receptor negative breast tumor cells and vitamin E counteracts this effect.     

Hormone action and chromatin remodelling

In this study, we analyzed the influence of vitamin E succinate (5-80 microM), 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 microM) of vitamin E succinate, whereas high concentrations (80 microM) 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 microM) greatly enhanced its cellular content, as compared to vitamin E acetate (80 microM), 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-Fe2+-induced oxidative stress (TBARS formation after cell oxidation decreased by about 15-fold or 1.6 fold, respectively, in the presence of 20 microM vitamin E succinate or 20 microM 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 microM), to 35.99 +/- 1.96% as compared to the control, but not by vitamin E acetate (80 microM), 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.     

Desferrioxamine and vitamin E protect against iron and MPTP-induced neurodegeneration in mice

To elucidate the neuroprotective effects of the iron chelator desferrioxamine (DFO) and the antioxidant vitamin E on excessive iron-induced free radical damage, a chronic iron-loaded mice model was established. The relationship between striatal iron content, oxidized to reduced glutathione ratio, hydroxyl radical (.OH) levels and dopamine concentrations were observed in DFO or vitamin E pretreated iron-loaded/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated C57BL/6 mice. The results demonstrated that both DFO and vitamin E inhibit the iron accumulation and thus reverses the increase in oxidized glutathione (GSSG), oxidized to reduced glutathione ratios, .OH and lipid peroxidation levels. The striatal dopamine concentration was elevated to normal value. Our data suggested that: (1) iron may induce neuronal damage and thus excessive iron in the brain may contribute to the neuronal loss in PD; (2) iron chelators and antioxidants may serve as potential therapeutic agents in retarding the progression of neurodegeneration.     

Cerebral metabolic monitoring experience in critical neurological patients

The effects of vitamin E on lipid peroxidation, intracellular free Ca2+ concentration ([Ca2+]i), and cell death were investigated in the postischemic immature cerebellum. Deprivation of oxygen and glucose for 10-min in a suspension of freshly dissociated granule cells from the cerebellum of 9-day-old male rat pups resulted in a recovery-induced consumption of cell nonenzymatic antioxidants (ascorbic acid, glutathione, and alpha-tocopherol) and development of membrane lipid peroxidation as measured by the thiobarbituric acid method. The rate of lipid peroxidation of the postischemic cells was stimulated, not reduced, by treatment of the cells with vitamin E (5-30 microM alpha-tocopherol phosphate). In flow-cytometric studies a 10-min period of ischemia resulted in a small increase in intracellular calcium concentration, lipid peroxidation products and cell death, but in the presence of alpha-tocopherol the same treatment caused a dramatic increase in cell death, accompanied by a large increase in [Ca2+]i and lipid peroxidation products. Pretreatment of the cells with a mixture of three antioxidants (vitamin C/rutin/ubiquinol-10, 10/5/1) or nickel (Ni2+) reduced the alpha-tocopherol-induced increases in [Ca2+]i, and cell death. Hydrogen peroxide (1 mM) and the water-soluble analogue of vitamin E, trolox (50 microM), mimicked the effect of vitamin E on lipid peroxidation in the postischemic cells. Pretreatment of the cells with the intracellular Ca2+ chelator BAPTA-AM, reduced both the alpha-tocopherol-induced increase in [Ca2+]i and cell death. The effect of vitamin E on [Ca2+]i was age dependent and decreased abruptly during maturation of the cerebellum between the first and second weeks of life. Results of in vitro treatment of the immature cerebellar cells with the water-soluble form of vitamin E (alpha-tocopherol phosphate) suggest that, after consumption of cellular co-antioxidants, vitamin E may be converted to an alpha-tocopheroxyl radical, which act as a toxic prooxidant as cellular bioenergetics deteriorate.     

