W. Ed Smith

Oxidation of vitamin E and vitamin C and inhibition of brain mitochondrial oxidative phosphorylation by peroxynitrite.

The effects of peroxynitrite (PN; product of the reaction between nitric oxide and superoxide) on mitochondrial respiration as well as oxidation of α‐tocopherol and ascorbic acid were studied. Mitochondria were isolated from brain hemispheres of 4‐month‐old male Fisher rats by standard centrifugation procedures utilizing Ficoll gradients. Treatment of brain mitochondria with PN caused a concentration‐dependent impairment of oxidative phosphorylation and depletion of the endogenous antioxidants α‐tocopherol and ascorbic acid. PN‐induced mitochondrial dysfunction was characterized by 1) decreases in state 3 respiration and oxidative phosphorylation, 2) loss of respiratory control [ratio of ADP‐stimulated (state 3) to basal (state 4) respiration], and 3) uncoupling of oxidative phosphorylation. PN did not function as a pure uncoupler, insofar as the increase in state 4 respiration was accompanied by a larger decrease in state 3 respiration. This contrasts with the uncoupling action of the protonophore carbonyl cyanide m‐chlorophenylhydrozone, which increases both state 3 and state 4 respiration. PN‐induced reduction in respiratory control and oxidative phosphorylation closely paralleled the oxidation of membrane tocopherol and were preceded by loss of ascorbate. α‐Tocopherol (the most potent biological lipid antioxidant) may have a unique role in protecting mitochondrial membranes from oxidative stress. The two antioxidant nutrients α‐tocopherol and ascorbate (which interact with each other and glutathione) may be intimately involved in protecting mitochondria in situations in which excessive release of superoxide and nitric oxide occurs under normal and/or pathological conditions.

Alpha and Gamma Tocopherols in Cerebrospinal Fluid and Serum from Older, Male, Human Subjects

Objective: The major forms of vitamin E in human physiological fluids are alpha and gamma tocopherols which exhibit different biological activities under a variety of assay conditions. The goal of this study was to obtain indirect information about the transport of tocopherols across the blood/spinal fluid barrier by comparing the concentrations of alpha and gamma tocopherols in serum and cerebrospinal fluid (CSF). Methods: CSF and serum samples were obtained simultaneously from 28 human, male subjects excluding those with known pathology during the performance of spinal anesthesia procedures. The samples were centrifuged and frozen, and analyzed for tocopherols by HPLC with electrochemical detection. Results: The concentrations of alpha and gamma tocopherols in CSF correlated significantly with their respective concentrations in serum. This would be expected since these nutrients have to be supplied by diet to serum followed by transport to the brain. The ratios of alpha to gamma tocopherols in the CSF and serum were highly correlated. High concentrations of alpha in serum tended to suppress gamma in both serum and CSF. Conclusions: These data suggest that the processes involved in the entry of tocopherol from blood to the CSF do not discriminate between the alpha and gamma tocopherols. In contrast, alpha tocopherol is highly preferred during the packaging of plasma lipoproteins by the liver. Our data also suggest that alpha and gamma tocopherols will be available to the human brain via transport from blood.

Apolipoprotein E exerts selective and differential control over vitamin E concentrations in different areas of mammalian brain

Apolipoprotein E (apoE) is known to be a risk factor for the incidence of Alzheimers disease (AD). In addition, vitamin E has been reported to have a role in the treatment of AD. We examined the potential interrelationship between vitamin E and apoE in brain. As the first step, we determined the concentrations of α‐tocopherol in selected brain regions of apoE‐deficient mice at different ages. The mice were fed normal rodent chow. All regions of the brain in apoE‐deficient mice contained less α‐tocopherol than control samples at 2.5 months of age, the initial time of study. This trend continued for 9.5 months for most regions except the spinal cord and cerebellum. Tocopherol levels in these latter regions of apoE‐deficient animals increased to control levels during the study. Serum α‐tocopherol and cholesterol levels were high in the apoE‐deficient animals; however, the CNS cholesterol levels were the same in apoE‐deficient and control mice. This suggests that 1) the decline in brain α‐tocopherol in apoE deficiency is not due to overall alterations in lipid metabolism; and 2) the processing of α‐tocopherol in brain follows a separate pathway than that of cholesterol. Subcellular concentrations of α‐tocopherol were unaltered by apoE deficiency indicating that intracellular handling of tocopherol is not affected by apoE. ApoE may be an important protein controlling vitamin E levels in specific brain regions. Further understanding of the interactions between apoE and vitamin E could be important in the appropriate use of vitamin E in AD.

