Search

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. [ABSTRACT FROM AUTHOR]

The article presents an abstract of the research "Alterations in Vitamin E Content in Different Regions of the Brains of Apo-E Knockout Mice," which will be presented at the American Society for Neurochemistry's 30th Annual Meeting to be held in New Orleans, Louisiana on March 14-17, 1999. Earlier studies have shown that high doses of vitamin E show some promise as a therapeutic agent in the treatment of Alzheimer's disease.

Background: Alzheimer's disease (AD) has been associated with both oxidative stress and excessive glutamate activity. A clinical trial was designed to compare the effectiveness of (i) alpha-tocopherol, a vitamin E antioxidant; (ii) memantine (Namenda), an N-methyl-D-aspartate antagonist; (iii) their combination; and (iv) placebo in delaying clinical progression in AD., Methods: The Veterans Affairs Cooperative Studies Program initiated a multicenter, randomized, double-blind, placebo-controlled trial in August 2007, with enrollment through March 2012 and follow-up continuing through September 2012. Participants with mild-to-moderate AD who were taking an acetylcholinesterase inhibitor were assigned randomly to 2000 IU/day of alpha-tocopherol, 20 mg/day memantine, 2000 IU/day alpha-tocopherol plus 20 mg/day memantine, or placebo. The primary outcome for the study is the Alzheimer's Disease Cooperative Study/Activities of Daily Living Inventory. Secondary outcome measures include the Mini-Mental State Examination; the Alzheimer's Disease Assessment Scale, cognitive portion; the Dependence Scale; the Neuropsychiatric Inventory; and the Caregiver Activity Survey. Patient follow-up ranged from 6 months to 4 years., Results: A total of 613 participants were randomized. The majority of the patients were male (97%) and white (86%), with a mean age of 79 years. The mean Alzheimer's Disease Cooperative Study/Activities of Daily Living Inventory score at entry was 57 and the mean Mini-Mental State Examination score at entry was 21., Conclusion: This large multicenter trial will address the unanswered question of the long-term safety and effectiveness of alpha-tocopherol, memantine, and their combination in patients with mild-to-moderate AD taking an acetylcholinesterase inhibitor. The results are expected in early 2013., (Published by Elsevier Inc.)

Hereditary canine spinal muscular atrophy (HCSMA) is an inherited motor neuron disease affecting a kindred of Brittanies. We have examined the clinicopathologic abnormalities in 57 animals with HCSMA, including 43 affected adult dogs and 14 homozygote pups. We also measured selected biochemical indices of oxidative stress: serum vitamin E (alpha-tocopherol) and Se concentrations; serum concentrations of Cu, Zn, Mg, and Fe; and total superoxide dismutase and glutathione peroxidase activities in red blood cells. Dogs with HCSMA had the following abnormalities: regenerative anemia, hypoglobulinemia, hypochloremia, and abnormally high creatine kinase and liver alkaline phosphatase activities. Serum Cu concentration was significantly (P = .01) increased in adult dogs with HCSMA compared to control dogs. Serum vitamin E concentrations tended to be lower in adult dogs with HCSMA compared to controls, and were significantly (P = .01) lower in homozygote pups compared to control pups.

Oxidative stress is a putative factor in the pathogenesis of many human disorders of the central nervous system. Therefore, antioxidants such as vitamin E have become attractive as therapeutic agents in the treatment of several diseases. In addition, vitamin E seems to play a specific role in the nervous system. As a result, vitamin E has been used in pharmacologic doses in the treatment of disorders such as Parkinson disease, Alzheimer disease, and tardive dyskinesia. One investigation showed that the use of 2000 IU all-rac-alpha-tocopheryl acetate is beneficial in the treatment of Alzheimer disease. Similar doses of vitamin E, however, were not beneficial for delaying the progression of Parkinson disease. In other studies, dosages >/=400 IU vitamin E/d were found to be beneficial in the treatment of tardive dyskinesia, although this finding was not confirmed in a larger cooperative study conducted by the Veterans Administration. Even though the efficacy of vitamin E in the management of cardiovascular disease has been shown, the potential role of vitamin E in the treatment of cerebrovascular disease remains essentially unknown. The experience from 2 large clinical trials involving the oral intake of 2000 IU vitamin E/d suggests that vitamin E is relatively safe at this dosage for periods <2 y. However, the safety and efficacy of supplemental vitamin E over periods of many years in the prevention of neurologic diseases has not been adequately explored.

The nervous system is protected from free-radical-induced oxidative damage by a number of antioxidants. These include small-molecular-weight substances such as vitamins C and E as well as the large protein molecules superoxide dismutase, glutathione peroxidase, and others. These antioxidants are often localized in specific portions of the cell. They also show considerable biological interactions with one another. These factors modulate their antioxidant activity. Oxidative stress has been implicated in the pathogenesis of some disorders of the brain; hence, antioxidants have become attractive therapeutic agents. Among the antioxidants, vitamin E has shown some promise in the treatment of Alzheimer's disease and cardiovascular disease. No investigations have been conducted to examine whether vitamin E or other nutritional antioxidants can be used prophylactically to prevent the incidence of degeneration or other pathologic processes in the brain.

