Original Contributions
Vitamin E regulates mitochondrial hydrogen peroxide generation

https://doi.org/10.1016/S0891-5849(99)00121-5Get rights and content

Abstract

The mitochondrial electron transport system consumes more than 85% of all oxygen used by the cells, and up to 5% of the oxygen consumed by mitochondria is converted to superoxide, hydrogen peroxide, and other reactive oxygen species (ROS) under normal physiologic conditions. Disruption of mitochondrial ultrastructure is one of the earliest pathologic events during vitamin E depletion. The present studies were undertaken to test whether a direct link exists between vitamin E and the production of hydrogen peroxide in the mitochondria. In the first experiment, mice were fed a vitamin E–deficient or–sufficient diet for 15 weeks, after which the mitochondria from liver and skeletal muscle were isolated to determine the rates of hydrogen peroxide production. Deprivation of vitamin E resulted in an approximately 5-fold increase of mitochondrial hydrogen peroxide production in skeletal muscle and a 1-fold increase in liver when compared with the vitamin E–supplemented group. To determine whether vitamin E can dose-dependently influence the production of hydrogen peroxide, four groups of male and female rats were fed diets containing 0, 20, 200, or 2000 IU/kg vitamin E for 90 d. Results showed that dietary vitamin E dose-dependently attenuated hydrogen peroxide production in mitochondria isolated from liver and skeletal muscle of male and female rats. Female rats, however, were more profoundly affected by dietary vitamin E than male rats in the suppression of mitochondrial hydrogen peroxide production in both organs studied. These results showed that vitamin E can directly regulate hydrogen peroxide production in mitochondria and suggest that the overproduction of mitochondrial ROS is the first event leading to the tissue damage observed in vitamin E–deficiency syndromes. Data further suggested that by regulating mitochondrial production of ROS, vitamin E modulates the expression and activation of signal transduction pathways and other redox-sensitive biologic modifiers, and thereby delays or prevents degenerative tissue changes.

Introduction

Since vitamin E was discovered over 70 years ago [1], a number of species-dependent deficiency symptoms of vitamin E have been reported [2], [3]. The essentiality of vitamin E for humans was established in the late 1960s. The recognition was largely derived from clinical investigations involving premature infants [3], [4]. Studies of children and adults with specific causes of fat malabsorption and patients with familial isolated vitamin E deficiency syndrome have conclusively shown that neurologic dysfunction is associated with vitamin E deficiency and that vitamin E is an essential nutrient necessary for the optimal development and maintenance of human nervous system integrity and function [5], [6], [7]. Recent reports on the benefit of vitamin E supplements in reducing the risk of cardiovascular disease, Alzheimer’s disease, and other degenerative disorders [8], [9], [10], [11], [12], [13], [14] have raised public interest in the vitamin to a new height. However, the mechanism by which vitamin E can delay or protect against the development of tissue degeneration remains to be delineated.

Mitochondria, the intracellular organelle that produce adenosine triphosphate, constitute the greatest source of steady state oxidants. The mitochondrial electron transport system consumes more than 85% of all the oxygen used by the cells, and it is estimated that between 1 and 5% of the oxygen consumed by mitochondria is converted to superoxide, hydrogen peroxide, and other ROS under normal physiological conditions [15], [16], [17]. Proximal to a large flux of ROS, mitochondria DNA is particularly susceptible to oxidative damage and mutation because it lacks protective histones and an effective repair system [18], [19], [20]. Mitochondrial DNA, for example, has a 16-fold higher oxidized base than nuclear DNA [21], and the accumulation of mitochondrial DNA damage products increases with age [22], [23]. In addition to oxidative damage to lipid and protein in mitochondria, many studies have found that increased oxidative lesions, deletions, point mutations, and aberrant forms in mitochondrial DNA of postmitotic tissues on aging and have suggested that mitochondrial respiratory chain defects and DNA mutations are two key contributors to human aging and neurodegenerative diseases [24], [25], [26], [27], [28], [29], [30].

As the major producer of ROS in the cell, it is important that mitochondrion has the highest concentration of vitamin E and that most of it is in the inner membrane [31]. It has long been recognized that the disruption of mitochondrial ultrastructure is one of the earliest pathologic events observed in the skeletal muscle of vitamin E–deficient animals [32]. Also, de novo synthesis of xanthine oxidase, which serves as a source of superoxide, is markedly increased in the skeletal muscle of vitamin E–deficient rabbits [33]. This activity has also been shown to be significantly higher in the liver of vitamin E–deficient rats [34]. These findings suggest an increased generation of superoxide during vitamin E deficiency and a direct involvement of excessive ROS as a cause of developing vitamin E–deficient syndromes. The studies reported herein sought to determine whether vitamin E has a direct functional effect over the production of ROS in mitochondria. Results clearly showed that dietary vitamin E markedly reduces hydrogen peroxide production in the mitochondria of liver and skeletal muscle derived from two rodent species and that this suppression was closely related to the vitamin E concentration of the diet. The results obtained from this research indicate that vitamin E regulates mitochondrial generation of ROS. This unique and direct function of vitamin E may in turn modulate signal transduction pathways and other redox-sensitive biologic modifiers, and thereby prevent or delay the development of degenerative diseases.

