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Lipid peroxidation in liver, plasma, and erythrocytes of rats chronically treated with ethanol
BIOCHEMICAL
MEDICINE
34,
370-372
(1985)
Lipid Peroxidation in Liver, Plasma, and Erythrocytes of Rats Chronically Treated with Ethanol M~JDAT UYSAL, G~L~IN AYKA~, NECLA KOC;AK-TOKER, AHMET SIV~\S. S~HA YALCIN, AND HIKMET Oz Department of Biochernistryv, Istanbul Fucrrlty qf Mediritw. Twkej Kecieved
September
24,
Univc~r.si/,voj fstunhfd. Copd. /.rrurth~rl. 1984
Increased hepatic lipid peroxide levels have been observed in humans due to long-term alcohol consumption ( 1,2). In addition, it has been reported that serum lipid peroxide levels were also elevated in chronic alcoholics and there is a correlation between hepatic and serum lipid peroxide levels (1). Further, Goebel eb al. (3) and Goebel and Schneider (4) have suggested that hemolytic implications in alcoholic liver disease may be due to increased lipid peroxidation together with an alteration of erythrocyte lipid composition. Indeed, Bidder and Jaeger (5) have reported that lipid peroxide production by erythrocytes from alcoholic individuals is significantly increased compared to non alcoholic subjects. However, there is no evidence whether or not plasma and erythrocyte lipid peroxide levels in rats are influenced by chronic ethanol administration. The primary aim of the present study was to investigate plasma and erythrocyte lipid peroxide levels together with liver lipid peroxidation status in an experimental model. MATERIALS
AND METHODS
Male Wistar rats weighing 200-230 g were used. One group was provided with water containing 20% (v/v) ethanol, the second one with an isocaioric amount of glucose in its drinking water. The duration of ethanol consumption lasted for 1-2 months. The ingestion of ethanol amounted to 8.5 g/kg body wt/day. They received a standard diet ad libitum. Experiments were done with chronically ethanol-administered rats following an overnight fast. Rats were killed by decapitation and livers were homogenized with 0.15 M KC1 to make a 10% homogenate. Liver lipid peroxide determination was performed as described by Uchiyama and Mihara (6). Heparinized blood was taken by heart puncture. The erythrocytes were separated from the plasma by centrifugation. Plasma lipid peroxide levels were determined according to Takeuchi er al. (7). Erythrocyte susceptibility to lipid peroxidation was determined with the Stocks and Dormandy (8) procedure, as modified by Bidder and Jaeger (5). The final 370 0006-2944i8.F
$3.00
CopYright u IYXS by Acadenuc Prer\. Inc All rights of reproduction in any form reserved.
ETHANOL
371
AND RAT LIVER LIPID PEROXIDATION
composition of the incubation mixture was 6.5 mM H202, 1.6 mM sodium azide, and erythrocyte suspension in phosphate-buffered saline (14 mg hemoglobin per ml incubation mixture). Lipid peroxidation was assayed by measurement of malondialdehyde (MDA) production during a 2-hr incubation period at 37°C. Values were expressed as nanomoles of MDA per gram of hemoglobin. Hemoglobin concentration of erythrocyte suspension was measured by Drabkin’s reagent. RESULTS AND DISCUSSION Studies of chronic ethanol consumption by rats have continued the controversy by providing evidence in favor of (9-13) and against (14-17) involvement of lipid peroxidation. The widely used indicator of lipid peroxidation in these studies is the determination of MDA in preincubated liver homogenates with thiobarituric acid (TBA) (11,12,15,17). However, it has been suggested that a TBA-reacting substance value after preincubation of tissue homogenates does not correlate well with in vivo peroxidation, and that a TBA test done with fresh homogenates gives a true value (18). For this reason, in the present study, lipid peroxides were measured in fresh liver homogenates following chronic ethanol ingestion in rats. Ingestion of ethanol for 1 and 2 months led to a significant increase in hepatic lipid peroxide levels (19.2 and 53.4%, respectively) (Table 1). Our results are in accordance with other investigators who have reported that lipid peroxidation has been stimulated after chronic ethanol consumption in rats (9-13). However, the influence of chronic ethanol exposure on plasma and erythrocyte lipid peroxide levels in rats has not been previously examined. We have observed a slight, but significant increase (10.4%) in plasma lipid peroxide levels after 1 month of ethanol treatment, whereas an obvious increase (40.8%) is seen after 2 months. In addition, the erythrocyte susceptibility to lipid peroxidation following ethanol ingestion for 2 months was significantly elevated (22.7%) but no significant TABLE I The Liver, Plasma and Erythrocyte Lipid Peroxide Levels following Ethanol Ingestion for 1 and 2 Months Treatment Hepatic lipid peroxide (532-520 nm) x10’
Plasma lipid peroxide (nmole MDA/ml plasma)
Erythrocyte lipid peroxide (nmole MDA/g Hb/2 hr)
I month
2 months
Glucose Ethanol Effect (%) P
99 2 8.6 118 t 12.8 + 19.2 0.01
101 -+ 8.6 155 + 12.9 + 53.4 0.001
Glucose
4.8 + 0.39 5.3 t 0.45 + 10.4 0.05
4.9 k 0.52 6.9 -t 0.44 + 40.8 0.001
Ethanol Effect (%) P Glucose Ethanol Effect (%) P
995 + 79.0 1010 2 74.3 k 1.5 NS
1015 5 83.7 1246 f 102.2 + 22.7 0.01
Nore. Values represent means and their standard deviations. Each group contained eight rats. Experiments were performed following an overnight (16 hr) fast.
