ALCOHOL-INDUCED ALTERATIONS IN BLOOD AND ERYTHROCYTE MEMBRANE IN DIABETICS

M. Paramahamsa, S. Aparna and N. Varadacharyulu,*

Department of Biochemistry, Sri Krishnadevaraya University, Anantapur 515003, A. P., India

Received 4 August 2000; in revised form 5 June 2001; accepted 5 July 2001


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
— Three groups consisting of 12 volunteers each, of alcoholic type 2 diabetics (non-insulin-dependent diabetes mellitus, NIDDM), non-alcoholic type 2 diabetics (NIDDM) and controls (teetotallers) were selected for assessing changes in lipids of plasma and erythrocyte membranes. Significant changes in cholesterol and phospholipids were observed in alcoholic diabetics suggesting a regulatory compensatory mechanism to resist the fluidizing action of ethanol as well as rigidification of erythrocyte membrane. A significant increase in lipid peroxidation of erythrocytes was observed, indicating the damage associated with disrupted organization, structure and function of membranes in alcoholic diabetics.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Chronic excessive consumption of alcohol (ethanol) may lead to deleterious effects upon many organs and metabolism (Gatti et al., 1993Go; Xu et al., 1998Go; Farren and Tipton, 1999Go). However, moderate intake of alcohol was reported to be beneficial in many disease conditions, including diabetes mellitus (Klatsky et al., 1992Go; Goldberg et al., 1999Go). Alcohol enhances insulin sensitivity in type 2 diabetics (Eagles and Martin, 1998Go; Wiel, 1998Go). Diabetes is associated with complications such as coronary heart disease, retinopathy, neuropathy and microangiopathy, and these complications have been attributed to membrane damage (Selvam and Anuradha, 1988Go; Gatti et al., 1993Go; Wei et al., 2000Go). As consumption of alcohol was widely prevalent in humans, including diabetics, the present study was undertaken to investigate alterations in lipids of plasma and red cell membranes in type 2 diabetics and the effect of chronic alcohol consumption in diabetics.


    MATERIALS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Subjects for study
Three groups of 12 male volunteers, aged 38–50 years with similar dietary habits, were selected for the study. Group I consisted of type II diabetics, group II were diabetics consuming alcohol three or four times a week and on each occasion ~250 ml of different brands of liquor containing 40–50% ethanol for the past 5–10 years, and group III were teetotallers (non-alcoholics and non-diabetics). Volunteers involved in the present experimentation were well informed and their consent was obtained. The use of all other drugs was discontinued for 2–3 days prior to each study and the subjects were kept under a physician's observation.

Sampling and assay methods
Blood samples were collected into EDTA-containing tubes by venepuncture and analysed immediately for plasma phospholipids and cholesterol by the methods of Connerty et al. (1961) and Zlatkis et al. (1953) repectively. Red cell preparation was according to the method described by Beutler (1975) and the extent of lipid peroxidation in red cells was measured by assaying malondialdehyde (MDA) formed according to the procedure of Buege and Aust (1978). Erythrocyte membrane preparations were obtained by the method of Dodge et al. (1963) and analysed for phospholipids and cholesterol (Jain et al., 1998). Membrane protein was assayed by the method of Lowry et al. (1951).

Chemicals
Thiobarbituric acid was obtained from Sigma Chemical Company, St Louis, MO, USA. All other chemicals were of analytical grade from Spectrochem Pvt. Ltd, Mumbai, India and Qualigens Fine Chemicals, Mumbai, India.

Statistical analysis
The data obtained in the present study were subjected to Duncan's multiple range test.


    RESULTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The results are presented in Table 1Go. Plasma cholesterol and phospholipids were significantly higher in the diabetic and alcoholic diabetic groups, as compared to controls. However, there was no significant difference in these parameters between the alcoholic and the non-alcoholic diabetic groups. The plasma cholesterol/phospholipid (C/P) ratio was 17% lower in the diabetics, compared to controls, whereas an intermediate ratio was observed in the alcoholic diabetics. The cholesterol content in erythrocyte membrane was found to be significantly higher in both diabetic groups when compared with controls, whereas no significant difference was noticed between the former two groups.


View this table:
[in this window]
[in a new window]
 
Table 1. Alterations in lipid constituents of plasma and erythrocyte membrane
 
However, the membrane phospholipids were 4-fold higher in the diabetics than in controls. By contrast, a significant decrease was observed in the alcoholic diabetics compared to controls and the diabetics. The membrane C/P ratio was accordingly 70% lower in the diabetics, but 71% higher in the alcoholic diabetics, than in controls. Erythrocyte lipid peroxidation was 4-fold and 6-fold higher respectively in the diabetics and the alcoholic diabetics, compared to the control group.


