Vitamin E Neurochemistry and Implications for Neurodegeneration in Parkinson’s Diseasea G. T. VATASSERY Veterans Affairs Medical Center Minneapolis, Minnesota 5541 7 and University of Minnesota Minneapolis, Minnesota 55455

NEUROCHEMISTRY OF VITAMIN E Vitamin E was discovered in 1922 as a nutritional factor that prevented resorption of the fetus in the laboratory rat.’ In recent years the antioxidant properties of vitamin E have been studied in great detail. As the major chain-breaking, lipid antioxidant, vitamin E would be expected to be important for functional integrity of all biological membranes. In fact, a deficiency of vitamin E results in a number of pathological changes in muscle, reproductive, cardiovascular, and nervous systems.2 The earliest report of an abnormality in the nerve tissue of vitamin E-deficient animals was from Pappenheimer and Goettsch3 who observed that chickens raised on a vitamin E-deficient diet developed cerebellar encephalomalacia. Einarson and Telford4 found that prolonged vitamin E deficiency in the rat caused demyelination of axons and gliosis in the gracilis and cuneate nuclei. Pentschew and Schwartz5 observed dystrophic afferent axons (spheroids) mainly in the dorsal columns, dorsal horns, Clarke’s column, and the gracile and cuneate nuclei. Lipopigment was also present within neuronal perikarya. The importance of vitamin E for maintaining the structural and functional integrity of the nervous system in humans has now been well-established. Soko16 has summarized clinical data on the role of vitamin E in nervous system diseases and points out that even though a dietary vitamin E deficiency is very rare in humans, a symptomatic vitamin E deficiency exists in association with abetalipoproteinemia, chronic cholestatic hepatobiliary diseases, cystic fibrosis, short bowel syndrome, and isolated vitamin E deficiency syndromes. The neuropathologic changes of vitamin E deficiency in humans are very similar to those in rats and rhesus monkeys? and the resulting neurological syndrome is characterized by areflexia, peripheral neuropathy, cerebellar involvement with gait and limb ataxia, and decreased propioception and vibration sense.’ Administration of vitamin E results in considerable improvements in symptomatology.6 In order to understand the basic mechanism of action of vitamin E, our laboratory has explored selected aspects of the neurochemistry of this nutrient. It is well-

‘The experimental work in the author’s laboratory is funded mainly by research funds from the Department of Veterans Affairs, Washington, DC. Partial support came from the Parkinson’s Disease Foundation and Grant IPOI AGO6309 from the National Institute on Aging. 97

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Gastroc Muscle Liver Dist Sciat Prox Sciat Lumb Cord Thor Cord Cerv Cord Medulla Pons Cerebellum Cortex 10

15

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Alpha Tocopherol (pg/g)

FIGURE 1. Concentration5 of alpha-tocopherol in different regions of rat nervouE system. Data from reference 9 was used to construct this figure.

known that anatomically discrete areas of the nervous system are quite distinct with respect to the type of cells and their biochemical properties. Therefore, one of the first experiments we did was t o determine the distribution of vitamin E in different regions of the nervous Various neuroanatomically defined areas of the nervous system in the rat were dissected out, and the tissue samples were analyzed for tocopherols by high-performance liquid chromatography with 1. fluorescent detection."' The results of a typical study are shown in FIGURE It can be seen that vitamin E is not highly localized in any specific region in rat brain. The distribution of vitamin E in different parts of the nervous system is in sharp contrast to that of a substance such as dopamine, whose concentration in putamen is about 44 times that in cerebral cortex and that functions as a neurotransmitter substance. ' I The distribution pattern, however, is compatible with a general antioxidant function for vitamin E in nerve tissue. Cerebellum and the three regions of spinal cord have the lowest concentrations of alpha-tocopherol. Therefore, it is conceivable that spinal cord and cerebellum are much more susceptibile to oxidant injury than other areas of the nervous system. compared the neuropathologic changes in vitamin E-deficient Nelson et d.'? rats and monkeys and described the process as a distal and dying-back type of axonopathy . Concentrations of alpha-tocopherol in the distal and proximal parts of the .;ciatic nerve were not significantly different (FIG.1). This suggests that susceptibility of the distal portion of peripheral nerves t o vitamin E deficiency must be due to reasons other than lower levels of tocopherol in the distal regions. Vitamin E deficiency and acrylamide neurotoxicity have some neuropathological Yimilarities. Three of the characteristics in common are axonopathy of the dying-back type, marked increase in 10 nm neurofilaments, and neuroaxonal dystrophy. I 3 Therefore, we examined the effect of acrylamide on concentrations of tocopherol in various regions of nerve tissue.y N o changes in vitamin E concentrations were observed, suggesting that a deficiency of vitamin E is not involved in the initial stages of pathogenesis of acrylamide-induced neuropathy. It should be noted that animals treated with acrylamide in drinking water (0.03% w h ) become quite ill relatively fast, and the experiment was conducted for only 24 days. The animals might have been able to supply nerve tissue with vitamin E derived from

