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Combined effect of selenium and ascorbic acid on alcohol induced hyperlipidemia in male guinea pigs
Comparative Biochemistry and Physiology Part C 137 (2004) 109–114
Combined effect of selenium and ascorbic acid on alcohol induced hyperlipidemia in male guinea pigs G.S. Asha, M. Indira* Department of Biochemistry, University of Kerala, Kariavattom, Thiruvananthapuram 695 581, Kerala, India Received 23 September 2003; received in revised form 5 December 2003; accepted 8 December 2003
Abstract Alcoholics usually suffer from malnutrition and are especially deficient in micronutrients like vitamin C, selenium and Zn. In the present study, combined effects of selenium and ascorbic acid on alcohol-induced hyperlipidemia were studied in guinea pigs. Four groups of male guinea pigs were maintained for 45 days as follows: control (1 mg ascorbate (AA)y 100 g body massyday), ethanol (900 mg ethanoly100 g body massq1 mg AAy100 g body massyday), seleniumq ascorbic acid w(25 mg AAq0.05 mg Se)y100 g body massydayx, ethanolqseleniumqascorbic acid w(25 mg AAq0.05 mg Seq900 mg ethanol)y100 g body massydayx. Co-administration of selenium and ascorbic acid along with alcohol reduced the concentration of all lipids, as also evidenced from the decreased activities of hydroxymethylglutaryl-CoA reductase and enhanced activities of plasma lecithin cholesterol acyl transferase and lipoprotein lipase. Concentrations of bile acids were increased. We conclude that the supplementation of Se and ascorbic acid reduced alcohol induced hyperlipidemia, by decreased synthesis and increased catabolism. 䊚 2004 Elsevier Inc. All rights reserved. Keywords: Ethanol; Selenium; Ascorbic acid; Hydroxymethylglutaryl-CoA (HMG-CoA); Lecithin cholesterol acyl transferase (LCAT); Lipoprotein lipase (LPL); Lipids; Bile acids
1. Introduction Many toxic effects of alcohol appear to be mediated through imbalances in micronutrients leading to secondary changes in biological and molecular functions of the cell. Levels of many nutrients such as vitamin C and selenium are lower in alcoholics (Rimm and Colditz, 1993). Vitamin C is a hypolipidemic agent. Results of the several studies have suggested that it can lower blood cholesterol in human subjects (Kothari and Jain, 1977). A positive correlation between vitamin *Corresponding author. Tel.: q91-2361344; fax: q91-471307158. E-mail address: [email protected] (M. Indira).
C and plasma HDL cholesterol has been proposed by Bates et al. (1977). Earlier studies showed that an exogenous supply of ascorbic acid reduced hyperlipidemia in most tissues (Suresh et al., 1997). Selenium is a micronutrient of fundamental importance to human health (Mahadev Rao, 2001). Hypercholesterolemia and cardiovascular disorders have been shown to be associated with Se deficiency (Kang et al., 2000). Se has a crucial role in controlling the effects of thyroid hormone on fat metabolism. It has been reported that deiodinase (DI), the enzyme responsible for converting T4 to T3, contains the rare aminoacid selenocysteine that is formed in the body using dietary Se (Behne et al., 1990). Kang et al. (2000) studied the effect of
1532-0456/04/$ - see front matter 䊚 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2003.12.002
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G.S. Asha, M. Indira / Comparative Biochemistry and Physiology Part C 137 (2004) 109–114
feeding a high fat diet and a Se supplemented high fat diet for 3 months on type I-59 monodeiodinase activity in rats. They found a significant increase in serum cholesterol and triglyceride levels in the high fat diet fed group as compared to the control when compared to the Se supplemented group. This was correlated with levels of 59-DI. A low serum Se level is associated with a decrease of liver microsomal activity and serum HDL-C concentration (Masukawa et al., 1983). Our earlier studies have shown that co-administration of ascorbic acid and Se is beneficial in reducing alcohol induced oxidative stress in guinea pigs (Asha et al., 2003). Therefore, in the present study, we investigated the supplementation of selenium and ascorbic acid on alcohol induced hyperlipidemia in guinea pigs. 2. Materials and methods Male guinea pigs (Cavia porcellus; Veterinary College, Mannuthy Strain) bred and reared in our animal house were used for the experiment. Animals were housed individually in wire netted cages at room temperature maintained at 25"1 8C with a lightydark cycle of 12 h. Weight matched guinea pigs were selected. A total of 24 guinea pigs were divided into four groups of six each: Controls 1 mg ascorbate (AA)y100 g body massyday; II Ethanol (900 mg ethanolq1 mg AA)y 100 g body massyday); III SeleniumqAA (25 mg AAq0.05 mg Naselenite)y100 g body massyday); and IV EthanolqseleniumqAA (25 mg AAq 0.05 mg Na-seleniteq900 mg ethanol)y100 g body massyday). I
