Α-tocopherol Transfer Protein Research Articles

Prolonged α-Tocopherol Deficiency Decreases Oxidative Stress and Unmasks α-Tocopherol-dependent Regulation of Mitochondrial Function in the Brain

Vitamin E is the major lipid-soluble chain-breaking antioxidant in mammals and plays an important role in normal development and physiology. Deficiency (whether dietary or genetic) results in primarily nervous system pathology, including cerebellar neurodegeneration and progressive ataxia (abnormal gait). However, despite the widely acknowledged antioxidant properties of vitamin E, only a few studies have directly correlated levels of reactive oxygen species with vitamin E availability in animal models. We explored the relationship between vitamin E and reactive oxygen species in two mouse models of vitamin E deficiency: dietary deficiency and a genetic model (tocopherol transfer protein, Ttp-/- mice). Both groups of mice developed nearly complete depletion of alpha-tocopherol (the major tocopherol in vitamin E) in most organs, but not in the brain, which was relatively resistant to loss of alpha-tocopherol. F4-neuroprostanes, an index of lipid peroxidation, were unexpectedly lower in brains of deficient mice compared with controls. In vivo oxidation of dihydroethidium by superoxide radical was also significantly lower in brains of deficient animals. Superoxide production by brain mitochondria isolated from vitamin E-deficient and Ttp-/- mice, measured by electron paramagnetic resonance spectroscopy, demonstrated a biphasic dependence on exogenously added alpha-tocopherol. At low concentrations, alpha-tocopherol enhanced superoxide flux from mitochondria, a response that was reversed at higher concentrations. Here we propose a mechanism, supported by molecular modeling, to explain decreased superoxide production during alpha-tocopherol deficiency and speculate that this could be a beneficial response under conditions of alpha-tocopherol deficiency.

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

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

The role of vitamin E in the diet.

Abstract The last decade has seen many developments in our understanding of vitamin E, its transport and metabolism, and its potential role in the prevention of chronic disease. Of the eight fat-soluble substances comprising vitamin E, α-tocopherol has emerged as central to vitamin E activity in the body, its concentration in the plasma being governed by α-tocopherol transfer protein and maintained in a narrow range. Although new insights into the role of habitual fat intake in determining human requirements for vitamin E have been gained, uncertainties remain and estimates of requirements differ considerably between authorities. Investigation of the potential of this potent chain-breaking antioxidant to prevent or delay conditions thought to be associated with oxidative stress, including cardiovascular disease, cancer, Alzheimer's disease and Parkinson's disease, has challenged nutritionists to reconsider optimal intakes of vitamin E. High-dose supplementation has proved ineffective in reducing risk and may even be associated with increased risk; yet, new evidence suggests that a lifetime diet rich in α-tocopherol may provide protection against heart disease and cancer. Intakes of α-tocopherol are low in some western populations, which may in part be the result of advice to consume low-fat diets, intended to help address rising rates of obesity. An increased focus on fat quality, consistent with advice stemming from the cardiovascular literature, will help ensure adequate intakes of vitamin E in the context of diets moderate in total fat.

Severe Vitamin E deficiency exacerbates acute hyperoxic lung injury associated with increased oxidative stress and inflammation

Hyperoxia causes acute lung injury along with an increase of oxidative stress and inflammation. It was hypothesized that vitamin E deficiency might exacerbate acute hyperoxic lung injury. This study used α-tocopherol transfer protein knockout (α-TTP KO) mice fed a vitamin E-deficient diet (KO E(-) mice) as a model of severe vitamin E deficiency. Compared with wild-type (WT) mice, KO E(-) mice showed a significantly lower survival rate during hyperoxia. After 72 h of hyperoxia, KO E(-) mice had more severe histologic lung damage and higher values of the total cell count and the protein content of bronchoalveolar lavage fluid (BALF) than WT mice. IL-6 mRNA expression in lung tissue and the levels of 8-iso-prostaglandin F2α (8-iso-PGF2α) in both lungs and BALF were higher in KO E(-) mice than in WT mice. It was concluded that severe vitamin E deficiency exacerbates acute hyperoxic lung injury associated with increased oxidative stress or inflammation.

