The influence of dietary selenium and vitamin E on glutathione peroxidase and glutathione in the rat

218

Biochimica et Biophysica Acta, 497 (1977) 218--224

© Elsevier/North-Holland Biomedical Press

BBA 28185 THE INFLUENCE OF DIETARY SELENIUM AND VITAMIN E ON GLUTATHIONE PEROXIDASE AND GLUTATHIONE IN THE RAT

DAVID L. SCOTT, JERRY KELLEHER and MONTY S. LOSOWSKY University Department o f Medicine, St. James's Hospital, Leeds LS9 7TF

(Received August 30th, 1976)

Summary The effect of dietary selenium (Se) and vitamin E supplementation on tissue reduced glutathione (GSH) and glutathione peroxidase activity has been studied in the rat. Increasing Se intake by 0.4 ppm gave significantly higher enzyme levels in all tissues studied, an effect not influenced by vitamin E intake. Further increasing Se to 4 ppm gave higher enzyme levels in red blood cells only, while in liver there was a significant decrease in enzyme activity probably reflecting Se hepatotoxicity. In the absence of Se supplements increasing dietary vitamin E to 100 mg/kg diet significantly increased enzyme activity but this effect was modified by simultaneous Se supplementation. Se intake had no effect on GSH levels. Rats on a high vitamin E intake 500 mg/kg had a significantly higher tissue GSH level. Dietary Se had a sparing effect on vitamin E, rats supplemented with Se having significantly raised plasma vitamin E levels. These results confirm the role of selenium in glutathione peroxidase and also show that vitamin E influences the activity of the enzyme.

Introduction There is a close metabolic relationship between dietary selenium and vitamin E (a-tocopherol), but the nature of this is not completely understood. In 1973 Rotruck et al. [1] found selenium as an essential cofactor in the enzyme glutathione peroxidase (EC 1.11.1.9), which catalyses the detoxification of peroxides by oxidation of reduced glutathione. This was the first characterisation of an important metabolic role for selenium. Later work has established the dependence o f tissue glutathione peroxidase activity on the dietary selenium intake in many animals, including the rat [2,3]. Vitamin E may also influence glutathione peroxidase activity [4,5] though the date so far is somewhat contradictory. The studies up to now have investigated vitamin E and

219 selenium separately and have not studied their possible interrelation in influencing glutathione peroxidase activity. The present study investigates the interrelationship between selenium, vitamin E and glutathione peroxidase. Glutathione peroxidase activity has been studied in various tissues in relation to separate and combined changes in the dietary intake o f selenium and vitamin E. In addition the effect of such dietary changes on tissue reduced glutathione (GSH) levels and plasma vitamin E levels have been studied. Methods 67 female Wistar rats were randomly divided into nine groups with 6--8 animals in each group, and were given synthetic diets from weaning. A basal diet was used, which contained the recommended daily requirements of all nutrients and minerals except vitamin E and selenium [6]. This diet was supplemented with vatimin E or selenium or both, selenium being added as sodium selenite at 0.4 and 4.0 ppm while DL<~-tocopherol acetate was added at 100 and 500 mg per kg. After 3 months on the diets the rats were killed b y cardiac puncture under anaesthesia and livers and kidneys removed. Portions of these tissues were homogenised in a glass homogeniser with teflon pestle, in 20--40 volumes of 0.9% saline in 0.02 M EDTA. Aliquots o f these homogenates were used for the estimation of reduced glutathione. The remainder o f the homogenates were centrifuged at 750 X g for 15 min, the supernatant then being centrifuged at 100 000 X g for 60 min and the soluble fraction used for enzyme assay. The blood was centrifuged and the plasma fraction retained. The red cells were washed t h r e e times in an equal volume of 0.9% saline in 0.02 M EDTA. The specimens were kept in an ice bath throughout and all the centrifugations were performed at 4 ° C. Glutathione peroxidase activity was assayed b y the method of Paglia and Valentine [ 7 ] on the soluble tissue fractions and the washed red cells. Enzyme activity is expressed as nmol of NADPH oxidised per min per mg protein (liver and kidney) or per 101° red blood cells. Reduced glutathione (GSH) was assayed on the day of preparation of specimens b y the method o f Sedlak and Lindsay [8] on whole tissue homogenates and washed red cell suspensions. The protein content o f these fractions was assayed by the Lowry et al. method [9]. The red cells were counted on a Coulter counter. Vitamin E and aspartate aminotransferase (EC 2.6.1.1) were determined on the stored plasma b y the methods of Hansen and Warwick [10] and Henry et al. [11], respectively. Results

