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Vitamin E protects guinea pig liver from lipid peroxidation without depressing levels of antioxidants
hr. J. Biochem.
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Bid.
Vol. 27, No. I I, pp. I 175-l 18I. 19% Copyright
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Vitamin E Protects Guinea Pig Liver from Lipid Peroxidation without Depressing Levels of Antioxidants S. CADENAS, G. BARJA”
C. ROJAS, R. PEREZ-CAMPO,
Department of Animal Biology-II Madrid 28040, Spain
M. LOPEZ-TORRES,
(Animal Physiology), Faculty of Biology, Complutense /Jnioersity,
Oxidative stress is considered a pathogenic factor in many disorders. The capacity of dietary vitamin E to increase global antioxidant capacity and to decrease lipid peroxidation was studied in the guinea pig, an animal that cannot synthesize ascorbate. Male guinea pigs were subj&ed for 5 weeks to three diets differing in vitamin E content in the presem~ of opt&man levels of vitamin C: group 15 (15 mg vitamin E/kg diet), group 150 (15Omg/kg), and group lso0 (1500 mg/kg). Hepatic vitamin E increased in the three groups in relatioa to the level of vihunin E in the diet. The increase in vitamin E between groups 15 and 150 was by a reduction in sensitivity to enzymatic lipid peroxidation. llds did not occur between groups 150 and 1500. The different liver vitamin E concentratioos did not a&t the antioxidant enzymes superoxide diintase, catahse, GSH-peroxidase and GSH-reductase, nor antioxidants vitamin C, GSH and ascorbate. It is concluded that dietary su vitamin E, at a level 6 times high than the minimum daily requirement for gumea pls% mcmases protection agahst kpatic lipid peroxidation without depressing e anthKidant defaces. Further increases in vitamia E to megadose levels dii not provide additional protection from oxidative stress. The results also suggest that optimum levels of both vitamin C and vitamia E, simultaueousiy needed for protection against oxidative stress, are much higher than the minimum daily requirements. Keywords:
Free radicals
Vitamin
E
Lipid peroxidation
Diet
Antioxidants
Int. J. Biochem. Cell Biol. (1995) 27, 1175-l 181
INTRODUCTION
Oxidative stress is now generally considered an important pathogenic factor in the development of many human diseases. Among the factors modifying oxidative stress, there is strong interest in the antioxidant vitamins E and C, the intake of which can be easily and safely controlled through the diet. In addition, there is strong epidemiological evidence to suggest that antioxidant vitamins such as vitamin E can protect against cardiovascular diseases (Gey, *To whom correspondence should be addressed. Received I1 January 1995; accepted 12 June 1995. Abbreviarions: MDA-malondialdehyde, SOD-superoxide dismutase, CAT-catalase, GF’x-glutathione peroxidase, GR-glutathione reductase. 1175
1990; Gey et al., 1991; Ferns et al., 1993) and cancer (Byers and Perry, 1992; Knekt, 1993). This is related to the capacity of antioxidants to decrease endogenous oxidative damage to lipids (Diplock, 1985; Sies and Murphy, 1991; Halliwell, 1993), proteins (Stadtman, 1991) and nucleic acids (Richter et al., 1988). The authors have recently shown that vitamin C is capable of reducing oxidative damage to both proteins and lipids in the liver of the guinea pig in uiuo (Barja et al., 1994). This species is an ideal in vivo model since it is the only laboratory animal that can synthesize neither ascorbate nor a-tocopherol; characteristics it shares with man. The effect of vitamin E on antioxidants and oxidative stress is studied in this research at the optimum levels of dietary vitamin C previously
1176
S. Cadenas
found for this particular tissue and animal (Barja et al., 1994). The aims of this investigation were to show if vitamin E is of further protective value in animals already subjected to optimum protection by the antioxidant vitamin C, and to test for possible synergism between these vitamins in vivo, a phenomenon already demonstrated in vitro. Previous work has shown that endogenous antioxidants are under homeostatic control in animal tissues. Vertebrates can respond to in vivo oxidative stress with strong inductions of endogenous antioxidant defences in the liver (Lopez-Torres et al., 1993). Any depression of these endogenous antioxidants following vitamin E supplementation, would seriously limit the efficacy of such treatment. In an effort to gain more knowledge about the optimum dose of vitamin E capable of protecting against oxidative damage without depressing these endogenous antioxidants, the livers of guinea pigs, provided with diets with three different levels of vitamin E for 5 weeks, were investigated for sensitivity to lipid peroxidation and changes in the activity of the main antioxidant enzymes (superoxide dismutase, catalase, GSHperoxidase and GSH-reductase), and the main non-enzymatic antioxidants vitamins C and E, and GSH.
MATERIALS
AND
METHODS
Dietary treatment
Male Dunkin-Hartley guinea pigs weighing approx. 250 g were obtained from Iffa-Creddo, Lyon, France. Three diets differing in vitamin E (d,l-cr-tocopherol acetate) content were prepared by U.A.R. (France) by adding different quantities of vitamin E to a vitamin E-free standard guinea pig diet (diet 114, U.A.R.): 1500 mg of vitamin E per kg diet (group 1500), 150 mg vitamin E/kg (group 150) or 15 mg of vitamin E/kg (group 15). These amounts were confirmed by HPLC analysis (for method see below) in two samples of each diet after arrival at the laboratory. The basal diet contained 18.5% protein, 2.9% fat, 46.9% carbohydrates, 8.4% mineral mix, 1.4% vitamin mix, 11% humidity and 10.9% non-nutritive bulk. The content of minerals and vitamins per kg of diet was: phosphorus, 8600 mg; calcium, 10,600 mg; potassium, 12,000 mg; sodium 3450 mg; magnesium, 3 130 mg; manganese, 100 mg; iron, 320 mg; copper, 26 mg; zinc, 85 mg; cobalt,
et ul.
