Apolipoprotein E serum concentration and polymorphism in six
European countries: the ApoEurope Project
F. Schiele
a, D. De Bacquer
b, M. Vincent-Viry
a, U. Beisiegel
c, C. Ehnholm
d,
A. Evans
e, A. Kafatos
f, M.C. Martins
g, S. Sans
h, C. Sass
a, S. Visvikis
a,i,
G. De Backer
b, G. Siest
a,i,*, The ApoEurope group
aLaboratoire du Centre de Me´decine Pre´
6enti6e,2,a6enue du Doyen Jacques Parisot,54500,Vandoeu6re-le`s-Nancy,France bDepartment of Public Health,Uni
6ersity of Gent,Gent,Belgium cMedizinische Klinik,Uni6ersita¨ts-Krankenhaus Eppendorf,Hamburg,Deutschland
dNational Public Health Institute,Department of Biochemistry,Helsinki,Finland eDepartment of Epidemiology and Public Health,the Queen’s Uni6ersity of Belfast,Belfast,UK
fUni6ersity of Crete,Faculty of Medicine,Heraklion,Crete,Greece gUnidade de Quimica Clinica,Instituto Nacional de Saude,Lisbon,Portugal
hPrograma Cronicat,Institute of Health Studies,Barcelona,Spain iUni6ersite´ Henri Poincare´(Nancy I)Inserm U-525,54000,Nancy,France
Received 5 April 1999; received in revised form 20 October 1999; accepted 13 December 1999
Abstract
As part of the ApoEurope Project, the apolipoprotein E (apo E) serum concentration and polymorphism were determined in 6934 healthy subjects aged 25 – 64 years recruited in six European countries: Finland; France; Greece; Northern Ireland; Portugal and Spain. Age and sex influenced apo E concentration with concentrations being significantly higher in men than in women for those aged between 25 and 44 years. The age effect differed between the sexes after the age of 44 years, displaying a linear increase in women and a plateau in men. As expected, the serum apo E concentration was highest ino2 carriers and lowest ino4 carriers
in each country with a significantly higher frequency of theo4 allele in the northern regions. The main finding of this study was
a clear increasing North – South gradient in serum apo E concentration independent of age, sex and apo E genotype. In subjects aged B45 years and with theo3/o3 genotype, apo E concentration was higher in the South-East (Greece) as compared to the
North by 20% for men and 32% for women. In addition to the genetic polymorphism, the geographical area is an important factor to take into account when studying serum apo E concentration in multicentre studies and defining reference values. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Apolipoprotein E polymorphism; Apolipoprotein E concentration; Multicentre study; Epidemiology; Cardiovascular risk
www.elsevier.com/locate/atherosclerosis
1. Introduction
Apolipoprotein (apo) E is an important component of plasma lipoproteins and influences lipoprotein metabolism through its action as a receptor ligand. Other important roles of apo E include immunomodu-lation and nerve regeneration. Apo E is polymorphic with three common alleles: o2, o3 and o4 [1]. More
recently polymorphisms in the promoter region have
been described [2,3]. The serum apo E concentration and cholesterol concentration are in part determined by the common apo E polymorphism; thus the o2 allele is
associated with lower serum cholesterol and higher apo E concentration, whereas theo4 allele is associated with
a high serum cholesterol and low apo E concentration [4]. A number of studies [5,6] have demonstrated that the o4 allele is associated with an increased risk of
coronary artery disease (CAD). It is unclear whether this effect is mediated through the effect of the apo E genotype on lipids or through other mechanisms [7]. Furthermore, the different rates of CAD observed in European countries may partly been explained by the
* Corresponding author. Tel.: +33-3-83-448720; fax: + 33-3-83-448721.
E-mail address:[email protected] (G. Siest).
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 o4
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 and/or 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.
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×gfor 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 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 B2 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 B24 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 mg/l). The detection limit of the method was 6.3 mg/l with an upper limit of 100 mg/l for the 114861 reference kit and 5.0 mg/l to 100mg/l for the 241918 reference kit. Sera were analysed without pre-treatment and diluted in double-distilled water when the apo E concentration exceeded 100 mg/l. Con-trol sera (lyophilised Daiichi ConCon-trol 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 mg/l. 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.
determined by PCR amplification and subsequent diges-tion with the restricdiges-tion 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, wasy=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 x2 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 x2 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%), percenpercen-tiles 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 ofa=0.05 was used to indicate statistical significance.