Retroviral vector-modified bone marrow stromal cells secrete biologically active factor IX in vitro and transiently deliver therapeutic levels of human factor IX to the plasma of dogs after reinfusion

Canine bone marrow stromal cells (BMSCs), transduced ex vivo with retroviral vectors, expressed and secreted biologically active human and canine coagulation factor IX (hFIX and cFIX) in vitro, and on autologous reinfusion expressed hFIX into the circulation of normal (nonhemophiliac) dogs. Human FIX, when expressed in vitro by BMSCs of two dogs at 1.22 and 1.39 microg/10(6) cells/24 hr in medium supplemented with vitamin K, respectively, exhibited 28.1 and 27.3% normal biological activity as determined on the basis of a one-stage clotting assay. BMSCs of two additional dogs expressed 1.54 and 4.81 microg of cFIX/10(6) cells/24 hr in vitamin K-supplemented medium and the expressed cFIX possessed 58.4 and 32.9% normal activity, respectively. Between 2.33 and 3.35 x 10(8) transduced BMSCs, expressing 1.22 and 2.61 microg of hFIX/10(6) cells/24 hr or 3.24 and 7.82 microg of cFIX/10(6) cells/24 hr were reintroduced into the four donor dogs by intravenous infusion. Human FIX was detected in plasma for 7 or 12 days after BMSC reinfusion, with peak levels of 85.8 and 233.0 ng/ml observed at 2 days. Canine anti-hFIX antibodies, which were detected as early as 2-4 days after reinfusion of BMSCs expressing hFIX, may have masked potentially longer duration expression in vivo. Peak plasma levels of hFIX represented 2.1 and 5.8% normal human hFIX levels. When adjusted for percent normal one-stage clotting activity determined in vitro, these levels represented 0.6 and 1.6% normal human hFIX activity levels. Thus, we have demonstrated that retroviral vector-modified BMSCs can deliver human therapeutic levels of hFIX to the circulation of dogs.     

Antifungal nickel(II) complexes derived from amino sugars against pathogenic yeast, Candida albicans

The effects of substances able to reduce peroxidative processes on thyroid hormone-induced electrophysiological changes in ventricular muscle fibres were examined. For this study, 60 day old euthyroid and hyperthyroid rats were used. One group of hyperthyroid rats was untreated and the others were treated with vitamin E, N-acetylcysteine, and cholesterol, respectively. Hyperthyroidism was elicited by 10 day treatment with daily i.p. injections of triiodothyronine (10 microg/100 g body weight). Vitamin E and N-acetylcysteine were administered for 10 days by daily i.m. injections (20 mg/100 g body weight) and daily i.p. injections (100 mg/100 g body weight), respectively. Cholesterol was administered by cholesterol-supplemented diet (4%) from day 30. Hyperthyroidism induced a decrease in the whole antioxidant capacity and an increase in both lipid peroxidation and susceptibility to oxidative stress. Vitamin E and N-acetylcysteine administration to hyperthyroid rats led to reduction in lipid peroxidation and susceptibility to oxidative stress and to increase in antioxidant level, while the diet addition of cholesterol decreased lipid peroxidation but did not modify the other parameters. The hyperthyroid state was also associated with a decrease in the duration of the ventricular action potential recorded in vitro. The vitamin E and N-acetylcysteine administration attenuated the thyroid hormone-induced changes in action potential duration, which was however, significantly different from that of the euthyroid rats. In contrast, cholesterol supplementation did not modify the electrical activity of hyperthyroid heart. These results demonstrate that the triiodothyronine effects on ventricular electrophysiological properties are mediated, at least in part, through a membrane modification involving a free radical mechanism. Moreover, they indicate that the antioxidant-sensitive shortening of action potential duration induced by thyroid hormone is likely independent of enhanced peroxidative processes in sarcolemmal membrane.     