1.61 mg; vitamin A 19,000 I.U.; vitamin D3, 2031 I.U.; thiamine, 22.5 mg; riboflavin, 21 mg; pantothenic acid, 123 mg; piridoxine, 10.7 mg; menadione, 55 mg; niacin, 1930 mg; folic acid, 7.3 mg; biotin, 0.275 mg; choline, 1740 mg; M-inositol 250mg; selenium 114pg; and vitamin C 660 mg. Guinea pigs were maintained for 5 weeks on the three experimental diets. They were caged inside aseptic air positive-pressure animal cabinets (A 130 SP, Flufrance, Cachan, France) equipped with an HEPA air filter (99.999% for particles > 0.3 pm) at the inlet. At the end of the dietary regime the animals were sacrificed by decapitation and liver samples were immediately collected and stored at -70°C a procedure known to preserve the activity of endogenous antioxidants over long periods of time (Jung et al., 1993). Lipid peroxidation
Peroxidation was stimulated in vitro by incubating supernatants of liver samples in the presence of 0.2 mM FeSO,, 5 mM ADP and 1 mM NADPH for 90 min at 25°C. At the end of the incubation period a thiobarbituric acid (TBA) assay was performed. TBA reacting substances (TBARS) were measured spectrophotometrically (Uchiyama and Mihara, 1978). The values represent the sensitivity of the tissue lipids to an oxygen radical challenge. Vitamins E and C, GSH and uric acid
Liver content of vitamin E (a-tocopherol) was measured by high performance liquid chromatography (HPLC) with U.V. detection at 292 nm. Samples of liver tissue were homogenized in 50 mM perchloric acid for simultaneous analysis of ascorbic and uric acids by ion pair HPLC with U.V. detection at 280 nm (Barja de Quiroga et al., 199 1). Liver samples were homogenized in 5% trichloroacetic acid with 0.01 N HCl. Total glutathione was measured by the spectrophotometric recycling assay (Tietze, 1969) in the presence of 5,5’-dithiobis (2-nitrobenzoic acid), NADPH and GSH-reductase at 412 nm. Antioxidant
enzymes
Liver samples were homogenized in 50mM phosphate buffer (pH 7.4). Superoxide dismutase activity was measured after 24 h of dialysis by quantifying its inhibition of pyrogall01 autoxidation at 420 nm (Marklund and Marklund, 1974). Catalase was measured
Vitamin
E and oxidative
following H,O, disappearance at 240 nm (Beers and Sizer, 1952). Glutathione peroxidase was measured following NADPH oxidation at 340 nm in the presence of excess glutathione reductase, GSH, and cumene hydroperoxide (Lawrence and Burk, 1976). GSH-reductase was assayed by following NADPH oxidation at 340 nm in the presence of GSSG (Massey and Williams, 1965). All enzymatic reactions were performed at 25°C. Protein concentrations were measured as described by Lowry et al. (1951). Statistical analysis The data were subjected to one way analysis of variance. Following ANOVA, Fisher’s least significant difference (LSD) test was used where necessary to analyze significance between paired groups. The 0.05 level was selected as minimal statistical significance in all analyses.
100 80
r(a) VITAMIN
stress
in guinea
RESULTS
After 5 weeks of dietary treatment with different quantities of vitamin E, the animals of the three experimental groups showed clearly different hepatic levels of vitamin E (Fig. la). Increasing the dietary vitamin E content by one order of magnitude from group 15 (15 mg of vitamin E/kg) to group 150 (150 mg of vitamin E/kg) resulted in a 125% increase in liver vitamin E content. When dietary vitamin E was further increased by another order of magnitude from group 150 to group 1500 (1500 mg vitamin E/kg) the vitamin E hepatic content increased again reaching 300% that of group 150. The level of vitamin E achieved in group 15 (12 pg/g tissue) shows that these animals were not vitamin E deficient. Initial mean body weight was close to 250 g and final weight around 558 g in the three groups. The growth rates of animals in all three groups remained unaffected both at 3 and 5
.r:.::12.. .:-..:: ...: :. *. ,. ‘.> :.y.-. ::::.;: :.I(.= ../.::: .I.’ :..:’ : VITAMIN
)
***
C (nmol/g)
(b)
I.200
60
1177
pig liver
T
c
800
t 40 20 :... . . . ..’.: . 0 ~ ‘:.‘.* I5
VITAMIN
GSH
”
150
E IN DIET
(nmol/mg
1,500
150
15
(mglkg)
VITAMIN
protein)
URIC
E IN DIET
ACID
1,500
(mg/kg)
(nmollg)
500
(4 400
T
I
”
15
VITAMIN
150
1.500
15
E IN DIET
(mglkg)
VITAMIN
Fig. I. Vitamin E, vitamin C, GSH, and uric acid concentration of treatment with three diets differing in vitamin E content. different vs group I5 (P < 0.05); ***significantly different
150
E IN DIET
1,500
(mglkg)
in the liver of guinea pigs after five weeks Values are means f SEM. *Significantly vs group 150 (P < 0.001). n = 5 7.