3. Results
3.1. Population description
The population sample stratified by age, sex, and centre, is presented in Table 1. The contribution of each centre to the total population sample was 7.6% in Greece, about 9 – 10% in Portugal and Northern Ireland, and 15% in Spain. It reached 28% in France and 30% in Finland. Forty-seven point five percent of all young men, 25 – 34 years old, came from the Belfast cohort; a majority of males and females 35 – 44 years old, came from the French cohort (representing 50.5 and 54.3%); 52 – 60% of the subgroups of males and females aged 45 – 64 years came from the Finnish cohort. The designs of recruit-ment, blood sampling and storage conditions used in the different centres are summarised in Table 2. Among the six participating centres, subjects were fasting overnight
Table 1
Number of subjects of the population sample in each centre stratified by age and sex
All subjects Spain
France Greece (Crete) Portugal
Finland
Age groups (years) Northern Ireland
Men
975 315 674 241 497
Table 2
Design of recruitment, blood sampling and storage conditions used in the six centres
Northern Ireland France
Centres Finland Spain Portugal Greece
Area of Belfast,
East and south North-east
Geographic areas Area of Area of Lisbon Crete
Barcelona Northern Ireland
Industrial workforce Stanislas cohort Monica-Cataloni Specific
Third Finmonica Specific
Recruitment
a
survey recruitment recruitment
11/1994–03/1995
Period 01/1992–03/1992 09/1993–08/1995 1993–1994 1996–1997 1997
Sampling
Fasting ]4 h ]6 h 12 h 12 h 12 h 12 h
Sitting Supine Sitting
Sitting Supine
Posture Supine
Storage
−80°C −196°C −80°C
–70°C −70°C
Temperature −80°C
2.6 years 1.5 year
Duration (up to) 5.5 years 3–4 years 6 months 4 months
Table 3
Prevalence of apo E polymorphism by participating centres
France
Finland Greece Northern Ireland Portugal Spain
Obser6ed frequencies
12 2
o2/o2 1 0 2 9
271
o3/o2 196 38 60 69 117
1211 304 395
1092 439
o3/o3 611
372 61 172
o4/o3 524 99 190
41 1 7
38 6
o4/o2 10
33 4 11 8 9
o4/o4 60
1.08 2.99
x2statistica 7.57 6.42 1.41 7.00
0.78 0.39 0.09 0.70
P-value 0.056 0.07
Relati6e allele frequencies
0.0866
o2 0.0617 0.0524 0.0519 0.0634 0.0766
o3 0.7598 0.7899 0.8622 0.7922 0.8395 0.8081
0.1235 0.0854 0.1558
0.1784 0.0971
o4 0.1152
a
x2statistic: between observed and expected values under Hardy–Weinberg equilibrium.
in four, and blood was drawn in the supine position in three. The storage temperature of serum samples until analysis was 5−70°C in all centres.
3.2. Distribution of Apo E polymorphism
The distribution of apo E genotypes was in Hardy – Weinberg equilibrium for all the participating centres in the ApoEurope Project (Table 3). In Finland, the sig-nificance of the deviation from the Hardy – Weinberg equilibrium was borderline due to the o2/o2
polymor-phism being underrepresented. However, regarding the number of x2 tests performed globally, the possibility that this was a chance-finding cannot be excluded. There is a clear North – South decreasing gradient in the prevalence of theo4/o3 genotype. The mirror image of
this gradient is present for theo3/o3 genotype and to a
lesser extent, for the o4/o4 genotype.
