PON1 Q192R polymorphism was detected using the following primers: sense 50 TATTGTTGCTGTGGGA- CCTGAG30 and reverse 50 CCTGAGAATCTGAGTAAA- TCCACT30 , (annealing temperature: 60 8C).

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 no-digested 238-bp fragment for the 192Q allele.

The analysis of PON1 promoter T(-107)C genotype frequencies showed a greater percentage of subjects with the (-107)CC genotype (27%) among octogenarians compared with the (-107)CC genotype found in the controls (14%).

PCR amplification of the PON1 T(-107)C polymorphism 0 was performed with the following primers: sense 5 AGCTAG- 0 0 CTGCGGACCCGGCGGGGAGGaG3 and reverse 5 GGC- TGCAGCCCTCACCACAACCC30 , (annealing temperature: 67 8C). The lower case base in PON1 (-107) upstream primer indicates a mismatch, introducing a restriction site for BseBI enzyme (New England Bio Labs, Cambridge, UK), because there is no specific restriction site cutting the DNA original sequence. After digestion, the (-107)C allele was identified by 28and212-bpfragments,whereasthe(-107)Talleleresultedin a no-digested 240-bp fragment.

Table 2

Alleles and genotypes distribution of PON1 promoter T(-107)C and coding region Leu55Met and Gln192Arg polymorphisms

Controls Octogenarians P Number Frequency Number Frequency PON1 T(-107)C genotype TT 64 0.32 24 0.24 0.01 TC 108 0.54 49 0.49 CC 28 0.14 27 0.27 PON1 L55M genotype Leu/Leu 84 0.42 44 0.44 n.s. Leu/Met 92 0.46 46 0.46 Met/Met 24 0.12 10 0.10 PON1 Q192R genotype Gln/Gln 20 0.10 10 0.10 n.s. Gln/Arg 92 0.46 46 0.46 Arg/Arg 88 0.44 44 0.44

DNA fragments were separated by electrophoresis on 4% high resolution agarose gel (agarose HR 3:1, Euro Clone) and identified by ethidium bromide staining. Then PON1 genotypes were confirmed by direct sequencing (377 DNA Sequencer, Applied Biosystems).

2.3. Statistical analysis

Categorical variables among paraoxonase genotypes were compared by x2 test. Allele frequencies were

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

Table 3

Serum PON1 activity and PON1 promoter T(-107)C and coding region Leu55Met and Gln192Arg polymorphisms

Multiple regression analysis of age

b SE T P-value Constant 230.568 7.914 23.863 0.001 T(-107)C 7.673 1.900 4.039 0.001 HDL-Cholesterol 0.750 0.067 11.228 0.001 PON 1 activity 0.048 0.011 4.355 0.001

Polymorphisms Genotypes PON1 activity (U/ml) Mean SD T(-107)C T/T (88) 139.25 72.17 C/T (157) 208.97 85.45 C/C (55) 372.36 117.05 ,0.001 Leu55Met Leu/Leu (128) 221.46 120.99 Leu/Met (138) 215.09 116.31 Met/Met (34) 220.97 121.28 n.s. Gln192Arg Gln/Gln (132) 226.60 114.55 Gln/Arg (138) 218.97 122.36 Arg/Arg (30) 180.30 144.43 n.s.

Regression coefficient ðbÞ; standard error of the regression coefficient (SE), t value of band its significance level (P-value).

These differences were significantly ðP , 0001Þ detect- able in the two groups (octogenarians and younger subjects). Linear regression analysis (Table 5) demonstrated that besides the T(-107)C promoter polymorphism, HDL-C and PON1 activity entered in the model, thus, indicating an association between the PON1 promoter T(-107)C poly- morphisms, HDL-C levels PON1 activity and longevity.

Mean values ^ SD. PON1 activity is given for substrate paraoxon. In brackets number of patients.

4. Discussion

No difference in the L55M and Q192R genotypes distribution was found in both groups.

For the first time, our results show that the PON1 gene promoter T(-107)C polymorphism could significantly affect the resistance to develop cardiovascular disorders in old subjects. A significant higher percentage of subjects with the (-107)CC genotype was found among the octogenarians compared with controls; the (-107)CC genotype was associated with higher PON1 activity and plasma HDL levels than (-107)TT genotype.

Linkage disequilibrium was observed between the PON1 promoter T(-107)C and L55M coding region (X2 ¼ 33: 34; : P , 0 001) polymorphisms and between L55M and Q192R coding region polymorphisms (X2 ¼ 14: 65; P , 0: 005).

No significant differences were detected among PON1 polymorphisms genotypes with respect to age, sex, BMI, smoking habit, diastolic and systolic blood pressure, lipid and glucose levels in both groups of subjects. There were significant differences among T(-107)C genotypes with respect to PON1 activity and HDL-C levels in both groups. The (-107)TT genotype showed the lowest values of PON1 activity, the (-107)CC genotype showed the highest ones and heterozygous genotypes had intermediate values (Table 3). Similarly, the (-107)CC genotype showed the highest levels : of HDL-C than (-107)TT genotype (P , 0 001; Table 4).

Until now, no investigation on the association between PON1 promoter T(-107)C polymorphism and atherosclero- tic disease has been performed in subjects aged 80 years and over. Indeed, the T(-107)C polymorphism has been found to be associated with CAD in younger but not in elder subjects (James et al., 2000, 2001). In this context, the PON1 function is particularly relevant as PON1 is sensitive to oxidative stress (Hayek et al., 1997) and could loose its activity with ageing (Beckman and Ames, 1998), thus, compromising the PON1 protective role on atherosclerosis and masking the impact of PON1 promoter polymorphism

HDL levels and PON1 promoter T(-107)C and coding region and Gln192Arg polymorphisms

Polymorphisms Genotypes HDL-C (mmol/l) Mean SD T(-107)C T/T (88) 1.31 0.28 C/T (157) 1.45 0.21 C/C (55) 1.72 0.29 ,0.001 Leu55Met Leu/Leu (128) 1.43 0.24 Leu/Met (138) 1.45 0.27 Met/Met (34) 1.43 0.24 n.s Gln192Arg Gln/Gln (132) 1.44 0.28 Gln/Arg (138) 1.43 0.22 Arg/Arg (30) 1.44 0.25 n.s.