S. Cadenas el al.
1178
500
LIPID PEROXIDATION
(nmol MDA/g)
rT
component of the endogenous antioxidant defence system, the antioxidant enzymes. Neither superoxide dismutase (SOD), catalase (CAT), GSH-peroxidase (GPx) or GSH-reductase (GR) were affected by the hepatic concentration of vitamin E (Fig. 3). DISCUSSION
0 15
VITAMIN
150
1,500
E IN DIET (mg/kg)
Fig. 2. Lipid peroxidation after 5 weeks of treatment with three diets differing in vitamin E content. Enzymatic lipid peroxidation (thiobarbituric acid assay) was measured after 90min of incubation of liver homogenates in the presence of 0.2 mM FeSO,, 5 mM ADP and 1 mM NADPH. Values are means +- SEM. ***Significantly different from group 15 (P < 0.05).
n = 7.
weeks (data not shown). The amount of food ingested per animal per day, which increased from 35 to 50 g during the 5 weeks of experimentation, was also unaffected by vitamin E treatment. Figure 2 shows that vitamin E dietary supplementation over 5 weeks profoundly affected enzymatic lipid peroxidation of liver homogenates stimulated by iron-ADP-NADPH. Lipid peroxidation decreased by 60% in groups 150 and 1500 with respect to that of group 15. This decrease was accompanied by a 125% increase in hepatic vitamin E content from group 15 to group 150. However, even though liver vitamin E concentration was further increased by 200% from group 150 to group 1500, enzymatic lipid peroxidation was similar in both groups. In spite of the strong difference in hepatic vitamin E concentration between the three groups, vitamin C content of the liver was not affected (Fig. lb). The same was true for GSH (Fig. lc). Uric acid levels showed a progressive trend to decrease as levels of vitamin E increased (Fig. Id) but these changes did not reach statistical significance. Thus, variation of vitamin E levels over very large ranges @-fold difference in hepatic vitamin E between extreme values) did not affect the rest of the nonenzymatic antioxidants in the guinea pig liver. The same was true for the other main
The different quantities of vitamin E added to the diet, and the duration of dietary supplementation, produced guinea pigs with three different hepatic concentrations of vitamin E. The guinea pigs of group 15 (15 mg of vitamin E/kg diet) ingested 0.6-0.7 mg of vitamin E per day. This is very close to the minimum vitamin E requirement for the growing guinea pig, 1 mg/day (Shimotori et af., 1939; National Research Council, 1978). Animals in this group showed normal growth, food intake and apparent behavior. Though their hepatic vitamin E level was low they still showed 12 pg vitamin E/g tissue. It is known that a diet totally deficient in vitamin E, followed over 8 weeks, is required in order to observe body weight loss, skeletal muscle degeneration, atrophy of the testes, and prostration in guinea pigs (National Research Council, 1978). Therefore, group 15 does not represent vitamin E-deficient animals, but rather guinea pigs with low vitamin E levels. Group 150 (150 mg vitamin E/kg) falls within the normal range used for routine maintenance of the guinea pig. Animals in this group ingested amounts of vitamin E over 6 times higher than the minimum daily requirement. Group 1500 (1500 mg vitamin E per kg) was designed to clarify if levels of vitamin E 64 times higher than the minimum daily requirement have any detrimental effect on antioxidants and oxidative stress. The three different levels of vitamin E used did not affect the rate of body growth, food intake, or general appearance of the animals. The dietary treatments were very effective at producing animals with different liver vitamin E concentrations. A 2.5- to 3-fold difference in liver vitamin E was found between groups 15 and 150 and between groups 150 and 1500. Thus, liver vitamin E responds to dietary treatment in guinea pigs in a way similar to that previously observed in rats (Vatassery et al., 1988; Ueda and Igarashi, 1990). Supplementation with vitamin E strongly inhibited NADPH-induced lipid peroxidation from group 15 to group 150. Further supplementation with vitamin E (group 1500) provided
Vitamin E and oxidative stress in guinea pig liver
no additional reduction in sensitivity to lipid peroxidation. This result shows that dietary levels of vitamin E higher than the minimum daily requirement of guinea pigs are needed to optimally prevent lipid peroxidation, though very great increases are of no value. Therefore, from the point of view of oxidative stress, the optimum vitamin E levels required are much higher than those needed to avoid overt deficiency. Several reports have described similar results in other laboratory rodents. A diet of 180 mg of vitamin E/kg, maintained for 24 days, decreased lipid peroxidation in rat liver compared to a diet of 86mg/kg whereas 1400 mg vitamin E/kg afforded no further protection (Gunther et al., 1992). Dietary supplementation with vitamin E also decreased susceptibility to lipid peroxidation in rat liver (Thompson and Lee, 1993; Williams et al., 1992), peroxidation of rat liver microsomes induced by iron-ascorbate (Palamanda and Kehrer, 1993), and in uiuo
r(a)
rat liver lipid peroxidation values (Yoshizawa et al., 1991; Wolf et al., 1993). No decrease in liver peroxidation was described, however, in the livers of mice treated with 200 mg of vitamin E/kg for 6-8 weeks over that of animals kept on a deficiency diet for the same length of time (Sutphin and Buckman, 1991). However, tissue levels of vitamin E attained were not measured in the above work. In a previous investigation the authors showed that vitamin C also decreases NADPHinduced lipid peroxidation in guinea pig liver (Barja et al., 1994). The optimum levels of vitamin C found in that work, 660mg of vitamin C/kg, were included in the basal diet of the three groups used in the present investigation. In spite of the presence of optimum vitamin C levels, the group fed the low vitamin E diet showed higher lipid peroxidation than the other two groups. This shows that optimum protection against liver lipid peroxidation requires
2. SOD (U/mg protein)
15-
CAT (Ulmg protein) 600 500
T
”
T
T
150
1,500
Y
15
VITAMIN
200
1179
150
1,500
E IN DIET (mg/kg)
r(cl
GPx (Ulmg protein)
1.5
15
VITAMIN
E IN DIET (mglkg)
GR (Ulmg protein) 5o T(d)
150
1,500
15
150
1,500
VITAMIN E IN DIET (mg/kg) VITAMIN E IN DIET (mg/kg) Fig. 3. Antioxidant enzymes in the liver of guinea pigs after 5 weeks of treatment with three diets differing in vitamin E content. SOD = superoxide dismutase, CAT = catalase, GPx = GSH-peroxidase, GR = GSH-reductase. Units (U) are ymole of H,O, (CAT), or nmole of NADPH (GPx and GR) transformed per min. Values are means + SEM. n = 7.
