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Vitamin E deficiency has different effects on brain and liver phospholipid hydroperoxide glutathione peroxidase activities in the rat
Neuroscience Letters 286 (2000) 87±90
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Vitamin E de®ciency has different effects on brain and liver phospholipid hydroperoxide glutathione peroxidase activities in the rat Jean-Marie Bourre a,*, Odile Dumont a, Michel CleÂment a, Lan Dinh a, Marie-TheÂrese Droy-Lefaix b, Yves Christen b a
INSERM U 26, HoÃpital Fernand Widal, 200 rue du Faubourg Saint-Denis, 75475 Paris Cedex 10, Paris, France b IPSEN, 24, rue Erlanger, 75781 Paris Cedex 16, France Received 11 February 2000; received in revised form 11 April 2000; accepted 11 April 2000
Abstract The effect of vitamin E de®ciency on glutathione peroxidase activity (GPX) and on the activity of a selenoenzyme (phospholipid hydroperoxide glutathione peroxidase (PHGPX) was measured in rat brain and liver. In brain, the activity of both enzymes was in the same range in homogenate and in microsomes. In contrast, in liver homogenate, PHGPX activity was approximately 20 times lower than that of GPX. Very interestingly, PHGPX activity was signi®cantly decreased in brain microsomes by vitamin E de®ciency, but slightly signi®cantly increased in liver microsomes. In contrast, GPX activity was not affected in brain by vitamin E de®ciency, but was signi®cantly lower in liver homogenate and microsomes. Thus, PHGPX activity is partially controlled by vitamin E in membranes, and PHGPX is probably an enzyme different from GPX. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Glutathione peroxidase; Phospholipid hydroperoxide glutathione peroxidase; Vitamin E; Desaturase; Polyunsaturated fatty acids; Brain; Liver
It is well known that the brain is especially susceptible to free radical damage since the membrane lipids are very rich in polyunsaturated fatty acids (PUFA). Free radicals contribute to neuronal loss in cerebral ischemia and hemorrhage, and may be involved in the degeneration of neurons during normal aging and also in various diseases such as Alzheimer's disease, epilepsy, schizophrenia, tardive dyskinesia, and Parkinson's disease. It has long been known that lipid peroxidation, which greatly in¯uences membrane ¯uidity, lipid composition, and enzymatic activities is correlated with brain damage. Free radicals are also implicated in the initiation of lipid peroxidation. Glutathione peroxidase (GPX) play a key role against peroxidation [2]. Therefore, antioxidant defences in the brain have justi®ably received considerable attention. The brain contains both enzymatic and non-enzymatic antioxidants that protect against free radical damage. The enzymatic antioxidants include catalase, superoxide dismutase, GPX, glutathione reductase and glucose-6-phosphate dehydrogenase. Known * Corresponding author. Tel.: 133-1-40-05-43-40; fax: 133-140-34-40-64.
non-enzymatic antioxidants include vitamin E, beta-carotene, estrogen, ascorbic acid, and glutathione. Another form of GPX isolated from pig liver by Ursini et al. [22] acts directly on peroxidized phospholipids integrated into biomembranes and is therefore called phospholipid hydroperoxide glutathione peroxidase (PHGPX). Apart from this important functional difference and the apparently monomeric nature of PHGPX, the two enzymes appear similar. The molecular mass of PHGPX is close to the subunit molecular mass of GPX, and both enzymes contain selenium in almost identical stoichiometric amounts [22]. The enzyme is present in brain [24], and in various organs such as testis, intestinal epithelium, heart, retina, and kidney. Individual glutathione peroxidases could have tissue-speci®c functions [2]. PHGPX has been cloned recently in mouse ®broblasts [1]. Expression of PHGPX is responsible for the protection of host cells from lipid hydroperoxide-mediated injury [17]. Moreover, PHGPX is more active than GPX in preventing the elevation of liver lipid peroxide levels in selenium-de®cient rats [9]. Interestingly, when lipid peroxidation was induced in liver microsomes by ascorbate and iron adenosine-dipho-
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 09 5- 8
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J.-M. Bourre et al. / Neuroscience Letters 286 (2000) 87±90
sphate, inhibition by PHGPX and glutathione occurred only if vitamin E was also present [12]. These results suggest cooperation between the peroxidase and the free radical scavenger, which raises the question of what is the mechanism of cooperation and the role of the vitamin E. Speculations have been made on the effect of vitamin E on GPX activity, both in vivo [4,16,18,20] and in cell culture [7]. The aim of the present work was to study the relationship between PHGPX activity and dietary vitamin E content. Moreover, due to the similarity between GPX and PHGPX it still remains uncertain whether PHGPX should be considered an enzyme in its own right or rather a variant or derivative of GPX. Female Sprague±Dawley rats from Iffa Credo (l'Arbresle, France) were fed a standard laboratory diet. Animals were maintained under standardized conditions of light (07:00± 19:00), temperature (22 ^ 18C, humidity (70%), and received water ad libitum. From