Inhibition of human oral squamous carcinoma cell (SCC-25) proliferation by prostaglandin E2 and vitamin E succinate

The primary objective of this investigation was to study the effect of D-alpha-tocopherol acid succinate (vitamin E succinate) and prostaglandin E2 (PGE2), individually and in combination, on the proliferation of human tongue squamous carcinoma cells (SCC-25) in vitro. Test compounds in varying concentrations were incubated with cells in serum-free Dulbecco's Modified Eagle's Medium-Ham's F-12 Medium (50:50), supplemented with 0.1% albumin for sixteen hours. Cell proliferation was measured by the incorporation of [3H] thymidine in acid-insoluble material (i.e. DNA). Prostaglandin E2 and vitamin E succinate, individually at 10(-9)-10(-6) M, caused significant dose-dependent inhibition in DNA synthesis. A combined dose of each compound at 10(-5) M resulted in significant additive inhibition which averaged 43.53% (p < 0.005). Addition of indomethacin (INDO) to cell cultures induced significant dose-dependent stimulation in DNA synthesis. Hence, we might suggest that the overall potential of vitamin E in controlling malignant cell proliferation in vivo could be due to its own effect combined with that of endogenous PGs which are normally produced in excessive amounts by malignant cells.     

Vitamin E prevents apoptosis in cortical neurons during hypoxia and oxygen reperfusion

Cerebral ischemia followed by oxygen reperfusion induces apoptosis in hippocampal neurons in stroke-prone spontaneously hypertensive rats (SHRSP) but not in Wistar Kyoto rats (WKY). The overproduction of oxygen-free radicals that occurs in the tissues of SHRSP is implicated in reoxygenation injury after hypoxia. Antioxidants inhibit reoxygenation injury in hippocampal slices, and temporal cortices in Alzheimer's disease increase sensitivity to oxygen-free radicals. Because this sensitivity may contribute to the development of the disease, we have studied hypoxia and oxygen reperfusion using cortical neurons isolated from WKY and SHRSP (at 15 days of gestation). We have tried to determine whether cortical neurons are damaged under these conditions, and whether neurons from SHRSP are more vulnerable than those from WKY. We have tried also to verify whether neuronal damage is minimized by vitamin E using the following techniques: (a) Trypan blue staining, (b) in situ staining of apoptosis, (c) ultrastructural examination, and (d) measurement of lactic dehydrogenase (LDH) activity in the bathing medium. Furthermore, we have examined the mechanisms involved in the development of neuronal damage and have studied ways of minimizing it. We demonstrated that 36 hours of hypoxia significantly increased the rate of cell death in SHRSP (p < 0.01), although 12 to 24 hours of hypoxia did not increase cell death in either WKY or SHRSP. In addition, 6 to 36 hours of hypoxia and 1.5 to 5 hours of oxygen reperfusion heavily damaged cells of both WKY and SHRSP, and most became apoptotic or necrotic. In contrast, cells incubated with 50 to 300 microg/ml of vitamin E remained intact, although 10 to 20 microg/ml of vitamin E did not totally preserve the cells. Moreover, vitamin E protected the neurons from high concentrations of sodium nitroprusside (nitric oxide donor) in a dose-dependent manner. Vitamin E, when added to the cells, increased in concentration in a time-dependent manner over a 24-hour period and in a dose-dependent manner below 200 microg/ml, and it was detected mostly in the mitochondria. We also demonstrated that serial treatments with allopurinol (a xanthine oxidase inhibitor) or superoxide dismutase preserved neurons during hypoxia and oxygen reperfusion. These data indicate that SHRSP neurons are weaker than WKY neurons in long-term hypoxia; oxygen radical generation occurs in the early minutes after reperfusion, and then the oxygen-free radicals cause heavy damage to the cells; and antioxidants including vitamin E react with the radicals, thereby preventing apoptosis and necrosis. Therefore, antioxidants appear to be the most important agents in lowering oxygen-free radical damage in cortical neurons.     