1180
S. Cadenas et al.
dietary supplementation with both vitamin C and E at levels higher than those needed to avoid deficiency. This is probably related to the proposed synergism between vitamin E and vitamin C antioxidant functions (Niki, 1991; Buettner, 1993). The antioxidant function of vitamin E is related to that of antioxidant enzymes such as GSH-peroxidases, GSH-reductase, and to GSH, a major non-enzymatic antioxidant (Asensi et al., 1994). One year of vitamin E deficiency increases GSH-peroxidase activity in various rat tissues (Kumar and Darad, 1988). Further, vitamin E supplementation restores normal levels of GPx in human smokers (Hoshino et al., 1990). However, 10 weeks of dietary vitamin E deficiency increased GSH levels by 50% in rat liver without changing GSH-peroxidase (Williams et al., 1992). A better understanding of the effect of dietary vitamin E on GSH-related antioxidants is therefore needed. Further understanding of the functioning of other endogenous enzymatic and nonenzymatic antioxidants, which are governed by homeostatic compensation in animal tissues, is also required. Long-term irreversible catalase inactivation is known to increase ascorbate, GSH and SOD contents by lOO%, and GSHreductase by 1000% in vertebrate liver (LopezTorres et al., 1993). However, it is possible that this homeostatic system also works in the opposite direction: excessive supplementation with a particular antioxidant might depress components of the endogenous antioxidant defence system. The results obtained in this investigation show that this does not occur in the guinea pig liver even after addition of megadose levels of vitamin E. Neither superoxide dismutase, catalase, GSH-peroxidase, GSHreductase, vitamin C, GSH or uric acid were affected by the three very different levels of dietary vitamin E. No similar information has previously been reported in the guinea pig, though these results agree with reports showing that vitamin E supplementation does not change superoxide dismutase, catalase or GSHperoxidase activity in the livers of mice (Sutphin and Buckman, 1991), and rats (Williams et al., 1992; Tiidus and Houston, 1994), nor GSHperoxidase activity and GSH content of chick liver (Kim and Combs, 1993). An absence of effect of dietary vitamin E on liver ascorbate concentration has also been described in the rat (Behrens and Madere, 1989). In a previous paper the authors showed that widely different
levels of dietary vitamin C supplementation caused no reactive depression of any enzymatic or non-enzymatic antioxidant in the guinea pig liver (Barja et al., 1994). In summary, these results show that high levels of the two main antioxidant vitamins, vitamins C and E, can be added to the diet of the guinea pig, (the only laboratory animal which, like man, is unable to synthesize these compounds), without compromising any alternative antioxidant defenses. These vitamins protect against lipid peroxidation at levels 6 times (vitamin E, this work) and 40 times (vitamin C, Barja et al., 1994) higher than the minimum daily requirements. Further supplementation above these levels continues to have no effect on endogenous antioxidants but is superfluous since no additional protection against lipid peroxidation is provided. The results of this work support the increasingly important hypothesis (Lachance and Langseth, 1994) that the optimum levels of some vitamins required for the maintenance of health are higher than those needed for prevention of deficiency syndromes, though megadose levels should be avoided. Acknowledgements-This work was supported by a grant (no 93/0145E) from the National Research Foundation of the Spanish Ministry of Health (FISss). Predoctoral fellowships were received by C. Rojas and S. Cadenas (F.P.I., Ministry of Education), and R. Perez-Campo (FISss).
