J. Biosci., Vol. 13, Number 3, September 1988, pp. 269–274. © Printed in India. Lipid peroxidation of hyperlipemic rat serum lipoproteins in chronic ethanol and acetaldehyde administration

RAMESH CHANDER, NARINDER K. KAPOOR* andCHANAN SINGH Division of Biochemistry, Central Drug Research Institute, Lucknow 226 001, India MS received 29 December 1987; revised 11 April 1988 Abstract. The levels of lipid peroxides in circulatory lipoproteins increased with chronic administration of ethanol or acetaldehyde. Low density lipoprotein showed a greater increase in its content of lipid peroxides than very low density lipoprotein or high density lipoprotein. However, very low density lipoprotein was more prone to lipid peroxidation in vitro than low density lipoprotein or high density lipoprotein. The effect of acetaldehyde was more marked than that of ethanol. Lipoproteins of control and hyperlipemic groups were partially protected against peroxidation by butyrated hydroxytoluene and serum high density lipoprotein of normal rats. Keywords. Lipid peroxidation; serum lipoproteins; hyperlipemia; alcoholism; high density lipoprotein; butyrated hydroxytoluene.

Introduction The chronic administration of ethanol or acetaldehyde is known to increase the levels of serum lipoproteins and causes the emergence of an abnormal lipoprotein, lipoprotein-X (Chander et al., 1987). Increased levels of serum lipid peroxide (LPO) were found in chronic alcoholism (Fink et al., 1985). ß-Lipoproteins (very low density lipoprotein, VLDL; and low density lipoprotein, LDL) and the process of lipid peroxidation in general play an important role in the pathogenesis of coronary vascular diseases and atherosclerosis (Morel et al., 1983; Mizukami et al., 1984), which are also known to be associated with alcoholism. However, information on lipid peroxidation of lipoproteins in alcoholism and the interrelationship among lipoproteins in this respect is hardly available in the literature. Since high density lipoprotein (HDL) is known to give protection against cytotoxicity of ß-lipoproteins in vitro (Hessler et al., 1979), it was considered of interest to ascertain if HDL could afford protection against lipid peroxidation of ß-lipoproteins during chronic administration of ethanol or acetaldehyde in rats. The studies described in this paper demonstrate partial protection from lipid peroxidation of ß-lipoproteins by HDL from normal rats. Materials and methods Heparin and dextran sulphate (molecular weight 500,000) were purchased from Loba-Chemie, Vienna, Austria, and Sigma Chemical Co., St. Louis, Missouri, USA, CDRI Communication No. 4181. *To whom all correspondence should be addressed. Abbreviations used: LPO, Lipid peroxide; VLDL, very low density lipoprotein; LDL, low density lipo- protein; HDL, high density lipoprotein; BHT, butyrated hydroxytoluene.

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270 Chander et al. respectively. Butyrated hydroxytoluene (BHT) and other chemicals used were of analytical grade.

Male adult rats of Charles Foster strain (150–200 g) inbred in the CDRI animal house were divided into 3 groups of 8 rats each. They were administered normal saline, 50% aqueous ethanol (3·76 g/kg body weight) and 20% acetaldehyde (1·3 g/kg body weight), respectively by gastric tubing once a day for 60 days. At the end of 20, 40 and 60 days of alcohol/acetaldehyde treatment, animals were taken from each group. The animals were fasted overnight, blood was withdrawn by retro-orbital plexus and the animals sacrificed to collect the liver. The serum was fractionated into VLDL, LDL and HDL by the polyanionic precipitation method (Burstein et al., 1982) using heparin, dextran sulphate and MnCl2 as reactants. Each fraction was dialysed against 0·1 Μ NaCl; containing 0·05% EDTA in the presence of N2 gas. LPO content of liver and serum lipoproteins was estimated by the thiobarbituric acid reaction (Ohkawa and Ohishi, 1978). Protein was estimated according to the method of Lowry et al. (1951). Lipid peroxidation of VLDL and LDL and protection by normal HDL were studied in vitro according to Hessler et al. (1979). Serum lipoproteins (100–200 µg protein) of control as well as hyperlipemic rats were mixed with normal rat serum HDL (N–HDL) solution (250– 500 µg protein). The same amounts of VLDL and LDL were mixed with 6–10 µ1 (containing 6–10 pmol) BHT. A set of control tubes without addition of N–HDL or BHT was also prepared. LPO content in all these sets were estimated at zero time as well as after incubation for 6 h at 37°C. Protection against lipid peroxidation was calculated by comparison of LPO levels at zero time and 6 h. Results The effects of chronic administration of ethanol and acetaldehyde on LPO levels of serum lipoproteins and liver are given in table 1. It may be seen that LPO levels in Table 1. LPO* of serum lipoproteins and liver in hyperlipemic rats.

*Expressed as nmol malondialdehyde 100 mg protein. All values are mean ± SD of 6 determinations. P < 0·01; aP < 0·05; bnot significant.

Lipid peroxidation of lipoproteins 271 control rats did not show any perceptible change during the experimental period of 60 days. The level of LPO was highest in case of HDL than in VLDL and LDL. The levels of LPO in total serum and the 3 lipoprotein fractions showed progressive increase in both treatments. LDL showed a greater increase in its content of LPO than VLDL and HDL in both alcohol and acetaldehyde-treated groups. Similarly LPO levels in liver in both groups also exhibited a progressive increase with ethanol or acetaldehyde treatment. The effect of acetaldehyde was more marked than that of ethanol in all cases.

