@Copyright 1985 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163-4984785/0809-0113502.00
Serum Selenium, Glutathione Peroxidase, Lipids, and Human Liver Microsomal Enzyme Activity A Double-Blind Controlled Trial of Selenium Supplementation P. V~ LUOMA,* H. KORPELA, E. A. SOTANIEM1,AND J. KUMPULAINEN Clinical Research Unit, Department of h4edicine, and Department of Physiology, University of Oulu, 90220 Oulu, and Agricultural Research Center Laboratory, 31600 Jokioinen, Finland Received February 19, 1985; Accepted March 27, 1985
ABSTRACT This study evaluated selenium status in relation to lipid peroxidation, liver microsomal function, and serum lipids in humans. Serum selenium concentration, glutathione peroxidase (GSH-Px) activity, liver microsomal enzyme activity, assessed by plasma antipyrine clearance (AP-CL) rate, and serum lipids were determined in 23 healthy subjects in a double-blind placebo-controlled trial of selenium supplementation. The low selenium concentration (74.0 -+ 14.2 p~g/L, mean ___ SD) is attributable to the low selenium content of the diet. Subjects with the lowest selenium levels (n = 11) had reduced serum GSH-Px activity, AP-CL rate, high-density lipoprotein cholesterol (HDL-C), and total cholesterol (T-C) as compared with subjects with higher selenium concentrations (n = 12). Low AP-CL rates were associated with low HDL-C:T-C ratios. Selenium supplementation, 96 }xg/d for 2 wk, increased serum selenium, GSH-Px activity, and the HDL-C:T-C ratio. The results suggest that a low serum selenium *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
1]3
Vol. 8, 1985
1 14
L u o m a et al.
level is associated with a decrease in liver microsomal enzyme activity and serum HDL-C and T-C concentrations. Selenium supplementation in subjects with low serum selenium may favorably influence relations between serum lipoproteins connected with the development of atherosclerotic vascular disease. Index Entries: Selenium, selenium supplementation, glutathione peroxidase, liver microsomal enzymes, antipyrine clearance, HDL cholesterol, cholesterol
INTRODUCTION Selenium is an essential trace element for h u m a n nutrition. It is a constituent of glutathione peroxidase (GSH-Px), an enzyme that protects cellular lipoprotein structures against oxidative damage (1). Inadequate selenium intake reduces GSH-Px activity and promotes lipid peroxidation (2,3). Experimental studies have shown that selenium deficiency influences the microsomal function in the liver (4), which plays a central role in the metabolism of xenobiotics and endogenous substances such as lipids (5). This suggests that selenium status may be reflected not only in GSH-Px activity, but also in liver microsomal function and lipid metabolism. The aim of the present study was to test this hypothesis. Serum selen i u m concentration, GSH-Px activity, liver microsomal e n z y m e activity in vivo assessed by plasma antipyrine clearance (AP-CL) rate (6,7), serum high-density lipoprotein cholesterol (HDL-C), total cholesterol (T-C), and triglycerides (TG) were determined before and after a doubleblind placebo-controlled trial of selenium supplementation in a group of healthy subjects.
SUBJECTS AND METHODS
Subjects Twenty-five healthy medical students volunteered to take part in the study. They were in double-blind fashion randomized for selenium supplementation and placebo treatment. Twenty-three of t h e m - - t e n in the selenium and 13 in placebo group--completed the trial. They were followed for serum selenium, GSH-Px activity, AP-CL rate, and lipids. The age of the 23 subjects (eight males, 15 females) ranged from 21 to 34 yr. Three of them were smokers, and three females were using oral contraceptive drugs. The participants had no regular physical exercise program, and no recent history of alchohol intake.