the 14th day of gestation, breeding female rats were fed a synthetic vitamin-E-de®cient diet or the same diet enriched with All-rac-a-tocopherol (0.050 g/kg diet) (APAE-INRA, Jouy-en Josas, France). Rats were decapitated and the liver and brain rapidly removed, weighed, and rinsed with homogenization medium. The liver was homogenized for 1 min in three volumes of 0.25 M sucrose, 20 mM Tris±HCl (pH 7.4). Following centrifugation at 10 000 £ g for 15 min and at 105 000 £ g for 1 h, the pellet was resuspended in 0.1 M Tris±HCl (pH 7.4) and 0.3 M KCl buffer solution and centrifuged again at 105 000 £ g for 1 h to obtain the microsomal fraction. Brain microsomes were prepared using the same method with homogenization for 1 min in ®ve volumes of 0.32 M sucrose, 0.1 M phosphate, NaCl 9/1000 buffer. The ®nal pellet containing brain microsomes was resuspended in 0.1 M Tris±HCl (pH 7.4) and 0.3 M KCl buffer solution. Liver and brain microsomes were stored at 2808C for no longer than 2 weeks before use. The substrate was synthesized according to Maiorino et al. [13]. Brie¯y, phosphatidylcholine polyunsaturated fatty acids were oxygenated with soybean lipoxidase in the presence of bile salts. Phosphatidylcholine contained mainly linoleic acid, as originating from soybean (Sigma, Ref. P7443). At the end of the reaction, phospholipids were separated from bile salts and extracted with methanol. The substrate was stored at 2208C for no longer than 2 weeks before analysis. The number of hydroperoxide groups was then evaluated by colorimetric chemical assay based on reduction of hydroperoxides with potassium iodide according to van Kuijk et al. [23]. Starch formed a coloured complex with the triiodide ion produced by reduction of the hydroperoxides in the presence of an acid catalyst, aluminium chloride. Calibration was performed with solutions of known concentrations of hydrogen peroxide. PHGPX was assayed as follows. One milligram of biological sample was added to a total volume of 2.5 ml containing 0.125 M Tris±HCl (pH 7.4), 6.25 mM EDTA, 0.125 mM
NADPH (Sigma), 1.25 mM NaN3 (Sigma), 3.75 mM reduced glutathione (Sigma), 5 ml of glutathione reductase (Sigma, speci®c activity 200 units/mg protein) and 15 ml of 20% (v/v) peroxide-free Triton X-100 (Sigma). After 5 min at 378C for temperature equilibration, complete reduction of glutathione and PHGPX activation, the reaction was started by addition of 40 nmol of substrate and the change in absorption at 340 nm was recorded (Kontron Uvikon 930 spectrophotometer). GPX was assayed by the method of Tappel [19]. Rats were maintained on control and vitamin-E de®cient diets during gestation and suckling and pups were fed the same diet as their dams for 4 weeks. At the end of the depletion period, four pups of each group were decapitated under light ether anesthesia. Livers and forebrains were rapidly removed and frozen. All the tissues were lyophilized and stored at 2308C until analysis of tocopherols. Tocopherols were determined by HPLC in serum and lyophilized tissues according to Ueda and Igarashi [21] with minor modi®cations [5,6]. The concentrations of alpha-tocopherol were calculated using calibration curves obtained with a 2500 Chromato-Integrator (Merck±Hitachi). Results are expressed as mg/g fresh weight. Protein was determined according to Lowry et al. [11]. Results are presented as means ^ SD for four experiments using four rats from each dietary group. Data were analyzed using Student's ttest for comparison between the two groups. Experimental protocols were approved and met French government guidelines (Ministry of Agriculture, authorization no. 03007, June 4, 1991). Dietary vitamin E deprivation reduced vitamin E content in both forebrain and liver, (69.7 and 96.8%, respectively) (Table 1). Fig. 1 shows GPX and PHGPX activities in brain and liver measured in homogenate and microsomal preparations in de®cient and control animal. In vitamin-E-de®cient animals, GPX was not affected in brain homogenate or in brain microsomes, but was signi®cantly decreased in liver homogenate and liver microsomes compared with controls. In vitamin-E-de®cient animals, PHGPX activity was significantly decreased in brain microsomes, but not in brain homogenate. In contrast, the activity was signi®cantly increased in liver microsomes, but not in liver homogenate. Thus, in brain, vitamin E de®ciency signi®cantly decreased microsomal PHGPX but not microsomal GPX. PHGPX and GPX were signi®cantly and inversely altered in liver from de®cient animals: PHGPX was signi®cantly increased in liver microsomes, whereas GPX was signi®cantly decreased in liver homogenate and microsomes. Table 1 Vitamin E content of brain and liver of control and vitamin-Ede®cient animals
Forebrain (vit E mg/g) Liver (vit E mg/g)
Vit E 1
Vit E 2
24.81 ^ 1.59 35.73 ^ 2.75
7.51 ^ 1.89 1.13 ^ 0.34
J.-M. Bourre et al. / Neuroscience Letters 286 (2000) 87±90
Fig. 1. Effect in rats of a vitamin-E-de®cient diet on speci®c activities of GPX and PHGPX in liver and brain. LH, liver homogenate; LM, liver microsomes; BH, brain homogenate; BM, brain microsomes; E 1, control diet; E 2, vitamin E de®cient diet. Data are means ^ SD of four experiments using four rats from each group. *P , 0:01 and **P , 0:001 compared with control diet (Student's t-test).