In order to study the independent effect of age, sex, and centre, on the prevalence of the apo E genotypes, a multivariate analysis was done and the results are
pre-sented in Table 4. The ORs and their 95% CI for occurrence of genotypeso2/o2+o3/o2 versuso3/o3 and
of o4/o4+o4/o3 versus o3/o3 are given by centre, age,
and sex. From these data, it seems that the o2/o2+o3/ o2 genotypes occur more frequently in France and in
Spain compared to Greece, independently of age and sex. Age by itself is not related to genotype, but in women, the o2/o2+o3/o2 genotypes are found more
frequently, even when the data were adjusted for centre and age. Similar results were obtained when ORs were calculated for occurrence of o2/o2 or o3/o2 genotypes
versus other genotypes (data not shown). To consider more closely the gender difference observed previously, age-adjusted ORs (women versus men) were performed after stratification by centre. The gender difference was present only in Finland (P=0.008), but not in other countries (P=0.12 to P=0.79). The prevalence of the
o4/o4+o4/o3 genotypes differs greatly between centres
compared to o3/o3 genotype or to other genotypes
and sex. These two variables were unassociated with the occurrence of the o4/o4+o4/o3 genotypes,
independently of the centre.
3.3. Apo E concentration by age group, sex, centre
In Table 5, the distribution of the apo E concentra-tion is given by centre, age and sex; the descriptive statistics are the number of observations, the mean
value, the SD, the median, the IR, the percentiles (2.5, 5.0, 95.0 and 97.5%), the geometrical mean with an asymmetrical 95% CI for the original apo E concentra-tions, and the mean and SD of the ln (apo E) concen-tration on which this geometrical mean and the 95% CI are based.
Fig. 1 shows the geometrical mean of the apo E concentrations obtained in men and women by age, adjusted for centre because of the large differences
Table 4
Multivariate adjusted odds ratios (OR) and (95% CI) for occurrence of phenotypeso2/o2 oro3/o2 versuso3/o3 and ofo4/o4 oro4/o3 versuso3/o3 by centre, age group and sex
o4/o4 oro4/o3 versuso3/o3 o2/o2 oro3/o2 versuso3/o3
OR (95%CI) OR (95%CI)
Centre Centre
1 1
Greece Greece
1.29 (0.83–2.00)
Northern Ireland Portugal 1.14 (0.81–1.61)
1.48 (1.08–2.03) Spain
Portugal 1.18 (0.78–1.79) 1.28 (0.88–1.85)
Finland France 1.52 (1.13–2.06)
Northern Ireland 2.24 (1.61–3.12)
Spain 1.50 (1.02–2.20)
1.78 (1.23–2.58)
France Finland 2.42 (1.80–3.24)
Significance P=0.006 Significance PB0.0001
Age(years) Age(years)
1 25–34
25–34 1
35–44 1.01 (0.77–1.34)
35–44 1.14 (0.91–1.43)
1.10 (0.83–1.47)
45–54 45–54 1.10 (0.87–1.39)
1.12 (0.81–1.55) 1.16 (0.90–1.50)
55–64 55–64
Significance P=0.83 Significance P=0.61
Gender Gender
1
Men 1 Men
Women 1.23 (1.04–1.44) Women 1.07 (0.94–1.21)
P=0.32 Significance
Significance P=0.01
Table 5
Distribution of the mean (SD), median (IR), percentiles, geometrical mean (CI) of apo E concentrationaby centre, age, and sex
Median (IR) Percentiles (%) GM (95% CI) ln (apo E) mean (SD)
N Mean (SD)
97.5 2.5 5.0 95.0
Centre
Finland 2087 33.8 (11.64) 38.4 (32.2–45.9) 21.4 24.0 59.6 65.1 38.2 (21.8–67.2) 3.64 (0.288) 67.1 37.7 (21.2–66.9) 3.63 (0.293) 39.4 (12.82)
France 1953 37.5 (31.3–44.9) 21.6 23.8 61.2
3.91 (0.311) 83.1 93.2 49.7 (27.0–91.5)
Greece 528 52.4 (20.50) 48.8 (40.6–59.4) 27.9 30.7
41.5 (22.7–76.0)
674 43.7 (15.21) 41.0 (34.3–49.9) 24.1 26.1 69.9 79.7 3.73 (0.308) Northern Ireland
77.9
637 43.6 (13.74) 41.9 (34.3–49.7) 25.2 26.9 67.0 41.7 (23.6–73.7) 3.73 (0.291) Portugal
67.0 75.1 45.0 (27.2–74.4) 3.81 (0.257) Spain 1055 46.5 (13.00) 44.9 (38.4–52.4) 27.2 29.6
Age(years)
25–34 800 40.6 (13.67) 38.4 (31.5–46.8) 20.9 24.2 64.5 74.8 38.6 (21.0–71.1) 3.65 (0.311) 3.68 (0.298) 64.1
25.0 39.6 (22.1–71.0)
22.7 74.5
39.4 (32.9–47.2) 41.5 (14.41)
2270 35–44
66.7 73.9 40.6 (22.4–73.5) 3.70 (0.303) 42.5 (14.38) 40.4 (33.7–49.0) 22.5
45–54 2256 25.2
66.7 74.4 42.5 (24.0–75.3) 3.75 (0.292) 55–64 1608 44.3 (13.35) 42.7 (35.6–50.8) 23.8 26.9
Sex
3706 43.3 (15.44) 40.8 (34.0–49.4) 23.3 25.7 68.6 41.2 (22.5–75.5) 3.72 (0.308)
Men 78.2
39.6 (22.4–70.1) 3.68 (0.292) Women 3228 41.3 (12.35) 39.9 (33.1–47.8) 22.1 24.6 63.1 69.1
Fig. 1. Geometrical means of the apo E concentration by age and sex adjusted for centre.