Biological Trace Element Research
Vol. 8, 1985
Se, HDL, and Human Liver /Hicrosomal Activity
115
Protocol Informed consent was obtained from all subjects and the research was carried out according to the provisions of the Declaration of Helsinki. The first group received selenium yeast tablets (96 ~g of selenium/d; Selena| Huhtamaki Ltd, Turku, Finland) and the second (control) group received placebo yeast tablets (Huhtamaki Ltd) for 2 wk. Samples for selenium assay, GSH-Px activity, and lipid determinations were drawn, and the antipyrine test (8) performed, at the beginning and end of the 2-wk trial after an overnight fast.
Analytical Methods The serum selenium concentration was determined with electrothermal atomic absorption spectrometry using the Perk,in-Elmer 5000 atomic absorption spectrometer equipped with a HGA-500 graphite furnace. A detailed description of the method and its validity has been reported earlier (9). Serum GSH-Px activity was measured by the method of G(inzler et al. (10) with some modifications, as used earlier (11). Tertbutyl hydroperoxide was used instead of hydrogen peroxide because much lower blanks and hence higher sensitivity were obtained. Serum GSH-Px activity was determined instead of the erythrocyte enzyme activity, because the former more rapidly reflects serum selenium concentrations (12). Serum HDL-C was determined by the dextran sulfate-magnesium chloride precipitation method described by Finley et al. (13). Cholesterol in the HDL fraction and serum T-C were measured using an enzymatic method (Boehringer kit, Cat. No. 187313, Boehringer, Mannheim GmbH, Mannheim) and TG by a fully enzymatic method (Boehringer kit, Cat. No. 12039). Liver function tests: Serum albumin and total bilirubin and the activities of aspartate aminotransferase (ASAT = GOT) and alkaline phosphatase (AP) were assayed using standard autoanalyzer techniques (SMAC analyzer, Technicon Instruments Corp).
Assessment of Hepatic/~Iicrosomal Function In Vivo The antipyrine elimination rate from plasma (AP-CL) was used as an index of hepatic microsomal enzyme function in vivo (6,7,14). Antipyrine in plasma was determined by a gas-liquid chromatographic method (15) as described elsewhere (7).
Calculations The body mass index (BMI) was determined as weight (kg)/height
(m) 2. The plasma antipyrine half-life (T/2) was read from the linear part of the time-concentration curve on a semilog graph. The AP-CL rate was
Biological Trace Element Research
VoL 8, !985
I 16
Luoma et aL
calculated from the equation: CL = D/(AUC total), w h e r e D is the oral dose of antipyrine a n d A U C total is the area u n d e r the d r u g c o n c e n t r a t i o n - t i m e curve (16). A U C was obtained by the trapezoidal rule a n d the area to infinite time a d d e d by integration (Ct/k), w h e r e Ct is the last d r u g concentration value a n d k the elimination rate c o n s t a n t calculated from the equation k = 0.693/(t/2). The Wilcoxon rank test for u n p a i r e d data, S t u d e n t ' s t-test for paired data a n d linear regression analysis w e r e e m p l o y e d to assess the significance of the results.