Evidence for role of transforming growth factor-beta in RRR-alpha-tocopheryl succinate-induced apoptosis of human MDA-MB-435 breast cancer cells

MDA-MB-435 human breast cancer cells treated with 10 micrograms/ml of RRR-alpha-tocopheryl succinate (vitamin E succinate, VES) for one, two, three, and four days exhibit 9%, 19%, 51%, and 73% apoptotic cells, respectively. Likewise, cells cultured for one, two, and three days with conditioned media (CM) obtained from MDA-MB-435 cells treated with VES exhibit 10%, 36%, and 74% apoptosis, respectively. A quantitative luciferase-based assay showed CM from VES-treated cells collected at 24 and 48 hours after treatment initiation to contain 75 and 32 pg of active transforming growth factor-beta (TGF-beta), respectively, per 10(6) cells. Although purified TGF-beta 1 is not an effective apoptotic agent for MDA-MD-435 cells, cotreatment of the cells for three days with suboptimal levels of VES (2.5 and 5 micrograms/ml) + 10 ng/ml of purified TGF-beta 1 enhanced apoptosis by 66% and 68%, respectively. Interference of the TGF-beta-signaling pathway by transient transfection of MDA-MB-435 cells with antisense oligomers to TGF-beta type II receptor (TGF-beta R-II) blocked VES-induced apoptosis. Likewise, addition of neutralizing antibodies to TGF-beta 1 or to all three mammalian isoforms of TGF-beta (TGF-beta 1, -beta 2, -beta 3) blocked VES- and CM-induced apoptosis. Furthermore, inhibitors of TGF-beta conversion from an inactive latent form to a biologically active form inhibited VES-induced apoptosis. In summary, the ability to reduce apoptosis by blocking TGF-beta or the TGF-beta receptor-signaling pathway with antisense oligomers or ligand-neutralizing antibodies or prevention of activation of TGF-beta indicates a role for TGF-beta signaling in VES-induced apoptosis.     

Novel fluorescein-based flow-cytometric method for detection of lipid peroxidation

The novel property of fluorescein to detect peroxyl radicals is demonstrated. On the basis of this observation, a fluorescein-based, flow-cytometric method to directly and continuously detect free radicals generated in cell membranes during lipid peroxidation has been developed. 5- and 6-Carboxyfluorescein (5-/6-CF) free in solution and fluorescein-labeled polylysine lose their fluorescence gradually upon addition of a peroxyl-radical-generating system (thermal decomposition of 2,2'-azobis(2-amidinopropane) [AAPH]). 5-/6-CF retains its fluorescence when exposed to AAPH in the presence of the peroxyl radical scavenger Trolox. When 5-/6-CF free in solution is incubated with red blood cells exposed to cumene hydroperoxide (CH), a similar loss of fluorescence occurs due to lipid peroxidation on RBC membranes, which is preventable by pretreatment of the cells with Trolox or vitamin E. Undecylamine-fluorescein (C11-fluor), a lipophilic fluorescein conjugate, has been incorporated into the membranes of RBC. Upon addition of CH, a decrease in fluorescence is fluorometrically observed that is proportional to the amount of hydroperoxide added and inhibited by preincubation with Trolox or vitamin E. Flow-cytometric studies are then performed to demonstrate that C11-fluor can monitor free radicals generated during lipid peroxidation on a cell-by-cell basis. When exposed to CH, a time-dependent shift of the flow-cytometric profile toward lower values is observed that is inhibited by Trolox or vitamin E. This approach in conjunction with multiparametric flow cytometry may allow examination of the biologic significance of lipid peroxidation by correlation to other cellular end points on single cells.     