Alpha-Tocopherol concentrations were determined in the CSF of patients with early untreated Parkinson's disease receiving 2,000 IU vitamin E orally per day. After treatment the concentrations increased significantly (p < 0.001) by 76+/-10 (SE)%. The net increases in CSF alpha-tocopherol concentrations after treatment showed a significant positive correlation with the number of days of vitamin E ingestion (p < 0.001). Thus, high-dose vitamin E treatment results in elevating CSF vitamin E levels and possibly brain vitamin E levels.

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.

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.

Objectives: To develop an HPLC method for the analysis of trazodone and its metabolite, 1-m-chlorophenyl-piperazine (m-CPP), in human plasma and red blood cells., Design and Methods: The analytes were extracted by heptane containing 1.5% isoamyl alcohol, back extracted into phosphoric acid and analyzed by reverse phase HPLC with UV detection., Results: In seven randomly selected, male, human subjects plasma concentrations (nmol/l) were 380-5841 for trazodone and 14-237 for m-CPP while those in packed red blood cells were 98-634 for trazodone and 15-155 for m-CPP. Plasma trazodone concentrations were 4-11-fold higher than those in red blood cells while those of m-CPP were about equal. This may be the first report on concentrations of trazodone and m-CPP in human red blood cells., Conclusions: This sensitive method can be used for monitoring trazodone and m-CPP in human plasma and red blood cells.

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.

Peroxynitrite is formed by the reaction of superoxide with nitric oxide, an important neurotransmitter. Incubation of rat brain synaptosomes with peroxynitrite resulted in the consumption of antioxidant substances such as alpha-tocopherol, ascorbate, and thiols. Membrane cholesterol was not oxidized under the same conditions. alpha-Tocopherol and ascorbate in synaptosomes were oxidized very rapidly by peroxynitrite. In contrast, previous reports in the literature have shown that peroxynitrite treatment did not oxidize tocopherol in human plasma. Peroxynitrite in sufficient concentrations oxidized all of the tocopherol and ascorbate in synaptosomes. Thus, the oxidant is able to diffuse to the different membranes in synaptosomes and oxidize tocopherol in all of them. alpha-Tocopherol is converted quantitatively to tocopherolquinone during the oxidation. Significant amounts of thiols (at least 30% of the total thiols) do not seem to be accessible to oxidation by peroxynitrite. However, the concentration of thiols is much higher than those of tocopherol and ascorbate. Addition of the hydroxyl radical quenchers benzoate or mannitol or the enzymes superoxide dismutase or catalase (alone or together) did not affect the oxidation of tocopherol and ascorbate by peroxynitrite, whereas cysteine and glutathione blocked the oxidation. Therefore, reactive oxygen species may not be directly involved as intermediates in oxidations induced by peroxynitrite. The latter is a potent oxidizing agent that can oxidize substances such as tocopherols, ascorbate, and thiols in the immediate vicinity of its formation. The antioxidant nutrients ascorbate and tocopherol could play important roles in protecting brain from oxidative damage induced by peroxynitrite.

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)

To investigate the relationship between clinical response and haloperidol blood concentrations in Alzheimer's dementia (AD) patients with behavior problems, 29 AD inpatients were assigned to a fixed oral dosage of haloperidol (0.5 mg, 1.0 mg, or 2.0 mg) every 12 hours for 3 weeks. BEHAVE-AD ratings and concentrations of plasma and RBC haloperidol and reduced haloperidol were obtained on Days 8, 15, and 22. Although no significant linear or curvilinear relationships were apparent between percent of change on BEHAVE-AD and plasma or RBC haloperidol concentrations, a good response (change ≥ 30%) was observed in 55% of the patients who entered the study., (Copyright © 1994. Published by Elsevier Inc. All rights reserved.)

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 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.

Carbamazepine and diphenhydramine interfered with the assays of haloperidol and its metabolite, reduced haloperidol, by reversed-phase HPLC. Retention times of haloperidol, reduced haloperidol, and the interfering drugs were very sensitive to the percentage of potassium phosphate buffer in the mobile phase as well as to the final pH of the eluant. Retention times were not very dependent upon ionic strength of the eluting solvent mixture. Haloperidol and reduced haloperidol in the range of 0.5-10 ng/mL were analyzed in the presence of 0.2 micrograms/mL diphenhydramine and 5 micrograms/mL of carbamazepine. The concentrations of all drugs used were in their expected therapeutic ranges. The isocratic chromatographic conditions were as follows: 25-cm x 4.6-mm C-18 column; mobile phase, 75% phosphate buffer (final concentration, 0.06M) and 25% acetonitrile; final pH, 3.5; flow rate, 2.5 mL/min; and detection by UV absorption at 220 nm. Additional changes in the percent buffer in the mobile phase may be useful in achieving separation of other interfering compounds.