Guinea pigs were fed with guinea pig feed (Lipton India Ltd.). Food and water were given ad libitum, and ascorbic acid, selenium and ethanol were administered as detailed above. Ascorbic acid and selenium freshly dissolved in distilled water and ethanol diluted in the ratio (1:1) were given orally by gastric tube for 45 days. Control and seleniumqascorbic acid groups were administered glucose solution equivalent to the caloric value of ethanol in group II and group IV. On the 46th day, the animals were killed and tissues were removed to ice cold containers for various estimations. The tissues were extracted according to the procedure
of Folch et al. (1957). Cholesterol, triglycerides, phospholipids and free fatty acids (FFA) were estimated in the tissues as described by Menon and Kurup (1976). Separation of serum HDL and LDLqVLDL was carried out according to the procedure of Warnick and Alberts (1978). The activity of hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase; EC 1.1.1.34) was assayed as described by Rao and Ramakrishnan (1975). Extraction of liver bile acids was carried out according to the procedure of Okishio et al. (1967) and quantified by the procedure described by Rober (1996). Activity of lipoprotein lipase (LPL; EC 3.1.1.34) was determined by the method of Liobera et al. (1979). The activity of plasma lecithin cholesterol acyl transferase (LCAT) was determined according to the procedure of Schoenheimer and Sperry (1934). Protein in the enzyme extract was determined after trichloroacetic acid precipitation by the method of Lowry et al. (1951). Data were statistically analyzed by One-way analysis of variance (ANOVA). All values were expressed as means"S.D. P values of 0.05 or less were considered significant. All analysis was performed by computer using the statistical package SPSS. 3. Results Concentration of cholesterol (Table 1) was significantly increased in liver, heart, kidney and brain of alcohol administered guinea pigs as compared to controls. Co-administration of Se and ascorbic acid along with alcohol decreased the concentration of cholesterol significantly in all the tissues when compared to the ethanol group. The concentration of phospholipids (Table 2) was significantly increased in the ethanol administered group when compared to controls. Supplementation of Se and ascorbic acid significantly decreased the concentration of phospholipids in all the tissues. Co-administration of Se and ascorbic acid along with alcohol decreased the levels of phospholipids significantly in all the tissues. Concentration of FFA (Table 3) was significantly increased in ethanol fed guinea pigs. FFA concentration was significantly decreased in all the tissues of the Se and ascorbic acid plus alcohol group, when compared to the ethanol group. Alcohol fed guinea pigs showed significant increase in the concentration of triglycerides (Table 4) in all the tissues as compared to controls. Se
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Table 1 Concentration of cholesterol in male guinea pigs Groups
Liver
Heart
Kidney
Brain
Control Ethanol Seqascorbic acid EthanolqSeq ascorbic acid
343.3"13.9 593.1"24.1a 341.0"13.9d 367.1"15.0c,e
200.0"8.1 273.1"11.1a 197.5"8.2d 215.6"8.5c,e
347.1"14.5 503.3"20.3a 324.1"13.0b,d 419.2"17.1c,e
866.7"35.1 1050.6"42.5a 857.2"34.7d 902.5"36.5e
Values are given in mgy100 g tissue, as mean"S.D. P-0.05 between control and ethanol groups. b P-0.05 between control and selenium groups. c P-0.05 between control and seleniumqethanol groups. d P-0.05 between ethanol and selenium groups. e P-0.05 between ethanol and seleniumqethanol groups. a
Table 2 Concentration of phospholipids in male guinea pigs Groups
Liver
Heart
Kidney
Brain
Control Ethanol Seqascorbic acid EthanolqSeq ascorbic acid
2211.9"89.8 6754.6"259.6a 2209.9"80.0d 2688.5"92.9ce
2302.1"93.6 2806.7"113.4a 2283.3"92.3d 2548.6"103.2ce
2426.7"98.1 2987.1"121.1a 2013.34"81.88bd 2777.6"112.5ce
1895.9"77.0 2189.5"88.7a 1569.39"62.5bd 2004.1"80.9ce
For explanation of superscripts see Table 1. Values are given in mgy100 g tissue, as mean"S.D. Table 3 Concentration of free fatty acids in male guinea pigs Groups
Liver
Heart
Kidney
Brain
Control Ethanol Seqascorbic acid EthanolqSeq ascorbic acid
260.5"106 308.1"12.2a 252.9"10.4d 278.7"11.2ce
144.1"5.8 187.1"7.8a 124.8"5.2bd 156.3"6.4ce
173.1"7.0 198.6"7.9a 144.6"5.9bd 176.3"7.0e
130.5"5.2 163.1"6.6 126.6"4.6d 150.6"6.1ce
For explanation of superscripts see Table 1. Values are given in mgy100 g tissue, as mean"S.D. Table 4 Concentration of triglycerides in male guinea pigs Groups
Liver
Heart
Kidney
Brain
Control Ethanol Seqascorbic acid EthanolqSeq ascorbic acid
351.0"14.2 491.1"19.9a 299.5"12.2bd 370.8"14.9ce
51.5"2.1 72.3"2.7a 49.9"2.0d 59.1"2.5ce
96.5"4.1 152.4"6.1a 89.5"3.6bd 101.1"4.1e
76.4"3.3 113.3"4.6a 69.7"2.9bd 78.5"3.3e
For explanation of superscripts see Table 1. Values are given in mgy100 g tissue, as mean"S.D.