Influence of structure on the antioxidant activity of indolinic nitroxide radicals

An in vitro study was carried out to verify whether the chain length of a substituent on an indolinic nitroxide could influence its antioxidant activity in different biological environments subjected to oxidative stress. Three distinct indolinic nitroxides were synthesized and compared with vitamin E and Trolox (a hydrophilic analogue of vitamin E), where the only difference between the nitroxides was the length of the hydrocarbon chain in the 2-position of indole, namely 2 (C2), 10 (C10), and 18 (C18) carbons. All the nitroxides were effective in preventing oxidation of bovine serum albumin, but to different extents, with the longer chain derivatives being more efficient. However, the C2 compound was the most efficient in preventing lipid peroxidation in microsomal membranes. The C2 and C18 compounds, Trolox, and vitamin E protected microsomal protein oxidation to the same extent at the highest concentration used (13 microM). The nitroxide with a C10 chain was less effective in this system. The influence of these compounds on the enzymatic activity of two mitochondrial proteins subjected to oxidative stress was also studied by means of oxygraph measurements. Mitochondrial rotenone-sensitive NADH oxidase and succinate oxidase responded differently to BuOOH-induced radical chemistry, and the compounds under study also protected the activity of the two enzymes but to different extents. The results clearly demonstrate that indolinic nitroxides are very efficient antioxidants, protecting both lipids and proteins from peroxidation. The indole structure influences the antioxidant efficacy in biological systems.     

Antigoitrogenic effect of combined supplementation with dl-alpha-tocopherol, ascorbic acid and beta-carotene and of dl-alpha-tocopherol alone in the rat

The effects of the vitamins dl-alpha-tocopherol, ascorbic acid and beta-carotene, free radical scavengers and lipid peroxidation inhibitors, were analyzed in male Wistar rats made goitrous by feeding a low iodine diet (< 20 micrograms iodine/kg) and perchlorate (1% in drinking water) for 4, 8, 16, and 32 days. Groups of control or goitrous rats received for at least 16 days before killing a diet containing 0.6% vitamin E (as dl-alpha-tocopherol acetate), 1.2% vitamin C (ascorbic acid) and 0.48% beta-carotene, either simultaneously (vitamin cocktail) or separately. This treatment led to a 5-fold increase of vitamin E in the thyroid gland, a 24-fold increase in the liver and a 3-fold increase in the plasma. In control rats, vitamin cocktail administration increased slightly the thyroid weight with little changes in thyroid function parameters. During iodine deficiency, administration of the vitamin cocktail or vitamin E alone reduced significantly the rate of increase in thyroid weight, and DNA and protein contents, as well as the proportion of [3H]thymidine labeled thyroid follicular cells, but not that of labeled endothelial cells. Plasma tri-iodothyronine, thyroxine, TSH levels, thyroid iodine content and concentration as well as relative volumes of glandular compartments were not modified. The proportion of necrotic cells rose from 0.5% in normal animals to about 2% after 16 days of goiter development. No significant protective effect of the vitamins was observed. These results suggest that these vitamins, particularly vitamin E, modulate one of the regulatory cascades involved in the control of thyroid follicular cell growth, without interfering with the proliferation of endothelial cells.     

Cytotoxicity and apoptosis produced by cytochrome P450 2E1 in Hep G2 cells

Two Hep G2 subclones overexpressing CYP2E1 were established with the use of transfection and limited dilution screening techniques. The Hep G2-CI2E1-43 and -47 (E47) cells (transduced Hep G2 subclones that overexpress CYP2E1) grew at a slower rate than parental Hep G2 cells or control subclones that do not express CYP2E1, but remained fully viable. When GSH synthesis was inhibited by treatment with buthionine sulfoximine, GSH levels rapidly declined in E47 cells but not control cells, which is most likely a reflection of CYP2E1-catalyzed formation of reactive oxygen species. Under these conditions of GSH depletion, cytotoxicity and apoptosis were found only with the E47 cells. Low levels of lipid peroxidation were found in the E47 cells, which became more pronounced after GSH depletion. The antioxidants vitamin E, vitamin C, or trolox prevented the lipid peroxidation as well as the cytotoxicity and apoptosis, as did transfection with plasmid containing antisense CYP2E1 or overexpression of Bcl-2. Levels of ATP were lower in E47 cells because of damage to mitochondrial complex I. When GSH was depleted, oxygen uptake was markedly decreased with all substrates in the E47 extracts. Vitamin E completely prevented the decrease in oxygen uptake. Under conditions of CYP2E1 overexpression, two modes of CYP2E1-dependent toxicity can be observed in Hep G2 cells: a slower growth rate when cellular GSH levels are maintained and a loss of cellular viability when cellular GSH levels are depleted. Elevated lipid peroxidation plays an important role in the CYP2E1-dependent toxicity and apoptosis. This direct toxicity of overexpressed CYP2E1 may reflect the ability of this enzyme to generate reactive oxygen species even in the absence of added metabolic substrate.     

