Table 1 - Biological variables in the sample population (men, 69.8±4.0 years

Variables Mean±SD Variables Mean±SD Age (years) 69.8±4.0 Triglycerides (mmol/L)a 1.38±1.46 BMI (kg/m2) 27.1±3.3 Apolipoprotein A1 (g/L) 1.66±0.24 WHR 0.97±0.06 Apolipoprotein B (g/L) 1.10±0.21 SBP (mmHg) 140.6±14.0 Lipoprotein (a) (g/L)a 0.18±0.24 DBP (mmHg) 79.1±8.0 Apolipoprotein E (mg/L)a 41.5±10.4 Hemoglobin (g/L) 148.5±12.5 ACE (U/L) 50.4±19.5 Glucose (mmol/L) 5.41±1.42 PON (nmol/mn/mL)a 191.0±97.5 (1) Total cholesterol (mmol/L) 5.68±0.83 PON (nmol/mn/mL)a 402.4±304.9 (2) HDL-Cholesterol (mmol/L)a 1.30±0.35 Arylesterase (μmol/mn/mL)a 103.6±30.2

are means±SD; a log-transformed for statistical testing. body mass index; WHR: waist-to-hip ratio; SBP: systolic blood pressure; DBP: diastolic blood pressure; ACE: angiotensin-converting enzyme; PON: paraox- PON : basal paraoxonase activity; PON : stimulated paraoxonase activity. (1) (2)

included in the model, g (a) is the regression curve of x on i i age, a is the CA of the individual, and β is the coefficient i of regression found for each variable x. AI is the difference i between BA and CA (AI= BA – CA). By construction, AI is independent of age: its mean is zero, whatever the age, in the reference group used to estimate the regression co- efficients. The following variables were used to define BA: hearing loss; WHR; SBP; number of healthy teeth; serum glucose concentration; serum total cholesterol concen- tration (TC); mean globular volume (MGV) of red blood cells; and hemoglobin value.

gene; Gln→Arg mutation in the PON gene at codon 192; and Ile→Thr mutation in the CBS gene at position 278.

Statistical analysis

Statistical analyses were performed by BMDP® statis- tical software (University of California, Los Angeles). One-way analysis of variance (ANOVA) was used to test the associations between AI and genetic polymorphisms, concentration of lipids and apolipoproteins, and activities of ACE and PON. Linear regression analysis was used to estimate the relationships between AI and serum con- stituents. The multiple comparison test of Tukey and Scheffé was used to assess differences in mean values of AI for each candidate gene. A p-value of 0.05 was re- tained as significant.

RESULTS

The clinical and biological characteristics of the sam- ple population are listed in Table 1. The mean age of the studied sample population (69.8±4.0 years old) was older than 65, the low-limitation age for the elder- ly. All biological and morphological parameters had values consistent with the generally good health of these subjects. The allele frequencies of the six gene polymorphisms were very close to those observed in oth- er French populations and were in Hardy-Weinberg equilibrium (Table 2) (23, 24).

Eight subjects with the apoE genotype were ex- cluded from this study because of the dominant effect of apoE ε2 on apoE ε4 allele (22). Since very few subjects had apoE ε2/ε2 or apoE ε4/ε4 genotypes, subjects were classified into three groups: subjects carrying apoE ε2/ε2 and apoE ε2/ε3 genotypes; subjects with apoE ε3/ε3 genotype; and subjects carrying apoE ε4/ε3 and apoE ε4/ε4 genotypes. We also grouped for LPL 447 polymorphism, subjects having G/G genotype with sub- jects having C/G genotype (since G allele has the same ef- fects on triglyceride concentration), and for PON poly- morphism (codominance of the two alleles), subjects hav- ing Arg/Arg and Gln/Arg genotypes. Serum HDL-C, TG, Lp(a), apoE concentrations, PON activities [PON and (1) PON ] and arylesterase activity were log-transformed (2) to improve normality for statistical testing.

Table 3 shows the mean value of the AI according to genotype. The genetic polymorphism of PON at codon 192 appeared to be significantly associated with the AI (p=0.0486) according to the three genotypes. It was found that the Arg allele is the least frequent and that the value of AI for those heterozygous subjects was outside the value of the AI interval defined for all Gln homozygotes, thus grouping Arg heterozygous and homozygous carri- ers. Comparing the mean value of AI according to the Gln carrier and Arg carrier groups, we found that the p-value was 0.0237. Since a positive value for this indicator meant that aging is advanced and a negative value re- flected that the aging process was retarded, the Arg car- riers (related to high paraoxonase activity) seemed to be

The statistical approach used to define the BA was based on a multiple regression of age by several quanti- tative variables showing a significant evolution with age (14). The BA of one individual was then calculated from this regression equation: BA= a+Σβ (x–g (a)), where x in- i i i i dicates the observed value of variable x for each variable i

Table 2 - Allele frequencies of candidate genes in the sample pop- ulation.

Table 4 - Regression analyses of serum variables with value of the aging indicator.

Gene/allele Allele Gene/allele Allele frequency frequency APOE ACE (I/D) ε2 0.114 I 0.438 ε3 0.800 D 0.562 ε4 0.086 CBS (Ile278Thr) LPL (C447G) Ile 0.924 C 0.870 Thr 0.076 G 0.130 MTHFR (C677T) PON (Gln192Arg) C 0.654 Gln 0.684 T 0.346 Arg 0.316

Serum constituents Correlation coefficients (N=159) Total cholesterol (mmol/L) 0.181* HDL-cholesterol (mmol/L) -0.126 (NS) Triglycerides (mmol/L) 0.234** Apolipoprotein A1 (g/L) -0.123 (NS) Apopolipoprotein B (g/L) 0.169* Lipoprotein (a) (g/L) -0.103 (NS) Apolipoprotein E (mg/L) 0.113 (NS) ACE (U/L) -0.083 (NS) PON (nmol/mn/mL) -0.268*** (1) PON (nmol/mn/mL) -0.257*** (2) Arylesterase (μmol/mn/mL) -0.165*

APOE: apolipoprotein E; LPL: lipoprotein lipase; PON: paraoxonase; ACE: angiotensin-converting enzyme; CBS: cystathionine β-synthase; MTHFR: methylenetetrahydrofolate reductase.

NS: not significant; *p<0.05, **p<0.01, ***p<0.001. ACE: angiotensin-converting enzyme; PON: paraoxonase; PON : basal (1) paraoxonase activity; PON : stimulated paraoxonase activity. (2)