When GPX and PHGPX activities were compared, activities were similar in brain homogenate and microsomes for both control and de®cient animals, as well as in liver microsomes for control animals. In liver microsomes the PHGPX activity was similar to GPX activity for de®cient animals. In contrast, in liver homogenate, PHGPX values were, respectively, about 20- and 17-fold lower than GPX values for control and de®cient animals. Unlike GPX, PHGPX can reduce membrane phospholipid hydroperoxides in situ without the necessity of prior hydrolysis by phospholipase A2. Thus, PHGPX has a more direct protective role and due to its interaction with vitamin E (also present in membrane) is probably more active. The cooperation between vitamin E and PHGPX in the protection against oxidative damage of membranes remains to be clari®ed. Both selenium and vitamin E are recognized as essential nutrients for prevention of oxidative damage. The obligatory presence of selenium for the enzymatic activity of the two seleno-dependent glutathione peroxidases is well known. In contrast, the role of vitamin E is still obscure. Moreover, it is believed that the major difference between GPX and PHGPX is that the reduction of phospholipid hydroperoxides in the membrane matrix is catalyzed uniquely by PHGPX. Others properties such as
89
kinetic mechanisms of the peroxidase reactions, selenium content and the similarity of molecular weight between PHGPX and the monomer of GPX suggest a structural relationship between these two enzymes. Therefore, we considered it to be important to study the role vitamin E plays in the activity of both glutathione peroxidase enzymes. GPX activity measured in brain microsomes could in fact correspond to the PHGPX activity. Moreover, GPX and PHGPX were not similarly distributed in different tissues: in contrast to liver, brain GPX activity in homogenate was the same as that of PHGPX in microsomes. This similar tissue distribution has already been reported [24]. Thus, very interestingly, PHGPX activity in liver and brain was not regulated in the same manner by vitamin E de®ciency. Microsomal PHGPX activity was reduced in brain but increased in liver. We have previously found that vitamin E de®ciency decreases GPX in liver [3], and we have attributed this to the impairment of selenium absorption during vitamin E de®ciency. Compared with GPX, PHGPX activity was lower in liver microsomes, but PHGPX activity was increased in vitamin E-de®cient microsomes. These results show that, in addition to the difference in their substrate speci®city and localization, these glutathione peroxidases have distinct functions. It seems likely that when vitamin E is withdrawn from the diet, PHGPX is required to prevent free radical generation by lipid hydroperoxides. Maiorino et al. [12] studied the interaction between vitamin E and PHGPX using the iron-dependent lipid peroxidation system. In this case, vitamin E has an effective antioxidant effect only if both PHGPX and glutathione are present, perhaps because PHGPX prevents formation of alkoxyl radicals against which vitamin E is a relatively weak antioxidant. This result could be linked to the in vitro alteration of delta-6-desaturase by vitamin E in rat brain and liver [8]. In that study we showed that delta-6-desaturase activity in brain microsomes was increased by vitamin E; in contrast, this activity was reduced in the liver. In our present study, vitamin E de®ciency could alter polyunsaturated fatty acid synthesis by delta-6-desaturation in the liver membrane. The effect of vitamin E depletion on PHGPX activity in liver microsomes was different from that of selenium, showing that selenium acts by a different mechanism from that of vitamin E, although they are both required for prevention of oxidative damage. Interestingly, GPX and PHGPX are regulated differently in rat by dietary selenium [10]. Selenium depletion has been observed in brain in a patient with epilepsy [15], and PHGPX is speculated to play a key role in the defense of neuronal cells against oxygen radical formation and peroxidation processes. The differences observed in this work between brain and liver microsomes could be a consequence of the distribution of GPX and PHGPX in this organelle, as it has been shown that PHGPX is predominant in the luminal phase of the endoplasmic reticulum, in contrast with GPX which predo-
