Hypertension was defined as BP ^ 140/90 or current use of anti hypertensive medication. Diabetes mellitus was defined according to the American Diabetes Association (Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus, 1997) or participant was taking insulin or oral hypoglycemic agents. Hypercholes- terolemia was defined on the basis of total cholesterol levels ^ 6 mmol/l.

mutations in the promoter region of PON1 gene (Leviev and James, 2000). In particular, the T(-107)C promoter poly- morphism has been shown to have a dominant effect on PON1 gene expression and enzymatic activity (Leviev and James, 2000), thereby it has been investigated as an independent predictor of atherosclerotic disease (James et al., 2000, 2001). The (-107)TT genotype, has been found to associate with low PON1 activity and has been found to be a risk factor for CAD (James et al., 2000, 2001). The aim of the present study was to find out whether PON1 gene promoter region T(-107)C, and coding region Q192R and L55M polymorphisms may influence the resistance to develop cardiovascular events in Sicilian healthy octogenarians.

Plasma lipids and blood glucose levels were measured by routine enzymatic methods. HDL-cholesterol was deter- mined after precipitation of the Apo B-containing lipopro- teins with magnesium phosphotungstate. LDL-cholesterol was calculated by the Friedewald formula.

Paraoxonase activity was determined with spectrophoto- metric assay with paraoxon (O,O-diethil-O-p-nitrophenyl- phosphate) as substrate. Serum was added to Tris–HCl buffer (100 mmol/l at pH 8) containing 2 mmol/l CaCl and 2 5 mmol/l paraoxon or 1 mmol phenyl acetate (Sigma

Table 1

Characteristics of the controls and octogenarians subjects

Controls n: ¼ Octogenarians P 200 sex (m/f) n: ¼ 100 sex 96/104 (m/f) 49/51 Mean SD Mean SD Age (years) 51.3 12.6 84.1 3.6 0.001 Body mass index (kg/m2) 24.4 0.4 24.2 0.5 n.s. SBP (mmHg) 133.3 11.3 133.9 12.1 n.s. DBP (mmHg) 69.3 10.9 67.8 10.6 n.s. Total cholesterol (mmol/l) 5.2 0.7 5.4 0.8 0.01 HDL-cholesterol (mmol/l) 1.4 0.2 1.6 0.2 0.001 Triglycerides (mmol/l) 1.4 0.3 1.5 0.3 n.s. LDL-C (mmol/l) 3 0.8 3 0.8 n.s. Glucose (mmol/l) 5.61 1.9 5.69 1.9 n.s. PON1 activity (U/l) 192 98.2 271.3 137.1 0.001 Diabetes (n%) 24 (12%) 11 (11%) n.s. Smokers (n%) 5 (7.5%) 7 (7%) n.s. Hypertension (n%) 25 (12.5%) 12 (12%) n.s. Hypercholesterolemia (n%) 40 (20%) 19 (19%) n.s.

2. Methods

2.1. Subjects and methods

The study population was composed by 100 healthy octogenarians (49 men and 51 women, mean age ^ 84.18 3.6 years) recruited from all the surroundings of Messina with the help of the local general practitioners and 200 healthy controls (96 men and 104 women, mean ^ age 51.34 12.6 years) recruited from the Hospital staff (doctors, nurses and medical orderly). All of them were unrelated and of Sicilian origin. The baseline examination consisted of a home interview and a clinic examination. Specific details of medical history were confirmed by contacting the participant’s physician. Informed consent was obtained from all subjects and the study was approved by Ethic Committee of the University of Messina.

Mean values ^ SD; SBP: systolic blood pressure; DBP: diastolic blood pressure; HDL: high density lipoprotein; LDL: low density lipoprotein. PON1 activity is given for substrate paraoxon.

Campo et al. / Experimental Gerontology 39 (2004) 1089–1094

Chemical Co). The hydrolysis of paraoxon was continuously monitored at 412 nm, 25 8C (molar extinction coefficient of p-nitrophenol at 412 nm: 16.900 mol/l21· cm21). Non-enzy- matic hydrolysis of paraoxon was subtracted from the total rate of hydrolysis. PON1 activity was expressed in U/l; 1 unit is equivalent to 1 mmol/min of p-nitrophenol.

calculated by the gene counting method Hardy–Weinberg equilibrium. Assumption of normal distribution for con- tinuous variables was tested by Kolmogorov–Smirnov test of goodness of fit. Non-normally distributed variables were compared by Kruskal–Wallis, non-parametric combination test and with multiple comparisons.

Multiple regression analysis was used to assess the contribution of age in the study. P-values lesser than 0.05 were considered to indicate statistical significance and P-values were based on two-sided test.

2.2. Genotype determination

The DNA was extracted from the whole blood according to standard procedures. PON1 genotypes were determined by PCR amplification and restriction analysis. PCRs reaction (total volume 25 ml) contained 150 ng DNA template, 200 mM dNTPs, 400 nM of each primer, 1.5 mM MgCl and 0.6 of Taq polymerase (Perkin–Elmer 2 cetus, USA) 35 cycles of amplification (3 min at 94 8C followed by 30 s at 95 8C, 20 s at specific primers annealing temperature and 50 s at 72 8C) with a final extension of 10 min at 72 8C were performed. PCR amplification of PON1 L55M polymorphism was performed using the 0 following primers: sense 5 CCTGCAATAATATGAAAC- AACCTG30 and reverse 50 TGAAAGACTTAAACTGCC- AGTC30 (annealing temperature: 63 8C).

3. Results

between controls and octogenarians. The old people group displayed significant higher levels of total cholesterol ðP , 0: 05Þ; HDL-C ðP , 0: 001Þ and PON1 activity ðP , 0: 001Þ: 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 com- parable between the two groups. All variables were similar between male and female subjects, except for PON1 activity ^ levels, 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 no- digested 172-bp fragment for the 55L allele.