Methods

Subjects

A total of 256 Japanese centenarians (190 women, 66 men) living in the Tokyo metropolitan area were recruited for the second term of Tokyo Centenarian Study from April 1999 to January 2001 by using national registry of centenarians published by Ministry of Health, Welfare, and Labor. Among them, 184 centenarians were living at home, and 72 were institutionalized, but none were in acute setting nor receiving tube feeding. Evaluations included physical examination, measurement of body weight and height, blood pressure, habitual food intake, alcohol consumption, smok- ing habits, physical and cognitive function, electrocardiogram, use of medication, and past medical history [23]. Blood chemistry an- alyses included screening for liver and renal dysfunction, malnu- trition, and inflammation. Fifty-three centenarians (20.6%) were receiving antihypertensive treatment, and ten had a history of can- cer treatment (two each for stomach, colon, prostate and breast, and one each for bladder uterus cancer). None was diabetic or used lipid-lowering drugs. The regionally matched younger con- trol group consisted of 190 healthy volunteers (104 women, 86 men; mean age 40.0±11.2 years, range 22–65). They were free from diseases such as CAD, stroke, diabetes, and cancer and had either no or moderate smoking and alcohol consumption. Written informed consent was obtained form all the participants or proxy. This study was approved by the ethics committee of Keio Univer- sity School of Medicine.

In addition to CETP, lecithin:cholesterol acyltransfer- ase, hepatic lipase (HL), lipoprotein lipase (LPL), and ATP-binding cassette transporter 1 (ABCA1) are partici- pants of reverse cholesterol transport. Certain polymor- phisms of the LPL gene have been shown to be associat- ed with atherogenic lipid profiles [11, 12, 13, 14, 15], CAD [13, 14], and diabetes [13]. Common variations in the ABCA1 gene are reported to be associated with plas- ma lipids and cardiovascular risk [16, 17]. These results suggest that genetic polymorphisms of those enzymes in reverse cholesterol transport could affect lipoprotein me- tabolisms and modulate risk for CAD presumably in concert with CETP. However, as far as we know, there have been a few studies that simultaneously investigate the interplay of participants in reverse cholesterol trans- port as a whole system [18, 19].

Table 1 presents the background characteristics in centenarians and controls. As previously described [23], lipid profiles in cente- narians were characterized by low levels of total cholesterol, low- density lipoprotein cholesterol, HDL-C, apoA1, and apo B and by elevated lipoprotein (a) levels. The CETP concentrations in cente- narians were significantly lower than those in controls (P<0.001), which are also compatible with our previous report [23]. Body mass index and serum albumin were also lower in centenarians. In centenarians no significant sex differences were found in the mean levels of plasma lipid, serum albumin, or body mass index; ac- cordingly, the data for both sexes were combined for subsequent analysis. In contrast, women in control group had significantly higher levels of HDL-C (67±12 vs. 53±14 mg/dl, P<0.0001), apoA1 (151±18 vs. 138±17 mg/dl, P=0.0018), and CETP mass (3.9±1.0 vs. 3.5±0.7 µg/ml, P=0.014), and lower levels of triglyce- ride (76±46 vs. 139±98 mg/dl, P=0.0002 by using logarithmically transformed value), apoB (80±20 vs. 98±26 mg/dl, P<0.0001), and body mass index (20.5±2.2 vs. 23.6±3.3, P<0.0001) as com- pare to male controls.

Recently there have been many findings from narians studies that have extensively investigated the sociations between genetic predisposition for fatal ε ease and longevity [20, 21, 22, 23]. For example, the allele of the apoE gene which is related not only to an crease risk for CAD but also to the development

Lipid measurements

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

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 (µg/ml) 3.1±0.9 3.7±0.9 <0.0001

Promoter polymorphism of the HL gene (C-514T) were deter- mined as developed by Couture et al. [29] with 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 PCR product was digested with 10 U NlaIII, resulting in two fragments of 215 and 70 bp for the T allele, a 285 bp for the C allele.

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