Apolipoprotein E allele frequencies in an Italian population: Relation to age and lipid profile

lG. Ruiu, 1M. Cassader, lR. Gambino, IN. Alemanno, IF. Demichieli, lAo Pagano, 2F. Veglia, IG. Pagano lIstituto di Medicina Interna, Universita di Torino, Torino, 2Unita di Epidemiologia, Istituto Scientifico Ospedale S. Raffaele, Milano, Italy

1G. Ruiu, 1M. Cassader, 1R. Gambino, !N. Alemanno, !F. Demichieli, 1A. Pagano, ?F. Veglia, and11m n........

lIstituto di Medicina Interna, Universita di Torino, Torino, 2Unita di Epidemiologia, Istituto Scientifico Ospedale S. Raffaele, Milano, Italy

complications (10-13) suggested that £4 frequency gradually decreases with age, and evidence of this change, coupled with an increase in £2, has been pro vided by a number of investigations (14-17).

ABSTRACT. Apo E phenotype and plasma Tg, Chol, LDL-Chol, HDL-Chol and Apo B levels were determined in a sample of 228 healthy Italian sub jects (124 men and 104 women) aged 18-93. The allele frequencies were: £2=0.070; £3=0.829; £4=0.101 (among the lowest values in the litera ture). Division of the sample into four age groups indicated that £4 frequency decreased with age to o in persons aged over 75. Covariance analysis of the influence of each allele on plasma lipids showed that £4 was significantly associated with the highest Chol, LDL-Chol and Apo B levels. These data are evidence of the influence of £4 on Chol metabolism in an Italian population. They also show that its fre quency decreases with age.

This study evidences the distribution of Apo E phenotypes, and their relationship to plasma lipids and £4 frequency patterns in a male and female Italian population divided into four age groups.

MATERIALS AND METHODS

MATERIALS AND METHODS Subjects

Plasma samples for Apo E phenotyping were col lected in the morning after an overnight fast from 124 male and 104 female subjects aged 18-93, divided in to 4 age groups: under 65, 66-70, 71-75 and over 75. The population consisted of medical students, hospital staff members and persons over 60 who were able-bodied inmates of a rest-home or regis tered for analyses at the laboratory of the San Gio vanni Hospital, Torino. Subjects with diabetes, or liver, kidney or thyroid diseases were excluded.

INTRODUCTION

The 299-amino-acid glycoprotein apolipoprotein (Apo) E is primarily a component of chylomicra and VLDL (1), and acts as a ligand for the LDL receptor (Apo BI00-E receptor) and the remnants receptor (Apo E receptor) (2). Human Apo E displays a ge netically-determined polymorphism (3, 4); three alle les on the same locus encode for the three E2, E3 and E4 isoforms to produce three homozygous and three heterozygous phenotypes. Differences in the iso electric points of the three isoforms allow pheno typing by isoelectrofocalization (IEF) (5, 6). Studies of allele distribution in several populations have revealed a lack of uniformity in £4 distribution (7). The major importance of this finding lies in the fact that the three alleles influence cholesterol (Chol) metabolism. Bearers of one or more £4 alleles display high total Chol and LDL-Chollevels, whereas the opposite is true for £2 (7-9). The observation of enhanced £4 fre quency in patients with atherosclerotic cardiovascular

Preparation of samples

1 mg/mL EDT A-Na2 plasma samples were cen trifuged for 30 minutes at 2500 rpm at 4°C in a J6B centrifuge (Beckman, Palo Alto, CA, U.S.A.), and stored at -20°C until processed. Chol and triglyc erides (Tg) were measured enzymatically. HDL-Chol was determined after precipitation of Apo B-contain ing lipoproteins with heparin and manganese chloride (18). LDL-Chol was calculated according to Friedewald et at. (19). Plasma Apo B was determined by the tur bidimetric method (Poli Diagnostici, Milano, Italy).

Apo E phenotyping

Plasma samples (10 ilL) were delipidated with ace tone and ethanol (2.5 mL) 1: 1 v/v for 4 hours at

Key words: Aging, Apo B, Apolipoprotein E phenotype, cholesterol, LDL-cholesterol. Correspondence: M. Cassader, M.D., Istituto di Medicina Interna, Corso A.M. Dogliotli 14, 10126 Torino, Italy. Received May 3, 1994; accepted in revised form January 23, 1995.

with rabbit anti-sheep IgG conjugated to horseradish peroxidase (1:500, v/v) (Pierce, Rockford, II, U.S.A.) for 1 hour at 37°C in washing buffer. After three more washes, it was developed at room tempera ture using 30 mg of 4-chloro-I-naphtol in 10 ml methanol, 40 ml Oulbecco's buffer solution (Sig ma), and 30 III 30% H 0 The reaction took place . 2 2 in 30 seconds and after 5-20 minutes was stopped with ddH 0. 2

-20°C, resuspended with acetone and ethanol (2.5 ml) for 1 hour, and washed in diethyl-ether (1 ml). The final precipitate was dried under nitrogen, dis solved in 200 III 0.1 mollL Tris-HCl, pH 10.2, containing 6 mollL urea, 1% SOS, 2% ampholytes pH 4-6.5, 1 III E-mercaptoethanol, and 5% sucrose, and left for 1 hour at room temperature before IEF. Polyacrylamide gel was poured onto a plastic sheet

covering the inner face of a glass plate (240 x 130 mm or 160 x 120). The spacer between the two glasses was 1 mm thick. The gel mixture contained 2% ampholytes (pH 4-6.5), and was prepared ac cording to Warnick (20).

Statistics