Table I shows the effects of dietary supplementation with selenium and vitamin E on tissue glutathione peroxidase activity. Red cell glutathione peroxidase activity showed a significant increase (P 0.001) when dietary selenium intake was increased by 0.4 ppm. This occurred in the absence of vitamin E supplements and also when vitamin E was added at

220 TABLE

I

THE EFFECT OF IDASE ACTIVITY

DIETARY

VITAMIN

E AND

SELENIUM

ON TISSUE

GLUTATHIONE

PEROX-

V i t a m i n E w a s added at 1 0 0 and 5 0 0 m g / k g o f the basal diet, and s e l e n i u m at 0 . 4 and 4 . 0 p p m . R e s u l t s are e x p r e s s e d as e n z y m e units +- S . E . N u m b e r s o f rats in each group axe given in p a r e n t h e s e s . Dietary supplement

Glutathione peroxidase activity

Vitamin E (mg/kg)

Selenium (ppm)

R e d cells units/1010 cells X 10 -3

Liver units/mg protein X 10-3

Kidney units/mg protein X 10-3

None None None 100 100 100 500 500 500

None 0.4 4.0 None 0.4 4.0 None 0.4 4.0

2.67 5.18 7.63 4.18 5.47 7.56 3.77 5.76 7.19

2.01 3.87 4.74 3.29 4.65 3.96 3.10 4.48 3.10

0.253 0.442 0.511 0.400 0.507 0.400 0.428 0.519 0.500

-+ 0 . 1 5 ± 0.21 ± 0.30 -+ 0 . 1 9 -+ 0 . 2 1 ± 0.28 -+ 0 . 2 3 + 0.33 ± 0.37

+ 0.13 ± 0.28 ± 0.20 ± 0.23 -+ 0 . 3 2 ± 0.20 +- 0 . 1 6 ± 0.24 ± 0.39

± 0.021 ± 0.024 -+ 0 . 0 4 0 ± 0.024 ± 0.041 +- 0 . 0 4 2 ± 0.043 ± 0.052 ± 0.056

(8) (8) (8) (8) (S) (6) (7) (7) (7)

100 or 500 mg/kg. Further supplementation with 4.0 ppm selenium led to a further significant increase in enzyme activity (P < 0.001), and this too occurred at each level of vitamin E intake. Kidney glutathione peroxidase activity was significantly increased by addition of 0.4 ppm Selenium to the diet in the absence of dietary vitamin E supplementation (P < 0.001) and when 100 mg vitamin E/kg was added to the diet (P < 0.05). When 500 mg vitamin E/kg was added there was a further increase in enzyme activity, which did not reach significance (P > 0.2). Further dietary supplementation with 4.0 ppm selenium led to no significant change in glutathione peroxidase activity at each level of vitamin E intake. Liver glutathione peroxidase activity increased significantly ( P < 0.005) when selenium was added to the diet at 0.4 ppm at each level o f vitamin E intake. Liver glutathione peroxidase activity showed a significant fall (P < 0.05) when dietary selenium supplementation was increased from 0.4 to 4.0 ppm in rats on the highest vitamin E intake. Rats on diets without additional vitamin E showed, in comparison, no significant change in enzyme activity when the dietary selenium content was similarly increased. Plasma aspartate aminotransferase activity, an index of hepatocellular damage, is related to dietary selenium intake, as shown in Table II. Rats on TABLE II THE EFFECT OF DIETARY TRANSFERASE LEVELS

SELENIUM

SUPPLEMENTATION

ON PLASMA

G r o u p s o f 2 2 - - 2 4 rats w e r e used. V a l u e s are given per group ± S . E . Selenium added (ppm)

Plasma aspartate a m i n o t r a n s f e r a s e ( u n i t s / m l )