Oxidation of cholesterol in synaptosomes and mitochondria isolated from rat brains

Cholesterol and α-tocopherol oxidations were studied in brain subcellular fractions isolated from cerebral hemispheres of 4-month-old, male Fischer 344 rats. The fractions were suspended in buffered media (pH 7.4, 37°C) and oxidized by adding (i) ferrous iron (Fe2+) with or without ascorbate or (ii) peroxynitrite (an endogenous oxidant produced by the reaction of superoxide and nitric oxide). Treatment of subcellular fractions with Fe2+ in the presence or absence of ascorbate produced primarily 7-keto- and 7-hydroxy-cholesterols and small amounts of 5α,6α-epoxycholesterol. Since brain contains high levels of ascorbate, any release of iron could result in oxysterol formation. Peroxynitrite oxidized α-tocopherol but not cholesterol. Hence, the toxicity of peroxynitrite or nitric oxide could not be due to cytotoxic oxysterols. When synaptosomes were incubated for 5 min in the presence of 0.5 to 2 μM Fe2+ and ascorbate, α-tocopherol was oxidized while cholesterol remained unchanged. Thus, α-tocopherol is functioning as an antioxidant, protecting cholesterol. Diethylenetriaminepentaacetic acid blocked production of oxysterols, whereas citrate, ADP and EDTA dit not. A significant percentage of mitochondrial cholesterol was oxidized by treatment with Fe2+ and ascorbate. Hence, mitochondrial membrane properties dependent on cholesterol could be particularly susceptible to oxidation. The oxysterols formed were retained within the membranes of synaptosomes and mitochondria. The 7-oxysterols produced are known to be inhibitors of membrane enzymes and also can modify membrane permeability. Hence, oxysterols may play an important role in brain tissue damage during oxidative stress.

Oxidative stress and inhibition of oxidative phosphorylation induced by peroxynitrite and nitrite in rat brain subcellular fractions.

Nitrite and nitrate, two endogenous oxides of nitrogen, are toxic in vivo. Furthermore, the reaction of superoxide (produced by all aerobic cells) with nitric oxide (NO) generates peroxynitrite, a potent oxidizing agent, that can cause biological oxidative stress. Using subcellular fractions from rat brain hemispheres we studied oxidative stress induced by these nitrogen compounds with special emphasis on nitrite. The consumption of Vitamin C (ascorbate) and Vitamin E (alpha tocopherol), two of the important nutritional antioxidants, was followed in synaptosomes (nerve-ending particles) and mitochondria along with changes in parameters of mitochondrial oxidative phosphorylation. Nitrite, but not nitrate, oxidized ascorbate without oxidizing alpha tocopherol in both synaptosomes and mitochondria whereas peroxynitrite oxidized both ascorbate and alpha tocopherol. Functionally, both nitrite and peroxynitrite inhibited mitochondrial oxidative phosphorylation. Nitrite was less potent than peroxynitrite when the effects of equal concentrations of the two were compared. However, since nitrite is much more stable than peroxynitrite the impact of nitrite as an oxidant in vivo could be as much or even more significant than peroxynitrite. Nitrate would not have similar action unless it is reduced to nitrite. It is possible that nitrite may impair oxidative phosphorylation through modulating levels of nitric oxide, changing the activity of heme proteins or a mild uncoupling of mitochondria.

Analysis of hydroxy and keto cholesterols in oxidized brain synaptosomes

A rapid method for the simultaneous determination of cholesterol and its oxidation products as well as α-tocopherol and tocopherolquinone in brain subcellular fractions is described. The samples are saponified and extracted with hexane. It is not necessary to remove cholesterol in the sample before analyzing for oxysterols. The hexane extract can be used for the assay of cholesterol compounds by capillary gas chromatography and tocopherol compounds by liquid chromatography using a procedure reported previously. Oxidation of synaptosomes by a mixture of Fe2+ plus ascorbate resulted in the production of 7-keto-, 7α-hydroxy-, 7β-hydroxy-, and 5α, 6α-epoxycholesterols. The identities of these products were confirmed with gas chromatography/mass spectrometry. Cholesterol oxidase treatment did not result in the formation of any of the above compounds. Thus the types and amounts of the products of oxidation of cholesterol were dependent upon the oxidizing agent. Extraction of the oxysterols under milder conditions without saponification using sodium dodecyl sulfate cannot be used since such treatment results in low recovery of oxysterols. Oxidation of synaptosomes by low concentrations of ferrous iron and ascorbate resulted in (i) low levels of oxidation of cholesterol which could be followed by estimating the production of oxysterols and (ii) oxidation of a substantial percentage of α-tocopherol. The proposed procedure will be useful in monitoring the oxidation of small quantities of membrane cholesterol in vitro.