existing between centres. The effect of age seems to be different by sex: in women, the effect seems to be positive and linear; in men, the apo E concentration increases linearly with age up to the age 40 – 44 years, but this increase seems to tail off thereafter. In order to study the curvilinear effect observed by age in men, regression analysis was performed, modelling the association be-tween the apo E concentration (after logarithmic trans-formation) and age for each of the centres separately. This analysis revealed that the age effect in men was indeed curvilinear with a quadratic term which was highly significant (PB0.0001) in those centres which covered the age range 30 – 64 years (France, Greece, Portugal and Spain). Following an identical procedure for modelling, the age-effect in women revealed that was no deviation from linearity as the quadratic term was not significant.
For that reason, the sex-related difference in apo E concentrations varies in two opposite directions: the mean of serum apo E concentration is higher in men than in women in the younger individuals, and lower in older ones, with equivalent concentration in middle aged men and women. Because of this differential age-effect by sex, we studied the gender difference in apo E concentration on the subgroup of 25 – 44 year old subjects for which the age-effect on apo E is comparable between the sexes. As there were no Northern-Irish women in the sample population and no Finnish men or women younger than 45 years, this subgroup analysis does not involve
North-ern Ireland or Finland. In France, Portugal, and Spain the concentration of apo E is significantly higher in men than in women: respectively 39.1 versus 35.8 mg/l (PB 0.0001) in France; 40.9 versus 37.8 mg/l (P=0.006) in Portugal; and 43.5 versus 40.8 mg/l (P=0.03) in Spain. The non-significant result in Greece (48.5 vs 46.3 mg/l; P=0.24) may be due to insufficient power.
Knowing the estimated functional relationship be-tween age and the apo E concentration in men and women, allows us to study possible differences between gender and centres adjusting for age as appropriate. Fig. 2 presents the apo E concentration by centre and gender, including all ages, and linearly adjusted for age in women and quadratically in men. The differences between cen-tres persist and are highly significant.
3.4. Association between the apo E concentration and
apo E genotypes
Table 6 shows the distribution of the apo E concentra-tion by apo E genotype for men and women of all participating centres in the ApoEurope Project. The concentration is significantly higher in the presence of the
o2 allele and this is true for both genders and is
independent of centre. After adjustment for centre and age the apo E concentration stratified by apo E genotype and by gender is 20 – 25% higher in men and women with the o2/o2 or o3/o2 genotype than those with
42.0 mg/l and 41.2 mg/l in men and 50 versus 41.8 mg/l and 39 mg/l in women.
Mean apo E concentrations by centre and sex ad-justed for apo E genotypes and age are shown in Fig. 3 for each of the participating centres. Large and signifi-cant differences in apo E concentration persist between countries in both genders.
3.5. Apo E serum concentrations by age, sex, and apo
E genotype in the different European regions
We calculated apo E serum concentrations (after exclusion of subjects with serum total cholesterol con-centrations ]11.0 mmol/l or triglyceride concentra-tions ]10.0 mmol/l) according to the following
Fig. 2. Mean apo E concentration by centre and sex after adjustment for age.