and ascorbic acid supplementation significantly reduced triglycerides in all the tissues when compared to the control. Co-administration of Se and ascorbic acid along with alcohol significantly
decreased the concentration of triglycerides in all the tissues compared to the ethanol group. The concentration of HDL-C (Table 5) was significantly decreased in alcohol fed guinea pigs
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Table 5 Concentration of serum lipoproteins in male guinea pigs Groups
HDL-C
LDLqVLDL-C
Control Ethanol Seqascorbic acid EthanolqSeq ascorbic acid
50.19"2.06 47.24"1.84a 51.23"2.09d 53.46"1.93ce
16.52"0.71 24.69"1.01a 10.26"0.42bd 18.27"0.69e
For explanation of superscripts see Table 1. Values are given in mgy100 ml serum, as mean"S.D.
when compared to controls. Co-administration of Se and ascorbic acid plus alcohol significantly increased HDL-C levels compared to the ethanol group. Concentration of LDLqVLDL was significantly increased in the alcohol fed group. Se and ascorbic acid supplementation reduced these levels in comparison with the alcohol and control groups. Co-administration of Se and ascorbic acid along with alcohol decreased the levels of LDLqVLDL significantly when compared to the ethanol group. LPL activity (Table 6) was significantly decreased in heart and adipose tissue of the alcohol fed group when compared to controls. Se and ascorbic acid supplementation significantly increased the activity of LPL in heart only com-
Fig. 1. Activity of HMG-CoA reductase in liver.
pared to the control group. Co-administration of Se and ascorbic acid plus alcohol increased its activity in both tissues when compared to the ethanol group. The activity of plasma LCAT was significantly decreased in the ethanol group, but significantly increased in both treated groups as compared to control and ethanol groups. Hepatic HMG-CoA reductase (Fig. 1) was significantly increased in the ethanol administered group as compared to controls (P-0.05). With Se and ascorbic acid supplementation the activity of HMG-CoA reductase was significantly decreased. Co-administration of Se and ascorbic acid along
Table 6 Activity of plasma LCAT* and LPLa in male guinea pigs Groups
Control Ethanol Seqascorbic acid EthanolqSeq ascorbic acid
LCAT Plasma 28.65"1.16 20.77"0.85a 71.57"2.90bd 47.76"1.94ce
LPL Adipose
Heart
160.52"6.53 149.35"5.71a 166.21"6.76d 160.21"5.59e
133.61"4.89 106.72"4.36a 167.23"6.17bd 150.27"5.72ce
For explanation of superscripts see Table 1. Values are mean"S.D. * % increase in the ratio of ester cholesterolycholesterol during incubation. a Units-mmoles of glycerol liberatedyhymg protein. Table 7 Concentration of total bile acids and neutral sterols in male guinea pigs Groups
Hepatic bile acids
Fecal bile acids
Neutral sterols
Control Ethanol Seqascorbic acid EthanolqSeq ascorbic acid
18.91"0.77 14.72"0.61a 20.97"0.85bd 19.35"0.79e
23.08"0.95 12.06"0.50a 25.92"1.17bd 24.64"1.01e
12.08"0.51 10.37"0.42a 19.29"0.88bd 18.95"0.77ce
For explanation of superscripts see Table 1. Values are given in mgy100 g tissue, as mean"S.D.