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Halliwell B. (1993) The role of oxygen radicals in human disease, with particular reference to the vascular system. Haemostasis 23, 118-126. Hoshino E., Shariff R., Van Gossum A., Allard J. P., Pichard C., Kurian R. and Jeejeebhoy K. N. (1990) Vitamin E suppresses increased lipid peroxidation in cigarette smokers. J. Parent. Ent. Nutr. 14, 3W305. Jung K., Kiihler S., Klotzek S., Becker S. and Henke W. (1993) Effect of storage temperature on the activity of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase in rat liver and kidney homogenates. Enzyme Prot. 47, 1499155. Kim Y. S. and Combs G. F. (1993) Effects of dietary selenium and vitamin E on glutathione concentrations and glutathione S-transferase activities in chick liver and plasma. Nutr. Res. 13, 455463. Knekt P. (1993) Epidemiology of vitamin E: evidence for anticancer effects in humans. In Vitamin E in Health and Disease (Edited by Packer L. and Fuchs J.), pp. 5 13-527. Dekker, New York, NY. Kumar D. A. and Darad R. (1988) Physiological antioxidants and antioxidative enzymes in vitamin E-deficient rats. Toxicol. Lett. 44, 47-54. Lachance P. and Langseth L. (1994) The RDA concept: time for a change? Nutr. Rev. 52, 266270. Lawrence R. A. and Burk R. F. (1976) Glutathione peroxidase activity in selenium-deficient rat liver. Biochem. biophys.
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Bid.
Vol. 27, No. I I, pp. I 175-l 18I. 19% Copyright
Pergamon
c 1995 Elsevier
Science Ltd
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Vitamin E Protects Guinea Pig Liver from Lipid Peroxidation without Depressing Levels of Antioxidants S. CADENAS, G. BARJA”
C. ROJAS, R. PEREZ-CAMPO,
Department of Animal Biology-II Madrid 28040, Spain
M. LOPEZ-TORRES,
(Animal Physiology), Faculty of Biology, Complutense /Jnioersity,
Oxidative stress is considered a pathogenic factor in many disorders. The capacity of dietary vitamin E to increase global antioxidant capacity and to decrease lipid peroxidation was studied in the guinea pig, an animal that cannot synthesize ascorbate. Male guinea pigs were subj&ed for 5 weeks to three diets differing in vitamin E content in the presem~ of opt&man levels of vitamin C: group 15 (15 mg vitamin E/kg diet), group 150 (15Omg/kg), and group lso0 (1500 mg/kg). Hepatic vitamin E increased in the three groups in relatioa to the level of vihunin E in the diet. The increase in vitamin E between groups 15 and 150 was by a reduction in sensitivity to enzymatic lipid peroxidation. llds did not occur between groups 150 and 1500. The different liver vitamin E concentratioos did not a&t the antioxidant enzymes superoxide diintase, catahse, GSH-peroxidase and GSH-reductase, nor antioxidants vitamin C, GSH and ascorbate. It is concluded that dietary su vitamin E, at a level 6 times high than the minimum daily requirement for gumea pls% mcmases protection agahst kpatic lipid peroxidation without depressing e anthKidant defaces. Further increases in vitamia E to megadose levels dii not provide additional protection from oxidative stress. The results also suggest that optimum levels of both vitamin C and vitamia E, simultaueousiy needed for protection against oxidative stress, are much higher than the minimum daily requirements. Keywords:
Free radicals
Vitamin
E
Lipid peroxidation
Diet
Antioxidants
Int. J. Biochem. Cell Biol. (1995) 27, 1175-l 181
INTRODUCTION
Oxidative stress is now generally considered an important pathogenic factor in the development of many human diseases. Among the factors modifying oxidative stress, there is strong interest in the antioxidant vitamins E and C, the intake of which can be easily and safely controlled through the diet. In addition, there is strong epidemiological evidence to suggest that antioxidant vitamins such as vitamin E can protect against cardiovascular diseases (Gey, *To whom correspondence should be addressed. Received I1 January 1995; accepted 12 June 1995. Abbreviarions: MDA-malondialdehyde, SOD-superoxide dismutase, CAT-catalase, GF’x-glutathione peroxidase, GR-glutathione reductase. 1175
1990; Gey et al., 1991; Ferns et al., 1993) and cancer (Byers and Perry, 1992; Knekt, 1993). This is related to the capacity of antioxidants to decrease endogenous oxidative damage to lipids (Diplock, 1985; Sies and Murphy, 1991; Halliwell, 1993), proteins (Stadtman, 1991) and nucleic acids (Richter et al., 1988). The authors have recently shown that vitamin C is capable of reducing oxidative damage to both proteins and lipids in the liver of the guinea pig in uiuo (Barja et al., 1994). This species is an ideal in vivo model since it is the only laboratory animal that can synthesize neither ascorbate nor a-tocopherol; characteristics it shares with man. The effect of vitamin E on antioxidants and oxidative stress is studied in this research at the optimum levels of dietary vitamin C previously
1176
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found for this particular tissue and animal (Barja et al., 1994). The aims of this investigation were to show if vitamin E is of further protective value in animals already subjected to optimum protection by the antioxidant vitamin C, and to test for possible synergism between these vitamins in vivo, a phenomenon already demonstrated in vitro. Previous work has shown that endogenous antioxidants are under homeostatic control in animal tissues. Vertebrates can respond to in vivo oxidative stress with strong inductions of endogenous antioxidant defences in the liver (Lopez-Torres et al., 1993). Any depression of these endogenous antioxidants following vitamin E supplementation, would seriously limit the efficacy of such treatment. In an effort to gain more knowledge about the optimum dose of vitamin E capable of protecting against oxidative damage without depressing these endogenous antioxidants, the livers of guinea pigs, provided with diets with three different levels of vitamin E for 5 weeks, were investigated for sensitivity to lipid peroxidation and changes in the activity of the main antioxidant enzymes (superoxide dismutase, catalase, GSHperoxidase and GSH-reductase), and the main non-enzymatic antioxidants vitamins C and E, and GSH.
