prevalence of apo E genotypes. The role of the apo E serum concentration as a risk factor for CAD has not
been investigated. During recent years several studies have demon-
strated that apo E genotype is a determinant of suscep- tibility
to Alzheimer’s
disease AD
with o
4 homozygotes at increased risk for review see [1]. This
finding has provided important clues to the pathogene- sis of AD. The role of the apo E serum concentration
as a risk factor for AD has been studied but the results have been controversial [8,9]. A prerequisite for the
proper investigation of serum apo E concentrations as a risk marker is an understanding of the factors deter-
mining serum apo E concentration in health and the establishment of serum reference values
The ApoEurope Project, supported by the European Community, consists of three parts: part I, epidemiol-
ogy of apo E concentration; part II, role of apo E in cardiovascular disease; part III, role of apo E in
Alzheimer’s disease. The specific objectives of this pa- per part I of ApoEurope are: a, to describe the
distribution of the serum apo E concentration across different European populations; b, to study some
factors affecting interindividual variation in serum apo E concentration: apo E genotype, age, sex, and geo-
graphical area; and c, to provide apo E genotype related values for serum apo E concentration in four
European regions. For these purposes, six research units were selected for collaboration.
2. Materials and methods
2
.
1
. Geographical distribution of the sampling locations Subjects were selected in different geographic areas
of Europe for participation in the ApoEurope Project Part I. In four centres existing databases were used
and in two centres new data were collected. Six centres participated: Crete Greece; Helsinki Finland; Belfast
Northern Ireland; Nancy France; Lisbon Portugal; and Barcelona Spain. Four European regions were
individualized: a, North Finland and Northern Ire- land; b, Middle France; c, South Portugal and
Spain; and d, South East Greece. The total sample population of the six centres comprised 3706 men and
3228 women aged 25 – 64 years. All subjects gave their informed consent for participating in the different stud-
ies which were approved by the ethic committee of each centre.
2
.
2
. Subjects In Finland, subjects were a subsample of the third
FINMONICA [10] risk factor survey carried out from January to March 1992. A random sample of 3000
persons aged 45 – 64 years was drawn from the Finnish population register for the North Karelia county in
eastern Finland, the city of Turku and an area around the town of Loimaa in south-western Finland, and the
cities of Helsinki and Vantaa in the southern part of the country. The sampling was stratified so that the sample
size was 250 persons per area, per sex and per 10-year age group. Of these, 2087 persons 1004 males and 1083
females were included in part I of the ApoEurope Project.
The description of the subjects and study design in Belfast Northern Ireland, has been reported [11].
Briefly, 674 males aged 30 – 49 years were recruited from an industrial workforce comprising both manual
and non-manual workers. Subjects were screened be- tween November 1994 and March 1995.
In France, the population sample was that of the Stanislas Cohort Study [12]. The Stanislas cohort was
based on a sample from the general population invited to the Centre for Preventive Medicine in Vandoeuvre-
le`s-Nancy and represented 10 – 13 of the subjects visit- ing the Centre per day between September 1993 and
August 1995. The subjects came from the Vosges and from the south of the Meurthe-et-Moselle and were
4 – 61 years old. One thousand and three families were selected consisting of two parents accompanied by at
least two biological children. Only parents older than 25 years, constituting a genetically unrelated sample
population, were included in the ApoEurope Project i.e. 975 men and 978 women. All subjects were of Eu-
ropean origin and free of serious andor chronic illnesses.
The Portugal sample consisted in users of different laboratories of the National Institute of Health of
Lisbon. Subjects were invited every day in a random way from those attending for the blood collection from
8:30 to 10:00 a.m. Excluded subjects were AIDS seropositives. From subjects considered healthy, 241
males and 293 females aged between 25 and 64 years old were selected in 1996 – 1997 for the study.
In Spain, a two-stage random sample of the general population stratified by sex and 10-year age group was
selected. The sample was drawn from seven municipal population registers with probability sampling propor-
tional to population size. The participation rate was 65. The study was carried out in 1993 – 1994. The
population sample was representative of a geographical area with over 1 000 000 inhabitants in the metropoli-
tan area of the province of Barcelona, which is the same area covered by MONICA-Catalonia [13]. Survey
methods
followed the
WHO-MONICA protocol
[14,15]. A total of 497 males and 558 females were selected from MONICA-Catalonia.
