Although severe and relatively rare forms of primary dyslipidemias, such as familial hypercholesterolemia, are known to be associated with shortened life expect- ancy, much less is known about the relevance of the

Correspondence to Professor Reijo S. Tilvis, MD, Geriatric Unit, Second Department of Medicine, University of Helsinki, Haartmaninkatu 4, SF-00290 Helsinki, Finland. © 1994 American Heart Association, Inc.

the southwestern and eastern parts of Finland. The mean serum cholesterol, LDL cholesterol, HDL cholesterol, and triglyceride concentrations in the adult reference population8 were 5.59, 3.52, 1.53, and 1.20 mmol/L, respectively, and the corresponding values in the young reference population11 were 4.80, 2.96, 1.44, and 0.87 mmol/L, respectively.

more common genetic variation of lipid-regulatory genes to longevity. Previous studies have suggested a lower apoE4 frequency in the elderly.22'23 We hypothe- sized that any of the three apolipoprotein alleles that had been unequivocally linked to elevated blood lipid levels (see above) in young and adult Finns may affect life expectancy adversely. To test the validity of this hypothesis, we determined the frequencies of these three alleles in the Finnish centenarians and compared them with those in groups of younger Finns studied earlier. The study design also permitted us to test whether the previously established relations between plasma lipid levels and apolipoprotein polymorphisms also hold true for the oldest people in the population.

DNA Analysis

DNA was digested with the restriction enzymes Xba I or Sst I (Promega), fractionated by gel electrophoresis on 0.6% to 0.8% agarose, and transferred to nitrocellulose filters. Hybrid- ization, washing, and autoradiography were carried out as described previously.1821 The DNA probes pB23 and pSV2 2.2-kb apoA-I were gifts from Dr J.L. Breslow (New York, NY). The apoB alleles with and without the polymorphic Xba I site are designated as X2 and XI, respectively, and the apoC-III alleles with and without the polymorphic Sst I site as 52 and SI, respectively.

Methods

Subjects

In 38 cases the amount of available DNA did not permit Southern blot analysis of the apoB and apoC-III polymor- phisms. In these cases, the variable nucleotides were identified by a solid-phase minisequencing technique according to anen et al.30 The conditions for determination of the apoB Xba I genotypes have been described previously.31 For determina- tion of the apoC-III Sst I genotypes, three oligonucleotide primers were synthesized (nucleotide numbering refers to the sequence given in Reference 32): polymerase chain reaction (PCR) primer 1, nucleotides 313 to 333; PCR primer 2 (biotinylated), nucleotides 445 to 465 (complementary strand); and sequencing primer, nucleotides 349 to 369.

At the beginning of the Finnish Centenarian study in March 1991, all persons born in 1891 or earlier and alive in December 1990 (n=271) were identified from the Finnish national pop- ulation registry. By December 1991 one of us (J.L.) visited all centenarians alive at the time of the examination (n = 185). Of the centenarians, four refused to participate in the study and two were not willing to give a blood sample. Of the 179, 25% were living at home, 44% in homes for the elderly, and 31% in hospitals. Of the centenarians alive, 70% (6.3/100 000 popula- tion) lived in southern Finland, 20% (4.7/100 000 population) in central Finland, and 10% (3.3/100 000 population) in north- ern Finland.

The apoB and apoC-III allele frequencies in centenarians were compared with those in 307 young (age range, 9 to 21 years) and 179 adult (age range, 20 to 66 years) Finns.1718 The apoC-III Sst I alleles in the adult series18 were determined during the course of the present study by means of the Southern blot technique. The adult reference subjects18 were living in Helsinki and its surroundings, whereas the young subjects17 were from both the western and eastern parts of the country. The mean serum cholesterol, LDL cholesterol, HDL cholesterol, and triglyceride values in this adult reference group were 5.76, 3.76, 1.74, and 1.17 mmol/L, respectively; in the young group they were 4.74, 2.89, 1.47, and 0.84 mmol/L, respectively.

Every subject was studied in the morning as early as possible. A venous blood sample was drawn after an overnight fast into a tube containing EDTA. A portion of the EDTA- anticoagulated blood was frozen for subsequent preparation of leukocytic DNA24 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.

Determination of Plasma Lipids, Lipoproteins, and Apolipoproteins

Cholesterol and triglyceride concentrations were deter- mined by enzymatic methods (Boehringer Mannheim) using an AutoAnalyzer II apparatus (Technicon Instruments). High- density lipoprotein (HDL) cholesterol was determined after precipitation of apoB-containing lipoproteins by heparin/man- ganese chloride, followed by separation of HDL and HDL3 by 2 addition of dextran sulfate.25 LDL cholesterol was calculated using the Friedewald formula.26 The concentrations of apoA-I, apoA-II, and apoB were measured with commercial immu- noassay kits (Orion Diagnostica).

Statistical Analysis

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 significances of gene allele and genotype differences were tested by cross tabulation (BMDP 4F). Their confidence intervals were calcu- lated with a computer program based on the method of Gardner and Altman.33

Determination of ApoE Phenotypes

Results