Experimental Gerontology 39 (2004) 1089-1094 www.elsevier.com/locate/expgero Association between serum paraoxonase (PON1) gene promoter T(-107)C polymorphism, PON1 activity, and HDL levels in healthy Sicilian octogenarians. S. Campo, Maria A. Sardo, Giuseppe Trimarchi, Michele Bonaiuto, Luisa Fontana, Maria Castaldo, Antonio Bonaiuto, Carlo Saitta, Alessandra Bitto, Bruno Manduca, Stefania Riggio, Antonino Saitta* Department of Internal Medicine, 82 Via Camiciotti, Messina 98123, University of Messina, Italy; Institute of Statistical Science, 82 Via Camiciotti, Messina 98123, University of Messina, Italy. Received 14 November 2003; received in revised form 3 March 2004; accepted 5 March 2004 Abstract Age is associated with enhanced low-density lipoprotein (LDL) oxidation and atherosclerosis. Thus, subjects over 80 years without cardiovascular disease provide a model to investigate the protective factors against atherosclerosis. Serum paraoxonase (PON1), a high-density lipoprotein (HDL)-bound enzyme prevents LDL oxidation. The aim of the present study was to evaluate the contribution of the PON1 promoter T(-107)C and coding region Gln192Arg (Q192R) and Leu55Met (L55M) polymorphisms to the resistance to developing cardiovascular events in healthy Sicilian octogenarians. Distribution of PON1 genotypes and activity and biochemical parameters were compared between 100 octogenarians and 200 adults. Individuals in the elderly group displayed significantly higher levels of HDL-C (P<0.001) and PON1 activity (P<0.001). The analysis of PON1 genotype distribution showed a higher percentage of (-107)CC among octogenarians compared with controls. A significant difference among T(-107)C genotypes respect to PON1 activity and HDL-C levels occurred in both groups. The CC genotype was associated with higher PON1 activity and HDL levels compared to the TT genotypes. In conclusion, our results provide strong evidence that in healthy Sicilians, aging may be characterized by a low frequency of PON1 (-107)T risk allele and by a high frequency of favorable genotypes such as (-107)CC, influencing PON1 activity and HDL-C levels. q 2004 Elsevier Inc. All rights reserved. Keywords: Paraoxonase polymorphism; Octogenarians; PON 1; T(-107)C; Q192R; L55M 1. Introduction The frequency of atherosclerotic disease increases with age (Hofman et al., 1991; Fried et al., 1998). Thus, old subjects who achieve long life-spans in the absence of severe atherosclerosis-related disorders such as coronary artery disease (CAD), stroke, and peripheral artery disease can provide a model to investigate the protective factors increasing their resistance to develop atherosclerotic disease (Rowe and Kahn, 1987). Elderly persons have been suggested to have a greater risk for oxidative stress (Beckman and Ames, 1998) and low-density lipoprotein (LDL) oxidation (Jira et al., 1996), which may contribute to the increased incidence of atherosclerotic disease in this population (Steinberg et al., 1989). A decline in protective mechanisms against oxidative stress has also been found during aging (Azhar et al., 1995; Napoli et al., 1997; Lykkesfeldt et al., 1998). Moreover, conditions such as hypertension, smoking habit, diabetes, and hypercholesterolemia, which may enhance oxidative stress, have been associated with increased risk of atherosclerotic disease (Ross, 1999). Human serum paraoxonase (PON1), a high-density lipoprotein (HDL)-associated ester hydrolase enzyme, has been shown to protect LDL from oxidation by the hydrolysis of biologically active lipoperoxides (Mackness et al., 1991). Animal models, including PON1-knockout mice, demonstrated that PON1 deficiency was related to an increased susceptibility of LDL to oxidation and atherosclerosis. * Corresponding author. Tel.: - -; fax: - - E-mail address: asaitta@unime.it (A. Saitta). S. Campo et al. / Experimental Gerontology 39 (2004) 1089-1094 The role of PON1 in predicting atherosclerosis remains controversial. Several studies revealed a significant association betweencorrections are needed in the text:

2 cetus, USA) 35 cycles of amplification (3 min at 94°C followed by 30 s at 95°C, 20 s at specific primers annealing temperature and 50 s at 72°C) with a final extension of 10 min at 72°C were performed. PCR amplification of PON1 L55M polymorphism was performed using the following primers: sense 5' CCTGCAATAATATGAAACAA CCTG 3' and reverse 5' TGAAAGACTTAAACTGCCAGTC 3' (annealing temperature: 63°C). 

Between controls and octogenarians, the old people group displayed significantly higher levels of total cholesterol (P<0.05), HDL-C (P<0.001) and PON1 activity (P<0.001). No significant differences in mean BMI, blood pressure, LDL-C, triglyceride and glucose levels were found between the two groups. The number of smokers, diabetic, hypertensive and hypercholesterolemic patients was comparable between the two groups. All variables were similar between male and female subjects, except for PON1 activity levels, which were higher in males than females (241.23±123.74 vs 197.46±109.78 U/ml). PCR products were digested with Nla III (New England Bio Labs, Cambridge, UK) and the digestion resulted in 66- and 106-bp fragments for the 55M allele and in a non-digested 172-bp fragment for the 55L allele.

PON1 Q192R polymorphism was detected using the following primers: sense 5' TATTGTTGCTGTGGGACCTGAG 3' and reverse 5' CCTGAGAATCTGAGTAAATCCACT 3', (annealing temperature: 60°C). PON1 genotypes and alleles distribution are reported in Table 2. The frequencies of PON1 genotypes were comparable to those previously reported in Caucasian populations (Garin et al., 1997) and were in Hardy Weinberg equilibrium in both groups. PCR products were digested with Alw I (New England Bio Labs, Cambridge, UK) and resulted in 66- and 172-bp fragments for the 192R allele and in a non-digested 238-bp fragment for the 192Q allele.