G.S. Asha, M. Indira / Comparative Biochemistry and Physiology Part C 137 (2004) 109–114
with alcohol significantly reduced its activity when compared to the ethanol group. Concentration of total bile acids and neutral sterols (Table 7) were significantly decreased in the ethanol administered group. Co-administration of Se and ascorbic acid along with alcohol significantly increased the level of bile acids and neutral sterols when compared to the ethanol group. 4. Discussion Epidemiological studies suggests that high intake of vitamin C from food sources is associated with reduced incidence of stroke (Achenson and Williams, 1983; Vollset and Bjelke, 1983) and myocardial infarction (Fehily et al., 1993). At the same time hypercholesterolemia and cardiovascular disorders have been shown to be associated with Se deficiency (Kang et al., 2000). In the present study, we have studied the supplementation of Se and ascorbic acid on alcohol induced hyperlipidemia in guinea pigs. Guinea pigs, like humans are unable to synthesize vitamin C due to a common genetic defect. The ascorbic acid synthesizing enzyme L-gulono-g-lactone oxidase is absent. Guinea pigs were selected as experimental animals since the results can be extrapolated to human beings. Consistent with previous reports alcohol intake caused hyperlipidemia (Feinman and Leiber, 1999). However, exogenous Se and ascorbic acid supplied along with the ethanol reduced the alcohol-induced hyperlipidemia to an extent. This is due to decreased synthesis as evidenced by the lower activity of HMG-CoA reductase. There are reports on direct inhibition of HMG-CoA reductase activity by ascorbate; for example, when guinea pig hepatic microsomes were incubated with Naascorbate (Greene et al., 1985). This is in agreement with our results. Nassier et al. (1997) reported that hypercholesterolemia associated with Se deficiency was related to the increased HMGCoA reductase activity in liver microsomes. Catabolism is also increased since the excretion of fecal bile acids and neutral sterols were increased. This is also evidenced from the increased activities of plasma LCAT and LPL. These enzymes are involved in the transfer of cholesterol from tissues to liver for degradation. Hypolipidemic action of vitamin C has been well documented (Kothari and Jain, 1977). It has been observed that the rate of transformation of choles-
113
terol to bile acids in the liver is directly correlated to the ascorbate concentration of liver cells. Exogenous supply of ascorbic acid to guinea pigs leads to increased production of ascorbic acid-2sulfate. Ascorbic acid-2-sulfate has a hypocholesterolemic effect in hyperlipidemic animals (Radhakrishna Pillai et al., 1991) Selenium is an integral part of the deiodenase, which transforms T4 to T3. Thyroid hormone influences lipid metabolism. Bekpinar and Tugrul (1995) observed that Se lowers testicular lipids and they linked this to the action of enhanced deiodenase activity on Se supplementation. Excessive Se intake induces the synthesis of many selenoproteins (Behne et al., 1996). They may be modulating the various enzymes, and only further studies will reveal the clear picture. Here, we conclude that supplementation of Se and ascorbic acid reduced alcohol-induced hyperlipidemia by decreased synthesis and increased catabolism. Acknowledgments Financial support from the Department of Welfare, Government of Kerala is gratefully acknowledged. References Achenson, R.M., Williams, D.R.R., 1983. Does consumption of fruit and vegetables protect against stroke? Lancet II, 1191–1193. Asha, G.S., Suresh, M.V., Indira, M., 2003. Combined effect of ascorbic acid and selenium on alcohol induced stress in guinea pigs. Comp. Biochem. Physiol. C 134, 397–401. Bates, C.J., Mundal, A.R., Cole, T.J., 1977. HDL-Cholesterol and vitamin C status. Lancet II, 611–615. Bekpinar, S., Tugrul, Y., 1995. Influence of selenium supplementation in non-toxic doses on testis lipid peroxide and antioxidant levels in chronic alcohol-fed rats. Alcohol Alcoholism 30, 645–650. Behne, D., Kyriakopoulos, A., Meinhold, H., Kohrle, J., 1990. Identification of type I iodothyronine 5’-deiodinase as a selenoenzyme. Biochem. Biophys. Res. Commun. 173, 1143–1149. Behne, D., Kyriakopoulos, A., Weiss-Nowak, C., Kalcklosch, M., Westphal, C., Gessner, H., 1996. Newly found selenium containing proteins in the tissues of the rat. Biol. Trace Elem. Res. 55, 99–110. Fehily, A.M., Yarnell, J.W.G., Sweetnam, P.M., Elwood, P.C., 1993. Diet and incident ischemic heart disease: the Caerphilly study. Br. J. Nutr. 69, 303–314. Feinman, L., Leiber, C.S., 1999. Ethanol and lipid metabolism. Am. J. Clin. Nutr. 70, 791–792. Folch, J., Lees, M., Stoane Stanley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226, 497–509.
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Greene, Y.J., Harwood Jr, H.J., Stacpoole, P.W., 1985. Ascorbic acid regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesterol synthesis in guinea pig liver. Biochim. Biophys. Acta 834, 134–138. Kang, B.P.S., Uma, M., Bansal, M.P., 2000. Hyperlipidemia type-I-59-mono deiodenase activity: regulation by selenium supplementation. Indian J. Biochem. Biophys. 37, 183–187. Kothari, L.K., Jain, K., 1977. Effect of vitamin C administration on blood cholesterol level in man. Acta Biol. 28, 111–115. Liobera, M., Montes, A., Herrera, E., 1979. Lipoprotein lipase activity in liver of the rat fetus. Biochem. Biophys. Res. Commun. 91, 272–277. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275. Mahadev Rao, , 2001. Role of selenium in cancer. Amala Res. Bull. 21, 158–165. Masukawa, T., Goto, J., Iwata, H., 1983. Impaired metabolism of arachidonate in selenium deficient animals. Experientia 39, 405–406. Menon, P.V.G., Kurup, P.A., 1976. Dietary fiber and cholesterol metabolism-effect of fiber rich polysaccharide from black gram (Phaseolus mungo) on cholesterol metabolism in rats fed normal and atherogenic diet. Biomedicine 24, 248–253. Nassier, F., Moundras, C., Bayle, D., Serougne, C., Gueux, E., Rock, E., et al., 1997. Effect of selenium deficiency on hepatic lipid and lipoprotein metabolism in the rat. Br. J. Nutr. 78, 493–500.