0 0.4 4.0

90.0 + 12.0 8 6 . 5 -+ 1 3 . 5 198.0 + 43.0

ASPARTATE

AMINO-

221 diets supplemented with 4.0 ppm selenium have significantly higher aspartate aminotransferase activity than those on lower dietary selenium intakes (P < O.02). Glutathione peroxidase activity was also affected by the dietary vitamin E intake. Red cell glutathione peroxidase activity significantly increased ( P < 0.001) when the basal diet, in the absence of selenium supplementation was supplemented with 100 mg/kg vitamin E. Increasing the dietary vitamin E to 500 mg/kg caused no further significant change in enzyme activity. When selenium was added to the diet at either 0.4 or 4.0 ppm increasing the dietary vitamin E intake had no significant effect on enzyme activity. Both liver and kidney glutathione peroxidase activities were similarly affected by dietary vitamin E intake; there were significant increases in enzyme activities ( P < 0.001) when the basal diet was supplemented with 100 mg/kg vitamin E, and this effect was not seen in the presence of selenium at either level o f supplementation. Table III summarises the changes in tissue reduced glutathione levels on the different diets. Vitamin E intake affected liver and kidney GSH levels. In both tissues GSH levels were significantly higher (P ~ 0.05) with vitamin E supplements of 500 mg/kg diet than in those without additional vitamin E. Vitamin E at 100 mg/kg diet had no effect on GSH levels compared to the basal diet. Erythrocyte GSH concentration was not influenced by vitamin E intake. Changes in the dietary selenium intake had no effect on tissue GSH levels. The effects o f dietary vitamin E and selenium intakes on plasma vitamin E levels are shown in Table IV. Plasma vitamin E levels were low on the basal diet, adding 100 mg/kg vitamin E led to a significant increase in plasma vitamin E levels ( P < 0.001), and this was seen at each level of selenium intake. A further significant increase (P ~ 0.001) in plasma vitamin E occurred when the diet was supplemented with 500 mg/kg vitamin ,E. This effect, too was seen at each level o f selenium intake. Dietary selenium intake also affected the plasma vitamin E level. Rats on the basal diet, without added vitamin E and selenium have significantly lower plasma vitamin E levels (P ~ 0.001) than animals on the same diet supplemented with 4 ppm selenium. Dietary selenium supplementation at 4 ppm also significantly increased (P < 0.002) plasma vitamin E levels when the diet contained 100 mg/kg of vitamin E. The rise in plasma vitamin E level with increase in selenium intake was not significant when the diet was supplemented with 500 mg/kg vitamin E. TABLE

III

THE EFFECT Results shown

OF VITAMIN E INTAKE ON NON-PROTEIN THIOL GROUP CONCENTRATIONS -+ S . E . Figures in p a r e n t h e s e s r e p r e s e n t t h e n u m b e r o f rats in e a c h g r o u p .

Vitamin E a d d e d to diet (mg/kg diet)

Plasma vitamin E

Reduced glutathione (mM/g protein liver)

Reduced glutathione (mM/g protein kidney)

0 100 500

140 903 1398

4 9 . 2 -+ 1 . 8 4 7 . 4 -+ 1 . 9 54.6 ± 1.9

37.0 + 1.4 (24) 39.9 + 1.9 (22) 42.1 ± 2.1 (21)

222 TABLE IV THE EFFECTS OF DIETARY SELENIUM AND VITAMIN E ON PLASMA VITAMIN E LEVELS ' T h e m e a n p l a s m a v i t a m i n E level ( # g / 1 0 0 m l ) f o r e a c h g r o u p o f r a t s is g i v e n -+ S.E. T h e n u m b e r s o f r a t s in e a c h g r o u p axe given in parentheses. Vitamin E s u p p l e m e n t to basal diet (mg/kg)

Serum vitamin E s e l e n i u m s u p p l e m e n t s to b a s a l d i e t in p p m None

0.4

4.0

None 100 500

1 0 9 + 15 (8) 6 6 8 + 94 (8) 1 3 3 1 -+ 1 1 6 (S)

1 1 9 -+ 16 (S) 8 2 4 + - 53 ( 8 ) 1 4 0 3 -+ 91 (6)

192-+ 22 (7) 9 9 9 -+ 71 (7) 1 4 6 1 + 1 5 6 (7)