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dietary sources and storage tissues even if the rate of utilization or turnover of vitamin E had changed as a result of acryamide toxicity. Interestingly, a defect in the fast anterograde and retrograde axonal transport has been reported in vitamin E-deficient rats. l4 In another experiment, radioactive alpha-tocopherol (tritiated in the 5 methyl position) was injected intravenously in rats, and uptake by different areas of the brain was studied.8 The specific activities of alpha-tocopherol in the brain regions were determined, and results are given in FIGURE 2. Cerebellar gray matter is characterized by lower levels of unlabeled tocopherol and higher uptake of radioactive vitamin E from blood than other parts of the brain. Thus, cerebellum seems to be active in the metabolic utilization of vitamin E. This could be the reason for cerebellar damage during experimental vitamin E deficiency and for the incidence of cerebellar symptoms in clinical vitamin E deficiency. The depletion of tocopherol from brain regions during a vitamin E deficiency regimen was also studied by us.15 In this investigation weanling CD-1 mice were fed control or vitamin E-deficient diet for 20 weeks. Peripheral tissues such as liver, plasma, and testes were nearly depleted of vitamin E by the sixth week of deficiency (FIG.3). Significant amounts of tocopherol, however remained in all regions of the brain, even up to 20 weeks, indicating that brain is more resistant to vitamin E deficiency than peripheral tissues (FIG.4). Similar results were also reported by Gross-Sampson and Muller.I6 Concentration of tocopherol in cerebellum sustained a larger decline within 6 weeks of deficiency than cerebral hemispheres or medulla and pons. Cerebellar alpha-tocopherol concentrations remained lower than those of the other brain regions throughout the 20 weeks. We also found that selenium deficiency for 12 weeks did not affect brain tocopherol concentrations. There is considerable interest in the use of high doses of vitamin E for treatment or amelioration of various diseases. Therefore we studied the effect of dietary ingestion of pharmacological doses of vitamin E on tocopherol content of various

Hypothalamus Cerebellumwhite Cerebral peduncle Caudate putamen Cerebellum gray Thalamus Spinal cord Medulla Pons Cortex gray 5

0

(dpm/Fg

10

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Specific Activity alpha tocophero1)xO.OOl

FIGURE 2. Specific activities of alpha-tocopherol in different areas of rat brain after intravenous injection of tritium-labeled alpha-tocopherol. Data from reference 8 was used to construct this figure.

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15.

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05-

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8

!2

i6

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Time on Dlet In Weeks

FIGURE 3. Concentrations of alpha-tocopherol in selected peripheral tissues of mice fed a vitamin E-deficient diet for 20 weeks. (Vatassery c t With permission from Riruen Press.)

discrete areas in the rat brain." Four-week-old male rats were fed control or high vitamin E (1000 IUlkg) diets for four months. The serum, adipose tissue, and liver concentrations of tocopherol in the high vitamin E group were 2.2-, 2.2-, and 4.6fold, respectively, of the control levels. Vitamin E concentrations in cerebrum, cerebellum, and striatum, however, increased uniformly to I .4-fold of the values in controls. Thus, high levels of dietary vitamin E do increase brain vitamin E levels, and this could explain the therapeutic efficacy of oral doses of high vitamin E (400 IU to 10 gramdday) in diseases such as abetalipoproteinemia, cholestasis, congenital biliary atresia, and cystic fibrosis. Transport of vitamin E into the nervous system is of great practical importance in the therapeutic use of this nutrient. No specific lipoprotein is known to act as the sole carrier of tocopherol in blood. Vitamin E is distributed i n all lipoproteins with a large proportion in the low-density lipoprotein fraction.'* We have studied tocopherol transport into brain in humans by comparing levels of tocopherol in blood serum and cerebrospinal fluid (CSF). The existence of blood-brain as well as blood-CSF barriers has been documented well, and it is generally accepted that CSF is in equilibrium with the extracellular fluid of' brain. In our study, blood and CSF were collected from 40 adult, male, human subjects with no neurologic and metabolic abnormalities. The mean concentration of alpha-tocopherol in CSF of 29.2 k 9.5 (mean SD) nanomoles/liter was considerably lower than that of serum (26 f 8.1 pmol/liter). This low level of tocopherol in CSF parallels the very low amounts of lipids in CSF of 10-20 mgiliter, which is about 0.2% of the concentration in serum. Concentrations of tocopherols correlated significantly with both total protein and albumin concentrations, suggesting that tocopherol transport into CSF is linked with that of plasma proteins. An examination of the transport of another endogenous lipid, cholesterol, across the blood, brain, and