Table 6
Distribution of the mean (SD), median (IR), percentiles, geometrical mean (CI) of apo E concentrationaby apo E polymorphism and sex
ln (apo E) mean (SD) Mean (SD) Median (IR) Percentiles (%)
N GM (95% CI)
2.5 5.0 95.0 97.5
Men
34.7 229.6 229.6
o2/o2 13 100.9 (59.05) 78.2 (52.9–148.3) 34.7 85.7 (26.1–280.8) 4.45 (0.606)
367 50.3 (17.93) 3.87 (0.302)
o3/o2 47.0 (40.2–56.8) 28.5 31.1 78.6 94.9 47.9 (26.5–86.6)
2208 73.8
o3/o3 42.3 (13.56) 40.2 (33.8–48.3) 24.1 26.1 65.0 40.5 (23.1–71.1) 3.70 (0.287) 24.5 70.6 79.2 39.3 (20.9–74.2)
o4/o3 776 41.6 (15.23) 38.6 (31.8–47.4) 22.0 3.67 (0.324)
28.6
48 48.4 (12.65) 45.0 (41.2–55.5) 30.5 67.7 80.4 46.9 (28.6–76.9) 3.85 (0.253) o4/o2
37.6 (30.4–45.6) 17.1 21.8 63.6 79.9 36.9 (18.5–73.9) 3.61 (0.353) 60
o4/o4 39.3 (14.95)
Women
35.6 112.2 112.2 60.6 (31.6–116.1) 4.10 (0.332) 35.6
63.7 (21.28)
13 59.7 (52.0–76.4)
o2/o2
30.0 66.3 73.3 46.1 (27.8–76.2)
o3/o2 384 47.6 (12.00) 46.4 (39.4–54.8) 26.8 3.83 (0.257)
24.9 60.7 66.6 39.0 (22.7–67.3)
o3/o3 1844 40.6 (11.42) 39.4 (32.8–46.7) 22.3 3.66 (0.277)
3.60 (0.293) 36.5 (20.6–64.8)
64.8 58.2 23.1 o4/o3 642 38.1 (11.68) 37.0 (30.1–44.0) 21.1
46.0 (39.9–53.8) 29.7 31.4 74.5 95.3 46.9 (27.4–80.5) 3.85 (0.275) 55
o4/o2 48.9 (15.93)
32.3 (26.3–38.1)
34.1 (11.29) 21.0 55.9 67.8 32.4 (17.4–60.3) 3.48 (0.316)
65 16.5
o4/o4
Fig. 3. Mean apo E concentration by centre after adjustment for apo E genotype and sex.
criteria: region, age, sex, and apo E polymorphism. The countries were grouped in four regions from North to South: North; Middle; South and South-East. Males and females were divided in two age classes: B45 years old and ]45 years old, and were classified in three groups according to their apo E genotypes: subjects witho3/o3 genotype; carriers of theo2 allele (o2/o3 and o2/o2) and carriers of the o4 allele (o4/o4, and o4/o3).
Subjects with the o2/o4 genotype were excluded. The
resulting data are presented in Table 7 for percentiles 2.5, 5.0, 50.0, 95.0 and 97.5. Serum apo E concentration is highest, irrespective of sex, age, and geographical area in o2 carriers; subjects with the o3/o3 genotype
having intermediate values too2 ando4 carriers.
How-ever, in men aged ]45 years, values for apo E were very similar betweeno3 ando4 carriers from the South
and South-East. Moreover, a clear North – South gradi-ent in serum apo E concgradi-entration is observed in each genotype, sex, and age groups. Subjects from the North exhibited the lowest values and subjects from South-East the highest values. In subjects with the o3/o3
genotype, the increase in apo E concentration at the 50th percentile was up to 20% for men B45 years and 32% for women in the same age range according to region. This North – South gradient leads to a shift of apo E distribution towards higher values in the South and South-East.
4. Discussion
The measurement of serum apo E concentration is, at present, rarely used in clinical chemistry as compared to the use of apo AI and B determinations. The apo E concentration is determined in part by the genetic vari-ation at the apo E locus; however, a large amount of variability remains unexplained by this genetic factor, suggesting that other genetic and environmental com-ponents are major determinants of serum apo E con-centration. This paper deals with the study of the influence of the apo E genotype, age, sex, and geo-graphical area on serum apo E concentration in view to provide values useful in the interpretation of clinical data.