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minate in cytosolic phase, as well as in mitochondrial inner membrane [14]. The changes in activity observed could be related to activation/inactivation processes or changes in protein expression. In conclusion, the present work investigated the relationship between vitamin E and PHGPX. It speculates that PHGPX can substitute for vitamin E to prevent lipid peroxidation in liver. In contrast, in brain, PHGPX activity was not changed by vitamin E de®ciency. Further studies are required to elucidate the mechanism of this substitution. This work was supported by INSERM and IPSEN. The authors are most grateful to A. Strickland for reviewing the manuscript, and to D. Fauconnier for help in feeding the animals. [1] Borchert, A., Schnurr, K., Thiele, B.J. and Kuhn, H., Cloning of the mouse phospholipid hydroperoxide glutathione peroxidase gene, FEBS Lett., 446 (1999) 223±227. [2] Brigelius-Flohe, R., Tissue-speci®c functions of individual glutathione peroxidases, Free Radic. Biol. Med., 27 (1999) 951±965. [3] ChaudieÁre, J., CleÂment, M., GeÂrard, D. and Bourre, J.M., Brain alterations induced by vitamin E de®ciency and intoxication with methyl ethyl ketone peroxide, Neurotoxicology, 9 (1988) 173±180. [4] Chen, L.H. and Thacker, R.R., Effect of ascorbic acid and vitamin E on biochemical changes associated with vitamin E de®ciency in rats, Int. J. Vitam. Nutr. Res., 57 (1987) 385± 390. [5] CleÂment, M., Dinh, L. and Bourre, J.M., Uptake of dietary RRR-alpha- and RRR-gamma-tocopherol by nervous tissues, liver and muscle in vitamin-E-de®cient rats, Biochim. Biophys. Acta, 1256 (1995) 175±180. [6] CleÂment, M. and Bourre, J.M., Graded dietary levels of RRRgamma-tocopherol induce a marked increase in the concentrations of alpha- and gamma-tocopherol in nervous tissue, heart, liver and muscle of vitamin-E-de®cient-rats, Biochim. Biophys. Acta, 1334 (1997) 173±181. [7] Conti, M., Couturier, M., Lemonnier, A. and Lemonnier, F., Effects of alpha-tocopherol on antioxidant enzyme activity in human ®broblast cultures, Int. J. Vitam. Nutr. Res., 63 (1993) 71±76. [8] Despret, S., Dinh, L., CleÂment, M. and Bourre, J.M., Alteration of delta-6 desaturase by vitamin E in rat brain and liver, Neurosci. Lett., 145 (1992) 19±22. [9] Guan, J.Y., Komura, S., Ohishi, N. and Yagi, K., Difference in effects of classic phospholipid hydroperoxide glutathione peroxidases on liver lipid peroxide level in selenium-de®cient rats, Biochem. Mol. Biol. Int., 37 (1995) 1103±1110. [10] Lei, X., Evenson, J., Thompson, K. and Sunde, R., Glutathione peroxidase and phospholipid hydroperoxide
[11] [12]
[13] [14]
[15] [16]
[17]
[18] [19] [20] [21]
[22] [23]
[24]
glutathione peroxidase are differentially regulated in rats by dietary selenium, J. Nutr., 125 (1995) 1438±1446. Lowry, O.M., Rosebrough, H.H., Farr, A.L. and Randal, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 226 (1951) 497±509. Maiorino, M., Coassin, M., Roveri, A. and Ursini, F., Microsomal lipid peroxidation: effect of vitamin E and its functional interaction with phospholipid hydroperoxide glutathione peroxidase, Lipids, 24 (1989) 721±726. Maiorino, M., Gregolin, C. and Ursini, F., Phospholipid hydroperoxide glutathione peroxidase, Methods Enzymol., 186 (1990) 448±457. Marinho, H.S., Antunes, F. and Pinto, R.E., Role of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase in the reduction of lysophospholipid hydroperoxides, Free Radic. Biol. Med., 22 (1997) 871± 883. Ramaekers, V.T., Calomme, M., Vanden Berghe, D. and Makropoulos, W., Selenium de®ciency triggering intractable seizures, Neuropediatrics, 25 (1994) 217±223. Sardar, S., Chakraborty, A. and Chatterjee, M., Comparative effectiveness of vitamin D3 and dietary vitamin E on peroxidation of lipids and enzymes of the hepatic antioxidant system in Sprague±Dawley rats, Int. J. Vitam. Nutr. Res., 66 (1996) 39±45. Sun, Q., Kojima, H., Komura, S., Oshishi, N. and Yagi, K., Effect of selenium on human phospholipid hydroperoxide