*

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CSF barriers is of interest for comparison. The concentration of cholesterol like that of vitamin E is very low in CSF.I9The major apolipoproteins present in CSF are reported to be apolipoprotein (apo) E and apo A1 by Roheim et a/.,20 and they occur in the form of a lipoprotein fraction similar in size to plasma high-density lipoprotein (HDL). Concentrations of these apolipoproteins, however, were much higher than would have been expected if intact HDL molecules had been transferred. Thus, the mechanism of entry of apolipoproteins into CSF is somewhat complex and may not involve entry of whole intact lipoprotein particles. Pitus et al.?' have confirmed that lipoproteins containing apo E and apo Al are present in CSF. They observed that the central nervous system has a mechanism for lipid transport and cholesterol homeostasis similar to that of other tissues. In addition, Pardridge and MietusZ2have shown that neutral lipids such as steroid hormones can penetrate the blood-brain barrier as albumin-bound complexes. Therefore, it is reasonable to assume that tocopherols also enter CSF as protein complexes even though the exact nature of the complex is unknown. This mode of transport of vitamin E is in sharp contrast to that of other vitamins such as ascorbic acid. For example, Spector and Eellsz3in a review noted that ascorbic acid is secreted into CSF in much higher concentrations than blood and then diffuses into extracellular space of brain to be accumulated by brain cells. There is very little information in the literature on turnover and metabolism of vitamin E in nerve tissue. Burton, Ingold, and coworkers have developed a technique to measure exchange half-lives for tocopherol in tissues.24Animals were maintained on 36 mg/kg alpha-tocopherol in the diet for four weeks and then switched to a diet containing the same amount of deuterated alpha-tocopherol. The relative amounts of deuterated and nondeuterated tocopherols in tissues were determined by GC-MS. The amount of time required for tissues to have equal amounts of deuterated and nondeuterated tocopherols was calculated. The estimated time is the exchange half-life of vitamin E and is probably related to its turnover rate. The turnover or exchange half-lives of deuterated tocopherol in brains of rats and guinea pigs were 39 and 107 days, respectively. By contrast, the

I

-Medulla Pons ---- Cerebral Hemisphere .......... Cerebellum

I

I

I

I

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I2

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Time on Diet in Weeks FIGURE 4. Concentrations of alpha-tocopherol in three brain regions of mice fed a vitamin E-deficient diet for 20 weeks. (Vatassery et ~ 1 . With ' ~ permission from Raven Press.)

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half-lives in plasma were 6 and 4 days for rats and guinea pigs, respectively. This obsercation is consistent with our own data, which show that the rate of depletion of tocopherol from brain during vitamin E deficiency in mice is considerably slower than that of serum.I5 The low turnover rate of tocopherol in the nervous system makes it very difficult to follow the biological transformations of this nutrient. Therefore we have recently staried to use an in uitro model to study oxidation of tocopherols in biological samples. In a typical experiment, human blood platelets or red blood cell ghosts are incubated at room temperature and p H 7.4 with oxidants such as linoleic acid hydroperoxide. tertiary butyl hydroperoxide, or hydrogen peroxide. This results in oxidation of tocopherol with formation of tocopherol quinone.?5,2h We have applied this it7 uitro technique of oxidation to subcellular fractions from rat brain. Earlier work from our laboratory has shown that tocopherol appears to be concentrated in microsomal and mitochondrial fractions.8 Recently we have determined the levels of tocopherol and cholesterol in subcellular fractions isolated

la

r

-P m Homog

Myelin

Micro

Synapt

Mito

FIGURE 5. Concentration5 of alpha-tocopherol normalized with the respective cholesterol concen?rations in subcellular fractions from rat brain.

from rat brains by a centrifugation method involving Ficoll gradients.?’ Inasmuch as it is well-known that tocopherol levels are related to those of total lipid and choleslerol in tissues, the tocopherol concentrations were calculated as millimoles alpha-tocopherol per mole cholesterol.?xThe results are given in FIGURE 5. The data show quite clearly that relative to cholesterol, tocopherol is more concentrated in mitochondrial and microsomal fractions than in other fractions. The lower levels of cholesterol in mitochondria also make it strikingly higher in tocopherol when compared with the other subcellular organelles. As with platelets and red cell membranes, incubation of the subcellular fractions in 50 mM phosphate buffer at pH 7.4 and room temperature for 60 minutes in the presence of 10 p M linoleic acid hydroperoxide induced oxidation of t o ~ o p h e r o l . ~The ’ extents of oxidation of tocopherol in microsomes, mitochondria, and synaptosomes were 48, 47, and 22 percent, respectively. Thus microsomes and mitochondria, which take part in

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electron transport functions, were characterized by their susceptibility for oxidation of tocopherol. Linoleic acid hydroperoxide was a much more potent oxidizing agent than exogenous synthetic peroxides such as tertiary butyl hydroperoxide or hydrogen peroxide. In general, this in vitro incubation system is useful as a model for studies of the fate of vitamin E in cells, subcellular organelles, or membranes. Using this system we have shown that ascorbate and other synthetic antioxidants can block the oxidation of vitamin E.30 This is an in uitro confirmation of a wellestablished biological interaction between these compounds.

VITAMIN E IN NERVOUS SYSTEM DISORDERS At present there is considerable interest in the potential involvement of vitamin E in diseases of the nervous system. Tissue damage from peroxidative reactions has been postulated to play a role in the pathogenesis of some degenerative, toxic, or ischemic neurological diseases. Because literature on Parkinson's disease (PD) is the most convincing, it is interesting to examine the role of peroxidation and vitamin E in PD.