UYSAL ET AL.
372
difference in erythrocyte lipid peroxide levels was observed in rats treated with ethanol for 1 month (Table I). On the other hand, it has been reported that decreased erythrocyte glutathione peroxidase activity f 19) and slight hemolysis (20) were observed in rats chronically given ethanol. Thus, it may be considered that increased peroxidation may contribute to the hemolysis due to chronic ethanol intake in rats, as observed in humans. As a conclusion, elevated plasma lipid peroxide levels and erythrocyte SUSceptibility to lipid peroxidation may reflect stimulated lipiperoxidation in rat liver following chronic ethanol ingestion. SUMMARY
The effect of ingestion of water containing 20% ethanol for 1-2 months on lipid peroxide levels of liver, plasma, and erythrocyte was investigated in rats. Our results show that elevated plasma lipid peroxide levels and erythrocyte susceptibility to lipid peroxidation may reflect stimulated lipid peroxidation in rat liver following chronic ethanol ingestion. REFERENCES 1. Suematsu, T., Matsumur~, T.. Sato, N.. Mi~~moto, T.. Ooka, T., Kamada. T., and Abe. H. Ahho~. C&i. Exp. Res. 5, 427 119811. 2. Shaw, S., Rubin, K. P., and Lieber, C. S.. Digest. LG. Sri. 28, SXT (1983). 3. Goebel. K. M., Goebel, F. D., Schubotz, R., and Schneider. J.. J. Lab. C/in. Med. 94, I23 (1979). 4. Goebel, K. M., and Schneider, J., Aciu Biol. Med. Germ. 40, 571 (1981j. 5. Bidder, T. G., and Jaeger, P. D.. Life Sci. 30, 1021 (1982). 6. Uchiyama, M.. and Mihara, M.. Anal. Biochetn. 86, 271 il97Xi. 7. Takeuchi. N., Tanaka, F., Katayama, Y., Matsumiya. K.. and Yamamura. Y.. &p. &vonf~,i. 11, 179 (1976).
8. 9. IO. Il. 12. 13. 14. IS. 16. 17. 18. 19. 20.
Stocks, J., and Dormandy, T. L., Brir. J. ffumcctol. 20, 95 (1971). MacDonald, C. M., FEBS Lerr. 35, 227 (1973). Ki%ter, U.. Albrecht, D.. and Kappus, H., Toxicol. A&. Phc7rmsc~oi. 41, 639 t 1977). Reitz, R. C., Biochinz. Biophys. Acfu 380, 145 (1975). Remmer, H., Albrecht, D.. and Kappus. H.. I\j,,,Yn-.Schrnird~~h~~~~‘~ .G~ir. ~~~~~~}?f~~,~~~. 298, 107 11977). Sies, H., Koch. 0. R., Martino. E., and Boveds, A., FEBS Lrrl. 103, 2X7 (1979). Shaw, S.. Jayatilleke. E., Ross, W. A.. Gordon. E. R.. and Lieher, C. S.. J. Lnh. l‘lir7. :Md. 98, 417 (1981). Torrielli, M. V., Gabriel, L., and Dianzani, M. U.. 1. Pu~/zo/. 126, I1 (1978). Litov, R. E., Gee. D. L.. Downey. J. E.. and Tappel. A. I.,., Lipids 16, 32 (1981 I. Hannah, J. S., and Soares. J. H.. Jr., h’ltfr. Rep. inr. 19, 733 (1979). Mihara, M., Uchiyama, M., and Fukuzawa, K., Biochc~m. Med. 23, 302 (1980). Capel, 1. D., Willenbrock, S. J. F., Jenner, M.. and Williams, D. C.. Biochem. Phurmmwl. 29, 1737 (1980). Baraona, E.. and Lieber, C. S., Amer. .I. C/in. Nu/r. 22. 356 (1969).