MATERIALS
AND
METHODS
Dietary treatment
Male Dunkin-Hartley guinea pigs weighing approx. 250 g were obtained from Iffa-Creddo, Lyon, France. Three diets differing in vitamin E (d,l-cr-tocopherol acetate) content were prepared by U.A.R. (France) by adding different quantities of vitamin E to a vitamin E-free standard guinea pig diet (diet 114, U.A.R.): 1500 mg of vitamin E per kg diet (group 1500), 150 mg vitamin E/kg (group 150) or 15 mg of vitamin E/kg (group 15). These amounts were confirmed by HPLC analysis (for method see below) in two samples of each diet after arrival at the laboratory. The basal diet contained 18.5% protein, 2.9% fat, 46.9% carbohydrates, 8.4% mineral mix, 1.4% vitamin mix, 11% humidity and 10.9% non-nutritive bulk. The content of minerals and vitamins per kg of diet was: phosphorus, 8600 mg; calcium, 10,600 mg; potassium, 12,000 mg; sodium 3450 mg; magnesium, 3 130 mg; manganese, 100 mg; iron, 320 mg; copper, 26 mg; zinc, 85 mg; cobalt,
et ul.
1.61 mg; vitamin A 19,000 I.U.; vitamin D3, 2031 I.U.; thiamine, 22.5 mg; riboflavin, 21 mg; pantothenic acid, 123 mg; piridoxine, 10.7 mg; menadione, 55 mg; niacin, 1930 mg; folic acid, 7.3 mg; biotin, 0.275 mg; choline, 1740 mg; M-inositol 250mg; selenium 114pg; and vitamin C 660 mg. Guinea pigs were maintained for 5 weeks on the three experimental diets. They were caged inside aseptic air positive-pressure animal cabinets (A 130 SP, Flufrance, Cachan, France) equipped with an HEPA air filter (99.999% for particles > 0.3 pm) at the inlet. At the end of the dietary regime the animals were sacrificed by decapitation and liver samples were immediately collected and stored at -70°C a procedure known to preserve the activity of endogenous antioxidants over long periods of time (Jung et al., 1993). Lipid peroxidation
Peroxidation was stimulated in vitro by incubating supernatants of liver samples in the presence of 0.2 mM FeSO,, 5 mM ADP and 1 mM NADPH for 90 min at 25°C. At the end of the incubation period a thiobarbituric acid (TBA) assay was performed. TBA reacting substances (TBARS) were measured spectrophotometrically (Uchiyama and Mihara, 1978). The values represent the sensitivity of the tissue lipids to an oxygen radical challenge. Vitamins E and C, GSH and uric acid
Liver content of vitamin E (a-tocopherol) was measured by high performance liquid chromatography (HPLC) with U.V. detection at 292 nm. Samples of liver tissue were homogenized in 50 mM perchloric acid for simultaneous analysis of ascorbic and uric acids by ion pair HPLC with U.V. detection at 280 nm (Barja de Quiroga et al., 199 1). Liver samples were homogenized in 5% trichloroacetic acid with 0.01 N HCl. Total glutathione was measured by the spectrophotometric recycling assay (Tietze, 1969) in the presence of 5,5’-dithiobis (2-nitrobenzoic acid), NADPH and GSH-reductase at 412 nm. Antioxidant
enzymes
Liver samples were homogenized in 50mM phosphate buffer (pH 7.4). Superoxide dismutase activity was measured after 24 h of dialysis by quantifying its inhibition of pyrogall01 autoxidation at 420 nm (Marklund and Marklund, 1974). Catalase was measured
Vitamin
E and oxidative
following H,O, disappearance at 240 nm (Beers and Sizer, 1952). Glutathione peroxidase was measured following NADPH oxidation at 340 nm in the presence of excess glutathione reductase, GSH, and cumene hydroperoxide (Lawrence and Burk, 1976). GSH-reductase was assayed by following NADPH oxidation at 340 nm in the presence of GSSG (Massey and Williams, 1965). All enzymatic reactions were performed at 25°C. Protein concentrations were measured as described by Lowry et al. (1951). Statistical analysis The data were subjected to one way analysis of variance. Following ANOVA, Fisher’s least significant difference (LSD) test was used where necessary to analyze significance between paired groups. The 0.05 level was selected as minimal statistical significance in all analyses.
100 80
r(a) VITAMIN
stress
in guinea
RESULTS
After 5 weeks of dietary treatment with different quantities of vitamin E, the animals of the three experimental groups showed clearly different hepatic levels of vitamin E (Fig. la). Increasing the dietary vitamin E content by one order of magnitude from group 15 (15 mg of vitamin E/kg) to group 150 (150 mg of vitamin E/kg) resulted in a 125% increase in liver vitamin E content. When dietary vitamin E was further increased by another order of magnitude from group 150 to group 1500 (1500 mg vitamin E/kg) the vitamin E hepatic content increased again reaching 300% that of group 150. The level of vitamin E achieved in group 15 (12 pg/g tissue) shows that these animals were not vitamin E deficient. Initial mean body weight was close to 250 g and final weight around 558 g in the three groups. The growth rates of animals in all three groups remained unaffected both at 3 and 5
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C (nmol/g)
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60
1177
pig liver
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VITAMIN
GSH
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150
E IN DIET
(nmol/mg
1,500
150
15
(mglkg)
VITAMIN
protein)
URIC
E IN DIET
ACID
1,500
(mg/kg)
(nmollg)
500
(4 400
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15
VITAMIN
150
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E IN DIET
(mglkg)
VITAMIN
Fig. I. Vitamin E, vitamin C, GSH, and uric acid concentration of treatment with three diets differing in vitamin E content. different vs group I5 (P < 0.05); ***significantly different
150
E IN DIET
1,500
(mglkg)
in the liver of guinea pigs after five weeks Values are means f SEM. *Significantly vs group 150 (P < 0.001). n = 5 7.