Section snippets

Chemicals and reagents

Mono- and dibasic sodium phosphate and 30% hydrogen peroxide were purchased from Fisher Scientific (Cincinnati, OH, USA); high-performance liquid chromatography-grade methanol, hexane, and water were purchased from EM Science (Gibbstown, NJ, USA); 95% ethanol was obtained from Midwest Grain Products (Pekin, IL, USA; isobutyl alcohol was obtained from Mallinckroft Chemist (St. Louis, MO, USA); 1,1,3,3-tetramethoxypropane, β-nicotinamide adenine dinucleotide (reduced form), 2-thiobarbituric acid,

Results

After 15 weeks on the vitamin E–deficient diet, the status of vitamin E in this group of mice was compared with corresponding controls that received the same diet supplemented with 50 IU vitamin E/kg diet. Figure 1 shows that in the deficient group, the levels of vitamin E were markedly lower, whereas TBA reactant values were significantly (p < .05) higher in skeletal muscle. Plasma pyruvate kinase activity, a sensitive indicator of myodegeneration [3], [36], was significantly elevated in the

Discussion

Results from Fig. 2 clearly show that regardless of the presence or absence of added superoxide dismutase to the incubations, there was no effect in the production of hydrogen peroxide from mitochondria isolated from mouse liver and skeletal muscle. This suggests that mitochondrial superoxide dismutase provides sufficient activity to convert superoxide generated to hydrogen peroxide rapidly; is also suggests that in situ superoxide, rather than hydrogen peroxide, was the original ROS generated.

Acknowledgements

This work was supported in parts by the University of Kentucky Agricultural Experiment Station.

References (76)

  • A. Bjorneboe et al.

    Effect of dietary deficiency and supplement with all-rac-alpha-tocopherol on hepatic content in rats

    J. Nutr.

    (1991)
  • C.K. Chow

    Increased activity of pyruvate kinase in plasma of vitatmin E–deficient rats

    J. Nutr.

    (1975)
  • S.K. Bhattacharya et al.

    Isolation of skeletal muscle mitochondria from hamsters using an ionic medium containing ethylenediaminetetraacetic acid and Nagarse

    Anal. Biochem.

    (1991)
  • P.A. Hyslop et al.

    A quantitative fluorometric assay for the determination of oxidant production by polymorphonuclear leukocytesits use in the simultaneous fluorometric assay of cellular activation processes

    Anal. Biochem.

    (1984)
  • L.J. Hatam et al.

    A high performance liquid chromatographic method for the determination of tocopherol in plasma and cellular elements of the blood

    J. Lipid Res.

    (1979)
  • T. Bucher et al.

    Pyruvate kinase from muscle

    Methods Enzymol.

    (1955)
  • C.K. Chow

    Nutritional influence on cellular antioxidant defense systems

    Am. J. Clin. Nutr.

    (1979)
  • G. Buettner

    The packing order of free radicals and antioxidantslipid peroxidation, alpha-tocopherol, and ascorbate

    Arch. Biochem. Biophys.

    (1993)
  • G.L. Squadrito et al.

    Oxidative chemistry on nitric oxidethe roles of superoxide, peroxynitrite, and carbon dioxide

    Free Radic. Biol. Med.

    (1998)
  • O. Augusto et al.

    Spin-trapping studies of peroxynitrite decomposition and 3- morpholinosydnonimide N-ethylcarbamide autooxidationdirect evidence for metal-independent formation of free radical intermediates

    Arch. Biochem. Biophys.

    (1994)
  • M.G. Traber et al.

    Vitamin Ebeyond antioxidant function

    Am. J. Clin. Nutr.

    (1995)
  • O. Cachia et al.

    α-Tocopherol inhibits the respiratory burst in human monocytes. Attenuation of p47phox membrane translocation and phosphorylation

    J. Biol. Chem.

    (1998)
  • E.M. Klann et al.

    A role for superoxide in protein kinase C activation and induction of long-term potentiation

    J. Biol. Chem.

    (1998)
  • G. Shklar et al.

    Vitamin E inhibits experimental carcinogenesis and tumor angiogenesis

    Eur. J. Cancer B. Oral Oncol.

    (1996)
  • R.K. Studer et al.

    Antioxidant inhibition of protein kinase C-signaled increased in transforming growth factor-beta in sesangial cells

    Metabolism

    (1997)
  • K. Tran et al.

    Vitamin E suppresses diacylglycerol level in thrombin-stimulated endothelial cells through an increase of diacylglycerol kinase activity

    Biochim. Biophys. Acta

    (1994)
  • H.M. Evans et al.

    On the existence of a hitherto unrecognized dietary factor essential for reproduction

    Science

    (1922)
  • M.L. Scott

    Studies on vitamin E and related factors in nutrition and metabolism

  • L. Machlin

    Vitamin E

  • J.G. Bieri et al.

    Vitamin E

    Vitam Horm

    (1986)
  • R.J. Sokol

    Vitamin E deficiency and neurological disorders

  • L.H. Kushi et al.

    Dietary antioxidant vitamins and death from coronary heart disease in postmenopausal women

    N. Engl. J. Med.

    (1996)
  • M. Sano et al.

    A controlled trial of selegiline, alpha-tocopherol, or both as treatment for Alzheimer’s disease. The Alzheimer’s Disease Cooperative Study

    N. Engl. J. Med.

    (1997)
  • S.N. Meydani et al.

    Recent developments in vitamin E and immune response

    Nutr. Rev.

    (1998)
  • M.C. De Rijk et al.

    Dietary antioxidants and Parkinson disease. The Rotterdam Study

    Arch. Neurol.

    (1997)
  • B. Chance et al.

    Hydroperoxide metabolism in mammalian organs

    Physiol. Rev.

    (1979)
  • H. Nohl et al.

    Do mitochondria produce oxygen radicals in vivo?

    Eur. J. Biochem.

    (1978)
  • M.K. Shigenaga et al.

    Oxidative damage and mitochondrial decay in ageing

    Proc. Natl. Acad. Sci. USA

    (1994)
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