Synergism of lactate and succinate as metabolites utilized by Veillonella to inhibit the growth of Salmonella typhimurium and Salmonella enteritidis in vitro

The inhibition of salmonellae growth by a Veillonella bacterium isolated from the cecal contents of adult chickens was examined. The Veillonella isolate was grown on an agar medium supplemented with 175 mumol of lactate or succinate/ml. Either 0, 100, 125, 150, or 175 mumol of succinate/ml was added to the lactate medium; either 0, 100, 125, 150, or 175 mumol of lactate/ml was added to the succinate medium; and the pH of all media was adjusted to 6.0. Agar overlays of Veillonella cultures grown on the media were inoculated with Salmonella typhimurium or S. enteritidis. The largest zones of inhibition of salmonellae growth were produced by Veillonella cultures grown on medium supplemented with 175 mumol/ml of both lactate and succinate. The widths of the zones of inhibition decreased as the concentration of lactate was reduced in the succinate medium and as the concentration of succinate was reduced in the lactate medium. Analyses of lactate broth and succinate broth inoculated with Veillonella indicated that inhibition of salmonellae growth on the agar media was related to the production of volatile fatty acids by Veillonella, the presence of residual succinate in the media, and the final pH of the media.     

The effect of excessive dietary vitamin A on performance and vitamin E status in swine fed diets varying in dietary vitamin E

A study was conducted to evaluate the effects of high dietary vitamin A on vitamin E status and performance of growing-finishing pigs fed diets supplemented with varying levels of vitamin E. Treatments consisted of corn-soybean meal-based diets supplemented with retinyl acetate to provide 2,000 or 20,000 IU of vitamin A/kg of diet and with DL-alpha-tocopheryl acetate to provide 0, 15, or 150 IU of added vitamin E/kg in a 2 x 3 factorial arrangement. The trial involved 84 crossbred pigs (26 kg initial BW) allotted to pens of two pigs each (one gilt, one barrow). Serum was obtained from all pigs on d 0, 3, 7, 21, 35, 63, and 77 of the 83- or 90-d feeding period. Tissue samples (liver, leg, and neck muscle, backfat, and leaf fat) were collected from one pig (barrow) in each pen at the end of the feeding period. Average daily gain and gain:feed were .93 kg and .30, respectively, without treatment differences (P > .10). Serum alpha-tocopherol increased linearly (P < .01) by d 3 with increasing level of dietary vitamin E supplementation. High dietary vitamin A resulted in a small decrease (P < .01) in serum alpha-tocopherol on d 3, but serum alpha-tocopherol concentration was not affected (P > .10) on other days. Tissue alpha-tocopherol increased linearly (P < .001) as dietary vitamin E increased in all tissues examined. No consistent evidence was found to indicate that a high level of dietary vitamin A interfered with performance or with blood serum or tissue alpha-tocopherol concentrations in growing-finishing swine.     