Parkinson's Disease Parkinson's disease is a common age-associated neurodegenerative disease that affects up to 1 in 40 persons in a full lifetime. Damage is to basal ganglia in the brain and is characterized neuropathologically by loss of dopaminergic neurons from substantia nigra and the presence of Lewy bodies. The latter can be described as acidophilic, cytoplasmic neuronal inclusions that are of variable size, contain proteins, and have a central core. They tend to occur in the pigmented cells of substantia nigra and locus ceruleus, although they can be found at other areas as well. The cardinal clinical features are resting tremor, rigidity, bradykinesia, and postural instability. Even though considerable progress has been made in the treatment of this disease with levodopa, the basic mechanism of pathogenesis of this disease remains unknown. In recent years a hypothesis has been proposed that oxidative damage to neurons from oxygen free radicals may be partly responsible for its i n ~ i d e n c e .This ~ ' postulate has led to several trials of the use of antioxidant compounds such as vitamin E in the therapeutic management of PD. There are at least six reasons to suggest that among the various anatomic regions of the nervous system, basal ganglia may be particularly susceptible to damage from free radical-induced oxidative reactions.

High Concentrations and Metabolism of Dopamine in Basal Ganglia

Dopaminergic neurons in substantia nigra project to caudat nucleus, putamen, and globus pallidus. The high concentration of dopamine in these latter three regions would be expected to lead to a local increase in dopamine metabolism. The normal catabolism of dopamine by monoamine oxidase produces hydrogen peroxide, which can liberate hydroxyl radicals in the presence of iron. The hydroxyl radical is probably the most potent of all free radical oxidants that are important in biological systems. Interestingly, activity of monoamine oxidase is

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known to increase with age,32and this parallels the increase in incidence of PD with age.

Alfrrtiiions in T ~ L I C Metcrls B kt'ith Spwid Rrfrrerrce to Iron

Basal ganglia also contain the highest levels of iron in brain. Spatz3' studied the distribution of iron in brain and noted that brain could be divided into four regions based upon the concentration of iron. Areas of highest concentrations were globus pallidus and substantia nigra, which was followed by red nucleus, putamcn, thalamus, and caudate nucleus as the second group. Structures of the extrapvramidal motor system are thus remarkably high in iron content. Hallgren and Sourander have confirmed this o b ~ e r v a t i o n As . ~ ~with other tissues, iron in brain i \ stored as the protein complex f e r r i t i ~Recent ~ . ~ ~ investigations have shown that excessive amounts of total iron are present in sustantia nigra in PD at autopsy.7'.3hIf such high levels of iron were present in brains of PD patients, the biological response would be an increase in levels of ferritin, which would bind excess iron. Ferritin levels, however, were found to be decreased in basal ganglia from PD brain^.^' Thus, these data suggest an abnormality of iron metabolism in the extr-apyramidal system in PD. It should be pointed out that the findings are not entirely unequivocal inasmuch as Riederer et (11. have reported that ferritin levels are increased in substantia nigra of P D brains.3x In addition to iron, other metals may alho be involved in PD. Chronic manganese toxicity is known to produce a syndrome resembling PD even though the toxic effects seem to involve more diffuse brain abnormalities." More recently, it has been observed that aluminum is present in abnormal amounts in substantia nigra of PD brains."O Numerous investigations have shown that heavy metals such as iron serve a s catalysts in peroxidation reactions. Therefore, it is quite likely that an abnormality in metabolism 01' heavy metals, especially iron, could predispose basal ganglia to oxidant injury in PD.

Uccrrrrrnce of' Neitvoinelanin rind I t s Role

(IS ( I

SourciJ of Frre Radicals

The cytoplasm of pigmented neurons in the brain stem region such as substantia nigra and locus ceruleus contains a brown-black pigment termed neuromelanin. Biosynthesis of neuromelanin involves autooxidation of dopamine and noradrenaline to quinones. which are converted t o indole quinone derivatives and then t o n e ~ r o m e l a n i n . Graham ~' proposed that a defect in compartmentalization, transport, o r degradation of catecholamines may predispose the aminergic cells to oxidative damage from redox-cycling effects of cytotoxic q ~ i n o n e s . In ~ ' addition, the polymerization process leading to eumelanin is considered to involve free radical intermediates. A few oxygen-centered free radical species of semiquinones have been postulated to occur during this p r o ~ e s s . ~In' any event, it is reasonable to assume that pigmented cells of substantia nigra are quite vulnerable to oxidative stress due to the occurrence and biosynthesis of neuromelanin in these cells. Interestingly. Fornstedt and others have found that depigmentation and degeneration of dopaminergic neurons in substantia nigra is correIated to enhanced rates of autooxidation of cat echo la mine^.^^ It is also important to recognize that trace metals. especially iron and copper, have high affinities for neuromelanin, which has some properties of an i ~ n - e x c h a n g e r . ~ ~