S. Cadenas el al.
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500
LIPID PEROXIDATION
(nmol MDA/g)
rT
component of the endogenous antioxidant defence system, the antioxidant enzymes. Neither superoxide dismutase (SOD), catalase (CAT), GSH-peroxidase (GPx) or GSH-reductase (GR) were affected by the hepatic concentration of vitamin E (Fig. 3). DISCUSSION
0 15
VITAMIN
150
1,500
E IN DIET (mg/kg)
Fig. 2. Lipid peroxidation after 5 weeks of treatment with three diets differing in vitamin E content. Enzymatic lipid peroxidation (thiobarbituric acid assay) was measured after 90min of incubation of liver homogenates in the presence of 0.2 mM FeSO,, 5 mM ADP and 1 mM NADPH. Values are means +- SEM. ***Significantly different from group 15 (P < 0.05).
n = 7.
weeks (data not shown). The amount of food ingested per animal per day, which increased from 35 to 50 g during the 5 weeks of experimentation, was also unaffected by vitamin E treatment. Figure 2 shows that vitamin E dietary supplementation over 5 weeks profoundly affected enzymatic lipid peroxidation of liver homogenates stimulated by iron-ADP-NADPH. Lipid peroxidation decreased by 60% in groups 150 and 1500 with respect to that of group 15. This decrease was accompanied by a 125% increase in hepatic vitamin E content from group 15 to group 150. However, even though liver vitamin E concentration was further increased by 200% from group 150 to group 1500, enzymatic lipid peroxidation was similar in both groups. In spite of the strong difference in hepatic vitamin E concentration between the three groups, vitamin C content of the liver was not affected (Fig. lb). The same was true for GSH (Fig. lc). Uric acid levels showed a progressive trend to decrease as levels of vitamin E increased (Fig. Id) but these changes did not reach statistical significance. Thus, variation of vitamin E levels over very large ranges @-fold difference in hepatic vitamin E between extreme values) did not affect the rest of the nonenzymatic antioxidants in the guinea pig liver. The same was true for the other main
The different quantities of vitamin E added to the diet, and the duration of dietary supplementation, produced guinea pigs with three different hepatic concentrations of vitamin E. The guinea pigs of group 15 (15 mg of vitamin E/kg diet) ingested 0.6-0.7 mg of vitamin E per day. This is very close to the minimum vitamin E requirement for the growing guinea pig, 1 mg/day (Shimotori et af., 1939; National Research Council, 1978). Animals in this group showed normal growth, food intake and apparent behavior. Though their hepatic vitamin E level was low they still showed 12 pg vitamin E/g tissue. It is known that a diet totally deficient in vitamin E, followed over 8 weeks, is required in order to observe body weight loss, skeletal muscle degeneration, atrophy of the testes, and prostration in guinea pigs (National Research Council, 1978). Therefore, group 15 does not represent vitamin E-deficient animals, but rather guinea pigs with low vitamin E levels. Group 150 (150 mg vitamin E/kg) falls within the normal range used for routine maintenance of the guinea pig. Animals in this group ingested amounts of vitamin E over 6 times higher than the minimum daily requirement. Group 1500 (1500 mg vitamin E per kg) was designed to clarify if levels of vitamin E 64 times higher than the minimum daily requirement have any detrimental effect on antioxidants and oxidative stress. The three different levels of vitamin E used did not affect the rate of body growth, food intake, or general appearance of the animals. The dietary treatments were very effective at producing animals with different liver vitamin E concentrations. A 2.5- to 3-fold difference in liver vitamin E was found between groups 15 and 150 and between groups 150 and 1500. Thus, liver vitamin E responds to dietary treatment in guinea pigs in a way similar to that previously observed in rats (Vatassery et al., 1988; Ueda and Igarashi, 1990). Supplementation with vitamin E strongly inhibited NADPH-induced lipid peroxidation from group 15 to group 150. Further supplementation with vitamin E (group 1500) provided
Vitamin E and oxidative stress in guinea pig liver
no additional reduction in sensitivity to lipid peroxidation. This result shows that dietary levels of vitamin E higher than the minimum daily requirement of guinea pigs are needed to optimally prevent lipid peroxidation, though very great increases are of no value. Therefore, from the point of view of oxidative stress, the optimum vitamin E levels required are much higher than those needed to avoid overt deficiency. Several reports have described similar results in other laboratory rodents. A diet of 180 mg of vitamin E/kg, maintained for 24 days, decreased lipid peroxidation in rat liver compared to a diet of 86mg/kg whereas 1400 mg vitamin E/kg afforded no further protection (Gunther et al., 1992). Dietary supplementation with vitamin E also decreased susceptibility to lipid peroxidation in rat liver (Thompson and Lee, 1993; Williams et al., 1992), peroxidation of rat liver microsomes induced by iron-ascorbate (Palamanda and Kehrer, 1993), and in uiuo
r(a)