glutathione peroxidase expression and host cell susceptibility to lipid hydroperoxide-mediated injury, Biochem. Mol. Biol. Int., 42 (1997) 957±963. Tangney, C.C., McCloskey, K.M. and Aye, P.L., Effects of hyperoxia and diet on murine tissue levels of vitamin E and polyunsaturated fatty acids, Lipids, 23 (1988) 707±712. Tappel, A.L., Glutathione peroxidase and hydroperoxides, Methods Enzymol., 52 (1991) 506±512. Tiidus, P.M. and Houston, M.E., Antioxidant and oxidative enzyme adaptations to vitamin E deprivation and training, Med. Sci. Sports Exerc., 26 (1994) 354±359. Ueda, T. and Igarashi, O., Determination of vitamin E in biological specimens and foods by HPLC. Pretreatment of samples and extraction of tocopherols, J. Micronutr. Anal., 7 (1990) 79±96. Ursini, F., Maiorino, M. and Gregolin, C., The selenoenzyme phospholipid hydroperoxide glutathione peroxidase, Biochim. Biophys. Acta, 839 (1985) 62±70. van Kuijk, F.J.G.M., Handelman, G.J. and Dratz, E.A., Consecutive action of phospholipase A2 and glutathione peroxidase is required for reduction of phospholipid hydroperoxides and provides a convenient method to determine peroxide values in membranes, J. Free Radic. Biol. Med., 1 (1985) 421±427. Zhang, L., Maiorino, M., Roveri, A. and Ursini, F., Phospholipid hydroperoxide glutathione peroxidase: speci®c activity in tissues of rats of different age and comparison with other glutathione peroxidases, Biochim. Biophys. Acta, 1006 (1989) 140±143.
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Vitamin E de®ciency has different effects on brain and liver phospholipid hydroperoxide glutathione peroxidase activities in the rat Jean-Marie Bourre a,*, Odile Dumont a, Michel CleÂment a, Lan Dinh a, Marie-TheÂrese Droy-Lefaix b, Yves Christen b a
INSERM U 26, HoÃpital Fernand Widal, 200 rue du Faubourg Saint-Denis, 75475 Paris Cedex 10, Paris, France b IPSEN, 24, rue Erlanger, 75781 Paris Cedex 16, France Received 11 February 2000; received in revised form 11 April 2000; accepted 11 April 2000
Abstract The effect of vitamin E de®ciency on glutathione peroxidase activity (GPX) and on the activity of a selenoenzyme (phospholipid hydroperoxide glutathione peroxidase (PHGPX) was measured in rat brain and liver. In brain, the activity of both enzymes was in the same range in homogenate and in microsomes. In contrast, in liver homogenate, PHGPX activity was approximately 20 times lower than that of GPX. Very interestingly, PHGPX activity was signi®cantly decreased in brain microsomes by vitamin E de®ciency, but slightly signi®cantly increased in liver microsomes. In contrast, GPX activity was not affected in brain by vitamin E de®ciency, but was signi®cantly lower in liver homogenate and microsomes. Thus, PHGPX activity is partially controlled by vitamin E in membranes, and PHGPX is probably an enzyme different from GPX. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Glutathione peroxidase; Phospholipid hydroperoxide glutathione peroxidase; Vitamin E; Desaturase; Polyunsaturated fatty acids; Brain; Liver
It is well known that the brain is especially susceptible to free radical damage since the membrane lipids are very rich in polyunsaturated fatty acids (PUFA). Free radicals contribute to neuronal loss in cerebral ischemia and hemorrhage, and may be involved in the degeneration of neurons during normal aging and also in various diseases such as Alzheimer's disease, epilepsy, schizophrenia, tardive dyskinesia, and Parkinson's disease. It has long been known that lipid peroxidation, which greatly in¯uences membrane ¯uidity, lipid composition, and enzymatic activities is correlated with brain damage. Free radicals are also implicated in the initiation of lipid peroxidation. Glutathione peroxidase (GPX) play a key role against peroxidation [2]. Therefore, antioxidant defences in the brain have justi®ably received considerable attention. The brain contains both enzymatic and non-enzymatic antioxidants that protect against free radical damage. The enzymatic antioxidants include catalase, superoxide dismutase, GPX, glutathione reductase and glucose-6-phosphate dehydrogenase. Known * Corresponding author. Tel.: 133-1-40-05-43-40; fax: 133-140-34-40-64.
