Alpha-tocopherol in rat brain subcellular fractions is oxidized rapidly during incubations with low concentrations of peroxynitrite.

The reaction of superoxide (a reactive oxygen species) and nitric oxide (one of the nitrogen oxides with numerous biological functions) results in the production of peroxynitrite. The characteristics of oxidation of alpha-tocopherol (vitamin E) in synaptosomes (nerve ending particles) and mitochondria by peroxynitrite were studied. The subcellular fractions were isolated from brain hemispheres of 4-month-old male Fischer 344 rats by standard centrifugation procedures involving Ficoll gradients. Peroxynitrite treatment oxidized alpha-tocopherol in <5 s. This reaction was selective because another membrane component, cholesterol, was not oxidized at the same time, as observed in our previous studies. Mitochondrial alpha-tocopherol was more susceptible to peroxynitrite-induced oxidation than synaptosomal tocopherol. Measurable and significant (P < 0.05) oxidation of tocopherol occurred when mitochondria or synaptosomes were incubated with peroxynitrite in concentrations as low as 5 or 10 micromol/L, respectively. The oxidation could be readily monitored by estimating the production of tocopherolquinone. Oxidation of tocopherol induced by ferrous iron and ascorbate was much slower and the yield of tocopherolquinone lower than by peroxynitrite. The fast and selective oxidation of alpha-tocopherol by peroxynitrite suggests that vitamin E may play an important role in preventing membrane oxidation induced by peroxynitrite. Literature reports indicate the existence of threshold concentrations of tocopherol below which functional alterations occur. Tocopherol oxidation by peroxynitrite could reduce tocopherol concentrations in tissues and subcellular structures to these threshold levels by different concentrations of peroxynitrite. Hence the sensitivity of tissues to peroxynitrite could vary over a wide range.

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 degrees 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 approximately 10 microM. 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.

Oxidation of cholesterol in synaptosomes and mitochondria isolated from rat brains.

Cholesterol and alpha-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 degrees C0 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 5 alpha, 6 alpha-epoxycholesterol. Since brain contains high levels of ascorbate, and release of iron could result in oxysterol formation. Peroxynitrite oxidized alpha-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 microM Fe2+ and ascorbate, alpha-tocopherol was oxidized while cholesterol remained unchanged. Thus, alpha-tocopherol is functioning as an antioxidant, protecting cholesterol. Diethylenetriaminepentaacetic acid blocked production of oxysterols, whereas citrate, ADP and EDTA did 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 plan an important role in brain tissue damage during oxidative stress.

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 alpha-to-copherol 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-, 7 alpha-hydroxy-, 7 beta-hydroxy-, and 5 alpha, 6 alpha-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 alpha-tocopherol. The proposed procedure will be useful in monitoring the oxidation of small quantities of membrane cholesterol in vitro.

In vitro oxidation of vitamin E, vitamin C, thiols and cholesterol in rat brain mitochondria incubated with free radicals.

The kinetics of oxidation of endogenous antioxidants such as vitamins C and E and thiols as well as membrane cholesterol in isolated rat brain mitochondria were studied. Oxidation was induced by incubating the mitochondria at 37 degrees C with the free radical generators 2,2' azobis (2'-amidinopropane) dihydrochloride (ABAPH) and 2,2' azobis (2,4-dimethyl) valeronitrile (ABDVN) which undergo thermal decomposition to yield free radicals. An approximate order for the in vitro ease of oxidation was: ascorbate >> alpha-tocopherol > sulfhydryls >> cholesterol. However, small amounts of ascorbate were present in the mitochondria when alpha-tocopherol and sulfhydryl compounds were getting oxidized. This observation is different from those with more homogeneous biological substrates like blood plasma or serum. The order of oxidation of the various compounds is a function of not only the redox potentials but also the (a) concentrations of the oxidized and reduced species, (b) compartmentation of the compounds and (c) enzymatic and nonenzymatic systems for the repair or regeneration of the individual antioxidants. Even though ascorbate levels are quite low within mitochondria this nutrient may play a major role as a first line of defense against oxidative stress. The lipid-soluble ABDVN was much more potent in oxidizing membrane alpha-tocopherol and thiols than the water-soluble ABAPH. With both free radical generators the rate of oxidation of the antioxidants consisted of two phases. The initial phase, that is more rapid, may represent a pool of antioxidant that is involved in immediate antioxidant protection of the organelle with the slower compartment being responsible for replenishing the faster pool whenever needed.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased susceptibility to oxidation of vitamin E in mitochondrial fractions compared with synaptosomal fractions from rat brains.

The in vitro oxidation of vitamin E (alpha tocopherol) in rat brain synaptosomes and mitochondria by 2,2'-azo-bis-(2'-amidinopropane) dihydrochloride (ABAPH), a free radical generator, was studied. Subcellular fractions (300 micrograms total protein) were suspended in different buffers at pH 7.4 and incubated at 37 degrees C. In the presence of 0.5 mM ABAPH the mitochondrial alpha tocopherol began to get oxidized after a lag time or induction time of 15 min compared with a lag time of 30 min for the synaptosomal fraction. Thus the reserve of reducing compounds that are responsible for delaying tocopherol oxidation is less in mitochondria than in synaptosomes. More tocopherolquinone was produced during incubations without ABAPH compared with incubations in the presence of ABAPH suggesting that the mechanism of oxidation of tocopherol differs under these two conditions. When mitochondria were incubated in buffer without oxidants the production of tocopherolquinone preceded that of thiobarbituric acid reactive substances, an indicator of peroxidation of fatty acids. Therefore, alpha tocopherol is active as an anti-oxidant in mitochondrial membranes and the production of alpha tocopherolquinone could be a monitor of mild membrane oxidation under in vitro conditions. The ease of oxidation of mitochondrial tocopherol suggests a general vulnerability of the mitochondrial membranes to oxidation. Adding vitamin E or its water soluble analogs during in vitro experiments may improve the stability and viability of mitochondria. Furthermore, antioxidant protection by vitamin E may be crucial for the maintenance of tissues, such as brain, whose function is critically dependent upon the availability of high energy phosphates.(ABSTRACT TRUNCATED AT 250 WORDS)

A liquid chromatographic method for the simultaneous determination of alpha-tocopherol and tocopherolquinone in human red blood cells and other biological samples where tocopherol is easily oxidized during sample treatment.

A liquid chromatographic method for the simultaneous determination of alpha-tocopherol and tocopherolquinone in human red blood cells is described. Tocopherols in the red cell membrane are very susceptible to oxidation during sample processing. Red cell samples are saponified in the presence of a mixture of butylated hydroxytoluene, ascorbic acid, and pyrogallol and then extracted with hexane. The tocopherol compounds are separated on a C-18 column using a mobile phase containing 12% acetonitrile, 83% methanol, and 5% buffer (NaH2PO4.H2O, 7.5 mM final concentration) and are detected electrochemically. The mixture of antioxidants is essential to avoid loss of the tocopherol compounds during processing of samples. The use of acetonitrile in the mobile phase results in the separation of tocopherolquinone from delta-tocopherol. The proposed method may be generally suitable for the analysis of biological samples where tocopherols are especially vulnerable to oxidation. The levels of tocopherolquinone and delta-tocopherol in normal red cells are quite small (less than 1% of alpha-tocopherol). The ratio of tocopherol and tocopherolquinone concentrations might serve as a useful index of the redox status of red cell membranes, particularly under in vitro conditions.

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.

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 incubated in 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 microM 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 direct in 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.