Okishio, T., Nair, P.P., Gordon, M., 1967. Studies on bile acids: the microqualitative separation of cellular bile acids by gas liquid chromatography. Biochem. J. 102, 654–658. Radhakrishna Pillai, G., Indira, M., Vijayammal, P.L., 1991. Role of exogenous ascorbic acid in the tissue status of ascorbic acid 2-sulfate in guinea pigs. Indian J. Exp. Biol. 29, 1127–1129. Rao, A.V., Ramakrishnan, S., 1975. Indirect assessment of hydroxymethylglutaryl-CoA reductase activity in liver tissue. J. Clin. Chem. 21, 1523–1527. Rimm, E., Colditz, G., 1993. Smoking, alcohol and plasma levels of carotenes and vitamin E. Ann. NY Acad. Sci. 686, 323–333. Rober, H.P., 1996. The enzymatic assay of bile acids and related 3 alpha-hydroxy steroids. Its application to serum and other biological fluids. Method. Enzymol. 15, 280–286. Schoenheimer, R., Sperry, W.M., 1934. A micromethod for the determination of free and combined cholesterol. J. Biol. Chem. 106, 745–760. Suresh, M.V., Lal, J.J., Sreeranjit Kumar, C.V., Indira, M., 1997. Interaction of ethanol and ascorbic acid on lipid metabolism in guinea pigs. Indian J. Exp. Biol. 35, 1065–1069. Vollset, S.E., Bjelke, E., 1983. Does consumption of fruit and vegetables protect against stroke? Lancet 2, 742. Warnick, R.G., Alberts, J., 1978. A comprehensive evaluation of the heparin manganese precipitating procedure for estimating high-density lipoprotein cholesterol. J. Lipid Res. 119, 65–69.
Combined effect of selenium and ascorbic acid on alcohol induced hyperlipidemia in male guinea pigs G.S. Asha, M. Indira* Department of Biochemistry, University of Kerala, Kariavattom, Thiruvananthapuram 695 581, Kerala, India Received 23 September 2003; received in revised form 5 December 2003; accepted 8 December 2003
Abstract Alcoholics usually suffer from malnutrition and are especially deficient in micronutrients like vitamin C, selenium and Zn. In the present study, combined effects of selenium and ascorbic acid on alcohol-induced hyperlipidemia were studied in guinea pigs. Four groups of male guinea pigs were maintained for 45 days as follows: control (1 mg ascorbate (AA)y 100 g body massyday), ethanol (900 mg ethanoly100 g body massq1 mg AAy100 g body massyday), seleniumq ascorbic acid w(25 mg AAq0.05 mg Se)y100 g body massydayx, ethanolqseleniumqascorbic acid w(25 mg AAq0.05 mg Seq900 mg ethanol)y100 g body massydayx. Co-administration of selenium and ascorbic acid along with alcohol reduced the concentration of all lipids, as also evidenced from the decreased activities of hydroxymethylglutaryl-CoA reductase and enhanced activities of plasma lecithin cholesterol acyl transferase and lipoprotein lipase. Concentrations of bile acids were increased. We conclude that the supplementation of Se and ascorbic acid reduced alcohol induced hyperlipidemia, by decreased synthesis and increased catabolism. 䊚 2004 Elsevier Inc. All rights reserved. Keywords: Ethanol; Selenium; Ascorbic acid; Hydroxymethylglutaryl-CoA (HMG-CoA); Lecithin cholesterol acyl transferase (LCAT); Lipoprotein lipase (LPL); Lipids; Bile acids
1. Introduction Many toxic effects of alcohol appear to be mediated through imbalances in micronutrients leading to secondary changes in biological and molecular functions of the cell. Levels of many nutrients such as vitamin C and selenium are lower in alcoholics (Rimm and Colditz, 1993). Vitamin C is a hypolipidemic agent. Results of the several studies have suggested that it can lower blood cholesterol in human subjects (Kothari and Jain, 1977). A positive correlation between vitamin *Corresponding author. Tel.: q91-2361344; fax: q91-471307158. E-mail address: [email protected] (M. Indira).
