Controls Nona- genarians Young adults Middle-aged adults Age (years) 34 (28-37)” 49 (44-53) 93 (92-95) n 124 136 95 Sex (M/F) 71153 85/51 8/87

 4Median value (lower and upper quartiles in parentheses).

Plasma lipids and lipoproteins by age categories

Young adults Middle-aged Nonagenarians (n = 124) adults (n = 95) (n = 136) Total cholesterol (mM) 4.9 (4.4-5.6) 5.6 (5.1-6.4)* 5.2 (4.4-5.9) LDL cholesterol (mM) 3.2 (2.6-3.6) 3.7 (3.l-4.6)* 3.3 (2.6-3.9) HDL cholesterol (mM) 1.3 (1.0-1.6) 1.3 (1.1-1.6) I.1 (0.9-l.3)* Total triglycerides (mM) 0.9 (0.7-1.4) I.0 (0.8-1.5) 1.4 (I.]-2.0)*

Median values (lower and upper quartiles in parentheses). *P < 0.01 for the difference as compared to the other groups.

was successful in 89 nonagenarians, 86 middle- aged adults and 102 young adults. The apo B gene probes were the genomic EcoRI fragment (pAB 3SC) used to detect the X&I RFLP [19] and the 2-kb Hind III fragment (pBH 2) to detect the EcoRI RFLP [7,20]. The allele resulting in the for- mation of an 8.5 kb X&I fragment is designated X- (absence of the cutting site) and that generating a 3.5 kb fragment X+ (presence of the cutting site). Probing EcoRI digests with pBH2 resulted in hybridizing fragments of IO.5 kb and

Plasma Lp(a) concentrations were determined by means of a two-site immununoradiometric assay (Pharmacia Diagnostica AB, Uppsala, Sweden). The coeflicient of variation within Lp(a) assays was 2.7% and 0.7% for samples with mean Lp(a) values of 3.7 and 43.3 mg/dI respectively. The coefficient of variation between assays was 7.9% and 5.5% for samples with mean Lp(a) values of 9.0 and 75.0 mgldl respectively.

Data storage and statistical analysis were per- formed with the use of SAS (Statistical Analysis System Institute Inc., Cary, NC). The results are given as medians and quartiles for the lipid and Ii- poprotein variables. For the statistical analysis,

DNA was extracted from white blood cells [ 181, digested with the restriction enzymes, electrophor- esed on agarose gels and transferred to nylon membranes by Southern blotting. DNA isolation

150 , Young Middle-aged Nona- adults adults genarians

1.0 - E2 ,” z % f 0.5 - E3 1 2 7 E4 0 - Young Middle-aged Nona- adults adults gensrians

Fig. 2. The allele frequencies of apo E in the three age groups. The proportional areas (sub-bars) indicate the frequencies of ~2 (0). e3 (4 and ~4 (m alleles. The frequency of t4 allele is significantly lower in the nonagenarians compared with the

Fig. I. Plasma Lp(a) concentrations in the three age groups. The boxes show the median and middle quartiles of the Lp(a) concentrations, while the whiskers represent the lowest and

E phenotype categories in the various age groups

ApoE phenotype 212 213 214 313 314 414 Young adults 0 9 2 12 35 6 (n = 124) (0.0) (7.3) (1.6) (58. I) (28.2) (4.8) Middle-aged adults :0.7, 4 3 1-l 45 6 (n = 136) (2.9) (2.2) (56.6) (33.1) (4.4) Nonagenarians 0 8 3 66 17 I (n = 95) (0.0) (8.4) (3.2) (69.5) (17.9) (1.1)

Percentages in parentheses.

homozygous and heterozygous apo E phenotypes were combined into E categories as follows: E2 = phenotypes E2/2, E2/3, E2/4; E3 = E3/3; E4 = E4/4, E4/3. Data were first tested by univariate procedure. The means of normally dis- tributed groups were compared using analysis of variance (ANOVA). Groups of skewed, not nor- mally distributed data (such as Lp(a) concentra- tions) were compared by the Kruskal-Wallis test. The gene-counting method was used for the esti- mation of gene frequencies. The x2 test was used to assess the distribution of allele frequencies between the groups.

The median concentration of plasma Lp(a) was similar in all age groups, being 8.4, 8.0 and 10.0 mg/dl in the young adults, middle-aged adults and nonagenarians respectively (Fig. 1). In the nona- genarians, plasma total and LDL-cholesterol levels tended to be lower in men (3.9 mM and 2.2 mM respectively) than in women (5.3 and 3.4 mM respectively), but the differences were not statistically significant.

3. Results

The frequency of the ~4 allele of apo E was significantly lower in the nonagenarians than in the middle-aged and young adults (x2 = 8.4 and x2 = 5.3 respectively, P < 0.05)(F ig. 2). When only females were included, the frequency of the ~4

Plasma lipid and lipoprotein levels in various age groups are presented in Table 2. ma total and LDL cholesterol were higher in

EcoRI genotype distribution and allele frequency in the various age groups

EcoRI genotype EcoRI allele frequency R+/R+ R-k/R- R-IR- R+ R- Young adults 69 27 6 0.809 0.191 (n = 102) (67.7) (26.5) (5.9) Middle-aged adults 43 37 6 0.715 0.285 (n = 86) (50.0) (43.0) (7.0) Nonagenarians 67 18 4* 0.854 0.146* (n = 89) (75.3) (20.2) (4.5)

Percentages in parentheses. R+ and R-, presence and absence of the EcoRI cutting site of the apo B gene respectively. *P < 0.05 for the difference of the distribution from that of the younger age categories.

Xbal genotype distribution and allele frequency in the various age groups

 Percentages in parentheses. X+ and X-, presence and absence of the Xbal cutting site of the apo B gene respectively.

allele was almost significantly lower in the nona- genarias (n = 87) than in the middle-aged (n = 51) and young adult groups (n = 53) (P < 0.06 for both comparisons). Also, the frequency of the ~4 allele was lower in the female nonagenarians than in the female subjects of both control groups (the middle-aged and young adult groups) (n = 104) (x2 = 5.0, P < 0.05).