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Selective Destruction of Substantia Nigra by Neurotoxins

In 1979, Davis et published a case report of a patient who developed symptoms of PD as a result of drug abuse involving synthetic meperidine analogues. Later, Langston et ~ 1reported . ~ the ~ occurrence of Parkinsonian syndrome in young drug abusers who were accidentally injecting themselves with the meperidine analogue: l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP). This compound is metabolized by monoamine oxidase B in glial cells to 1-methyl-Cphenylpyridinium (MPP+), which is then avidly picked up by pigmented cells of substantia nigra. It is assumed that cell death follows through functional damage to mito~hondria.~’ The incidence of a Parkinson-like syndrome by injestion of MPTP led many investigators to look for an environmental toxin responsible for the incidence of PD. No specific neurotoxin, however, has been identified so far. The effect of vitamin E upon MPTP toxicity has been controversial. Perry et found vitamin E treatment to be beneficial, whereas Gong et ~ 1failed . to ~ observe ~ any protection. Nonetheless, vitamin E treatment has been shown to ameliorate neurotoxic effects of some synthetic compounds such as alkyl mercury c o m p o ~ n d s . ~ ~ Mitochondria1 Abnormalities

Defects in the electron transport enzymes in the mitochondria have also been reported in PD. Decreases in activity of complex I have been observed in the platelets of living PD patients5’ and in postmortem brain.52 Using mitochondria prepared from the striata of patients who died of PD and an immunoblotting technique, Mizuno et a/.” have found that some of the subunits of complex I are decreased in PD. One of the consequences of such enzyme deficiencies is an inefficient oxidative phosphorylation. This could then lead to enhanced production and leakage of superoxide from mitochondria and result in membrane and cellular oxidation. Decline in Antioxidant Protection

A selective and marked reduction in glutathione levels in substantia nigra of advanced PD patients was reported by Perry and Y ~ n gAmbani . ~ ~ et al.ss found that activities of catalase and nonspecific peroxidase in PD substantia nigra and striatum was reduced by 30 to 60%, whereas the other parts of brain showed normal levels. Activity of glutathione peroxidase has been shown to be slightly reduced in basal ganglia in PD brains.s6 Vitamin E Therapy in PD

The data presented above suggest that enhanced peroxidative damage to neurons could be a cause of PD and that antioxidants like vitamin E may have a therapeutic role in treatment of PD. The most comprehensive clinical trial involving use of vitamin E in PD is the multicenter study called deprenyl and tocopherol antioxidant therapy of parkinsonism (DATATOP). The study was designed to test the efficacy of vitamin E and/or deprenyl in decreasing the rate of progression of

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PD. Deprenyl is a specific inhibitor of monoamine oxidase B and would be expected to reduce the production of hydrogen peroxide during catabolism of monoamines in brain. DATATOP patients were given an oral dose of 2000 IU of vitamin E as alpha-tocopherol acetate per day. The study was recently completed, and results will be published soon. In the mean time, Fahn who is one of the principal investigators of DATATOP has conducted an open label trial of high doses of oral vitamins E and C (3200 IU/day and 3000 mgiday, respectively) in a group of 15 early I’D patients.” It was observed that the period of time that elapsed before it became necessary to begin treatment with levodopa was extended by 2.5 years in patients taking supplemental antioxidants. In a less controlled study that involved comparison of PD patients who were self-administering vitamin E with those who found that P D patients taking vitamin E had less severe were not, Factor et d.’* disease.

SUMMARY AND CONCLUSIONS 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.

ACKNOWLEDGEMENT The author wishes to thank Mr. W. Ed Smith and Mr. Hung Quach for expert technical assistance. REFERENCES

EVANS, H. M. & K . S. BISHOP.1922. On the existence of a hitherto unrecognized dietary factor essential for reproduction. Science 56: 650-661. 2 . SCOT,M. L. 1969. Studies on vitamin E and related factors in nutrition and metabolism. I n Fat Soluble Vitamins. H. F. Deluca & J . W. Suttie, Eds.: 335-368. The University of Wisconsin Press. Madison, WI. A. M. & M. GOETTSCH.1931. A ccrcbellar disordcr in chicks, appar3. PAPPENHEIMER, ently of nutritional origin. J. Exp. Med. 53: 11-26, 1960. Effect of vitamin E deficiency on the central 4. E I N A R S O N ,L. & I. R. TELFORD. nervous system in various laboratory animals. Danske Videnskabernes Selskab 11: I.

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1-81. PENTSCHEW, A . & K . SCHWARZ. 1962. Systemic axonaldystrophy in vitamin Edeficient adult rats. Acta Neuropath. 1: 313-334.

