and Alzheimer’s disease (AD) (5) with unfavored longevity (6), whereas ε2 is regarded as a protective factor resulting in longevity, according to a French centenarian population study (7), although 1% of apoE ε2/ε2 carriers suffer from hypertriglyceridemia (3). ACE is an enzyme which catalyzes the generation of angiotensin II and also inactivates bradykinin, the two peptides playing a key role in modu- lating vascular tone. Serum ACE levels are under genetic control, and up to 50% of the variation is due to the I/D polymorphism of the ACE gene, highest concentrations be- ing found in DD homozygous (8). Although ACE D allele has been proposed as a genetic risk marker for CVD (9), this allele was once found to be frequent in centenarians but later ruled out (7). Mild hyperhomocysteinemia is a risk factor for CVD. Genetic mutation in MTHFR and CBS genes may account for reduced enzyme activities and el- evated plasma homocysteine levels (10). LPL is an enzyme mediating the TG hydrolysis of chylomicrons and VLDL in the circulation. Several polymorphisms have been identi- fied at its gene locus, and the polymorphism at position 447 (Ser→Ter) has been found to be associated with serum TG concentration and/or CVD (11). The PON gene is polymorphic, and the common mutation is re- sponsible for (Gln→Arg) amino acid substitution in position 192 of the protein, the Gln isoform being associated with low serum paraoxonase activity, whereas the Arg iso- form is associated with high activity and quicker peroxide hydrolysis. Earlier studies have demonstrated an associa- tion between this polymorphism and CVD. This relation- ship may be partly related to the association with lipid and lipoprotein in HDL (12), which suggests that serum paraoxonase contributes to the ability of HDL to attenuate oxidative changes (13).

posed of 256 men aged 69.8±4.0 (range 53-86) years. Subjects were unrelated and free of chronic diseases and known pathological states. Clinical measurements such as weight, height, systolic blood pressure (SBP), diastolic blood pressure (DBP) and waist-to-hip ratio (WHR) were systematic measurements of the general health exami- nation provided by CMP; body mass index (BMI) was calculated by Quetelet’s formula: weight/height2 (kg/m2).

Laboratory procedures

For biological measurements of serum and genomic DNA extraction, venous blood was collected from supine subjects by venipuncture after overnight fasting. Sera were frozen in liquid nitrogen (-196°C) until analysis. Serum TC, TG and glucose were measured using standard enzy- matic methods (Merck, Darmstadt, Germany) automated on an AU 5021 apparatus (Olympus-Merck, Nogent-sur- Marne, France). Determination of serum apoE concentra- tion was performed by immunoturbidimetry on a Cobas-Mi- ra (Roche-Diagnostics, Meylan, France) using reagents supplied by Daiichi Pure Chemicals Co. Ltd. (Tokyo, Japan). HDL-cholesterol was measured by the phospho- tungstate method with reagents supplied by Roche- Boehringer (Meylan, France), followed by measurement of TC on the same Cobas-Mira as above. Serum lipoprotein(a) [Lp(a)], apoA1 and apoB were determined by im- munonephelometry on a Behring nephelometer analyzer, with Dade-Behring reagents (Paris la Défense, France). Serum PON activities and serum ACE activity were mea- sured on a Cobas-Fara analyzer (Roche-Boehringer, Mey- lan, France). Of the two, the former used 1 mM paraoxon and the latter 0.76 mM synthetic substrate furylacryloyl- phenylalanylglycyl-glycine as substrates (15, 16). The activity of arylesterase was assessed using 1 mM phenylacetate as substrate on a Uvikon 943 double beam UV/VIS spec- trophotometer (Kontron, France) according to the method of Eckerson et al. (15). The basal activity of PON was called PON , and the NaCl-stimulated activity of PON, (1) PON . Hematological data, such as mean globular volume (2) of red blood cells (MGV) and hemoglobin, were measured

An association study is an acceptable method of in- vestigating the biological determinants of human longevi- ty (7). However, new methods still need to be sought, since there are not many healthy centenarians available to compose a large sample. Thus, we first developed a sta- tistical model estimating an individual’s theoretical age based upon biological parameters, as an indicator of bi- ological (rather than chronological) aging (14). This indi- cator was applied to evaluate whether the specific alleles of these six candidate genes and the studied serum con- stituents were associated with early aging in a healthy French sample population.

Determination of genetic polymorphisms

Nuclear blood cells of EDTA blood samples were stored at -80°C until DNA extraction, according to the method of Miller et al. (17). The common apoE polymorphism was de- termined by HhaI digestion following polymerase chain re- action (PCR), with the method of Hixson and Vernier (18). ACE I/D polymorphism genotyping was performed according to Evans et al. (19). Genotyping MTHFR C677T was done according to Frosst et al. (20). Then these three DNA polymorphism determinations were verified by the multiplex assay of Cheng et al. (21). In addition, three other genotypes were determined by this assay: C→G mutation (Ser447→Stop codon) in exon 9 of the LPL

Subjects

METHODS

Subjects

The sample population came from the Centre de Médecine Préventive (CMP) in Nancy (France), where subjects were consecutive attenders for health screening ev- ery five years. They were recruited between September 1996 and October 1998 according to the possibility of da- ta collection, and all subjects gave their informed consent for inclusion in this study. The sample population was com-

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