Neuroleptic drugs such as haloperidol (H) induce a rapid increase in neurotensin/neuromedin N (NT/N) gene expression in the dorsolateral striatum (DLSt) and nucleus accumbens (NA) in young adult rats. This effect may be mediated by post-receptor effectors that are activated by dopamine D2 receptor antagonism. The regional pattern of induction of neurotensin gene expression correlates with the side effect profile of particular neuroleptics. As motor side effects of H differ in aged animals, we hypothesized that the regional expression of the neurotensin gene may differ between young and old animals. We administered H or saline acutely to 3, 14, and 25 month-old Fischer 344 rats, followed by in situ hybridization and quantitative autoradiography for NT/N mRNA. There was a significant age effect on the H-induced NT/N mRNA response in the DLSt, but not the NA, of older animals. In addition to the blunted NT/N mRNA response, significant decreases in D2 receptor mRNA were observed in the lateral striatum of another group of young, middle-aged, and aged rats. Age-related blunting of the NT/N mRNA response to H in the DLSt may be due in part to a decrease in D2 receptors in this structure.

The turnover rate of vitamin E is slow in nerve tissue. Therefore, we have developed in vitro techniques to study the biochemical reactions of this nutrient in brain. Subcellular fractions were isolated from the cerebral hemispheres of 4-month-old, male, Fisher 344 rats. Aliquots of fractions (500 micrograms protein) were suspended in 50 mM phosphate buffer at pH 7.4 and incubated at room temperature (20-22 degrees) or 37 degrees for 2 hr in the presence or absence of the following oxidizing agents: 1 mM tertiary butyl hydroperoxide, 10 microM linoleic acid hydroperoxide, 0.5 to 50 mM 2,2'-azobis (2-amidinopropane) dihydrochloride (ABAPH) or 0.1 to 2 mM 2,2'-azobis (2,4-dimethyl) valeronitrile (ABDVN). The latter two compounds generate free radicals upon heating. After oxidation, the subcellular fractions were sedimented, saponified and assayed for tocopherol by liquid chromatography. Linoleic acid hydroperoxide was the most potent oxidizing agent, suggesting that endogenous fatty acid peroxides (e.g. eicosanoid intermediates) are very powerful oxidizing agents. Vitamin E may play an important role in providing antioxidant protection for membranes against excessive oxidation induced by these peroxides. Tocopherol in mitochondria and microsomes was much more susceptible to oxidation than synaptosomal tocopherol. The possible reasons for this observation are: (a) mitochondria and microsomes may contain less of the other reducing agents such as sulfhydryl compounds than synaptosomes, and/or (b) the electron transport structures in the former two subcellular fractions may be facilitating oxidation of tocopherol induced by free radicals. A portion of tocopherol remained unoxidized in all subcellular fractions even at high concentrations of ABAPH, suggesting that tocopherol exists in labile and nonlabile biochemical compartments or complexes.

Recently there has been a great deal of interest in the potential therapeutic use of supplemental vitamin E in amelioration of diseases of the nervous system. Even though many studies have provided encouraging results, the mechanism of any beneficial effect remains elusive. Experimental studies suggest that the presence of high levels of vitamin E in tissues prior to injury is essential for biological efficacy because administration of the vitamin after insult is often ineffective. The rationale for this phenomenon is unknown at present. Some of the remaining areas of investigation include the biochemical interaction of vitamin E with other biological antioxidant substances such as vitamin C and sulfhydryl compounds; the relative potencies of different molecular forms of tocopherols, such as trienols and various optical isomers; and the optimal dosage and mode of administration of the most potent tocopherol molecule. Future research on these and other topics will shed more light on the effective use of vitamin E in neurodegeneration.

Red blood cell (RBC) and plasma concentrations of haloperidol (H) and reduced haloperidol (RH) were determined in nine patients (five men, four women) who had been receiving monthly injections of haloperidol decanoate (HD) for at least 3 months. A very sensitive high performance liquid chromatography method was used to measure H and RH, which could be detected at levels as low as 0.1 ng/ml. Blood samples for H and RH were drawn at the end of weeks 1, 2, 3, and 4 of a 4-week cycle. Monthly HD dosages ranged from 50 mg to 200 mg. At each time point RBC H did not differ significantly from plasma H. RBC RH and plasma RH did not change significantly over time and were consistently lower than RBC H and plasma H. However, RBC RH levels were higher than plasma RH levels. Also, the ratios of RBC RH/H were higher than were the ratios of plasma RH/H, which reflect an accumulation of RH intracellularly. HD dosage was highly correlated with both RBC H and plasma H, but RBC RH and plasma RH were not significantly related to dosage at any time point. Significant correlations were observed between HD dosage and prolactin concentration at week 1 and between prolactin concentration and RBC H and plasma H at weeks 1 and 4. Blood concentrations of H and RH and the corresponding standardized areas under the curve were not significantly related to smoking.

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.