non-enzymatic antioxidants include vitamin E, beta-carotene, estrogen, ascorbic acid, and glutathione. Another form of GPX isolated from pig liver by Ursini et al. [22] acts directly on peroxidized phospholipids integrated into biomembranes and is therefore called phospholipid hydroperoxide glutathione peroxidase (PHGPX). Apart from this important functional difference and the apparently monomeric nature of PHGPX, the two enzymes appear similar. The molecular mass of PHGPX is close to the subunit molecular mass of GPX, and both enzymes contain selenium in almost identical stoichiometric amounts [22]. The enzyme is present in brain [24], and in various organs such as testis, intestinal epithelium, heart, retina, and kidney. Individual glutathione peroxidases could have tissue-speci®c functions [2]. PHGPX has been cloned recently in mouse ®broblasts [1]. Expression of PHGPX is responsible for the protection of host cells from lipid hydroperoxide-mediated injury [17]. Moreover, PHGPX is more active than GPX in preventing the elevation of liver lipid peroxide levels in selenium-de®cient rats [9]. Interestingly, when lipid peroxidation was induced in liver microsomes by ascorbate and iron adenosine-dipho-
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 09 5- 8
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J.-M. Bourre et al. / Neuroscience Letters 286 (2000) 87±90
sphate, inhibition by PHGPX and glutathione occurred only if vitamin E was also present [12]. These results suggest cooperation between the peroxidase and the free radical scavenger, which raises the question of what is the mechanism of cooperation and the role of the vitamin E. Speculations have been made on the effect of vitamin E on GPX activity, both in vivo [4,16,18,20] and in cell culture [7]. The aim of the present work was to study the relationship between PHGPX activity and dietary vitamin E content. Moreover, due to the similarity between GPX and PHGPX it still remains uncertain whether PHGPX should be considered an enzyme in its own right or rather a variant or derivative of GPX. Female Sprague±Dawley rats from Iffa Credo (l'Arbresle, France) were fed a standard laboratory diet. Animals were maintained under standardized conditions of light (07:00± 19:00), temperature (22 ^ 18C, humidity (70%), and received water ad libitum. From the 14th day of gestation, breeding female rats were fed a synthetic vitamin-E-de®cient diet or the same diet enriched with All-rac-a-tocopherol (0.050 g/kg diet) (APAE-INRA, Jouy-en Josas, France). Rats were decapitated and the liver and brain rapidly removed, weighed, and rinsed with homogenization medium. The liver was homogenized for 1 min in three volumes of 0.25 M sucrose, 20 mM Tris±HCl (pH 7.4). Following centrifugation at 10 000 £ g for 15 min and at 105 000 £ g for 1 h, the pellet was resuspended in 0.1 M Tris±HCl (pH 7.4) and 0.3 M KCl buffer solution and centrifuged again at 105 000 £ g for 1 h to obtain the microsomal fraction. Brain microsomes were prepared using the same method with homogenization for 1 min in ®ve volumes of 0.32 M sucrose, 0.1 M phosphate, NaCl 9/1000 buffer. The ®nal pellet containing brain microsomes was resuspended in 0.1 M Tris±HCl (pH 7.4) and 0.3 M KCl buffer solution. Liver and brain microsomes were stored at 2808C for no longer than 2 weeks before use. The substrate was synthesized according to Maiorino et al. [13]. Brie¯y, phosphatidylcholine polyunsaturated fatty acids were oxygenated with soybean lipoxidase in the presence of bile salts. Phosphatidylcholine contained mainly linoleic acid, as originating from soybean (Sigma, Ref. P7443). At the end of the reaction, phospholipids were separated from bile salts and extracted with methanol. The substrate was stored at 2208C for no longer than 2 weeks before analysis. The number of hydroperoxide groups was then evaluated by colorimetric chemical assay based on reduction of hydroperoxides with potassium iodide according to van Kuijk et al. [23]. Starch formed a coloured complex with the triiodide ion produced by reduction of the hydroperoxides in the presence of an acid catalyst, aluminium chloride. Calibration was performed with solutions of known concentrations of hydrogen peroxide. PHGPX was assayed as follows. One milligram of biological sample was added to a total volume of 2.5 ml containing 0.125 M Tris±HCl (pH 7.4), 6.25 mM EDTA, 0.125 mM
