Canine motor neuron disease: clinicopathologic features and selected indicators of oxidative stress.

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.

High doses of vitamin E in the treatment of disorders of the central nervous system in the aged.

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.

Vitamin E and other endogenous antioxidants in the central nervous system.

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 in CSF of subjects taking high-dose vitamin E in the DATATOP study. Parkinson Study Group.

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.

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.

Determination of trazodone and its metabolite, 1-m-chlorophenyl-piperazine, in human plasma and red blood cell samples by HPLC.

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.

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.

Oxidation of vitamin E, vitamin C, and thiols in rat brain synaptosomes by peroxynitrite.

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.

In vitro oxidation of vitamins C and E, cholesterol, and thiols in rat brain synaptosomes.

Free radical-induced oxidation of vitamins C, E, sulfhydryl compounds, and cholesterol in brain synaptosomes from Fisher 344 rats was studied. The synaptosomes were incubated at 37 degrees C with 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH), which undergoes thermal decomposition to yield free radicals. After incubation, the synaptosomes were sedimented, saponified, and extracted with hexane to isolate tocopherol and cholesterol. Ascorbate and tocopherol were assayed by liquid chromatography, cholesterol by gas chromatography, and total sulfhydryls by spectrophotometry. Under the in vitro conditions used in this study, the approximate order for the ease of oxidation of the various compounds was: ascorbate >>tocopherol > sulfhydryl compounds >>> cholesterol. However, tocopherol and sulfhydryl oxidation occurred even before all of the ascorbate had been consumed. Therefore, the fate of a specific antioxidant at a particular cellular location cannot be predicted with complete accuracy using the in vitro order for ease of oxidation shown here. Ascorbate may play a major role in protecting brain against oxidative damage because: (i) ascorbate concentration is high in brain, (ii) it can regenerate vitamin E from its radical oxidation product, and (iii) it is one of the first antioxidants to be consumed during oxidative reactions.

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.

Resolution of the interference from carbamazepine and diphenhydramine during reversed-phase liquid chromatographic determination of haloperidol and reduced haloperidol.

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.

Blunting of the neurotensin mRNA response to haloperidol in the striatum of aging rats: possible relationship to decline in dopamine D2 receptor expression.

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.

Oxidation of alpha-tocopherol in subcellular fractions from rat brain and its possible involvement in nerve function.

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.

Vitamin E. Neurochemistry and implications for neurodegeneration in Parkinson's disease.

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.