Improved glucose metabolism in mice lacking α-tocopherol transfer protein

Conflicting evidence suggests a possible role for vitamin E in mammalian glucose metabolism and the protection from type 2 diabetes. The alpha-tocopherol transfer protein (alpha-TTP) mediates the transfer of alpha-tocopherol (alpha-TOH) from hepatocytes to very-low-density lipoproteins, thereby controlling plasma levels of alpha-TOH. The aim of this study was to investigate the putative impact of alpha-TTP knock-out on glucose metabolism in mice. Mice deficient for alpha-TTP and wild-type control littermates were fed a diet containing 200 mg alpha-tocopheryl acetate per kg to ameliorate alpha-TOH deficiency in knock-out mice. We investigated fasting and postprandial plasma glucose, insulin and triglyceride levels of both groups of mice at different ages. All genotypes and age groups were further subjected to glucose and insulin tolerance tests, and number of insulin-producing islets of Langerhans were determined. Plasma alpha-TOH levels of knock-out mice were 34% the levels of wild-type controls: Any signs of alpha-TOH deficiency were absent at any age. Unexpectedly, serum glucose levels both in the fasted and in the fed state were lower in alpha-TTP-deficient mice at any age. Removal rates for intraperitoneally injected glucose were found to be significantly increased in young alpha-TTP-deficient mice. This improved glucose tolerance was caused by increased insulin secretion in response to an intraperitoneal glucose challenge due to an increased number of pancreatic islets, as well as by increased sensitivity to intraperitoneally injected insulin, both significantly promoting glucose metabolism in alpha-TTP-deficient mice. Our findings suggest that alpha-TTP-deficiency in states of alpha-TOH supplementation unexpectedly promotes glucose tolerance in mice due to both increased insulin secretion and insulin action, suggesting differential roles of alpha-TTP and alpha-TOH in the pathogenesis of type 2 diabetes mellitus.

Vitamin E Regulatory Mechanisms

Dietary and supplemental vitamin E is absorbed and delivered to the liver, but of the various antioxidants with vitamin E activity, only alpha-tocopherol is preferentially recognized by the alpha-tocopherol transfer protein (alpha-TTP) and is transferred to plasma, while the other vitamin E forms (e.g., gamma-tocopherol or tocotrienols) are removed from the circulation. Hepatic alpha-TTP is required to maintain plasma and tissue alpha-tocopherol concentrations. The liver is the master regulator of the body's vitamin E levels in that it not only controls alpha-tocopherol concentrations, but also appears to be the major site of vitamin E metabolism and excretion. Vitamin Es are metabolized similarly to xenobiotics; they are initially omega-oxidized by cytochrome P450s, undergo several rounds of beta-oxidation, and then are conjugated and excreted. As a result of these various mechanisms, liver alpha-tocopherol and other vitamin E concentrations are closely regulated; thus, any potential adverse vitamin E effects are limited.

Antioxidant effects of 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran and α-tocopherol in hyperlipidemic mice as evaluated by hydroxyoctadecadienoic acid and 7-hydroxycholesterol

It has been hypothesized that oxidative modification of low density lipoprotein plays a key role in the pathogenesis of atherosclerosis. In order to elucidate the role of lipid oxidation and its inhibition in vivo, apolipoprotein E and α-tocopherol (αT) transfer protein double knockout (ApoE−/−α-TTP−/−) and apolipoprotein E knockout (ApoE−/−) mice fed with a vitamin E-depleted diet and a diet containing 0.002wt.% αT, respectively, were used with or without the treatment of a synthetic antioxidant 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran (BO-653, 0.2wt.%). The lipid oxidation markers of total hydroxylinoleic acid (tHODE), 8-iso-prostaglandin F2α, and 7-hydroxycholesterol (t7-OHCh) in the blood, liver, and brain were inclusively measured with or without an excessive cholesterol-feeding (Ch-diet). The tHODE levels were elevated by Ch-diet in the plasma and brain of ApoE−/−α-TTP−/− mice and in the liver of ApoE−/− mice without BO-653. The levels of t7-OHCh in the liver were also increased due to the Ch-diet, and the ratio of t7-OHCh to the parent compound cholesterol was reduced to the control levels by BO-653.In summary, it was demonstrated by biomarkers, tHODE and t7-OHCh, that the added BO-653 in their diets exerted antioxidative effects in vivo under the condition of reduced vitamin E.