    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The observed hypercholesterolaemia and hyperphospholipidaemia in diabetics in the present study agree with the findings of earlier reports (Ben et al., 1991Go; Zargar et al., 1995Go; Wei et al., 2000Go). Though cholesterol can be synthesized from acetate, its biosynthesis in diseased conditions might be augmented by some unknown mechanism(s) and/or the catabolic process may not keep balance with it, resulting in accumulation of cholesterol in blood. An elevation in plasma phospholipids in diabetics has been well documented and has been attributed to diet coupled with abnormal fat metabolism in diabetes (Rao et al., 1967Go; Zargar et al., 1995Go). Furthermore, no significant increase in plasma cholesterol and phospholipids was observed in the alcoholic diabetics, compared with the diabetics, in the present study. While the plasma and erythrocyte membrane phospholipid contents were respectively 46 and 300% higher in the diabetics, plasma phospholipids were 60% higher and membrane phospholipids were 28% lower in the alcoholic diabetics, compared to controls. Jain et al. (1988) observed no change in the plasma C/P ratio in alcoholics. The findings of the present study in alcoholic diabetics, compared to diabetics, could not be attributed to an influence of alcohol on these two parameters in plasma.

The similar increases observed in the present study in the cholesterol contents of erythrocyte membranes of diabetics and alcoholic diabetics can be correlated to enhanced cholesterol levels in plasma in these two groups. There is evidence to indicate an accelerated transfer of cholesterol from plasma to erythrocyte membranes in many disease conditions, including diabetes, and also to ethanol consumption (Daniels and Goldstein, 1982Go). The decreased phospholipid content observed in alcoholic diabetic erythrocyte membranes seems to be independent of plasma phopholipids. However, a decrease in phospholipids has been reported in other membranes, such as hepatic (Cairns and Peters, 1983Go), mitochondrial (Schilling and Reitz, 1980Go) and erythrocyte membranes (Jain et al., 1988Go) in non-diabetic alcoholics. The role of membrane-bound lipase in altering phospholipid composition in the alcoholic diabetics in the present study cannot be ruled out.

A marked decrease in C/P ratio in diabetic erythrocyte membranes was observed in the present study, which may indicate fluidization of membranes, which is in agreement with the report of Selvam and Anuradha (1988) in non- alcoholic diabetics. It is interesting to note that the increased C/P ratio in alcoholic diabetics indicates rigidification of membranes and the possibility of operation of a regulatory compensatory mechanism in chronic alcoholic diabetics to resist the fluidization of the membrane.

The observed increase in erythrocyte lipid peroxidation of diabetics is in agreement with an earlier report (Selvam and Anuradha, 1988Go). Ethanol-induced lipid peroxidation is well documented (Hrelia et al., 1986Go; Gatti et al., 1993Go; Morell et al., 1998Go). The enhanced free radical generation by ethanol has been reported to be mediated by 2,3-butanediol and 1,2-propanediol, the two novel metabolites of alcohol in alcoholics (Xu et al., 1998Go; Wei et al., 2000Go). Although results from the present study indicate the beneficial effects of alcohol consumption by diabetics in bringing about an anti-fluidizing effect on membranes, further enhancement of lipid peroxidation in alcoholic diabetics suggests further damage to membrane organization and function.


    ACKNOWLEDGEMENTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The authors are grateful to Professor K. Venkateshwarlu of Microbiology and Professor P. Ramakrishna Rao of Biochemistry, S K University Anantapur, India for suggestions and Dr Baba Younus, Physician for help. This study was supported, in part, by the University Grants Commission (F.3-11/97), New Delhi, India.


    FOOTNOTES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
* Author to whom correspondence should be addressed. Back


    REFERENCES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Ben, G., Luigi, G., Gnudi, L., Maran, A., Gigante, A., Duner, E., Lori, E., Tiengo, A. and Avogaro, A. (1991) Effect of chronic alcohol intake on carbohydrate and lipid metabolism in subjects with type II (noninsulin-dependent) diabetes. American Journal of Medicine 90, 70–76.[ISI][Medline]

Beutler, E. (1975) The preparation of red cells for assay. In Red Cell Metabolism. A Manual of Biochemical Methods, 2nd edn, Beutler, E. ed., pp. 8–11. Grune & Stratton, New York.

Buege, J. A. and Aust, S. D. (1978) Microsomal lipid peroxidation. In Methods in Enzymology, Vol. 52, Fleischer, S. and Packer, L. eds, pp. 302–310. Academic Press, New York.