Lipid peroxidation in vivo during vitamin E and selenium deficiency in the rat as monitored by ethane evolution

Ethane evolution was monitored from vitamin E and selenium (Se)-deficient rats to determine if lipid peroxidation occurs in vivo when these rats develop fatal organ lesions. Weanling rats were fed a vitamin E and Se-deficient, or supplemented, diet for 40 to 90 days. Each was then prefasted for 4 hours and fasting was continued for 24 to 40 hours while ethane was collected. Approximately 50% of the doubly-deficient rats died as a result of fasting. Pathological signs included hematuria, lung hemorrhage, and liver necrosis. Ethane evolution increased exponentially 10 to 20 hours before death and then declined 2 hours before death. Rats that survived (at least 5 days after ethane collection) evolved 7.4+/-1.3 nmoles ethane/100 g body weight/24 hours compared to 100+/-6 for rats that died. Supplementation of the basal diet with vitamin E (200 IU/kg), Se (0.2 ppm, as Na2SeO3), or both, completely prevented mortality and reduced ethane evolution values to 0.4+/-0.2, 3.1+/-0.4, or 0.2+/-0.2, respectively. These experiments indicate that lipid peroxidation occurs in vivo as a result of vitamin E and Se deficiency, and the peroxidation process greatly accelerates during the terminal phase of the fatal disease.     

Vitamin E decreases the occurrence of malformations in the offspring of diabetic rats

An association between excess oxygen radical activity and disturbed embryogenesis in diabetic pregnancy has been suggested. In the present study, the protective capacity of vitamin E with different treatment regimens was investigated in early and late pregnancy of streptozotocin-induced diabetic rats. Daily gavaging of 0.2 g/kg or 0.8 g/kg of vitamin E exerted moderate protective effects. In contrast, treatment with a diet enriched with 2% (wt/wt) of vitamin E, yielding an approximate daily dosage of 2 g/kg of vitamin E, clearly restored both embryonic and fetal morphology. High-performance liquid chromatography measurement showed that maternal diabetes decreased embryonic content of vitamin E. When pregnant diabetic animals were supplemented with vitamin E, increased concentrations of the vitamin were found in maternal, embryonic, and fetal tissues. Thus, despite marked accumulation of vitamin E in maternal tissues, the compound apparently reached the conceptus. Thiobarbituric acid reactive substances (TBARS) were estimated as a measure of lipid peroxidation, and no changes were observed in maternal tissue, embryonic tissue, placenta, and fetal brain in the untreated diabetic group. In contrast, a fivefold increase of TBARS was found in fetal liver, a rise that was reduced with vitamin E treatment of the diabetic pregnant rats and completely normalized with 2% vitamin E in the diet. Congenital malformations caused by experimental diabetes can be prevented by antioxidants in vivo. These findings further corroborate the notion that an imbalance in the metabolism of free oxygen radicals is involved in the embryonic maldevelopment of diabetic pregnancy, and suggest a direction for prophylactic treatment in the future.     

Lipid peroxidation of lung surfactant by bacteria

The epithelium of the lung is lined with extracellular pulmonary surfactant. This is the surface that invading bacteria first come into contact with when they enter the alveoli. As bacteria become established and interact with this layer, various characteristics of surfactant may become altered. We studied free radical production by three bacterial species, group B streptococci, Escherichia coli, and Pseudomonas aeruginosa, as well as the effect of two concentrations of lung surfactant (Curosurf at 0.04 and 0.4 mg/ml) on this production estimated by the nitro blue tetrazolium reduction test. We also measured the lipid peroxidation of surfactant at various incubation times (0-20 h), using a LPO-586 test kit. In addition, the effect of vitamin E as an antioxidant in a concentration of 0.5 microM was determined by the lipid peroxidation test. We found that the nitro blue tetrazolium reduction by the three bacterial species and lipid peroxidation of lung surfactant increased with time. Vitamin E reduced the lipid peroxidation of this surfactant. By measuring bacterial growth at various incubation times we showed that lung surfactant was bactericidal to group B streptococcal and E. coli strains and that P. aeruginosa strains were resistant to surfactant. We conclude that bacteria, probably by their production of reactive oxygen species, cause lipid peroxidation of lung surfactant.     