RESULTS The 23 subjects were h e a l t h y medical s t u d e n t s (age 24 + 3 yr; BMI 22 + 2 kg/m2; m e a n --- SD). Their s e r u m s e l e n i u m at the b e g i n n i n g of the s t u d y (74.0 + 14.2 ~g/L) r a n g e d from 54 to 104 ~g/L. S e r u m GSH-Px activity was 399 + 54 U/L a n d the AP-CL rate 56.6 -+ 18.0 mL/min. S e r u m HDL-C (46 + 8 mg/dL), T-C (185 -+ 34 mg/dL), a n d TG (62 -+ 18 mg/dL) concentrations, a n d the HDL-C:T-C ratio (0.25 - 0.05) w e r e normal. The results of conventional liver function tests varied w i t h i n the n o r m a l range. For the evaluation of s e l e n i u m status in relation to o t h e r p a r a m e t e r s , the 23 subjects were arbitrarily classified into two g r o u p s according to p r e t h e r a p y s e l e n i u m concentrations (< 70 ~g/L, n = 11, G r o u p I vs -> 70 p,g/L, n = 12, G r o u p II; Table 1). Low s e r u m s e l e n i u m levels w e r e associated w i t h low GSH-Px activity, AP-CL rate, HDL-C, a n d T-C. S e r u m TG a n d the HDL-C : T-C ratio in G r o u p I did not diverge from those in G r o u p II. S e l e n i u m concentrations at the b e g i n n i n g of s t u d y correlated m o s t closely to GSH-Px activity (r = 0.565; p < 0.01). The a s s e s s m e n t of s e r u m lipids in relation to liver m i c r o s o m a l e n z y m e activity s h o w e d that the lowest AP-CL rates (n = 11) were associated with lower H D L - C : T - C ratios (0.23 + 0.05 vs 0.27 -+ 0.03; p < 0.01) than higher rates (n = 12). Ser u m HDL-C, T-C, a n d TG in the former AP-CL g r o u p (n = 11) did n o t deviate from values in the latter one (n = 12). S e l e n i u m s u p p l e m e n t a t i o n , but n o t placebo treatment, increased ser u m s e l e n i u m level, GSH-Px activity a n d the HDL-C:T-C ratio (Table 2). it did n o t affect the AP-CL rate or HDL-C, T-C, a n d TG concentrations.
DISCUSSION S e r u m s e l e n i u m concentration is related to selenium intake (17), a n d varies b e t w e e n individuals a n d p o p u l a t i o n s (18). The h e a l t h y subjects s t u d i e d here were on a typical Finnish diet, and the s e r u m s e l e n i u m concentrations reflect their low dietary selenium intake (19). The findings s h o w that low s e r u m s e l e n i u m concentrations in healthy subjects are associated with increased lipid peroxidation as assessed by GSH-Px activity, r e d u c e d liver microsomal e n z y m e activity, a n d low HDL-C a n d T-C Biological Trace Element Research
VoL 8, 1985
Se, HDL, a n d H u m a n Liver ~4icrosomal Activity
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Serum Selenium, Glutathione Peroxidase, Lipids, and Human Liver Microsomal Enzyme Activity A Double-Blind Controlled Trial of Selenium Supplementation P. V~ LUOMA,* H. KORPELA, E. A. SOTANIEM1,AND J. KUMPULAINEN Clinical Research Unit, Department of h4edicine, and Department of Physiology, University of Oulu, 90220 Oulu, and Agricultural Research Center Laboratory, 31600 Jokioinen, Finland Received February 19, 1985; Accepted March 27, 1985
ABSTRACT This study evaluated selenium status in relation to lipid peroxidation, liver microsomal function, and serum lipids in humans. Serum selenium concentration, glutathione peroxidase (GSH-Px) activity, liver microsomal enzyme activity, assessed by plasma antipyrine clearance (AP-CL) rate, and serum lipids were determined in 23 healthy subjects in a double-blind placebo-controlled trial of selenium supplementation. The low selenium concentration (74.0 -+ 14.2 p~g/L, mean ___ SD) is attributable to the low selenium content of the diet. Subjects with the lowest selenium levels (n = 11) had reduced serum GSH-Px activity, AP-CL rate, high-density lipoprotein cholesterol (HDL-C), and total cholesterol (T-C) as compared with subjects with higher selenium concentrations (n = 12). Low AP-CL rates were associated with low HDL-C:T-C ratios. Selenium supplementation, 96 }xg/d for 2 wk, increased serum selenium, GSH-Px activity, and the HDL-C:T-C ratio. The results suggest that a low serum selenium *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
1]3
Vol. 8, 1985
1 14
L u o m a et al.
