Original Contribution LIPID PEROXIDATION CONTRIBUTES TO IMMUNE REACTIONS ASSOCIATED WITH ALCOHOLIC LIVER DISEASE ELISA MOTTARAN,* STEPHEN F. STEWART, † ROBERTA ROLLA,* DARIA VAY,* VALENTINA CIPRIANI,* MARIAGRAZIA MORETTI,* MATTEO VIDALI,* MASSIMO SARTORI, ‡ CRISTINA RIGAMONTI, ‡ CHRISTOPHER P. DAY, † and EMANUELE ALBANO* *Department of Medical Sciences, University of East Piedmont, Novara, Italy; † Centre for Liver Research, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, UK; and ‡ Medical Clinic, University of East Piedmont, Novara, Italy (Received 6 August 2001; Accepted 4 October 2001) Abstract—Increasing evidence indicates the involvement of immune reactions in the pathogenesis of alcoholic liver disease. We have investigated whether ethanol-induced oxidative stress might contribute to immune response in alcoholics. Antibodies against human serum albumin modified by reaction with malondialdehyde (MDA), 4-hy- droxynonenal (HNE), 2-hexenal, acrolein, methylglyoxal, and oxidized arachidonic and linoleic acids were measured by ELISA in 78 patients with alcoholic cirrhosis and/or hepatitis, 50 patients with nonalcoholic cirrhosis, 23 heavy drinkers with fatty liver, and 80 controls. Titers of IgG-recognizing epitopes derived from MDA, HNE, and oxidized fatty acids were significantly higher in alcoholic as compared to nonalcoholic cirrhotics or healthy controls. No differences were instead observed in the titers of IgG-recognizing acrolein-, 2-hexenal-, and methylglyoxal-modified albumin. Alcoholics showing high IgG titers to one adduct tended to have high titers to all the others. However, competition experiments showed that the antigens recognized were structurally unrelated. Anti-MDA and anti-HNE antibodies were significantly higher in cirrhotics with more severe disease as well as in heavy drinkers with cirrhosis or extensive fibrosis than in those with fatty liver only. We conclude that antigens derived from lipid peroxidation contribute to the development of immune responses associated with alcoholic liver disease. © 2001 Elsevier Science Inc. Keywords—Oxidative stress, Ethanol, Aldehyde adducts, Alcohol toxicity, Liver injury, Free radicals INTRODUCTION The etiology of alcoholic liver disease is poorly under- stood, but recent evidence points towards a possible contribution of immunological reactions against hepatic antigens [1,2]. The development of such reactions has been ascribed to the formation of new antigens as a result of the interaction between liver proteins and reactive metabolites originating from ethanol metabolism [3,4]. In particular, antibodies recognizing protein adducts of acetaldehyde and hydroxyethyl free radicals have been identified in the sera of alcohol-fed rats and in patients with alcoholic liver disease [5– 8]. Nonetheless, the pos- sible contribution of other antigens to immunological reactions in alcoholic liver disease cannot be excluded. Increasing evidence indicates that oxidative damage is an important feature of human alcoholic liver disease [9]. Protein modifications induced by oxidative damage, and particularly the formation of protein adducts with lipid peroxidation products such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE) can be detected in the liver of ethanol-fed rats [10,11]. Furthermore, Tuma and col- leagues have demonstrated that the livers of ethanol-fed rats contain mixed adducts derived from the combined reaction of acetaldehyde and MDA, also known as ma- londialdehyde-acetaldehyde adducts [12]. Aldehyde-modified proteins are highly immunogenic and both monoclonal and polyclonal antibodies towards epitopes derived from different lipid peroxidation prod- ucts have been produced following animal immunization with these adducts [13,14]. Recent studies have shown that chronic ethanol exposure in both experimental ani- mals and humans is associated with the development of specific IgG-recognizing epitopes derived from MDA Address correspondence to: Prof. Emanuele Albano, Department of Medical Science, University “Amedeo Avogadro” of East Piedmont, Via Solaroli 17, 28100 Novara, Italy; Tel: +39 (0321) 660642; Fax: +39 (0321) 620421; E-Mail: albano@med.unipmn.it. Free Radical Biology & Medicine, Vol. 32, No. 1, pp. 38 – 45, 2002 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/02/$–see front matter PII S0891-5849(01)00757-2 38