Acceleration of Age-Related Changes in the Retina in α-Tocopherol Transfer Protein Null Mice Fed a Vitamin E–Deficient Diet

To assess the role of vitamin E (VE) in age-related changes in the retinal tissues by using a mouse model of severe VE deficiency. Pups of alpha-tocopherol transfer protein null (a-TTP(-)(/)(-)) mice were fed a VE-deficient diet for 4 or 18 months (VE (-) group). Wild-type C57BL/6 mice were fed a 0.002% alpha-tocopherol-supplemented diet (VE (+) group). In various ocular tissues, the VE levels were measured by high-performance liquid chromatography; the fatty acid composition by gas chromatography (GC); and the hydroxyoctadecadienoic acid and 8-iso-prostaglandin F(2)(alpha) levels, which are biomarkers for lipid peroxidation, by GC-mass spectrometry. The retinal structure was assessed by light, electron, and fluorescence microscopy. The alpha-tocopherol level in the retinas obtained from 4-month-old VE (-) animals was 71-fold lower than that in the retinas obtained from the VE (+) group. In addition, gamma-tocopherol was not detected; thus, the VE (-) group demonstrated a more severe VE deficiency than ever reported. In this group, the concentration of n-3 polyunsaturated fatty acids decreased (0.3- to 0.9-fold), whereas that of other classes of fatty acids was unchanged or increased. At 18 months of age, the number of the outer nuclear layer (ONL) nuclei was observed to be 17% lower in the VE (-) than in the VE (+) group (P < 0.05). Electron microscopy revealed larger amounts of matrix between the ONL nuclei indicating the Müller cell hypertrophy, greatly expanded rod outer segment discs, and a larger number of inclusion bodies in the retinal pigment epithelium (RPE; P < 0.05) in the VE (-) group. Fluorescence microscopy revealed that the autofluorescence signal was increased in the RPE layer in this group. When the observations of the 18-month-old animals were compared to those of the 4-month-old animals, the hydroxyoctadecadienoic acid and 8-iso-prostaglandin F(2)(alpha) levels were found to be increased in the retina and RPE obtained from both the VE (-) and VE (+) groups; however, the age-related increases were more remarkable in the VE (-) group (2.6- to 43.5-fold) than in the VE (+) group (0.8- to 8.7-fold). The combined use of a-TTP(-)(/)(-) mice and a VE-deficient diet leads to a severe deficiency of VE, enhances lipid peroxidation in the retina, and accelerates degenerative damage of the retina with age.

Dietary zinc restriction in rats alters antioxidant status and increases plasma F 2 isoprostanes

Approximately 12% of Americans do not consume the estimated average requirement for zinc and could be at risk for zinc deficiency. Since zinc has proposed antioxidant function, inadequate zinc consumption may lead to an enhanced susceptibility to oxidative stress through several mechanisms, including altered antioxidant defenses. In this study, we hypothesized that dietary zinc restriction would result in lower antioxidant status and increased oxidative damage. We fed weanling Sprague–Dawley rats ( n=12 per group) a zinc-adequate (50 mg/kg of zinc) diet, a zinc-deficient (<0.05 mg/kg of zinc) diet or a pair-fed diet for 3 weeks and then assessed their antioxidant status and oxidative stress parameters. Rats were zinc deficient as indicated by a significant ( P<.05) reduction in body weight (49%) and 19% lower ( P<.05) hepatic zinc (20.6±2.1 mg/kg) as compared with zinc-adequate rats (24.6±2.2 mg/kg). Zinc deficiency resulted in elevated ( P<.05) plasma F 2 isoprostanes. Zinc deficiency-mediated oxidative stress was accompanied by a 20% decrease ( P<.05) in the ferritin-reducing ability of plasma assay and a 50% reduction in plasma uric acid ( P<.05). No significant change in plasma ascorbic acid or in plasma α-tocopherol and γ-tocopherol was observed. However, hepatic α-tocopherol and γ-tocopherol concentrations were decreased by 38% and 27% ( P<.05), respectively, as compared with those in zinc-adequate rats. Hepatic α-tocopherol transfer protein levels were unaltered ( P>.05) by zinc deficiency, but cytochrome P450 (CYP) 4F2 protein levels were elevated ( P<.05) as compared with those in zinc-adequate rats. Collectively, zinc deficiency increased oxidative stress, which may be partially explained by increased CYP activity and reductions in hepatic α-tocopherol and γ-tocopherol and in plasma uric acid.