level is associated with a decrease in liver microsomal enzyme activity and serum HDL-C and T-C concentrations. Selenium supplementation in subjects with low serum selenium may favorably influence relations between serum lipoproteins connected with the development of atherosclerotic vascular disease. Index Entries: Selenium, selenium supplementation, glutathione peroxidase, liver microsomal enzymes, antipyrine clearance, HDL cholesterol, cholesterol
INTRODUCTION Selenium is an essential trace element for h u m a n nutrition. It is a constituent of glutathione peroxidase (GSH-Px), an enzyme that protects cellular lipoprotein structures against oxidative damage (1). Inadequate selenium intake reduces GSH-Px activity and promotes lipid peroxidation (2,3). Experimental studies have shown that selenium deficiency influences the microsomal function in the liver (4), which plays a central role in the metabolism of xenobiotics and endogenous substances such as lipids (5). This suggests that selenium status may be reflected not only in GSH-Px activity, but also in liver microsomal function and lipid metabolism. The aim of the present study was to test this hypothesis. Serum selen i u m concentration, GSH-Px activity, liver microsomal e n z y m e activity in vivo assessed by plasma antipyrine clearance (AP-CL) rate (6,7), serum high-density lipoprotein cholesterol (HDL-C), total cholesterol (T-C), and triglycerides (TG) were determined before and after a doubleblind placebo-controlled trial of selenium supplementation in a group of healthy subjects.
SUBJECTS AND METHODS
Subjects Twenty-five healthy medical students volunteered to take part in the study. They were in double-blind fashion randomized for selenium supplementation and placebo treatment. Twenty-three of t h e m - - t e n in the selenium and 13 in placebo group--completed the trial. They were followed for serum selenium, GSH-Px activity, AP-CL rate, and lipids. The age of the 23 subjects (eight males, 15 females) ranged from 21 to 34 yr. Three of them were smokers, and three females were using oral contraceptive drugs. The participants had no regular physical exercise program, and no recent history of alchohol intake.
Biological Trace Element Research
Vol. 8, 1985
Se, HDL, and Human Liver /Hicrosomal Activity
115
Protocol Informed consent was obtained from all subjects and the research was carried out according to the provisions of the Declaration of Helsinki. The first group received selenium yeast tablets (96 ~g of selenium/d; Selena| Huhtamaki Ltd, Turku, Finland) and the second (control) group received placebo yeast tablets (Huhtamaki Ltd) for 2 wk. Samples for selenium assay, GSH-Px activity, and lipid determinations were drawn, and the antipyrine test (8) performed, at the beginning and end of the 2-wk trial after an overnight fast.
Analytical Methods The serum selenium concentration was determined with electrothermal atomic absorption spectrometry using the Perk,in-Elmer 5000 atomic absorption spectrometer equipped with a HGA-500 graphite furnace. A detailed description of the method and its validity has been reported earlier (9). Serum GSH-Px activity was measured by the method of G(inzler et al. (10) with some modifications, as used earlier (11). Tertbutyl hydroperoxide was used instead of hydrogen peroxide because much lower blanks and hence higher sensitivity were obtained. Serum GSH-Px activity was determined instead of the erythrocyte enzyme activity, because the former more rapidly reflects serum selenium concentrations (12). Serum HDL-C was determined by the dextran sulfate-magnesium chloride precipitation method described by Finley et al. (13). Cholesterol in the HDL fraction and serum T-C were measured using an enzymatic method (Boehringer kit, Cat. No. 187313, Boehringer, Mannheim GmbH, Mannheim) and TG by a fully enzymatic method (Boehringer kit, Cat. No. 12039). Liver function tests: Serum albumin and total bilirubin and the activities of aspartate aminotransferase (ASAT = GOT) and alkaline phosphatase (AP) were assayed using standard autoanalyzer techniques (SMAC analyzer, Technicon Instruments Corp).
Assessment of Hepatic/~Iicrosomal Function In Vivo The antipyrine elimination rate from plasma (AP-CL) was used as an index of hepatic microsomal enzyme function in vivo (6,7,14). Antipyrine in plasma was determined by a gas-liquid chromatographic method (15) as described elsewhere (7).