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SOKOL,R . J. 1989. Vitamin E and neurologic function in man. Free Radical Biol. Med. 6: 189-207. MULLER, D. P. R., J . K. LLOYD& 0. H. WOLFF.1983. Vitamin E and neurological function. Lancet 1 (8318): 225-228. VATASSERY,G. T . , C. K. ANGERHOFER, C. A . KNOX& D. S . DESHMUKH. 1984. Concentrations of vitamin E in various neuroanatomical regions and subcellular fraction. and the uptake of vitamin E by specific areas of rat brain. Biochim. Biophys. Acta 792: 118-122. G. T., C. K. ANGERHOFER, R. C. ROBERTSON & M. I. SABRI.1986. VATASSERY, Vitamin E concentrations in different regions of the spinal cord and sciatic nerve of the rat. Neurochem. Res. 11: 1419-1424. VATASSERY, G. T. & W. E. S M I T H1987. . Determination of alpha tocopherolquinone (vitamin E-quinone) in human serum, platelets. and red cell membrane samples. Anal. Biochem. 167: 411-417. SOURKES, T. L. 1981. Parkinson’s disease and other disorders of the basal ganglia. In Basic Neurochemistry. G. J. Siegel, R. W. Albers, B. W. Agranoff & R. Katzman, Eds.: 719-733. Little Brown and Company. Boston. NELSON,J . S . , C. D. FITCH,V. W. FISCHER, G. 0 . BROUN, JR. & A. C. CHOU.1981. Progressive neuropathological lesions in vitamin E-deficient rhesus monkeys. J. Neuropathol. Exp. Neurol. 40: 166-186. Kosic, K. S. & D. J . SELKOE. 1983. Experimental models of neurofilamentous pathology. In Neurofilament. C. A . Marotta, Ed.: 155-195. University of Minnesota Press. Minneapolis, Minnesota. SOUTHAM, E., P. K. THOMAS, R. H. M. KING.M. A . GROSS-SAMPSON & D. P. R. MULLER. 1991. Experimental vitamin E deficiency in rats. Brain 114: 915-936. VATASSERY, G . T., C. K. ANGERHOFER & F. J. PETERSON. 1984. Vitamin E concentrations in the brains and some selected peripheral tissues of selenium-deficient and vitamin E-deficient mice. J . Neurochem. 42: 554-558. MULLER, D. P. R. & M. A. GOSS-SAMPSON. 1990. Neurochemical, neurophysiological and neuropathological studies in vitamin E deficiency. Crit. Rev. Neurobiol. 5: 239-263. VATASSERY, G. T., M. F. BRIN,S. FAHN,H. J. KAYDEN & M. J. TRABER. 1988. Effect of high doses of vitamin E o n the concentrations of vitamin E in several brain regions, plasma, liver, and adipose tissue of rats. J . Neurochem. 51: 621-623. BJORNSON, L. K., H. J. KAYDEN, E. MILLER & A . N. MOSHELL. 1976. The transport of alpha tocopherol and beta carotene in human blood. J . Lipid Res 17: 343-352. FISHMAN. R. A . 1980. Cerebrospinal fluid in diseases of the nervous system. WB Saunders. Philadelphia. ROHEIM, P. S . , M. CAREY, T. FORTE& G. L. VEGA.1979. Apolipoproteins in human cerebrospinal fluid. Proc. Natl. Acad. Sci. USA 76: 4646-4649. PiTus. R. E . , J . K. BOYLES, S . H. LEE,D. H U I& K. H. WEISGRABER. 1987. Lipoproteins and their receptors in the central nervous system. J. Biol. Chem. 262: 14352-14360. PARDRIDGE, W. M. & L. J. MIETUS.1979. Transport of steroid hormones through the rat blood-brain barrier. J . Clin. Invest. 64: 145-154. SPECTOR, R. & J . EELLS.1984. Deoxynucleoside and vitamin transport into t h e central nervous system. Fed. Proc. Fed. Am. Soc. Exp. Biol. 43: 196-200. BURTON, G. W. & M. G . TRABER.1990. Vitamin E: antioxidant activity, biokinetics, and bioavailability. Annu. Rev. Nutr. 10: 357-382. VATASSERY, G. T . 1987. In uitro oxidation of alpha tocopherol (vitamin E) in human platelets upon incubation with unsaturated fatty acids, diamide, and superoxide. Biochim. Biophys. Acta 926: 160-169. VATASSERY, G. T. 1989. Oxidation of vitamin E in red cell membranes by fatty acids, hydroperoxides and selected oxidants. Lipids 24: 299-304. LAI . J . C. K. & J . €3. CLARK.1989. Isolation and characterization of synaptic and nonsynaptic mitochondria from mammalian brain. In Neuromethods, Volume I 1 : Carbohydrates and Energy Metabolism. A. A. Boulton & G. B . Baker, Eds.: 43-97. Humana Press. Clifton, NJ.