Bioactivity of vitamin E

More than 80 years after the discovery of the essentiality of vitamin E for mammals, the molecular basis of its action is still an enigma. From the eight different forms of vitamin E, only alpha-tocopherol is retained in the body. This is in part due to the specific selection of RRR-alpha-tocopherol by the alpha-tocopherol transfer protein and in part by its low rate of degradation and elimination compared with the other vitamers. Since the tocopherols have comparable antioxidant properties and some tocotrienols are even more effective in scavenging radicals, the antioxidant capacity cannot be the explanation for its essentiality, at least not the only one. In the last decade, a high number of so-called novel functions of almost all forms of vitamin E have been described, including regulation of cellular signalling and gene expression. alpha-Tocopherol appears to be most involved in gene regulation, whereas gamma-tocopherol appears to be highly effective in preventing cancer-related processes. Tocotrienols appear to be effective in amelioration of neurodegeneration. Most of the novel functions of individual forms of vitamin E have been demonstrated in vitro only and require in vivo confirmation. The distinct bioactivities of the various vitamers are discussed, considering their metabolism and the potential functions of metabolites.

Autosomal recessive cerebellar ataxias

Autosomal recessive cerebellar ataxias (ARCA) are a heterogeneous group of rare neurological disorders involving both central and peripheral nervous system, and in some case other systems and organs, and characterized by degeneration or abnormal development of cerebellum and spinal cord, autosomal recessive inheritance and, in most cases, early onset occurring before the age of 20 years. This group encompasses a large number of rare diseases, the most frequent in Caucasian population being Friedreich ataxia (estimated prevalence 2–4/100,000), ataxia-telangiectasia (1–2.5/100,000) and early onset cerebellar ataxia with retained tendon reflexes (1/100,000). Other forms ARCA are much less common. Based on clinicogenetic criteria, five main types ARCA can be distinguished: congenital ataxias (developmental disorder), ataxias associated with metabolic disorders, ataxias with a DNA repair defect, degenerative ataxias, and ataxia associated with other features. These diseases are due to mutations in specific genes, some of which have been identified, such as frataxin in Friedreich ataxia, α-tocopherol transfer protein in ataxia with vitamin E deficiency (AVED), aprataxin in ataxia with oculomotor apraxia (AOA1), and senataxin in ataxia with oculomotor apraxia (AOA2). Clinical diagnosis is confirmed by ancillary tests such as neuroimaging (magnetic resonance imaging, scanning), electrophysiological examination, and mutation analysis when the causative gene is identified. Correct clinical and genetic diagnosis is important for appropriate genetic counseling and prognosis and, in some instances, pharmacological treatment. Due to autosomal recessive inheritance, previous familial history of affected individuals is unlikely. For most ARCA there is no specific drug treatment except for coenzyme Q10 deficiency and abetalipoproteinemia.

Deletion of vitamin E enhances phenotype of Alzheimer disease model mouse

Increased oxidative damage is a prominent and early feature in Alzheimer disease (AD). However, whether it is a primary cause or merely a downstream consequence in AD pathology is still unknown. We previously generated α-tocopherol transfer protein knockout ( Ttpa −/−) mice, in which lipid peroxidation in the brain was significantly increased by complete depletion of α-tocopherol (α-Toc). Here we crossed AD transgenic ( APPsw) model mice (Tg2576) with Ttpa −/− mice. The resulting double-mutant ( Ttpa −/− APPsw) mice showed earlier and more severe cognitive dysfunction in the Morris water maze, novel-object recognition, and contextual fear conditioning tests. They also showed increased amyloid β-peptide (Aβ) deposits in the brain by immunohistochemical analysis, which was ameliorated with α-Toc supplementation. In this report we provide clear evidence indicating that chronic lipid peroxidation due to α-Toc depletion enhances AD phenotype in a mouse model.

α-Tocopherol regulation of hepatic cytochrome P450s and ABC transporters in rats

To test the hypothesis that supra-elevated hepatic α-tocopherol concentrations would up-regulate mechanisms that result in increased hepatic α-tocopherol metabolism and excretion, rats received daily subcutaneous α-tocopherol injections (10 mg/100 g body wt) and then were sacrificed on Day 0 or 12 h following their previous injection on Days 3, 6, 9, 12, 15, and 18. Liver α-tocopherol concentrations increased from 12 ± 1 nmol/g (mean ± SE) to 819 ± 74 (Day 3), decreased at Day 9 (486 ± 67), and continued to decrease through Day 18 (338 ± 37). α-Tocopherol metabolites and their intermediates increased and decreased similarly to α-tocopherol albeit at lower concentrations. There were no changes in known vitamin E regulatory proteins, i.e., hepatic α-tocopherol transfer protein or cytochrome P450 (CYP) 4F. In contrast, both CYP3A and CYP2B, key xenobiotic metabolizing enzymes, doubled by Day 6 and remained elevated, while P450 reductase increased more slowly. Consistent with the decrease in liver α-tocopherol concentrations, a protein involved in biliary xenobiotic excretion, p-glycoprotein, increased at Day 9, doubling by Day 15. Thus hepatic α-tocopherol concentrations altered hepatic proteins involved in metabolism and disposition of xenobiotic agents.