Calculations The body mass index (BMI) was determined as weight (kg)/height
(m) 2. The plasma antipyrine half-life (T/2) was read from the linear part of the time-concentration curve on a semilog graph. The AP-CL rate was
Biological Trace Element Research
VoL 8, !985
I 16
Luoma et aL
calculated from the equation: CL = D/(AUC total), w h e r e D is the oral dose of antipyrine a n d A U C total is the area u n d e r the d r u g c o n c e n t r a t i o n - t i m e curve (16). A U C was obtained by the trapezoidal rule a n d the area to infinite time a d d e d by integration (Ct/k), w h e r e Ct is the last d r u g concentration value a n d k the elimination rate c o n s t a n t calculated from the equation k = 0.693/(t/2). The Wilcoxon rank test for u n p a i r e d data, S t u d e n t ' s t-test for paired data a n d linear regression analysis w e r e e m p l o y e d to assess the significance of the results.
RESULTS The 23 subjects were h e a l t h y medical s t u d e n t s (age 24 + 3 yr; BMI 22 + 2 kg/m2; m e a n --- SD). Their s e r u m s e l e n i u m at the b e g i n n i n g of the s t u d y (74.0 + 14.2 ~g/L) r a n g e d from 54 to 104 ~g/L. S e r u m GSH-Px activity was 399 + 54 U/L a n d the AP-CL rate 56.6 -+ 18.0 mL/min. S e r u m HDL-C (46 + 8 mg/dL), T-C (185 -+ 34 mg/dL), a n d TG (62 -+ 18 mg/dL) concentrations, a n d the HDL-C:T-C ratio (0.25 - 0.05) w e r e normal. The results of conventional liver function tests varied w i t h i n the n o r m a l range. For the evaluation of s e l e n i u m status in relation to o t h e r p a r a m e t e r s , the 23 subjects were arbitrarily classified into two g r o u p s according to p r e t h e r a p y s e l e n i u m concentrations (< 70 ~g/L, n = 11, G r o u p I vs -> 70 p,g/L, n = 12, G r o u p II; Table 1). Low s e r u m s e l e n i u m levels w e r e associated w i t h low GSH-Px activity, AP-CL rate, HDL-C, a n d T-C. S e r u m TG a n d the HDL-C : T-C ratio in G r o u p I did not diverge from those in G r o u p II. S e l e n i u m concentrations at the b e g i n n i n g of s t u d y correlated m o s t closely to GSH-Px activity (r = 0.565; p < 0.01). The a s s e s s m e n t of s e r u m lipids in relation to liver m i c r o s o m a l e n z y m e activity s h o w e d that the lowest AP-CL rates (n = 11) were associated with lower H D L - C : T - C ratios (0.23 + 0.05 vs 0.27 -+ 0.03; p < 0.01) than higher rates (n = 12). Ser u m HDL-C, T-C, a n d TG in the former AP-CL g r o u p (n = 11) did n o t deviate from values in the latter one (n = 12). S e l e n i u m s u p p l e m e n t a t i o n , but n o t placebo treatment, increased ser u m s e l e n i u m level, GSH-Px activity a n d the HDL-C:T-C ratio (Table 2). it did n o t affect the AP-CL rate or HDL-C, T-C, a n d TG concentrations.
DISCUSSION S e r u m s e l e n i u m concentration is related to selenium intake (17), a n d varies b e t w e e n individuals a n d p o p u l a t i o n s (18). The h e a l t h y subjects s t u d i e d here were on a typical Finnish diet, and the s e r u m s e l e n i u m concentrations reflect their low dietary selenium intake (19). The findings s h o w that low s e r u m s e l e n i u m concentrations in healthy subjects are associated with increased lipid peroxidation as assessed by GSH-Px activity, r e d u c e d liver microsomal e n z y m e activity, a n d low HDL-C a n d T-C Biological Trace Element Research
VoL 8, 1985
Se, HDL, a n d H u m a n Liver ~4icrosomal Activity
u
~] 7
I
~
("40
f'.lO
c~c~
c~c~
D -G o C"IC~ ',D~"~
t~lP', ',DO'I
.5 9 r~