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C. H. DAHM,JR. & M. T. SEARCY. 1972.Relationship 28. HORWITT,M. K., C. C. HARVEY, between tocopherol and serum lipid levels for determination of nutritional adequacy. Ann. N.Y. Acad. Sci. 203: 223-236. G. T., W. E. SMITH & H. T. QUACH.1990.In uitro oxidation of vitamin 29. VATASSERY, E in brain subcellular fractions. FASEB J. 4 A1051. G. T., W. E. SMITH & H. T. QUACH.1989.Ascorbic acid, glutathione and 30. VATASSERY, synthetic antioxidants prevent the oxidation of vitamin E in platelets. Lipids 24: 1043- 1047. 31. COHEN,G. 1985.Oxidative stress in the nervous system. I n Oxidative Stress. H. Sies, Ed.: 383-402. Academic Press. New York. 32. FINCH, C. E. 1977. Neuroendocrine and autonomic aspects of aging. In Handbook of the Biology of Aging. C. E. Finch & L. Hayflick, Eds.: 262-280. Van Nostrand Reinhold Company. New York. 33. SPATZ,H. 1922.Uber den eisennachweis im gehern, besonders in zentren des extrapyramidal-motorischen systems. Z. Gesamte Neurol. Psychiat. 77: 261-290. B. & P. SOURANDER. 1958.The effect of age on the non-haemin iron in the 34. HALLGREN, human brain. J. Neurochem. 3: 41-51. 35. DEXTER,D. T., F. R. WELLS,F. ACID, Y. ACID, A. J. LEES,P. JENNER& C. D. 1987.Increased nigral MARSDEN. - iron content in postmortem Parkinson brain. Lancet 2: 1219-1220. H. HEINSEN.H. BECKMAN. G. P. REYNOLDS. G. HEBEN36. SOFIC.E.. P. RIEDERER. STRETT & M. B. H . YOUDIM.1988. Increased iron (111) and total iron content in postmortem substantia nigra of parkinsonian brain. J. Neural. Transm. 74: 199205. M. VIAILHET,M. RUBERG,F. ACID, Y. ACID, A. J. 37. DEXTER,D. T., A. CARAYON, LEES, F. R. WELLS,P. JENNER& C. D. MARSDEN.1990. Decreased ferritin levels in brain in Parkinson’s disease. J. Neurochem. 55: 16-20. P., E. SOFIC, W. D. RAUSCH,B. SCHMIDT, G. P. REYNOLDS, K. JELLINGER 38. RIEDERER, & M. B. H. YOUDIM. 1989.Transition metals, ferritin, glutathione, and ascorbic acid in Parkinson’s disease. J. Neurochem. 52: 515-520. A. 1984. Manganese and extrapyramidal disorders. (A critical review and 39. BARBEAU, tribute to Dr. George C. Cotzias.) Neurotoxicology (Little Rock) 5: 13-36. E. C., J. P. BRANDEL, P. GALLE,F. JAVOY-ACID & Y. ACID. 1991.Iron and 40. HIRSCH, aluminum increase in the substantia nigra of patients with Parkinson’s disease: an Xray microanalysis. J. Neurochem. 56: 446-451. D. G. 1979.On the origin and significance of neuromelanin. Arch. Pathol. 41. GRAHAM, Lab. Med. 103: 359-362. 42. RILEY,P. 1988.Radicals in melanin biology. Ann. N.Y. Acad. Sci. 551: 111-120. B., A. BRUN,E. ROSENGREN & A . CARLSSON. 1989.The apparent autoxi43. FORNSTEDT, dation rate of catechols in dopamine-rich regions of human brains increases with the degree of depigmentation of substantia nigra. J. Neural Transm. [P-D Section] 1: 279-295. 44. PILAS,B., T. SARNA,B. KALYANARAMAN & H. M. SWARTZ. 1988. The effect of melanin on iron-associated decomposition of hydrogen peroxide. Free. Radical Biol. Med. 4: 285-293. 45. DAVIS,G. C., A. C. WILLIAMS,S. P. MARKEY,M. H. EBERT,E. D. CAINE,C. M. REICHERT & I. J. KOPIN. 1979. Chronic parkinsonism secondary to intravenous injection of meperidine analogues. Psychiatry Res. 1: 249-254. 3. W. 13P. BALLARD.1983. Chronic parkinsonism in humans due to a 46. LANGSTON, product of mepiridine analog synthesis. Science 219: 979-980. 41. HEIKKILA, R. E., M. V. KINDT& P. K. SONSALLA. 1987.MPTP and animal models of Parkinson’s disease. i n Handbook of Parkinson’s Disease. W. C. Koller, Ed.: 274. Marcel Dekker Inc. New York. 48. PERRY,T. L., V. W. YONG,R. M. CLAVIER,K. JONES,J. M. WRIGHT,J. G. FOULKS & R. A. WALL.1985. Partial protection from the dopaminergic neurotoxin N-methyl4-phenyl-l,2,3,6,-tetrahydropyridineby four different antioxidants in the mouse. Neurosci. Lett. 60: 109-1 14. R. V. ACUFF& R. M. KOSTRZEWA. 1991. Vitamin 49. GONG,L., E. A. DAIGNEAULT,