NADPH (Sigma), 1.25 mM NaN3 (Sigma), 3.75 mM reduced glutathione (Sigma), 5 ml of glutathione reductase (Sigma, speci®c activity 200 units/mg protein) and 15 ml of 20% (v/v) peroxide-free Triton X-100 (Sigma). After 5 min at 378C for temperature equilibration, complete reduction of glutathione and PHGPX activation, the reaction was started by addition of 40 nmol of substrate and the change in absorption at 340 nm was recorded (Kontron Uvikon 930 spectrophotometer). GPX was assayed by the method of Tappel [19]. Rats were maintained on control and vitamin-E de®cient diets during gestation and suckling and pups were fed the same diet as their dams for 4 weeks. At the end of the depletion period, four pups of each group were decapitated under light ether anesthesia. Livers and forebrains were rapidly removed and frozen. All the tissues were lyophilized and stored at 2308C until analysis of tocopherols. Tocopherols were determined by HPLC in serum and lyophilized tissues according to Ueda and Igarashi [21] with minor modi®cations [5,6]. The concentrations of alpha-tocopherol were calculated using calibration curves obtained with a 2500 Chromato-Integrator (Merck±Hitachi). Results are expressed as mg/g fresh weight. Protein was determined according to Lowry et al. [11]. Results are presented as means ^ SD for four experiments using four rats from each dietary group. Data were analyzed using Student's ttest for comparison between the two groups. Experimental protocols were approved and met French government guidelines (Ministry of Agriculture, authorization no. 03007, June 4, 1991). Dietary vitamin E deprivation reduced vitamin E content in both forebrain and liver, (69.7 and 96.8%, respectively) (Table 1). Fig. 1 shows GPX and PHGPX activities in brain and liver measured in homogenate and microsomal preparations in de®cient and control animal. In vitamin-E-de®cient animals, GPX was not affected in brain homogenate or in brain microsomes, but was signi®cantly decreased in liver homogenate and liver microsomes compared with controls. In vitamin-E-de®cient animals, PHGPX activity was significantly decreased in brain microsomes, but not in brain homogenate. In contrast, the activity was signi®cantly increased in liver microsomes, but not in liver homogenate. Thus, in brain, vitamin E de®ciency signi®cantly decreased microsomal PHGPX but not microsomal GPX. PHGPX and GPX were signi®cantly and inversely altered in liver from de®cient animals: PHGPX was signi®cantly increased in liver microsomes, whereas GPX was signi®cantly decreased in liver homogenate and microsomes. Table 1 Vitamin E content of brain and liver of control and vitamin-Ede®cient animals
Forebrain (vit E mg/g) Liver (vit E mg/g)
Vit E 1
Vit E 2
24.81 ^ 1.59 35.73 ^ 2.75
7.51 ^ 1.89 1.13 ^ 0.34
J.-M. Bourre et al. / Neuroscience Letters 286 (2000) 87±90
Fig. 1. Effect in rats of a vitamin-E-de®cient diet on speci®c activities of GPX and PHGPX in liver and brain. LH, liver homogenate; LM, liver microsomes; BH, brain homogenate; BM, brain microsomes; E 1, control diet; E 2, vitamin E de®cient diet. Data are means ^ SD of four experiments using four rats from each group. *P , 0:01 and **P , 0:001 compared with control diet (Student's t-test).
When GPX and PHGPX activities were compared, activities were similar in brain homogenate and microsomes for both control and de®cient animals, as well as in liver microsomes for control animals. In liver microsomes the PHGPX activity was similar to GPX activity for de®cient animals. In contrast, in liver homogenate, PHGPX values were, respectively, about 20- and 17-fold lower than GPX values for control and de®cient animals. Unlike GPX, PHGPX can reduce membrane phospholipid hydroperoxides in situ without the necessity of prior hydrolysis by phospholipase A2. Thus, PHGPX has a more direct protective role and due to its interaction with vitamin E (also present in membrane) is probably more active. The cooperation between vitamin E and PHGPX in the protection against oxidative damage of membranes remains to be clari®ed. Both selenium and vitamin E are recognized as essential nutrients for prevention of oxidative damage. The obligatory presence of selenium for the enzymatic activity of the two seleno-dependent glutathione peroxidases is well known. In contrast, the role of vitamin E is still obscure. Moreover, it is believed that the major difference between GPX and PHGPX is that the reduction of phospholipid hydroperoxides in the membrane matrix is catalyzed uniquely by PHGPX. Others properties such as
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kinetic mechanisms of the peroxidase reactions, selenium content and the similarity of molecular weight between PHGPX and the monomer of GPX suggest a structural relationship between these two enzymes. Therefore, we considered it to be important to study the role vitamin E plays in the activity of both glutathione peroxidase enzymes. GPX activity measured in brain microsomes could in fact correspond to the PHGPX activity. Moreover, GPX and PHGPX were not similarly distributed in different tissues: in contrast to liver, brain GPX activity in homogenate was the same as that of PHGPX in microsomes. This similar tissue distribution has already been reported [24]. Thus, very interestingly, PHGPX activity in liver and brain was not regulated in the same manner by vitamin E de®ciency. Microsomal PHGPX activity was reduced in