Nonfasting venous blood (20 ml) was collected from all subjects. Plasma was immediately separated by centrifugation at 4°C.

Centenarians (n=256) Controls (n=190) Pa Sex: M/F 190/66 104/86 Mean age (years) 100.9±1.6 40.0±11.1 <0.0001 Body mass index 19.2±3.6 21.9±3.2 <0.0001 Albumin (g/dl) 3.6±0.4 4.6±0.3 <0.0001 Total cholesterol (mg/dl) 163±32 184±36 <0.0001 Triglyceridea (mg/dl) 93.39 108.82 0.048 Low-density lipoprotein cholesterol (mg/dl) 93±26 102±32 <0.0001 High-density lipoprotein cholesterol (mg/dl) 52±14 60±14 0.0002 Apolipoprotein A1 (mg/dl) 126±24 145±19 <0.0001 Apolipoprotein B (mg/dl) 85±21 89±25 0.0034 Lipoprotein(a)a (mg/dl) 25±16 16±13 <0.0001 Cholesteryl ester transfer protein mass (g/ml) 3.1±0.9 3.7±0.9 <0.0001

Promoter polymorphism of the HL gene (C-514T) was determined as developed by Couture et al. [29] with the following primer: 5-TCT AGG ATC ACC TCT CAA TGG GTC A-3 and 5-GGT GGC TTC CAC GTG GCT GCC TAA G-3.

The concentrations of total cholesterol and triglyceride were determined by automated enzymatic procedures. The concentrations of HDL-C were determined after precipitation of apoB-containing lipoproteins with phosphotungstic Mg2+. Low-density lipoprotein cholesterol was calculated from the Friedewald formula. The concentrations of apoA1 and apoB were determined by turbidimetric immunoassay. The concentrations of lipoprotein(a) were measured by latex agglutination immunoassay. Plasma CETP concentration was measured by enzyme-linked immunosorbent assay using a commercial kit [26] (CETP ELISA, Chugai, Tokyo).

The R219K polymorphism of the ABCA1 gene was recently identified by Clee et al. [16]. The PCR primer was as follows: 5-GTA TTT TTG CAA GGC TAC CAG TTA CAT TTG ACA A-3 and 5-GAT TGG CTT CAG GAT GTC CAT GTT GGA A-3. PCR was conducted with denaturation at 95°C for 5 min, annealing at 60°C for 1 min and primer extension at 72°C for 1.5 min for a total of 30 cycles. After digestion with EcoNI, the resulting fragment is a 177-bp for the R allele and 107 and 100 bp fragments for the K allele.

DNA mutation and polymorphism cell by phenol extraction. CETP mutation in intron 14 (Int14A) was determined by polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) method as described by Inazu et al. [4Consistently associated with HDL-C concentrations (P=0.0003 and t-test, respectively) and with markedly lower CETP concentration (P<0.0001 and t-test, respectively). In regard to the genotype, the B2 allele was significantly associated with lower CETP mass and higher HDL-C in controls (P=0.0387 and P=0.029, ANOVA), and a similar but nonsignificant trend was observed in centenarians. The B2 allele was also associated with lower low-density lipoprotein cholesterol (P=0.024). There was a significant association of the P( ) allele of the LPL gene with higher HDL-C concentrations in centenarians, but this relationship was not observed in controls. There was no significant association between ABCA1 genotype parameters either in centenarians or in controls. Neither the LPL HindIII polymorphism nor the promoter polymorphism had a significant association with lipid parameters in centenarians and are not shown. When men and women were analyzed separately, the same results as shown in Table were observed in centenarians of both sexes; however, statistical power was less in male centenarians. There was a significant association between alleles frequencies of genes involved in reverse cholesterol transport between centenarians and controls (Fig. 1). Comparison of alleles frequencies between centenarians and controls were calculated by 2 test. No significant difference was observed.

Two common CETP deficiencies, an intron 14 splicing defect [4] and an exon 15 missense mutation [5], have been reported in the Japanese population. In this study, there was no subject who was identified as homozygous CETP deficiency. There was no difference in frequencies of either type of heterozygous CETP deficiencies between centenarians and controls.

The distributions of the Taq1B polymorphism of the CETP gene, the LPL PvuII and HindIII genotype, the C-514T polymorphism of the HL gene and recently identified R219K variant of the ABCA1 gene did not differ between centenarians and controls. For CETP mutations, unpaired t-tests were used (TC total cholesterol, LDL-C low-density lipoprotein cholesterol, HDL-C high-density lipoprotein cholesterol, TG triglyceride, CETP cholesteryl ester transfer protein).

Table 3 displays body mass index (BMI), albumin, and lipid fractions in centenarians in relation to deficiency and Taq1B genotype of CETP and LPL PvuII genotype. Statistical significance for LPL PvuII and ABCA1 R219K genotype were calculated using ANOVA. Genotype n Sex: BMI Albumin TC LDL-C HDL-C TGa CETP M/F (g/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) (g/ml) CETP deficiency Deficiency 18 12/6 20.3±3.3 3.7±0.4 171±32 89±28 61±22** 109±44 1.8±0.4** Wild type 238 178/60 19.1±3.6 3.6±0.4 162±32 93±26 51±13** 91±39 3.3±0.8**

CETP Taq1B B1B1 73 51/22 19.4±4.7 3.6±0.4 169±36 100±30* 51±13 94±41 3.3±1.1 B1B2 132 101/31 19.0±3.0 3.5±0.4 159±29 90±24* 51±13 93±39 3.1±0.9 B2B2 51 38/13 19.4±3.2 3.6±0.5 162±34 89±26* 55±17 92±40 3.0±0.8