Each subject was studied early in the morning after an overnight fast. A venous blood sample was drawn into a tube containing EDTA. A portion of the EDTA-anticoagulated blood was frozen for subsequent preparation of leukocytic DNA in a low-speed transportable centrifuge to prepare plasma. Ice-cooled plasma samples were sent to Helsinki within 24 hours for further analyses. This study was approved by the Ethical Committee of the Helsinki University Central Hospital.

The determination of plasma lipids, lipoproteins, and apolipoproteins were done using enzymatic methods (Boehringer Mannheim) with an AutoAnalyzer II apparatus (Technicon Instruments). High-density lipoprotein (HDL) cholesterol was determined after precipitation of apoB-containing lipoproteins by heparin/manganese chloride, followed by separation of HDL and HDL3 by the addition of dextran sulfate. LDL cholesterol was calculated using the Friedewald formula. The concentrations of apoA-I, apoA-II, and apoB were measured with commercial immunoassay kits (Orion Diagnostica).

Statistical analyses were carried out using the BMDP software package (BMDP Statistical Software Inc). Plasma lipid and lipoprotein levels in different apolipoprotein genotypes were compared by ANOVA (BMDP 7D). The statistical significance of gene allele and genotype differences were tested by cross-tabulation (BMDP 4F). Their confidence intervals were calculated with a computer program based on the method of Gardner and Altman.

The determination of ApoE phenotypes showed that the Finnish centenarians had relatively low plasma total (4.6 mmol/L) and LDL cholesterol (2.4 mmol/L) levels and high HDL cholesterol (1.5 mmol/L) levels (Table 1). No significant sex differences were found in the mean plasma lipid or apolipoprotein levels. The sex difference in the phospholipid levels, even though statistically significant, was similar to those in the HDL and LDL cholesterol levels. For subsequent analysis of plasma lipoproteins in different genotypes, the data for both sexes were combined.

The ApoE phenotype distribution and allele frequencies in centenarians were compared with those in 615 adult Finns (age range, 20 to 55 years) and 1577 young Finns (age range, 3 to 18 years) described previously. In the former study, the participants were mainly from the vicinity of Helsinki, the capital of the country, but the latter study included volunteers from both western and eastern parts of the country. The frequency of the ApoE e2 allele was significantly higher, and that of the e4 allele was lower among centenarians than in younger Finnish populations.

The apoC-III 52 allele occurred among centenarians with a relatively high frequency (12.9%), which showed a difference bordering on significance (P<0.05) when compared with either of the younger reference cohorts. In contrast, thereData from Reference 18 (n=176; age group, 20-66 years) were used to determine ApoC-III alleles. ApoE alleles were determined using data from Reference 11 (n=1577; age group, 3-18 years). For ApoB and ApoC-III alleles, data from Reference 17 (n=307; age group, 9-21 years) were used. P<.05, P<.01, and P<.001 were compared with the centenarians. 

4. Serum Upld Fractions in Finnish Centenarians In Relation to Their Apolipoprotein Genotype Combinations) 
Genotype n* Cholesterol LDL-C HDL-C HDLj-C Triglyce 
ApoE e2/e2 + e2/e3 24 4.33±1.10 2.14±0.88 1.41±0.36 0.48±0.30+ 1.72±0.07 
e1/e3+e3/e3 125 4.57±0.93 2.33±0.84 1.48±0.41 0.55±0.29+ 1.66±0.81 
e3/e4 + e4/e4 30 4.82±0.90 2.57±0.75 1.60±0.41 0.67±0.30t 1.42±9.42 
ApoB X1X1 S7 4.48±0.86 2.30±0.68 1.46±0.36 0.55±0.23 1 .68 ±0 .89 
X1X2 M 4 .62 ±0 .99 2.40±0.78 1 .48 ±0 .40 0.55±0.31 1.70±0.74 
X2X2 4.73±1.04 2.53±0.98 1.58±0.49 0.62±0.35 1.38±0.49 
ApoC-lll S1S1 130 4.60±0 .92 2 .40 ±0 .75 1 .49 ±0 .42 0 .55 ±0 .30 1 .64 ±0 .79 
S1S2 38 4.62±1.02 2.35±0.89 1.55±0.36 0.61±0.26 1.59±0.62 
S2S2 3 4.63±1.84 2.62±1 .31 1 .20±0 .37 0 .31 ±0 .32 1 .78 ±1 .19 

LDL-C indicates low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; ApoE, apolipoprotein E; ApoB, apolipoprotein B; and ApoC-lll, apolipoprotein C-lll. Values are meanSD, expressed in millimoles per liter.
*DNA samples for ApoB and ApoC-lll genotyping were available from only 171 subjects.
P<.05 indicates statistical difference among three groups.

Differences in the geographic origin of the subjects may have influenced the data obtained in this study in various ways, such as differences in the age groups and selection criteria used for the reference groups, as well as differences in the distribution of apoE phenotypes across different regions of Finland. However, previous studies have shown that the distribution of the apoE phenotypes is relatively uniform across different regions of Finland. It was assumed that the subjects living in the Helsinki region as adult control subjects were representative of the whole Finnish population. 

Subjects with the ApoB X1X1 genotype and E2 phenotype had lower plasma cholesterol levels (3.43 mmol/L; P<.05) than those with other genotype/phenotype combinations, while subjects with other combinations had intermediate plasma cholesterol levels (Figure). The ApoB gene was not associated with significant variations in plasma cholesterol or triglyceride levels in the centenarians (Table 4). No significant association was found between the polymorphisms of the apolipoprotein genes examined and plasma phospholipid, apoA-I, apoA-II, or apoB concentrations (data not shown).

Discussion