Cairns, S. R. and Peters, T. J. (1983) Biochemical analysis of hepatic lipid in alcoholic diabetic and control subjects. Clinical Science 65, 645–648.[ISI][Medline]

Connerty, H. V., Briggs, A. R. and Eaton, E. H., Jr (1961) Determination of serum phospholipids, lipid phosphorous. In Practical Clinical Biochemistry, 4th edn, Varley, H. ed., pp. 319–320. CBS Publishers, India.

Daniels, C. K. and Goldstein, D. B. (1982) Movement of free cholesterol from lipoproteins on lipid vesicles into erythrocytes. Acceleration by ethanol in vitro. Molecular Pharmacology 21, 694–700.[Abstract]

Dodge, J. T., Mitchell, C. and Hanahan, D. J. (1963) The preparation and chemical characteristics of haemoglobin free ghosts of human erythrocytes. Archives of Biochemistry and Biophysics 100, 119–130.[ISI]

Eagles, C. J. and Martin, U. (1998) Non-pharmacological modification of cardiac risk factors: part 3. Smoking cessation and alcohol consumption. Journal of Clinical Pharmacology and Therapeutics 23, 1–9.

Farren, C. K. and Tipton, K. F. (1999) Trait markers for alcoholism: clinical utility. Alcohol and Alcoholism 34, 649–665.[Abstract/Free Full Text]

Gatti, P., Viani, P., Cervato, G., Testolin, G., Simonetti, P. and Cestaro, B. (1993) Effects of alcohol abuse: studies on human erythrocyte susceptibility to lipid peroxidation. Biochemistry and Molecular Biology International 30, 807–817.[ISI][Medline]

Goldberg, D. M., Soleas, G. J. and Levesque, M. (1999) Moderate alcohol consumption: the gentle face of Janus. Clinical Biochemistry 32, 505–518.[ISI][Medline]

Hrelia, S., Lercker, G., Biagi, P. L., Bordoni, A., Stefanini, F., Zunarelli, P. and Rossi, C. A. (1986) Effect of ethanol intake on human erythrocyte membrane fluidity and lipid composition. Biochemistry International 12, 741–750.[ISI][Medline]

Jain, S., Taranath Shetty, K., Ray, R. and Janakiramaiah, N. (1988) Erythrocyte lipids in alcohol dependence. Indian Journal of Medical Research 88, 530–535.[ISI][Medline]

Klatsky, A. L., Armstrong, M. A. and Friedman, G. D. (1992) Alcohol and mortality. Annals of Internal Medicine 117, 646–654.[ISI][Medline]

Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265–275.[Free Full Text]

Morell, F. B., Soriano, F. M., Colell, A., Checa, C. F. and Romero, F. J. (1998) Chronic ethanol feeding induces cellular antioxidants decrease and oxidative stress in rat peripheral nerves. Effect of S-adenosyl-l-methionine and N-acetyl-cysteine. Free Radical Biology and Medicine 25, 365–368.[ISI][Medline]

Rao, M. B., Chitre, R. G. and Patel, J. C. (1967) Some biochemical findings in diabetes and their significance relating to atherosclerosis. Indian Journal of Medical Sciences 22, 171–174.

Schilling, R. J. and Reitz, R. C. (1980) A mechanism for ethanol induced damage to liver mitochondrial structure and function. Biochimica et Biophysica Acta 603, 266–277.[ISI][Medline]

Selvam, R. and Anuradha, C. V. (1988) Lipid peroxidation and antiperoxidative enzyme changes in erythrocytes in diabetes mellitus. Indian Journal of Biochemistry and Biophysics 25, 268–272.[ISI][Medline]

Wei, M., Gibbons, L. W., Mitchell, T. L., Kampert, J. B. and Blair, S. N. (2000) Alcohol intake and incidence of type 2 diabetes in men.Diabetes Care 23, 18–22.[Abstract]

Wiel, V. D. A. (1998) Alcohol and insulin sensitivity. Netherlands Journal of Medicine 52, 91–94.[ISI][Medline]

Xu, D., Amardeep, S. D., Abelmann, A., Croft, K., Peters, T. J. and Palmer, T. N. (1998) Alcohol-related diols cause acute insulin resistance in vivo. Metabolism 47, 1180–1186.[ISI][Medline]

Zargar, A. H., Wandroo, F. A., Wadhwa, M. B., Laway, B. A., Masoodi, S. R. and Shah, N. A. (1995) Serum lipid profile in non-insulin-dependent diabetes mellitus associated with obesity. International Journal of Diabetes in Developing Countries 15, 9–13.

Zlatkis, A., Zak, B. and Boyle, A. J. (1953) A new method for the direct determination of serum cholesterol. Journal of Laboratory and Clinical Medicine 4, 486–492.