Effect of Oxidative Stress Induced by L‐Dopa on Endogenous Antioxidants in PC‐12 Cells

Abstract:  The mechanism of action of many drugs of abuse involves the dopaminergic pathway. One method of increasing dopamine in brain is by ingestion of L‐dopa (3,4‐dihydroxy‐L‐phenylalanine). Interestingly, both dopamine and L‐dopa cause oxidative stress which is also a factor in drug‐induced damage. Oxidative stress can be reduced by the antioxidant activities of vitamins C (ascorbate) and E (tocopherols). However, the interactions between L‐dopa and tocopherols and ascorbate are not well understood. In this article, PC‐12 cells (as models of neurons) were cultured for 21 hours with ascorbate (400 μM) and/or alpha tocopherol (25 μM) and in the presence or absence of L‐dopa (250 μM). After incubation, cells were harvested and analyzed for the various biochemical components by HPLC. As expected, the addition of L‐dopa resulted in an almost threefold increase in cellular dopamine content. Addition of alpha tocopherol resulted in marked increase in cellular tocopherol. Similarly, addition of ascorbate substantially increased its cellular concentration. These increases were strongly attenuated by the presence of L‐dopa in the medium. The data indicate that: (a) the uptake systems for ascorbate and tocopherols in these cells are inhibited by L‐dopa and/or (b) L‐dopa treatment causes an increase in the rate of utilization of the two nutrients. Thus L‐dopa modulates the cellular dynamics of ascorbate and tocopherol altering cellular antioxidant protection.

Brains of apolipoprotein E deficient mice fed vitamin E deficient diets show alteration in handling alpha tocopherol injected into the cerebral ventricles

Abnormal function of apolipoprotein E (apoE) has been implicated in the incidence of some neurological disorders including dementia. Our recent experiments have shown that apoE deficiency alters the dynamics of alpha tocopherol (vitamin E) handling by brain. In the current investigation, we examined the uptake and retention of tritium-labeled alpha tocopherol that was injected into the lateral cerebral ventricles of apoE-deficient and wild type mice that were fed vitamin E-deficient diet. Eighteen weeks-old, male mice were fed vitamin E-deficient diets for 28 weeks. Labeled cholesterol was injected with the radioactive tocopherol and the cholesterol counts were used as internal standard. After an equilibration time of 48 h, radioactive alpha tocopherol levels in most brain regions were higher in apoE deficient animals when compared with the wild type. Along with our other data, this suggests that the clearance of vitamin E is slower in apoE-deficient brains. Nearly all of the injected alpha tocopherol was unchanged in the brains of both apoE-deficient and wild type animals (even with the additional dietary stress of vitamin E deficiency) suggesting low turnover rate of tocopherol in brain. The data strongly suggest that apoE is a key protein involved with the transport and/or retention of alpha tocopherol in brain.

Deletion of apolipoprotein E gene modifies the rate of depletion of alpha tocopherol (vitamin E) from mice brains.

Our previous reports show that apolipoprotein E (apoE) influences the dynamics of alpha tocopherol (vitamin E) in brain. In this investigation, the patterns of depletion of alpha tocopherol from tissues of apoE deficient and wild type mice were compared after the animals were fed vitamin E deficient diets. Alpha tocopherol concentrations in specific regions of the brain and peripheral tissues at different times were determined by HPLC with electrochemical detection. ApoE deficiency significantly retarded the rate of depletion of alpha tocopherol from all regions of the brain. In addition, comparison of the rates of depletion of alpha tocopherol in both apoE deficient and wild type animals showed that cerebellum behaved differently from other areas such as cortex, hippocampus and striatum. This reinforces the uniqueness of cerebellum with regard to vitamin E biology. Patterns of depletion of tocopherol from peripheral tissues were different from brain. Serum tocopherol was higher in apoE deficient animals and remained higher than wild type during E deficiency. Depletion of liver tocopherol also tended to be unaffected by apoE deficiency. Our current and previous observations strongly suggest that apoE has an important role in modulating tocopherol concentrations in brain, probably acting in concert with other proteins as well.