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E supplements fail to protect mice from acute MPTP toxicity. Neuropharmacol. Neurotoxicol. 2: 544-546. KASUYA. M. 1975. The effect of vitamin E on the toxicity of alkyl mercurials on nerve tissue in culture. Toxicol. Appl. Pharmacol. 32: 347-354. PARKER,W. D., S. J . BOYSON& J . K. PARKS.1989. Abnormalities of the electron transport chain in idiopathic Parkinson’s Disease. Ann. Neurol. 26: 719-723. D. DEXTER,J . B. CLARK,P. J E N N E R & C. D. SCHAPIRA, A. H. V . . J . M. COOPER, MARSDEN.1990. Mitochondria1 complex 1 deficiency in Parkinson’s Disease. J. Neurochem. 54: 823-827. S. T A K A M I YK. A , S U Z U K T. I , SATO,H . OYA,T. MIZUNO, Y.. S. OHTA,M. TANAKA. OZAWA& Y. KAGAWA. 1989. Deficiencies in complex 1 subunits of the respiratory chain in Parkinson’s Disease. Biochem. Biophys. Res. Commun. 163: 1450-1455. PERRY,T. L. & V . W. YONG.1986. Idiopathic Parkinson’sdisease,progressive supranuclear palsy and glutathione metabolism in the substantia nigra of patients. Neurosci. Lett. 67: 269-274. 1975. Brain peroxidase and catalase A M B A NL. I , M., M. H. VANWOERT& S. MURPHY. in Parkinson’s disease. Arch. Neurol. (Chicago) 32: 114-1 18. KISH,S. J . , C. MORITO& 0. HORNYKIEWICZ. 1985. Glutathione peroxidase activity in Parkinson disease brain. Neurosci. Lett. 58: 343-346. F A H N S. , 1991. An open trial of high-dosage antioxidants in early Parkinson’s disease. Am. J . Clin. Nutr. 53: 380s-382s. FACTOR, S. A., J. R. SANCHEZ-RAMOS & W . J. WEINER.1990. Vitamin E therapy in Parkinson’s disease. Adv. Neurol. 53: 457-461.

DISCUSSION A. MILUNSKY (Bostorz University School of Medicine): I’d like your comments about vitamin E and tardive dyskinesia. Are those studies meritorious, and is there a role for vitamin E? G . T. VATASSERY (University of Minnesotu, Minneapolis, M N ) : The studies are valid. The problem is that once the sequence of events has started, there seems to be no way of reversing them. It would be very interesting to treat with vitamin E prophylactically before the incidence of any of the symptoms of the disease.. The studies need further confirmation. F. COLBY( N e w York, N Y ) : There is a disease that is becoming more and more widespread that has certain mechanistic parallels t o Parkinson’s disease, and that is Alzheimer’s disease. In late 1989 or early 1990, there was a symposium on Alzheimer’s disease, at the University of Rotterdam, where they found certain definite vitamin effects. Did your data show any relationship between Alzheimer’s disease and Parkinson’s disease? VATASSERY:We are looking into that now. We have some spinal fluid data from the Alzheimer’s patients that w e are investigating. J. CLAUSEN(University of Roskilde, Denmnrk): We have an animal model and also similar human experiments with Parkinson’s disease induced by MPTP, which is formed by free radicals. Have you any experiments on supplementation of vitamin E in animal experiments producing MPTP? VATASSERY: MPTP is a so-called designer drug, synthesized t o get around the legal problems of synthesizing heroin and selling it on the market. What happened was that there were a few young people who ingested MPTP and became parkinsonian in their early twenties. One study indicates that if an animal is

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pretreated with vitamin E, it seems to make the MPTP less toxic, but I’ve also seen one or two other studies that indicate that this is not true. At the moment I’d have to say we do not know the answer. N . E. CRAFT(Nationul Institute of Standards and Technology, Gaithersburg, MD):Did you also look at other forms of the vitamin that were present in tissues? Did you see any differences in concentration of gamma-tocopherol versus alphatocopherol? VATASSERY: No. Most of our animal experiments have used diets that contained only alpha-tocopherol. D. MENZEL(University of California, Iruine, C A ) : Do you know of any levels of vitamin E that have been determined in central nervous tissue of those cyclic fibrosis patients that demonstrate neurocerebellar spinal cord defect? VATASSERY: No, I am not aware of any, and as a matter of fact with regard to cystic fibrosis, the only report that I know of comes from the University of Minnesota by Drs. Sung, Warwick, and co-workers, who have followed this particular disease for a number of years. They have been giving vitamin E as a therapeutic regimen for the patients. They report a substantial betterment of the neuropathologic changes. Namely, they find much less axonal dystrophy in the patients. I’m not aware of any studies, however, that deal with the actual vitamin E levels in cystic fibrosis patients at autopsy. R. FEEHAN (Elizabeth, N J ) : Are there any indications for using these antioxidants in conjunction with vitamin B I 2in MS patients or in other patients undergoing any demyelinization process? VATASSERY:That’s a very interesting question, because one of the pathological changes in vitamin E deficiency is demyelination in certain areas of the brain. I do not know whether in cases of demyelinating processes such as multiple sclerosis, vitamin E would have an effect.