Biochemical Consequences of Heritable Mutations in the α-Tocopherol Transfer Protein

Tocopherol transfer protein (TTP) regulates vitamin E status by facilitating the secretion of tocopherol from liver to circulating lipoproteins. Heritable mutations in the ttpA gene, encoding for TTP, result in ataxia with vitamin E deficiency (AVED) syndrome, typified by low vitamin E levels and a plethora of neurological disorders. The molecular mechanisms by which TTP facilitates tocopherol secretion are presently unknown. We recently showed that vitamin E is taken up by hepatocytes through an endocytic process and that, shortly following uptake, the vitamin is found primarily in lysosomes. We showed further that TTP is localized to late endocytic vesicles and that it facilitates the intracellular trafficking of tocopherol from lysosomes to the plasma membrane. To gain insight into the molecular mechanisms that underlie TTP actions, we studied the physiological impact of three naturally occurring heritable mutations in the ttpA gene (the R59W, R221W, and A120T substitutions). We found that these mutations impair the ability of TTP to facilitate the secretion of vitamin E from cells. Furthermore, the degree of impairment corresponded to the severity of the AVED pathology associated with each mutation. In cells that express mutated TTP proteins, vitamin E did not traffic to the plasma membrane and remained "trapped" in lysosomes. In addition, we observed that substitution mutations that cause the AVED syndrome impart a marked instability on the TTP protein. These observations suggest that the physiological role of TTP is anchored in its ability to direct vitamin E trafficking from the endocytic compartment to transport vesicles that deliver the vitamin to the site of secretion at the plasma membrane.

Protective effect of vitamin E supplements on experimental atherosclerosis is modest and depends on preexisting vitamin E deficiency

Vitamin E has failed to protect humans from cardiovascular disease outcome, yet its role in experimental atherosclerosis remains less clear. A previous study ( Proc. Natl. Acad. Sci. USA 97:13830–13834; 2000) showed that vitamin E deficiency caused by disruption of the α-tocopherol transfer protein gene ( Ttpa) is associated with a modest increase in atherosclerosis in apolipoprotein E gene deficient ( Apoe −/−) mice. Here we confirm this finding and report that in Apoe −/− Ttpa −/− mice dietary α-tocopherol (αT) supplements restored circulating and aortic levels of αT, and decreased atherosclerosis in the aortic root to a level comparable to that seen in Apoe −/− mice. However, such dietary supplements did not decrease disease in Apoe −/− mice, whereas dietary supplements with a synthetic vitamin E analog (BO-653), either alone or in combination with αT, decreased atherosclerosis in Apoe −/− and in Apoe −/− Ttpa −/− mice. Differences in atherosclerosis were not associated with changes in the arterial concentrations of F 2-isoprostanes and cholesterylester hydro(pero)xides, nor were they reflected in the resistance of plasma lipids to ex vivo oxidation. These results show that vitamin E at best has a modest effect on experimental atherosclerosis in hyperlipidemic mice, and only in situations of severe vitamin E deficiency and independent of lipid oxidation in the vessel wall.

γ-Tocopherol attenuates MPTP-induced dopamine loss more efficiently than α-tocopherol in mouse brain

Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mouse has been widely used as a rodent model of Parkinson's disease. In this study, α-tocopherol (αT) transfer protein knockout (heteromutant type, α-TTP (+/−)) mice were used to evaluate the protective effects of αT and γ-tocopherol (γT) against MPTP-induced neurotoxicity. The intraperitoneal administration of MPTP to mice induced a decrease in the striatal levels of dopamine (DA) 3 days after the administration in both α-TTP (+/−) and wild-type mice; these mice were fed an αT-deficient diet for 3 weeks before the MPTP administration. The DA levels in the α-TTP (+/−) mice, which had been fed a γT-fortified diet (0.10 wt.%) for 3 weeks and were administered with MPTP, were recovered to those of the control, whereas there was no significant protective effect of αT despite the considerably higher striatal concentration of αT than γT. The immunohistochemical study also revealed that γT exerted a protective effect against neurodegenerative toxicity of MPTP. Collectively, this is the first report showing that the protective effect of γT is stronger than that of αT against the MPTP-induced damage of dopaminergic neurons in the mouse.