rat liver lipid peroxidation values (Yoshizawa et al., 1991; Wolf et al., 1993). No decrease in liver peroxidation was described, however, in the livers of mice treated with 200 mg of vitamin E/kg for 6-8 weeks over that of animals kept on a deficiency diet for the same length of time (Sutphin and Buckman, 1991). However, tissue levels of vitamin E attained were not measured in the above work. In a previous investigation the authors showed that vitamin C also decreases NADPHinduced lipid peroxidation in guinea pig liver (Barja et al., 1994). The optimum levels of vitamin C found in that work, 660mg of vitamin C/kg, were included in the basal diet of the three groups used in the present investigation. In spite of the presence of optimum vitamin C levels, the group fed the low vitamin E diet showed higher lipid peroxidation than the other two groups. This shows that optimum protection against liver lipid peroxidation requires
2. SOD (U/mg protein)
15-
CAT (Ulmg protein) 600 500
T
”
T
T
150
1,500
Y
15
VITAMIN
200
1179
150
1,500
E IN DIET (mg/kg)
r(cl
GPx (Ulmg protein)
1.5
15
VITAMIN
E IN DIET (mglkg)
GR (Ulmg protein) 5o T(d)
150
1,500
15
150
1,500
VITAMIN E IN DIET (mg/kg) VITAMIN E IN DIET (mg/kg) Fig. 3. Antioxidant enzymes in the liver of guinea pigs after 5 weeks of treatment with three diets differing in vitamin E content. SOD = superoxide dismutase, CAT = catalase, GPx = GSH-peroxidase, GR = GSH-reductase. Units (U) are ymole of H,O, (CAT), or nmole of NADPH (GPx and GR) transformed per min. Values are means + SEM. n = 7.
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dietary supplementation with both vitamin C and E at levels higher than those needed to avoid deficiency. This is probably related to the proposed synergism between vitamin E and vitamin C antioxidant functions (Niki, 1991; Buettner, 1993). The antioxidant function of vitamin E is related to that of antioxidant enzymes such as GSH-peroxidases, GSH-reductase, and to GSH, a major non-enzymatic antioxidant (Asensi et al., 1994). One year of vitamin E deficiency increases GSH-peroxidase activity in various rat tissues (Kumar and Darad, 1988). Further, vitamin E supplementation restores normal levels of GPx in human smokers (Hoshino et al., 1990). However, 10 weeks of dietary vitamin E deficiency increased GSH levels by 50% in rat liver without changing GSH-peroxidase (Williams et al., 1992). A better understanding of the effect of dietary vitamin E on GSH-related antioxidants is therefore needed. Further understanding of the functioning of other endogenous enzymatic and nonenzymatic antioxidants, which are governed by homeostatic compensation in animal tissues, is also required. Long-term irreversible catalase inactivation is known to increase ascorbate, GSH and SOD contents by lOO%, and GSHreductase by 1000% in vertebrate liver (LopezTorres et al., 1993). However, it is possible that this homeostatic system also works in the opposite direction: excessive supplementation with a particular antioxidant might depress components of the endogenous antioxidant defence system. The results obtained in this investigation show that this does not occur in the guinea pig liver even after addition of megadose levels of vitamin E. Neither superoxide dismutase, catalase, GSH-peroxidase, GSHreductase, vitamin C, GSH or uric acid were affected by the three very different levels of dietary vitamin E. No similar information has previously been reported in the guinea pig, though these results agree with reports showing that vitamin E supplementation does not change superoxide dismutase, catalase or GSHperoxidase activity in the livers of mice (Sutphin and Buckman, 1991), and rats (Williams et al., 1992; Tiidus and Houston, 1994), nor GSHperoxidase activity and GSH content of chick liver (Kim and Combs, 1993). An absence of effect of dietary vitamin E on liver ascorbate concentration has also been described in the rat (Behrens and Madere, 1989). In a previous paper the authors showed that widely different
levels of dietary vitamin C supplementation caused no reactive depression of any enzymatic or non-enzymatic antioxidant in the guinea pig liver (Barja et al., 1994). In summary, these results show that high levels of the two main antioxidant vitamins, vitamins C and E, can be added to the diet of the guinea pig, (the only laboratory animal which, like man, is unable to synthesize these compounds), without compromising any alternative antioxidant defenses. These vitamins protect against lipid peroxidation at levels 6 times (vitamin E, this work) and 40 times (vitamin C, Barja et al., 1994) higher than the minimum daily requirements. Further supplementation above these levels continues to have no effect on endogenous antioxidants but is superfluous since no additional protection against lipid peroxidation is provided. The results of this work support the increasingly important hypothesis (Lachance and Langseth, 1994) that the optimum levels of some vitamins required for the maintenance of health are higher than those needed for prevention of deficiency syndromes, though megadose levels should be avoided. Acknowledgements-This work was supported by a grant (no 93/0145E) from the National Research Foundation of the Spanish Ministry of Health (FISss). Predoctoral fellowships were received by C. Rojas and S. Cadenas (F.P.I., Ministry of Education), and R. Perez-Campo (FISss).
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