Lipid peroxidation in vitro was examined in the serum and in serum lipoproteins after treatment of rats with ethanol and acetaldehyde for 60 days. The results (figures 1–4) show that VLDL was more prone to lipid peroxidation than LDL or HDL, and lipid peroxidation was higher in the acetaldehyde-fed group than in the ethanol-fed groups. Lipid peroxidation of lipoproteins was found to be partially inhibited by N–HDL and BHT. VLDL and LDL of control and hyperlipemic groups were protected against lipid peroxidation in vitro by BHT and N–HDL, the former being a more potent protector. VLDL was protected by BHT or N–HDL to a greater extent than LDL.

Figure 1. Protection from lipid peroxidation of hyperlipemic rat serum by normal HDL or BHT. C, Control; E, ethanol-fed; A, acetaldehyde-fed.

Figure 2. Protection from lipid peroxidation of hyperlipemic rat serum VLDL by normal HDL or BHT. C, Control; E, ethanol-fed; A, acetaldehyde-fed.

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Figure 3. Protection from lipid peroxidation of hyperlipemic rat serum LDL by normal HDL or BHT. C, Control; E, ethanol-fed; A, acetaldehyde-fed.

Figure 4. Protection from lipid peroxidation of hyperlipemic rat serum HDL by normal HDL or BHT. C, Control; E, ethanol-fed, A, acetaldehyde-fed.

Discussion The investigation showed enhancement in LPO content of serum lipoproteins and liver under conditions of chronic administration of ethanol and acetaldehyde. The role of antioxidants in modifying hepatic injury and hyperlipemia induced by chronic feeding of ethanol would suggest that the primary events in the deve- lopment of fatty liver and the damage undergone by it consist in the formation of LPO at selective subcellular sites (Di Luzio, 1973; Harta et al., 1983). Since liver is a major site of synthesis of lipoproteins, hepatic injury could be accompanied by abnormalities of lipoprotein biosynthesis and metabolism which may be reflected in the blood lipoprotein spectrum (Vadivelu and Ramakrishnan, 1986). Increased

Lipid peroxidation of lipoproteins 273 levels of LPO or VLDL and LDL (ß-lipoproteins) in treated rats are known to be cytotoxic to cells and tissues. Ross and Harker (1976) reported that during hyperlipemia, lipoproteins may initiate and maintain atheromatous lesions by endothelial cell injury and lipid accumulation. Recently it has been emphasised that hyperlipemic ß-lipoproteins are cytotoxic to cells and tissues presumably due to enhanced levels of associated LPO (Jurgens et al., 1986). Hypertriglyceridemic- VLDL and LDL are known to suppress the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase in cultured fibroblasts from human subjects; they also exert toxic effects on endothelial cells (Gianturco et al., 1980).

Our findings indicate that BHT (an antioxidant) as well as N–HDL provide protection to VLDL and LDL in vitro against lipid peroxidation. These results would seem to be consistent with recent epidemiological data linking the high LDL and low HDL concentration in circulation with the development of accelerated atherosclerosis (Narula and Wasir, 1985). The property of HDL to act as an antioxidant for VLDL and LDL may mean that the cytotoxicity of pathogenic ß- lipoproteins as observed in atherosclerosis and vascular diseases is due to their susceptibility to lipid peroxidation (Henriksen et al., 1979; Evensen et al., 1983). Acetaldehyde caused more pronounced lipid peroxidation than ethanol, which may be attributed to the fact that acetaldehyde is the immediate active metabolite of ethanol (Ramakrishnan 1984). Lipid peroxidation in alcoholism may augment the effects of atherosclerosis and vascular abnormalities. It seems that HDL plays a vital role in the inhibition of free radical-induced lipid peroxidation of lipids and lipoproteins.

In conclusion it may be added that apart from other measures which are generally adopted against the pathophysiology/ill-effects of alcoholism, the use of antioxidants, preferably of natural origin, viz., β-carotene, α-tocopherol, mannitol and ascorbic acid, as an additional measure is strongly indicated (Morgan, 1982). Such a protective measure may also prove to be useful in preventing atherosclerosis and other vascular diseases. Acknowledgement One of the authors (C. S.) is an Emeritus Scientist of the Council of Scientific and Industrial Research, New Delhi. References Burstein, M. and Legmann, P. (1982) in Monographs in atherosclerosis. Lipoprotein precipitations (ed.

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Dormandy, T, L. (1985) Lancet, 2, 291. Gianturco, S. H., Eskin, S. G., Navarro, L. T., Lahart, G. J., Smith, L. C. and Golto, A. M. Jr. (1980)

Biochim. Biophys. Acta, 618, 143. Harta, J., Nagata, M., Sasaki, E., Ishiguro, I. and Ohta, Y. (1983) Biochem. Pharmacol., 32, 1795. Henriksen, Τ., Evensen, S. A. and Carlander, Β. (1979) Scand. J. Clin. Lab. Invest., 39, 369. Hessler, J. R., Robertson, A. L. and Chisolm, G. M. (1979) Atherosclerosis, 32, 213. Jurgens, G., Lang, J. and Esterbauer, Η. (1986) Biochim. Biophys. Acta, 875, 103.

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