C and plasma HDL cholesterol has been proposed by Bates et al. (1977). Earlier studies showed that an exogenous supply of ascorbic acid reduced hyperlipidemia in most tissues (Suresh et al., 1997). Selenium is a micronutrient of fundamental importance to human health (Mahadev Rao, 2001). Hypercholesterolemia and cardiovascular disorders have been shown to be associated with Se deficiency (Kang et al., 2000). Se has a crucial role in controlling the effects of thyroid hormone on fat metabolism. It has been reported that deiodinase (DI), the enzyme responsible for converting T4 to T3, contains the rare aminoacid selenocysteine that is formed in the body using dietary Se (Behne et al., 1990). Kang et al. (2000) studied the effect of
1532-0456/04/$ - see front matter 䊚 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2003.12.002
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feeding a high fat diet and a Se supplemented high fat diet for 3 months on type I-59 monodeiodinase activity in rats. They found a significant increase in serum cholesterol and triglyceride levels in the high fat diet fed group as compared to the control when compared to the Se supplemented group. This was correlated with levels of 59-DI. A low serum Se level is associated with a decrease of liver microsomal activity and serum HDL-C concentration (Masukawa et al., 1983). Our earlier studies have shown that co-administration of ascorbic acid and Se is beneficial in reducing alcohol induced oxidative stress in guinea pigs (Asha et al., 2003). Therefore, in the present study, we investigated the supplementation of selenium and ascorbic acid on alcohol induced hyperlipidemia in guinea pigs. 2. Materials and methods Male guinea pigs (Cavia porcellus; Veterinary College, Mannuthy Strain) bred and reared in our animal house were used for the experiment. Animals were housed individually in wire netted cages at room temperature maintained at 25"1 8C with a lightydark cycle of 12 h. Weight matched guinea pigs were selected. A total of 24 guinea pigs were divided into four groups of six each: Controls 1 mg ascorbate (AA)y100 g body massyday; II Ethanol (900 mg ethanolq1 mg AA)y 100 g body massyday); III SeleniumqAA (25 mg AAq0.05 mg Naselenite)y100 g body massyday); and IV EthanolqseleniumqAA (25 mg AAq 0.05 mg Na-seleniteq900 mg ethanol)y100 g body massyday). I
Guinea pigs were fed with guinea pig feed (Lipton India Ltd.). Food and water were given ad libitum, and ascorbic acid, selenium and ethanol were administered as detailed above. Ascorbic acid and selenium freshly dissolved in distilled water and ethanol diluted in the ratio (1:1) were given orally by gastric tube for 45 days. Control and seleniumqascorbic acid groups were administered glucose solution equivalent to the caloric value of ethanol in group II and group IV. On the 46th day, the animals were killed and tissues were removed to ice cold containers for various estimations. The tissues were extracted according to the procedure
of Folch et al. (1957). Cholesterol, triglycerides, phospholipids and free fatty acids (FFA) were estimated in the tissues as described by Menon and Kurup (1976). Separation of serum HDL and LDLqVLDL was carried out according to the procedure of Warnick and Alberts (1978). The activity of hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase; EC 1.1.1.34) was assayed as described by Rao and Ramakrishnan (1975). Extraction of liver bile acids was carried out according to the procedure of Okishio et al. (1967) and quantified by the procedure described by Rober (1996). Activity of lipoprotein lipase (LPL; EC 3.1.1.34) was determined by the method of Liobera et al. (1979). The activity of plasma lecithin cholesterol acyl transferase (LCAT) was determined according to the procedure of Schoenheimer and Sperry (1934). Protein in the enzyme extract was determined after trichloroacetic acid precipitation by the method of Lowry et al. (1951). Data were statistically analyzed by One-way analysis of variance (ANOVA). All values were expressed as means"S.D. P values of 0.05 or less were considered significant. All analysis was performed by computer using the statistical package SPSS. 3. Results Concentration of cholesterol (Table 1) was significantly increased in liver, heart, kidney and brain of alcohol administered guinea pigs as compared to controls. Co-administration of Se and ascorbic acid along with alcohol decreased the concentration of cholesterol significantly in all the tissues when compared to the ethanol group. The concentration of phospholipids (Table 2) was significantly increased in the ethanol administered group when compared to controls. Supplementation of Se and ascorbic acid significantly decreased the concentration of phospholipids in all the tissues. Co-administration of Se and ascorbic acid along with alcohol decreased the levels of phospholipids significantly in all the tissues. Concentration of FFA (Table 3) was significantly increased in ethanol fed guinea pigs. FFA concentration was significantly decreased in all the tissues of the Se and ascorbic acid plus alcohol group, when compared to the ethanol group. Alcohol fed guinea pigs showed significant increase in the concentration of triglycerides (Table 4) in all the tissues as compared to controls. Se