Efficacious vitamin E treatment in a child with ataxia with isolated vitamin E deficiency

Vitamin E deficiency can cause a progressive spinocerebellar ataxia similar to Friedreich ataxia phenotype [2, 3]. Differential diagnosis between the two forms is based on serum level of vitamin E and is very important for prognosis because in patients with ataxia with isolated vitamin E deficiency (AVED) vitamin E supplementation allows a stabilization of the neurological conditions [3, 6] and may lead to clinical improvement [4]. We report the case of a young patient with AVED with a complete regression of ataxia after early and long-term vitamin E oral supplementation. Our case strongly suggests early recognition and treatment of vitamin E deficiency to improve disease outcome. AVED (OMIM #277460) is a rare autosomal recessive neurodegenerative disease caused by mutations in the αtocopherol transfer protein (TTPA) gene, which maps to locus 8q13.1-q13.3 on chromosome 8 [3]. The deficit reduces the capacity to incorporate α-tocopherol (the most biologically active form of vitamin E) into very low density lipoproteins secreted by the liver and therefore into plasma and tissues [1]; it affects particularly the nervous system, where neurons are more vulnerable to free-radical damage [5]. The disease was characterized clinically and the defective gene localized to proximal chromosome 8q by Ben Hamida et al. in 1993 [2]. Neurological findings include spinocerebellar degeneration and mild to moderate axonal sensory neuropathy (less severe than in Freidreich ataxia), with clinical picture of ataxia, areflexia, dysarthria, and impaired proprioception. AVED should be differentiated from other syndromes with vitamin E deficiency, which can be caused by intestinal and liver diseases, malabsorption or malnutrition [1]. An 8-year-old girl came to our attention for progressive ataxia since the age of 3 years. She was the only child of healthy consanguineous parents (3rd degree) with a family history of a not defined ataxia in the father’s line. Neurological examination disclosed spinocerebellar degeneration with clinical features of important trunkal ataxia, head tremor, dysmetria, areflexia, bilateral Babinski sign, pes cavus with retraction of Achilles tendon on left the side. Tests for cholestatic liver disease, fat malabsorption and abetalipoproteinemia showed no pathologic alterations. Brain and spine magnetic resonance imaging, fundus examination, electroencephalogram, electrocardiogram, as well as motor conduction velocity (MCV) on peroneal and median nerves resulted unrevealing. Alphafetoprotein (to rule out Ataxia-Telangiectasia) and molecular DNA analysis for Friedereich ataxia were both negative. Sensory conduction velocity (SCV) on sural and median nerves were suggestive of axonal sensory neuropathy and somatosensory evoked potentials (SEP) showed the alteration of bilateral somatosensory conduction. Alpha-tocopherol level was below the sensitivity of the test (normal values 0.5–2.0 mg/dl) and thus high-dose vitamin E supplementation was given at the dosage of 1200– 1500 mg/day until the serum concentration raised to 1.23 mg/dL. DNA analysis for the detection of the two most common mutations of the α-TTP gene in patients from Mediterranean population [6] showed homozygous 744delA mutation. After only six months we witnessed complete resolution of trunkal and limb ataxia as well as SCVand SEP normalization. Deep tendon reflexes remained abolished and the foot deformity persisted requiring surgical correction. We want to highlight the importance of the assay of vitamin E serum levels in Friedreich-like ataxia. In our case the truncating homozygous 744delA mutation caused the complete abolishment of α-TTP synthesis with a severe and early phenotype of the disease [1, 4]. However, early and high-dose oral supplementation could lead to complete L. Doria-Lamba (*) . E. De Grandis . E. Cristiani . I. Fiocchi . L. Montaldi . P. Grosso Operative Unit of Child Neuropsychiatry, Department of Neurosciences, Ophthalmology and Genetics, G Gaslini Institute, University of Genoa, L.go Gaslini 5, 16148 Genoa, Italy e-mail: lauradoria@ospedale-gaslini.ge.it Tel.: +39-010-5636702 Fax: +39-010-381303