brain but increased in liver. We have previously found that vitamin E de®ciency decreases GPX in liver [3], and we have attributed this to the impairment of selenium absorption during vitamin E de®ciency. Compared with GPX, PHGPX activity was lower in liver microsomes, but PHGPX activity was increased in vitamin E-de®cient microsomes. These results show that, in addition to the difference in their substrate speci®city and localization, these glutathione peroxidases have distinct functions. It seems likely that when vitamin E is withdrawn from the diet, PHGPX is required to prevent free radical generation by lipid hydroperoxides. Maiorino et al. [12] studied the interaction between vitamin E and PHGPX using the iron-dependent lipid peroxidation system. In this case, vitamin E has an effective antioxidant effect only if both PHGPX and glutathione are present, perhaps because PHGPX prevents formation of alkoxyl radicals against which vitamin E is a relatively weak antioxidant. This result could be linked to the in vitro alteration of delta-6-desaturase by vitamin E in rat brain and liver [8]. In that study we showed that delta-6-desaturase activity in brain microsomes was increased by vitamin E; in contrast, this activity was reduced in the liver. In our present study, vitamin E de®ciency could alter polyunsaturated fatty acid synthesis by delta-6-desaturation in the liver membrane. The effect of vitamin E depletion on PHGPX activity in liver microsomes was different from that of selenium, showing that selenium acts by a different mechanism from that of vitamin E, although they are both required for prevention of oxidative damage. Interestingly, GPX and PHGPX are regulated differently in rat by dietary selenium [10]. Selenium depletion has been observed in brain in a patient with epilepsy [15], and PHGPX is speculated to play a key role in the defense of neuronal cells against oxygen radical formation and peroxidation processes. The differences observed in this work between brain and liver microsomes could be a consequence of the distribution of GPX and PHGPX in this organelle, as it has been shown that PHGPX is predominant in the luminal phase of the endoplasmic reticulum, in contrast with GPX which predo-
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minate in cytosolic phase, as well as in mitochondrial inner membrane [14]. The changes in activity observed could be related to activation/inactivation processes or changes in protein expression. In conclusion, the present work investigated the relationship between vitamin E and PHGPX. It speculates that PHGPX can substitute for vitamin E to prevent lipid peroxidation in liver. In contrast, in brain, PHGPX activity was not changed by vitamin E de®ciency. Further studies are required to elucidate the mechanism of this substitution. This work was supported by INSERM and IPSEN. The authors are most grateful to A. Strickland for reviewing the manuscript, and to D. Fauconnier for help in feeding the animals. [1] Borchert, A., Schnurr, K., Thiele, B.J. and Kuhn, H., Cloning of the mouse phospholipid hydroperoxide glutathione peroxidase gene, FEBS Lett., 446 (1999) 223±227. [2] Brigelius-Flohe, R., Tissue-speci®c functions of individual glutathione peroxidases, Free Radic. Biol. Med., 27 (1999) 951±965. [3] ChaudieÁre, J., CleÂment, M., GeÂrard, D. and Bourre, J.M., Brain alterations induced by vitamin E de®ciency and intoxication with methyl ethyl ketone peroxide, Neurotoxicology, 9 (1988) 173±180. [4] Chen, L.H. and Thacker, R.R., Effect of ascorbic acid and vitamin E on biochemical changes associated with vitamin E de®ciency in rats, Int. J. Vitam. Nutr. Res., 57 (1987) 385± 390. [5] CleÂment, M., Dinh, L. and Bourre, J.M., Uptake of dietary RRR-alpha- and RRR-gamma-tocopherol by nervous tissues, liver and muscle in vitamin-E-de®cient rats, Biochim. Biophys. Acta, 1256 (1995) 175±180. [6] CleÂment, M. and Bourre, J.M., Graded dietary levels of RRRgamma-tocopherol induce a marked increase in the concentrations of alpha- and gamma-tocopherol in nervous tissue, heart, liver and muscle of vitamin-E-de®cient-rats, Biochim. Biophys. Acta, 1334 (1997) 173±181. [7] Conti, M., Couturier, M., Lemonnier, A. and Lemonnier, F., Effects of alpha-tocopherol on antioxidant enzyme activity in human ®broblast cultures, Int. J. Vitam. Nutr. Res., 63 (1993) 71±76. [8] Despret, S., Dinh, L., CleÂment, M. and Bourre, J.M., Alteration of delta-6 desaturase by vitamin E in rat brain and liver, Neurosci. Lett., 145 (1992) 19±22. [9] Guan, J.Y., Komura, S., Ohishi, N. and Yagi, K., Difference in effects of classic phospholipid hydroperoxide glutathione peroxidases on liver lipid peroxide level in selenium-de®cient rats, Biochem. Mol. Biol. Int., 37 (1995) 1103±1110. [10] Lei, X., Evenson, J., Thompson, K. and Sunde, R., Glutathione peroxidase and phospholipid hydroperoxide
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