MEDICINE
34,
370-372
(1985)
Lipid Peroxidation in Liver, Plasma, and Erythrocytes of Rats Chronically Treated with Ethanol M~JDAT UYSAL, G~L~IN AYKA~, NECLA KOC;AK-TOKER, AHMET SIV~\S. S~HA YALCIN, AND HIKMET Oz Department of Biochernistryv, Istanbul Fucrrlty qf Mediritw. Twkej Kecieved
September
24,
Univc~r.si/,voj fstunhfd. Copd. /.rrurth~rl. 1984
Increased hepatic lipid peroxide levels have been observed in humans due to long-term alcohol consumption ( 1,2). In addition, it has been reported that serum lipid peroxide levels were also elevated in chronic alcoholics and there is a correlation between hepatic and serum lipid peroxide levels (1). Further, Goebel eb al. (3) and Goebel and Schneider (4) have suggested that hemolytic implications in alcoholic liver disease may be due to increased lipid peroxidation together with an alteration of erythrocyte lipid composition. Indeed, Bidder and Jaeger (5) have reported that lipid peroxide production by erythrocytes from alcoholic individuals is significantly increased compared to non alcoholic subjects. However, there is no evidence whether or not plasma and erythrocyte lipid peroxide levels in rats are influenced by chronic ethanol administration. The primary aim of the present study was to investigate plasma and erythrocyte lipid peroxide levels together with liver lipid peroxidation status in an experimental model. MATERIALS
AND METHODS
Male Wistar rats weighing 200-230 g were used. One group was provided with water containing 20% (v/v) ethanol, the second one with an isocaioric amount of glucose in its drinking water. The duration of ethanol consumption lasted for 1-2 months. The ingestion of ethanol amounted to 8.5 g/kg body wt/day. They received a standard diet ad libitum. Experiments were done with chronically ethanol-administered rats following an overnight fast. Rats were killed by decapitation and livers were homogenized with 0.15 M KC1 to make a 10% homogenate. Liver lipid peroxide determination was performed as described by Uchiyama and Mihara (6). Heparinized blood was taken by heart puncture. The erythrocytes were separated from the plasma by centrifugation. Plasma lipid peroxide levels were determined according to Takeuchi er al. (7). Erythrocyte susceptibility to lipid peroxidation was determined with the Stocks and Dormandy (8) procedure, as modified by Bidder and Jaeger (5). The final 370 0006-2944i8.F
$3.00
CopYright u IYXS by Acadenuc Prer\. Inc All rights of reproduction in any form reserved.
ETHANOL
371
AND RAT LIVER LIPID PEROXIDATION
composition of the incubation mixture was 6.5 mM H202, 1.6 mM sodium azide, and erythrocyte suspension in phosphate-buffered saline (14 mg hemoglobin per ml incubation mixture). Lipid peroxidation was assayed by measurement of malondialdehyde (MDA) production during a 2-hr incubation period at 37°C. Values were expressed as nanomoles of MDA per gram of hemoglobin. Hemoglobin concentration of erythrocyte suspension was measured by Drabkin’s reagent. RESULTS AND DISCUSSION Studies of chronic ethanol consumption by rats have continued the controversy by providing evidence in favor of (9-13) and against (14-17) involvement of lipid peroxidation. The widely used indicator of lipid peroxidation in these studies is the determination of MDA in preincubated liver homogenates with thiobarituric acid (TBA) (11,12,15,17). However, it has been suggested that a TBA-reacting substance value after preincubation of tissue homogenates does not correlate well with in vivo peroxidation, and that a TBA test done with fresh homogenates gives a true value (18). For this reason, in the present study, lipid peroxides were measured in fresh liver homogenates following chronic ethanol ingestion in rats. Ingestion of ethanol for 1 and 2 months led to a significant increase in hepatic lipid peroxide levels (19.2 and 53.4%, respectively) (Table 1). Our results are in accordance with other investigators who have reported that lipid peroxidation has been stimulated after chronic ethanol consumption in rats (9-13). However, the influence of chronic ethanol exposure on plasma and erythrocyte lipid peroxide levels in rats has not been previously examined. We have observed a slight, but significant increase (10.4%) in plasma lipid peroxide levels after 1 month of ethanol treatment, whereas an obvious increase (40.8%) is seen after 2 months. In addition, the erythrocyte susceptibility to lipid peroxidation following ethanol ingestion for 2 months was significantly elevated (22.7%) but no significant TABLE I The Liver, Plasma and Erythrocyte Lipid Peroxide Levels following Ethanol Ingestion for 1 and 2 Months Treatment Hepatic lipid peroxide (532-520 nm) x10’
Plasma lipid peroxide (nmole MDA/ml plasma)
Erythrocyte lipid peroxide (nmole MDA/g Hb/2 hr)
I month
2 months
Glucose Ethanol Effect (%) P
99 2 8.6 118 t 12.8 + 19.2 0.01
101 -+ 8.6 155 + 12.9 + 53.4 0.001
Glucose
4.8 + 0.39 5.3 t 0.45 + 10.4 0.05
4.9 k 0.52 6.9 -t 0.44 + 40.8 0.001
Ethanol Effect (%) P Glucose Ethanol Effect (%) P
995 + 79.0 1010 2 74.3 k 1.5 NS
1015 5 83.7 1246 f 102.2 + 22.7 0.01
Nore. Values represent means and their standard deviations. Each group contained eight rats. Experiments were performed following an overnight (16 hr) fast.
