Protective Effect of Paraoxonase Activity in High-Density Lipoproteins against Erythrocyte Membranes Peroxidation: A Comparison between Healthy Subjects and Type 1 Diabetic Patients

0021-972X/04/$15.00/0 Printed in U.S.A. The Journal of Clinical Endocrinology & Metabolism 89(6):2957–2962 Copyright © 2004 by The Endocrine Society doi: 10.1210/jc.2003-031897 Protective Effect of Paraoxonase Activity in HighDensity Lipoproteins against Erythrocyte Membranes Peroxidation: A Comparison between Healthy Subjects and Type 1 Diabetic Patients G. FERRETTI, T. BACCHETTI, D. BUSNI, R. A. RABINI, AND G. CURATOLA Istituto di Biochimica (G.F., T.B., G.C.), Università Politecnica delle Marche, 60131 Ancona, Italy; Unità Operativa di Dietetica e Nutrizione Clinica (D.B.), Azienda Ospedaliera Umberto I, 60020 Ancona, Italy; and Unità Operativa di Diabetologia (R.A.B.), Istituto Nazionale di Riposo e Cura per Anziani, 60131 Ancona, Italy High-density lipoproteins (HDL) plays a key role in the protection against oxidative damage of lipoprotein and biological membranes. The aim of the present study was to investigate the relationship between the antioxidant role of HDL and the HDL-paraoxonase (PON) activity in healthy subjects and in type 1 diabetic patients. Moreover, the ability of HDL of controls and diabetic patients to protect and/or repair biological membranes from oxidative damage was studied. HDL were isolated from 31 type 1 diabetic patients and 31 sex- and age-matched healthy subjects and immediately used to evaluate lipid hydroperoxides and HDL-PON activity. Erythrocyte membranes obtained from healthy subjects were oxidized with 2,2-azo-bis(2-aminidinopropane)dihydrochloride and then incubated in the presence of HDL isolated from healthy or type 1 diabetic subjects, with measurements of membrane lipid hydroperoxides before and after the incubation. HDL from type 1 diabetic patients showed higher levels of lipid hydroperoxides and a lower activity of HDL-PON than healthy subjects. Moreover, HDL of type 1 diabetic patients H IGH-DENSITY LIPOPROTEINS (HDL) have been shown to be inversely correlated with the risk of atherosclerosis and coronary heart disease (1). The protective effect of HDL has been related to their role in the cholesterol reverse transport (from peripheral tissues to the liver) and to their ability to inhibit oxidation of low-density lipoproteins (LDL) and biological membranes (2, 3). Using different models of Cu2⫹-oxidized cells, HDL have been demonstrated to exert a protective role also against oxidative damage of cells (4, 5). It has been suggested that the antioxidant properties of HDL might be related to their ability to accept phospholipids containing hydroperoxides from oxidized membranes or lipoproteins (4). A transfer of hydroperoxides from oxidized LDL to HDL and an exchange of lipid peroxidation products between the lipoproteins were previously demonstrated (3). Several lines of evidence suggest that the antioxidant effect Abbreviations: AAPH, 2,2-Azo-bis(2-aminidinopropane)dihydrochloride; HbA1c, glycated hemoglobin A1c; HDL, high-density lipoprotein(s); LCAT, lecithin:cholesterol acyltransferase; LDL, low-density lipoprotein(s); ns, not significant; PON, paraoxonase. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community. protected less efficiently erythrocyte membranes against oxidative damage compared with HDL from healthy subjects. A negative correlation was found between HDL-PON activity and the levels of hydroperoxides of HDL, confirming the relationship between PON and lipid peroxidation and suggesting that subjects with low PON activity are more exposed to oxidative damage than subjects with high PON activity. The ability of HDL to protect erythrocyte membranes was positively correlated with HDL-PON activity and negatively correlated with the levels of lipid hydroperoxides of HDL of healthy subjects. These results confirm a linkage between PON activity and lipid peroxidation of lipoproteins and suggest that the ability of HDL to protect erythrocyte membranes might be related to the PON activity. It might be hypothesized that the decrease of PON activity in diabetic patients and the lower HDL protective action against membrane peroxidation could contribute to acceleration of arteriosclerosis in type 1 diabetes mellitus. (J Clin Endocrinol Metab 89: 2957–2962, 2004) of HDL is at least partially related to paraoxonase (PON), an enzyme associated with HDL surface (HDL-PON). In fact, previous studies have shown that PON is able to hydrolyze preformed lipid hydroperoxides and to delay or inhibit the initiation of oxidation induced by metal ions on lipoproteins (6, 7). The enzymatic activity of HDL-PON varies widely among healthy humans, and it has been suggested that subjects with low PON activity may have a greater risk of developing diseases in which oxidative damage and lipid peroxidation are involved, compared with subjects with high PON activity (8). Moreover, previous studies have shown a relationship between PON activity and the antioxidant properties of HDL (9, 10) and the susceptibility of HDL to atherogenic modifications induced in vitro, such as glycation and homocysteinylation (11, 12). Diabetes is associated with oxidative damage (13), and it has been suggested that the higher levels of lipid peroxidation products in plasma of diabetic patients (14) could be related to a higher susceptibility of plasma lipoproteins of diabetic patients to oxidation (15, 16) and/or to a decrease of plasma antioxidant defenses (17). A decrease of PON activity has been also observed in diabetic patients (18 –20). Abnormal HDL composition and altered HDL subclasses 2957 2958 J Clin Endocrinol Metab, June 2004, 89(6):2957–2962 distribution have also been observed in patients with type 1 diabetes (21). The compositional changes of HDL are reflected in modifications of functional activities, with a lower protection exerted by HDL from diabetic patients against LDL oxidation than HDL from healthy subjects (22) and a decreased capacity to induce cholesterol efflux from biological membranes (23). The ability of HDL of diabetic patients to protect biological membranes from oxidative damage has not been investigated previously. The aim of this study was to further investigate the relationship between the antioxidant role of HDL and the HDL-PON activity in healthy subjects and in type 1 diabetic patients. Moreover, we compared the ability of HDL of controls and diabetic patients to protect and/or repair biological membranes from oxidative damage. The compositional changes of red blood cell membranes oxidized in vitro with 2,2⬘-azo-bis-(2-amidinopropane) have been well characterized (24). Therefore, oxidized erythrocytes represent a useful model to investigate the protective effect exerted by HDL against oxidative damage of biological membranes. Subjects and Methods Subjects Thirty-one type 1 diabetic patients (16 women and 15 men, 42 ⫾ 8 yr old; duration of disease, 12 ⫾ 5 yr) and 31 healthy subjects (18 women and 13 men; 35 ⫾ 8 yr old) (...truncated)