G.S. Asha, M. Indira / Comparative Biochemistry and Physiology Part C 137 (2004) 109–114
111
Table 1 Concentration of cholesterol in male guinea pigs Groups
Liver
Heart
Kidney
Brain
Control Ethanol Seqascorbic acid EthanolqSeq ascorbic acid
343.3"13.9 593.1"24.1a 341.0"13.9d 367.1"15.0c,e
200.0"8.1 273.1"11.1a 197.5"8.2d 215.6"8.5c,e
347.1"14.5 503.3"20.3a 324.1"13.0b,d 419.2"17.1c,e
866.7"35.1 1050.6"42.5a 857.2"34.7d 902.5"36.5e
Values are given in mgy100 g tissue, as mean"S.D. P-0.05 between control and ethanol groups. b P-0.05 between control and selenium groups. c P-0.05 between control and seleniumqethanol groups. d P-0.05 between ethanol and selenium groups. e P-0.05 between ethanol and seleniumqethanol groups. a
Table 2 Concentration of phospholipids in male guinea pigs Groups
Liver
Heart
Kidney
Brain
Control Ethanol Seqascorbic acid EthanolqSeq ascorbic acid
2211.9"89.8 6754.6"259.6a 2209.9"80.0d 2688.5"92.9ce
2302.1"93.6 2806.7"113.4a 2283.3"92.3d 2548.6"103.2ce
2426.7"98.1 2987.1"121.1a 2013.34"81.88bd 2777.6"112.5ce
1895.9"77.0 2189.5"88.7a 1569.39"62.5bd 2004.1"80.9ce
For explanation of superscripts see Table 1. Values are given in mgy100 g tissue, as mean"S.D. Table 3 Concentration of free fatty acids in male guinea pigs Groups
Liver
Heart
Kidney
Brain
Control Ethanol Seqascorbic acid EthanolqSeq ascorbic acid
260.5"106 308.1"12.2a 252.9"10.4d 278.7"11.2ce
144.1"5.8 187.1"7.8a 124.8"5.2bd 156.3"6.4ce
173.1"7.0 198.6"7.9a 144.6"5.9bd 176.3"7.0e
130.5"5.2 163.1"6.6 126.6"4.6d 150.6"6.1ce
For explanation of superscripts see Table 1. Values are given in mgy100 g tissue, as mean"S.D. Table 4 Concentration of triglycerides in male guinea pigs Groups
Liver
Heart
Kidney
Brain
Control Ethanol Seqascorbic acid EthanolqSeq ascorbic acid
351.0"14.2 491.1"19.9a 299.5"12.2bd 370.8"14.9ce
51.5"2.1 72.3"2.7a 49.9"2.0d 59.1"2.5ce
96.5"4.1 152.4"6.1a 89.5"3.6bd 101.1"4.1e
76.4"3.3 113.3"4.6a 69.7"2.9bd 78.5"3.3e
For explanation of superscripts see Table 1. Values are given in mgy100 g tissue, as mean"S.D.
and ascorbic acid supplementation significantly reduced triglycerides in all the tissues when compared to the control. Co-administration of Se and ascorbic acid along with alcohol significantly
decreased the concentration of triglycerides in all the tissues compared to the ethanol group. The concentration of HDL-C (Table 5) was significantly decreased in alcohol fed guinea pigs
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Table 5 Concentration of serum lipoproteins in male guinea pigs Groups
HDL-C
LDLqVLDL-C
Control Ethanol Seqascorbic acid EthanolqSeq ascorbic acid
50.19"2.06 47.24"1.84a 51.23"2.09d 53.46"1.93ce
16.52"0.71 24.69"1.01a 10.26"0.42bd 18.27"0.69e
For explanation of superscripts see Table 1. Values are given in mgy100 ml serum, as mean"S.D.
when compared to controls. Co-administration of Se and ascorbic acid plus alcohol significantly increased HDL-C levels compared to the ethanol group. Concentration of LDLqVLDL was significantly increased in the alcohol fed group. Se and ascorbic acid supplementation reduced these levels in comparison with the alcohol and control groups. Co-administration of Se and ascorbic acid along with alcohol decreased the levels of LDLqVLDL significantly when compared to the ethanol group. LPL activity (Table 6) was significantly decreased in heart and adipose tissue of the alcohol fed group when compared to controls. Se and ascorbic acid supplementation significantly increased the activity of LPL in heart only com-
Fig. 1. Activity of HMG-CoA reductase in liver.
pared to the control group. Co-administration of Se and ascorbic acid plus alcohol increased its activity in both tissues when compared to the ethanol group. The activity of plasma LCAT was significantly decreased in the ethanol group, but significantly increased in both treated groups as compared to control and ethanol groups. Hepatic HMG-CoA reductase (Fig. 1) was significantly increased in the ethanol administered group as compared to controls (P-0.05). With Se and ascorbic acid supplementation the activity of HMG-CoA reductase was significantly decreased. Co-administration of Se and ascorbic acid along
Table 6 Activity of plasma LCAT* and LPLa in male guinea pigs Groups
Control Ethanol Seqascorbic acid EthanolqSeq ascorbic acid
LCAT Plasma 28.65"1.16 20.77"0.85a 71.57"2.90bd 47.76"1.94ce
LPL Adipose
Heart
160.52"6.53 149.35"5.71a 166.21"6.76d 160.21"5.59e
133.61"4.89 106.72"4.36a 167.23"6.17bd 150.27"5.72ce
For explanation of superscripts see Table 1. Values are mean"S.D. * % increase in the ratio of ester cholesterolycholesterol during incubation. a Units-mmoles of glycerol liberatedyhymg protein. Table 7 Concentration of total bile acids and neutral sterols in male guinea pigs Groups
Hepatic bile acids
Fecal bile acids
Neutral sterols
Control Ethanol Seqascorbic acid EthanolqSeq ascorbic acid
18.91"0.77 14.72"0.61a 20.97"0.85bd 19.35"0.79e
23.08"0.95 12.06"0.50a 25.92"1.17bd 24.64"1.01e
12.08"0.51 10.37"0.42a 19.29"0.88bd 18.95"0.77ce
For explanation of superscripts see Table 1. Values are given in mgy100 g tissue, as mean"S.D.