UYSAL ET AL.
372
difference in erythrocyte lipid peroxide levels was observed in rats treated with ethanol for 1 month (Table I). On the other hand, it has been reported that decreased erythrocyte glutathione peroxidase activity f 19) and slight hemolysis (20) were observed in rats chronically given ethanol. Thus, it may be considered that increased peroxidation may contribute to the hemolysis due to chronic ethanol intake in rats, as observed in humans. As a conclusion, elevated plasma lipid peroxide levels and erythrocyte SUSceptibility to lipid peroxidation may reflect stimulated lipiperoxidation in rat liver following chronic ethanol ingestion. SUMMARY
The effect of ingestion of water containing 20% ethanol for 1-2 months on lipid peroxide levels of liver, plasma, and erythrocyte was investigated in rats. Our results show that elevated plasma lipid peroxide levels and erythrocyte susceptibility to lipid peroxidation may reflect stimulated lipid peroxidation in rat liver following chronic ethanol ingestion. REFERENCES 1. Suematsu, T., Matsumur~, T.. Sato, N.. Mi~~moto, T.. Ooka, T., Kamada. T., and Abe. H. Ahho~. C&i. Exp. Res. 5, 427 119811. 2. Shaw, S., Rubin, K. P., and Lieber, C. S.. Digest. LG. Sri. 28, SXT (1983). 3. Goebel. K. M., Goebel, F. D., Schubotz, R., and Schneider. J.. J. Lab. C/in. Med. 94, I23 (1979). 4. Goebel, K. M., and Schneider, J., Aciu Biol. Med. Germ. 40, 571 (1981j. 5. Bidder, T. G., and Jaeger, P. D.. Life Sci. 30, 1021 (1982). 6. Uchiyama, M.. and Mihara, M.. Anal. Biochetn. 86, 271 il97Xi. 7. Takeuchi. N., Tanaka, F., Katayama, Y., Matsumiya. K.. and Yamamura. Y.. &p. &vonf~,i. 11, 179 (1976).
8. 9. IO. Il. 12. 13. 14. IS. 16. 17. 18. 19. 20.
Stocks, J., and Dormandy, T. L., Brir. J. ffumcctol. 20, 95 (1971). MacDonald, C. M., FEBS Lerr. 35, 227 (1973). Ki%ter, U.. Albrecht, D.. and Kappus, H., Toxicol. A&. Phc7rmsc~oi. 41, 639 t 1977). Reitz, R. C., Biochinz. Biophys. Acfu 380, 145 (1975). Remmer, H., Albrecht, D.. and Kappus. H.. I\j,,,Yn-.Schrnird~~h~~~~‘~ .G~ir. ~~~~~~}?f~~,~~~. 298, 107 11977). Sies, H., Koch. 0. R., Martino. E., and Boveds, A., FEBS Lrrl. 103, 2X7 (1979). Shaw, S.. Jayatilleke. E., Ross, W. A.. Gordon. E. R.. and Lieher, C. S.. J. Lnh. l‘lir7. :Md. 98, 417 (1981). Torrielli, M. V., Gabriel, L., and Dianzani, M. U.. 1. Pu~/zo/. 126, I1 (1978). Litov, R. E., Gee. D. L.. Downey. J. E.. and Tappel. A. I.,., Lipids 16, 32 (1981 I. Hannah, J. S., and Soares. J. H.. Jr., h’ltfr. Rep. inr. 19, 733 (1979). Mihara, M., Uchiyama, M., and Fukuzawa, K., Biochc~m. Med. 23, 302 (1980). Capel, 1. D., Willenbrock, S. J. F., Jenner, M.. and Williams, D. C.. Biochem. Phurmmwl. 29, 1737 (1980). Baraona, E.. and Lieber, C. S., Amer. .I. C/in. Nu/r. 22. 356 (1969).