In Greece Crete, subjects were recruited from four sources: a, employees of the University hospital of
Crete and the University of Crete, born and living in
Crete; b, a 10 random sample of men and women aged 50 – 65 years from two villages in the vicinity of
the University of Crete Medical School; c, third year medical students at the University of Crete; and d,
subjects from the Seven Countries Study Cohort, aged 40 – 60 and 75 – 97 years old. Subjects aged between 25
and 64 years, all examined in 1997, were included in the study resulting in 528 subjects 315 males and 213
females.
2
.
3
. Blood samples In Finland, blood samples were drawn from the
antecubital vein with the subject in a sitting position using minimal stasis. Tubes were centrifuged at room
temperature at 1400 × g for 30 min. Serum was har- vested with plastic Pasteur pipettes and divided in 0.5
ml aliquots for freezing and storage. The samples were snap frozen within 2 h of venipuncture in a mixture of
dry ice and alcohol. They were stored at – 70°C until analysed. The subjects were examined between 11:00
a.m. and 6:00 p.m. They were advised to fast totally for at least 4 h before the examination and to avoid fatty
meals earlier during the day. A low-fat breakfast was however allowed.
In Northern Ireland, after a fast of at least 6 h and usually a full overnight fast, a venous blood sample of
20 ml was taken from sitting subjects with minimal stasis. Of this 10 ml were anticoagulated with EDTA
and 10 ml were allowed to clot at room temperature in the dark. Serum was separated within 4 h and stored at
−
80°C until it was shipped to Nancy in June 1997. Buffy coats were collected from the EDTA tubes and
stored at − 80°C until the DNA was prepared. In France, blood was collected by venipuncture after
overnight fasting, either in Vacutainer tubes containing EDTA for DNA preparation or in Vacutainer tubes
containing a gel for serum separation Becton Dickin- son. Subjects were supine during the blood sampling.
Blood was centrifuged promptly at 1000 × g for 15 min at room temperature for serum and buffy coats prepa-
ration. The sera and buffy coats were frozen for no more than 18 months in liquid nitrogen − 196°C until
analysis or extraction of DNA.
In Portugal, recruited subjects were fasted overnight and blood samples were taken from supine subjects.
Blood was collected in two Vacutainer tubes, one con- taining EDTA for DNA extraction and the other con-
taining a
gel without
anticoagulant for
serum separation. This was done immediately and samples
were stored at − 70°C until analyses. In Spain, blood samples were drawn from the antecu-
bital vein with the subject in a sitting position and with tourniquet-use for B 2 min. Subjects had been fasting
overnight for 10 – 12 h and blood was drawn between 9 and 11 a.m. Blood was collected in Vacutainer tubes
with no anticoagulant. Tubes were left at room temper- ature 22°C for a maximum of 1 h and then cen-
trifuged in a refrigerated centrifuge at 2500 × g for 15 – 20 min. Samples were transported at 4°C to the
central laboratory where they were stored, after aliquoting, at − 80°C within B 24 h after venipunc-
ture. The samples were kept at − 80°C between 3 and 4 years until their air shipment on dry ice to the Nancy
laboratory for the ApoEurope study.
In Greece, the subjects were advised to fast overnight 12 h before sampling. Blood was drawn from subjects
in supine position and collected in 10 ml Vacutainer tubes containing EDTA for DNA preparation, or in 10
ml Vacutainer tubes without anticoagulant for serum separation. The tubes were centrifuged at room temper-
ature for 10 min at 4000 rpm and the samples were stored at − 80°C for : 4 months until shipment on dry
ice to Nancy.
2
.