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with alcohol significantly reduced its activity when compared to the ethanol group. Concentration of total bile acids and neutral sterols (Table 7) were significantly decreased in the ethanol administered group. Co-administration of Se and ascorbic acid along with alcohol significantly increased the level of bile acids and neutral sterols when compared to the ethanol group. 4. Discussion Epidemiological studies suggests that high intake of vitamin C from food sources is associated with reduced incidence of stroke (Achenson and Williams, 1983; Vollset and Bjelke, 1983) and myocardial infarction (Fehily et al., 1993). At the same time hypercholesterolemia and cardiovascular disorders have been shown to be associated with Se deficiency (Kang et al., 2000). In the present study, we have studied the supplementation of Se and ascorbic acid on alcohol induced hyperlipidemia in guinea pigs. Guinea pigs, like humans are unable to synthesize vitamin C due to a common genetic defect. The ascorbic acid synthesizing enzyme L-gulono-g-lactone oxidase is absent. Guinea pigs were selected as experimental animals since the results can be extrapolated to human beings. Consistent with previous reports alcohol intake caused hyperlipidemia (Feinman and Leiber, 1999). However, exogenous Se and ascorbic acid supplied along with the ethanol reduced the alcohol-induced hyperlipidemia to an extent. This is due to decreased synthesis as evidenced by the lower activity of HMG-CoA reductase. There are reports on direct inhibition of HMG-CoA reductase activity by ascorbate; for example, when guinea pig hepatic microsomes were incubated with Naascorbate (Greene et al., 1985). This is in agreement with our results. Nassier et al. (1997) reported that hypercholesterolemia associated with Se deficiency was related to the increased HMGCoA reductase activity in liver microsomes. Catabolism is also increased since the excretion of fecal bile acids and neutral sterols were increased. This is also evidenced from the increased activities of plasma LCAT and LPL. These enzymes are involved in the transfer of cholesterol from tissues to liver for degradation. Hypolipidemic action of vitamin C has been well documented (Kothari and Jain, 1977). It has been observed that the rate of transformation of choles-
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terol to bile acids in the liver is directly correlated to the ascorbate concentration of liver cells. Exogenous supply of ascorbic acid to guinea pigs leads to increased production of ascorbic acid-2sulfate. Ascorbic acid-2-sulfate has a hypocholesterolemic effect in hyperlipidemic animals (Radhakrishna Pillai et al., 1991) Selenium is an integral part of the deiodenase, which transforms T4 to T3. Thyroid hormone influences lipid metabolism. Bekpinar and Tugrul (1995) observed that Se lowers testicular lipids and they linked this to the action of enhanced deiodenase activity on Se supplementation. Excessive Se intake induces the synthesis of many selenoproteins (Behne et al., 1996). They may be modulating the various enzymes, and only further studies will reveal the clear picture. Here, we conclude that supplementation of Se and ascorbic acid reduced alcohol-induced hyperlipidemia by decreased synthesis and increased catabolism. Acknowledgments Financial support from the Department of Welfare, Government of Kerala is gratefully acknowledged. References Achenson, R.M., Williams, D.R.R., 1983. Does consumption of fruit and vegetables protect against stroke? Lancet II, 1191–1193. Asha, G.S., Suresh, M.V., Indira, M., 2003. Combined effect of ascorbic acid and selenium on alcohol induced stress in guinea pigs. Comp. Biochem. Physiol. C 134, 397–401. Bates, C.J., Mundal, A.R., Cole, T.J., 1977. HDL-Cholesterol and vitamin C status. Lancet II, 611–615. Bekpinar, S., Tugrul, Y., 1995. Influence of selenium supplementation in non-toxic doses on testis lipid peroxide and antioxidant levels in chronic alcohol-fed rats. Alcohol Alcoholism 30, 645–650. Behne, D., Kyriakopoulos, A., Meinhold, H., Kohrle, J., 1990. Identification of type I iodothyronine 5’-deiodinase as a selenoenzyme. Biochem. Biophys. Res. Commun. 173, 1143–1149. Behne, D., Kyriakopoulos, A., Weiss-Nowak, C., Kalcklosch, M., Westphal, C., Gessner, H., 1996. Newly found selenium containing proteins in the tissues of the rat. Biol. Trace Elem. Res. 55, 99–110. Fehily, A.M., Yarnell, J.W.G., Sweetnam, P.M., Elwood, P.C., 1993. Diet and incident ischemic heart disease: the Caerphilly study. Br. J. Nutr. 69, 303–314. Feinman, L., Leiber, C.S., 1999. Ethanol and lipid metabolism. Am. J. Clin. Nutr. 70, 791–792. Folch, J., Lees, M., Stoane Stanley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226, 497–509.
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