Influence of erythromycin resistance, inoculum growth phase, and incubation time on assessment of the bactericidal activity of RP 59500 (quinupristin-dalfopristin) against vancomycin-resistant Enterococcus faecium

RP 59500, a mixture of two semisynthetic streptogramin antibiotics (quinupristin and dalfopristin), is one of a few investigational agents currently in clinical trials with inhibitory activity against multiple-drug-resistant strains of Enterococcus faecium. We evaluated the bactericidal activity of this antimicrobial against 30 recent clinical isolates of vancomycin-resistant E. faecium, including 23 erythromycin-resistant (MIC, >256 microg/ml) and 7 erythromycin-intermediate (MIC, 2 to 4 microg/ml) strains. All isolates were inhibited by RP 59500 at 0.25 to 1.0 microg/ml. The bactericidal activity of RP 59500 was markedly influenced by the erythromycin susceptibility of the strains and by several technical factors, such as inoculum growth phase and time of incubation of counting plates. As determined by time-kill methods, RP 59500 at a concentration of 2 or 8 microg/ml failed to kill erythromycin-resistant organisms under any conditions. Bactericidal activity was observed against all seven erythromycin-intermediate isolates when log-phase inocula were used and the cells were counted after 48 h of incubation (mean reductions in viable bacteria for RP 59500 at concentrations of 2 and 8 microg/ml, 3.45 and 3.50 log10 CFU/ml, respectively), but killing was diminished when the plates were examined at 72 h (mean killing, 3.06 and 2.95 log10, CFU/ml, respectively). No bactericidal activity was observed when stationary-phase cultures were used. On the basis of these data, we expect that bactericidal activity of RP 59500 against the multiple-drug-resistant E. faecium strains currently encountered would be distinctly uncommon.     

Relationship of blood thromboxane-B2 (TxB2) with lipid peroxides and effect of vitamin E and placebo supplementation on TxB2 and lipid peroxide levels in type 1 diabetic patients

OBJECTIVE: To study the effect of vitamin E supplementation on platelet hyperaggregability in type 1 diabetic patients. RESEARCH DESIGN AND METHODS: Written informed consent according to the Institutional Review Board on Human Experimentation guidelines was obtained from diabetic patients (n = 29) and their age-matched normal siblings (n = 21) to participate in this study. Diabetic patients were supplemented with DL-alpha-tocopherol (vitamin E) capsule (orally, 100 IU/day) or placebo for 3 months in a double-blind clinical trial. Alternate diabetic patients were assigned to vitamin E or placebo during regular visits to the clinic. Fasting blood was collected from each diabetic patient before the start and after the vitamin E or placebo supplementation. Platelet aggregability was assessed by competitive enzyme-linked immunosorbent assay of the blood TxB2 (a stable thromboxane metabolite). Plasma vitamin E and MDA (malondialdehyde, a product of lipid peroxidation) was assessed by high-performance liquid chromatography. Data were analyzed statistically on 12 diabetic patients on vitamin E and 12 on placebo supplementation. RESULTS: Diabetic patients (n = 29) had 62% higher (P < 0.05) levels of TxB2 and 15% higher levels (P < 0.05) of MDA in comparison to normal subjects (n = 21). Plasma TxB2 levels had a significant correlation with MDA levels (r = 0.45, P < 0.02) but not with the HbA1 (r = -0.08), glucose (r = -0.13), duration of diabetes (r = -0.04), or age (r = 0.12) of diabetic patients. Vitamin E supplementation lowered MDA levels by 30% (P < 0.04), TxB2 levels by 51% (P < 0.03), and triglyceride levels by 22% (P < 0.04) in diabetic patients. There were no differences in these parameters before versus after placebo supplementation. CONCLUSIONS: The elevated blood level of TxB2 (hyperaggregability of platelets) is significantly related to the level of lipid peroxidation products (oxidative stress) in type 1 diabetic patients. Supplementation of modest doses of vitamin E (100 IU/day) significantly lowers blood TxB2 and lipid peroxidation products levels in type 1 diabetic patients.