4
. Analytical procedures All measurements of apo E concentration were done
centrally in Vandoeuvre-le`s-Nancy. Apo E concentra- tion was determined on serum samples by immunotur-
bidimetry using a kit from Daiichi, Tokyo, Japan Apo E Auto ‘Daiichi’, cat. Number 114861 for French sam-
ples and Apo E Auto. N ‘Daiichi’, cat. Number 241918 for all other samples, according to the manufacturer’s
recommendations [16]. Calibration curves were ob- tained by serial dilution of a serum standard Daiichi
High Level Standard, reference 125799, Tokyo, Japan, target value: 105 mgl. The detection limit of the
method was 6.3 mgl with an upper limit of 100 mgl for the 114861 reference kit and 5.0 mgl to 100mgl for
the 241918 reference kit. Sera were analysed without pre-treatment and diluted in double-distilled water
when the apo E concentration exceeded 100 mgl. Con- trol sera lyophilised Daiichi Control and pool sera
were included in each series of measurements. The within-series imprecision of apo E measurements was
tested on three different freshly prepared serum pools. It varied from 1.7 to 3.4 for mean values ranging
from 80.2 to 35.3 mgl. The day-to-day reproducibility was estimated to be 3.4 – 5.5 using two serum pools
kept frozen at − 20°C. For the commercially available Daiichi control serum kept at 4°C the reproducibility
was 4.2 1 month and 7.0 12 months. Because of the changes in the formulation between the two Daiichi
kits, the data for the French samples were mathemati- cally corrected using a regression equation obtained by
measurement of apo E concentration with the two kits: n = 192, r = 0.939, y cat. Number, 241918 = 0.90x
cat. Number 114861 − 3.13.
DNA extraction was performed according to the method of Miller et al. [17], after validation of the
procedure and DNA stability [18]. Apo E genotype was
determined by PCR amplification and subsequent diges- tion with the restriction enzyme HhaI as described by
Hixson and Vernier [19]. Genotyping was performed in a single site in Vandoeuvre-le`s-Nancy France for
Northern Ireland, France and Greece. The exactly same methods were transferred to Lisbon which carried out
their own determinations. Phenotyping was done in Hamburg Pr. U Beisiegel by isoelectric focusing [20] for
Spain and Finland.
2
.
5
. Effect of storage on serum apo E concentration The stability of apo E concentration during storage for
up to 3 months at − 80°C was assessed by comparing apo E concentration values measured in ten fresh serum
samples. In addition, we verified the effect of storage at −
196°C for up to 4 years on 34 samples from individuals from the Stanislas Cohort Study. Two measurements
were performed after 2 and 4 years of storage. At − 80°C no significant change in serum apo E concentration
occurred during for up to 3 months of storage. Similar results were obtained on EDTA plasma after storage at
−
80°C. Storage at − 196°C for up to 4 years did not significantly affect apo E serum concentration. The
regression equation obtained between two measurements made after 2 years and 4 years of storage at − 196°C,
was y = 0.986x + 0.383 with a coefficient of determina- tion of 0.9945.
2
.
6
. Statistical methods The distribution of the apo E polymorphism was tested
for Hardy – Weinberg equilibrium using x
2
testing. The independent association between the occurrence of cer-
tain apo E genotypes and centre, age, and gender, was expressed as multivariately adjusted odds ratios ORs
calculated using multiple logistic regression modelling. The statistical significance of the independent contribu-
tion of the latter three variables was evaluated using Wald x
2
testing. Individual ORs are accompanied by 95 confidence intervals CI in order to statistically
judge their difference from unity. Association between the same set of explanatory
variables and the apo E concentration was studied using multiple regression analysis after natural logarithmic
transformation. From these analyses, it was concluded that the effect of age on the apo E concentration in all
centres was significantly quadratic in men and linear in women. Hence, further age-adjustment was done
quadratically in men and linearly in women.
The distribution of apo E concentrations by centre, age, and gender, was characterised by the mean, standard
deviation SD, median, interquartile range IR, percen- tiles 25.0 – 75.0, percentiles 2.5, 5.0, 95.0 and 97.5,
the geometrical mean accompanied by an asymmetrical 95 CI, the mean of ln-transformed values [ln apo E]
and their SDs. A level of a = 0.05 was used to indicate statistical significance.
3. Results