Preparation of fluorescent tocopherols for use in protein binding and localization with the α-tocopherol transfer protein

Sixteen fluorescent analogues of the lipid-soluble antioxidant vitamin α-tocopherol were prepared incorporating fluorophores at the terminus of ω-functionalized 2- n-alkyl-substituted chromanols ( 1a– d and 4a– d) that match the methylation pattern of α-tocopherol, the most biologically active form of vitamin E. The fluorophores used include 9-anthroyloxy (AO), 7-nitrobenz-2-oxa-1,3-diazole (NBD), N-methyl anthranilamide (NMA), and dansyl (DAN). The compounds were designed to function as fluorescent reporter ligands for protein-binding and lipid transfer assays. The fluorophores were chosen to maximize the fluorescence changes observed upon moving from an aqueous environment (low fluorescence intensity) to an hydrophobic environment such as a protein’s binding site (high fluorescence intensity). Compounds 9d (anthroyloxy) and 10d (nitrobenzoxadiazole), having a C9-carbon chain between the chromanol and the fluorophore, were shown to bind specifically and reversibly to recombinant human tocopherol transfer protein (α-TTP) with dissociation constants of approximately 280 and 60 nM, respectively, as compared to 25 nM for the natural ligand 2 R,4′ R,8′ R-α-tocopherol. Thus, compounds have been prepared that allow the investigation of the rate of α-TTP-mediated inter-membrane transfer of α-tocopherol and to investigate the mechanism of α-TTP function at membranes of different composition.

Selective accumulation of α-tocopherol in Drosophila is associated with cytochrome P 450 tocopherol-ω-hydroxylase activity but not α-tocopherol transfer protein

Humans and other mammals actively discriminate among the various forms of vitamin E to selectively retain α-tocopherol, but the phylogenetic breadth of this trait is unknown. We sought to determine if the fruit fly, Drosophila melanogaster, similarly discriminates and if so by what mechanism. Larvae and adult flies fed diets containing predominantly γ- and δ-tocopherols were enriched in α-tocopherol. Inclusion in the diet of piperonyl butoxide (PBO), an insect cytochrome P 450 inhibitor and inhibitor of tocopherol-ω-hydroxylase activity, greatly elevated tissue levels of δ-tocopherol but not α-tocopherol. Drosophila microsomes exhibited tocopherol-ω-hydroxylase activity in the order of δ-T > γ-T ≫ α-T, a pattern consistent with the effect of PBO in vivo. To determine if selectivity involved α-tocopherol transfer protein (α-TTP), adult flies were fed an equimolar mixture of d 3-RRR- and d 6-all-racemic α-tocopherol. Flies exhibited a d 3/ d 6 ratio of 1.03, demonstrating an inability to discriminate on the basis of phytyl tail stereochemistry, a hallmark of α-TTP activity. We conclude that Drosophila preferentially accumulates α-tocopherol via a mechanism involving cytochrome P 450 tocopherol-ω-hydroxylase-mediated catabolism of other tocopherols, but not a mammalian-like α-TTP. The selective pressure favoring this trait and its remarkable conservation from insects to humans requires elucidation.

Lipid peroxidation induced by carbon tetrachloride and its inhibition by antioxidant as evaluated by an oxidative stress marker, HODE

We have recently proposed total hydroxyoctadecadienoic acid (HODE) as a biomarker for oxidative stress in vivo. The biological samples such as plasma, urine, and tissues were first reduced and then saponified to convert the oxidation products of linoleate to HODE. In the present study, this method was applied to measure the oxidative damage induced by the administration of carbon tetrachloride to mice and also to evaluate the capacity of antioxidant to inhibit the above damage. α-Tocopherol transfer protein knock out (α-TTP −/−) mice were used to evaluate antioxidant effect in the absence of α-tocopherol. The intraperitoneal administration of carbon tetrachloride to mice induced the increase in HODE in liver and plasma, which was followed by an increase in plasma glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT). F2-isoprostanes, another prevailing biomarker, were also increased similarly, but their concentration was approximately two to three orders of magnitude smaller than that of HODE. The lipophilic antioxidants such as γ-tocopherol, γ-tocotrienol and 2,3-dihydro-5-hydroxy-4,6-di- tert-butyl-2,2-dipentylbenzofuran (BO-653) were effective in suppressing the formation of HODE.