Discussion

Dietary vitamin E in rats influences glutathione peroxidase activity and this effect is related to dietary selenium intake. This can be contrasted with the stimulatory effect of added dietary selenium which is independent of dietary vitamin E intake. The response to vitamin E was similiar in all tissues studied and was maximal when 100 mg/kg was added to the vitamin E-deficient diet. Further increasing the vitamin E intake to 500 mg/kg diet, tended to decrease glutathione peroxidase activity but not significantly so. This agrees with the recent report o f Yang et al. [5], who further showed that at extremely high vitamin E intakes glutathione peroxidase activity was strikingly decreased. The changes in glutathione peroxidase activity produced by vitamin E are abolished by supplementing the basal diet with 0.4 and 4 ppm selenium. This suggests that vitamin E may only exert its effect at low selenium intakes. Chow et al. [4] have also studied the effect of vitamin E on glutathione peroxidase activity. In contrast to the present study and the previous report of Yang et al. [5], these authors failed to show any effect o f vitamin E on glutathione peroxidase activity, in livers or kidneys, but in muscle, and adipose tissue they showed a significant depression of enzyme activity. These results of Chow et al. [4] for liver and kidney may be related either to the selenium content o f their diet and also to the fact that the diet fed to their rats contained a high percentage of unsaturated fat (corn oil or cod liver oil) as compared to a lower percentage of a more saturated fat (lard) used in the present study and in that o f Yang et al. [5]. Thus a complex interrelation may exist between selenium, vitamin E and the type and quantity of dietary fat. Selenium affects enzyme activity similarly at all intakes of vitamin E. There are differences in the response to selenium of the three tissues studied. Red cell glutathione peroxidase activity increases continuously with increasing selenium intakes, even when this reaches a potentially toxic level, while the activity of glutathione peroxidase in liver and kidney does not increase when the selenium supplement is increased to 4 ppm. These differences in responsiveness of different tissues to dietary selenium have previously been noted by Hoekstra [12], the reasons are still not known. Selenium is an integral component of glutathione peroxidase [1] and the relationship between the intake of selenium and enzyme activity can readily be

223 explained. This does not apply to the relationship between vitamin E and glutathione peroxidase activity and shows further the complex relationship which exists between selenium and vitamin E. Some possible mechanisms to explain this relationship have recently been proposed by Yang et al. [5], including substrate induction, reduced oxidative stress, regulation of protein synthesis, and liver necrosis. However, the observation in the present study that dietary selenium supplementation abolishes the effect of vitamin E might suggest that vitamin E influences the bioavailability of selenium which is in agreement with the report of Caygill et al. [13], that subcellular uptake of dietary selenium in the liver was related to vitamin E intake. Such a function would be in keeping with both our findings, and the hypothesis of Diplock and Lucy [14] who have suggested a membrane-stabilising function of vitamin E and suggested that this, by preventing oxidation of selenide-containing proteins in subcellular membranes, determines the selenium content of the membrane. The observation that selenium at 4 ppm causes increased levels of plasma aspartate aminotransferase suggests that this dietary level of selenium produces liver damage. The consequent fall in liver glutathione peroxidase in some of the groups on high selenium may be a further indication of liver cell damage, though this remains to be proved. The fact that the fall in liver glutathione peroxidase at high selenium intakes was seen in those groups of rats on the high vitamin E intake may imply that the hepatotoxic effect of selenium is potentiated at high vitamin E intakes. The dietary intake of vitamin E is the major factor influencing plasma vitamin E levels. However, the effect of dietary selenium supplements on plasma vitamin E levels is marked, unless the plasma vitamin E level is very high. Desai and Scott [15] have reported similar findings. The mechanism for the increase in plasma vitamin E level with increasing dietary selenium are unclear but may be related to a requirement for selenium in the specific lipoprotein which transport vitamin E. These findings suggest that there are ways in which selenium and vitamin E inter-relate other than those proposed previously. The relationship between dietary vitamin E intake and liver and kidney reduced glutathione levels may represent another interaction between vitamin E and selenium. Glutathione peroxidase uses reduced glutathione as a specific substrate [16,12]. This suggests that vitamin E can act to influence the activity of the biological enzyme system of which glutathione peroxidase is a part by means additional to an effect on the activity of the enzyme. Furthermore, experiments with paracetamol hepatotoxicity [17,18] have shown that vitamin E and thiol group agents both protect against liver damage. The demonstration that reduced glutathione levels are raised by increased vitamin E intake may help to explain why such different compounds offer protection against this hepatotoxin. References 1 R o t r u c k , J . T . , P o p e , A.L.0 G a n t h e r , H . E . , H a f e m a n , D . G . , S w a n s o n , A.B. and H o e k s t r a , W . G . ( 1 9 7 3 ) Science 179, 588--590 2 H a f e m a n , D . G . , S u n d e , R . A . and H o e k s t r a , W . G . ( 1 9 7 4 ) J. N u t r . 1 0 4 , 5 8 0 - - 5 8 7 3 C h o w , C . K . and Tappel, A . L . ( 1 9 7 4 ) J. N u t r . 1 0 4 , 4 4 4 - - 4 5 1

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