Hung T. Quach

Ascorbic acid, glutathione and synthetic antioxidants prevent the oxidation of vitamin E in platelets

An earlier report from this laboratory showed that tocopherol in human platelets is oxidized when the platelets are incubatedin vitro in Tyrode medium with arachidonate (or other oxidants). Arachidonate is a more potent oxidizing agent in 50 mM potassium phosphate buffer at pH 7.4 with 0.1 mM ethylenediaminetetraacetic acid (EDTA) than in Tyrode medium. Forty to fifty percent of total platelet tocopherol was oxidized upon incubation with 40–50 μM arachidonate in the phosphate-buffered medium. The tocopherol oxidation took place within 15 min after the addition of arachidonate. Preincubation of platelets with ascorbate blocked the oxidation of tocopherol. This is one of the first directin vitro demonstrations of the vitamin E-sparing action of vitamin C in media containing biological cellular material. Other compounds which blocked the oxidation of platelet tocopherol were ascorbyl palmitate, propyl gallate, butylated hydroxytoluene, hydroquinone and glutathione. If ascorbate or glutathione was added after the tocopherol was oxidized to the quinone there was no reversal of the oxidation.

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.

Aging is associated with a decrease in synaptosomal glutamate uptake and an increase in the susceptibility of synaptosomal vitamin E to oxidative stress

We examined the influence of aging upon the uptake of glutamate by synaptosomes, and the oxidation of Synaptosomal vitamin E. Synaptosomes isolated from the cerebral hemispheres of Fischer 344 rats, 4 and 24 months old, were suspended at 37°C in buffer (pH 7.4) simulating extracellular fluid containing 10 mM glucose. The Km for the high affinity uptake of tritium labeled glutamate was ∼10 μM. The uptake of glutamate was lower in synaptosomes from older animals than those from younger animals for periods of up to 20 minutes. Upon incubation with a mixture of ferrous iron and ascorbate, more of the alpha tocopherol in synaptosomes derived from older rats was oxidized than in those derived from younger ones. Older animals may be more susceptible to excitotoxicity because: a) Synaptosomal reuptake of glutamate is less efficient and b) oxidative stress induced by various agents including glutamate may be higher in synaptosomes from the older animal.

Effect of vitamin E deficiency on the growth and secretory function of the rat prostatic complex.

Increased intake of vitamin E has been suggested to be protective against prostate cancer in men, but the effects of vitamin E on prostate growth and function remain poorly defined. The purpose of this study was to determine the effects of vitamin E deficiency on pubertal growth and maturation of the prostate in the rat. Animals were placed on a vitamin E deficient diet at 28 days of age and were followed for 15 and 26 weeks. Vitamin E deficient rats had a circulating vitamin E level of less than 1% of control animals and experienced a decrease in body and testis weight. The deficiency did not alter the weights of the ventral and dorsal lobes of the prostate. However, there was an increase in weight, DNA, and protein contents of the lateral lobe in control and vitamin E deficient rats from 15 to 26 weeks of treatment, but these increases were significantly lower in vitamin E deficient 26-week treated rats. The volume of secretion per milligram tissue was greater in the ventral than lateral or dorsal lobes. The volume of secretion and activity of the secretory 26 kDa protease in the ventral prostate was lower in vitamin E deficient rats at 15 weeks, but not at 26 weeks of treatment. In contrast, the relative protein content of lateral lobe secretion increased in both control and vitamin E deficient rats from 15 to 26 weeks of treatment. The lateral, but not ventral or dorsal, lobes of both control and vitamin E deficient rats were affected by chronic prostatitis as evidenced by infiltration of inflammatory cells. The lateral lobes also showed markedly elevated activities of the matrix metalloproteinases gelatinase A (MMP-2) and gelatinase B (MMP-9). These data indicate that vitamin E deficiency does not alter the growth of the prostatic lobes, nor the onset and extent of lateral lobe specific prostatitis, but it may delay some differentiated functions such as secretion of specific proteins in the ventral lobe. Thus, the effects of vitamin E in the prostate of the rat appear to be selective.

A sensitive assay of transthyretin (prealbumin) in human cerebrospinal fluid in nanogram amounts by ELISA

A sensitive ELISA method for determining transthyretin (prealbumin) in human cerebrospinal fluid (CSF) is described. The method utilizes goat antihuman transthyretin antibody (IgG fraction) for capture and peroxidase conjugated antibody for color development. The assay has a linear range of 1-4 ng transthyretin added per well. The within-day and between-day coefficients of variation are 5.1 and 6.1%, respectively. The concentration of transthyretin in CSF (ranging from 5 to 20 mg per L) correlated significantly with the corresponding serum concentrations (range 170-420 mg/l). This suggests that synthesis of transthyretin in the brain and peripheral tissues is under similar biological control in normal subjects. The transthyretin concentrations in CSF did not correlate with total CSF protein concentration or age of the subject.

Mice lacking α-tocopherol transfer protein gene have severe α-tocopherol deficiency in multiple regions of the central nervous system

Ataxia with vitamin E deficiency is caused by mutations in alpha-tocopherol transfer protein (alpha-TTP) gene and it can be experimentally generated in mice by alpha-TTP gene inactivation (alpha-TTP-KO). This study compared alpha-tocopherol (alpha-T) concentrations of five brain regions and of four peripheral organs from 5 months old, male and female, wild-type (WT) and alpha-TTP-KO mice. All brain regions of female WT mice contained significantly higher alpha-T than those from WT males. alpha-T concentration in the cerebellum was significantly lower than that in other brain regions of WT mice. These sex and regional differences in brain alpha-T concentrations do not appear to be determined by alpha-TTP expression which was undetectable in all brain regions. All the brain regions of alpha-TTP-KO mice were severely depleted in alpha-T. The concentration of another endogenous antioxidant, total glutathione, was unaffected by gender but was decreased slightly but significantly in most brain regions of alpha-TTP-KO mice. The results show that both gender and the hepatic alpha-TTP, but not brain alpha-TTP gene expression are important in determining alpha-T concentrations within the brain. Interestingly, functional abnormality (ataxia) develops only very late in alpha-TTP-KO mice in spite of the severe alpha-tocopherol deficiency in the brain starting at an early age.

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.