All subjects were ambulatory individuals free from serious diseases. The population samples were recruited from heterogeneous population frames i.e. random samples, workers, couples, students. . . Nevertheless, apo E genotypes were in Hardy – Weinberg equilibrium in all centres, which is in opposition to important selection biases. Another argument is the fact that apo E concentration is not strongly influenced by socio-eco-nomic and life-style variables such as smoking, drink-ing, dietary habits, and physical activity [21,22].
F
.
Schiele
et
al
.
/
Atherosclerosis
152
(2000)
475
–
488
Table 7
Serum apo E concentration by sex, age, regionaand apo E genotype, number of subjects, percentiles (%) 2.5, 5.0, 50.0, 95.0, 97.5
Apo E genotypes:o4/o3+o4/o4 Apo E genotype:o3/o3
Apo E genotypes:o2/o2+o3/o2+o4/o2
Percentiles
N Percentiles N Percentiles N
MenB45years
109.1 353 25.1 26.6 39.6 62.9 67.2 166 23.5
84.1 25.3
59 37.5 71.8 78.4
North 31.1 31.8 46.9
78.2
116 27.8 31.1 46.6 125.4 458 23.2 25.5 38.3 59.3 66.1 142 20.5 21.9 36.2 66.3 80.2 Middle
155.7 203
South 31 27.9 28.9 47.7 77.9 25.7 26.4 40.6 62.8 75.11 56 24.1 25.0 40.0 74.1 95.8 229.6 71 26.9 33.9 47.5 85.9 85.9 19 16.7
229.6 16.7
42.6 38.6 101.2 101.2
42.6 96.0 Greece 8
WomenB45years
141 25.3 28.8 61.3 66.6 490 20.4 22.3 35.2 50.5 55.6 166 19.8 21.0 30.8 47.1 52.7
Middle 44.1
77.0 265 24.4 25.5 38.9 52.8 59.1 84 22.4
76.4 23.8
South 51 27.6 31.4 46.2 37.9 56.7 66.1
102.1 64 27.1 31.4 46.6 62.0 78.9 14 19.9 19.9 37.9 92.1 92.1 Greece 9 48.4 48.4 53.5 102.1
Men]45years
103 24.6 28.7 74.2 79.7 580 20.9 24.2 38.7 62.7 70.8 303 21.8 25.1 38.3 63.2 75.8
North 44.0
102.7 159 25.8 28.1 39.3 60.0 66.8 50 21.8
Middle 39 26.9 34.0 47.0 89.0 22.1 33.4 56.8 67.7
94.9 253 27.5 30.2 44.1 62.5 67.4 78 26.4
85.6 29.0
33.9 45.5 74.7 100.1
South 57 34.7 51.1
110.3
15 34.4 34.4 54.3 110.3 128 30.5 31.8 47.1 82.3 92.2 21 27.9 29.9 49.5 70.6 74.6 Greece
Women]45years
65.1 554 21.4 24.1 38.4 56.1 60.6 298 21.1
63.4 22.9
29.3 44.8 37.3 57.1 63.2
North 140 27.0
66.7
28 23.9 33.8 47.8 75.6 103 23.1 25.2 36.4 53.2 64.2 46 20.3 21.3 33.5 52.2 57.7 Middle
South 74 34.4 37.7 56.4 77.9 81.4 328 28.4 31.9 46.4 66.4 70.4 88 28.0 28.4 45.0 61.1 66.8 73.9 40 33.1 36.1 62.5 84.4 99.3 11 30.6 30.6
73.9 56.3
Greece 9 47.5 47.5 62.4 78.0 78.0
storage. Our data demonstrate that sera can be storedat −80°C for at least up to three months and at −196°C for up to 4 years without deterioration. This is in accordance with previous reports [23 – 25].
Among the pre-analytical factors influencing serum lipid and lipoprotein concentrations, previous works have shown that fasting and length of fast before blood sampling have a noticeable effect on these constituents [26,27]. The low-fat breakfast allowed to the Finns may result in insulin secretion and thus decreases in VLDL concentration. This could be partially responsible for the lower total apo E concentration since VLDL contains a proportion of the plasma total apo E. However, Re´gis – Bailly et al. [28] reported a significant increase (PB 0.0001) in total apo E concentration 2 h after a test meal without excess fat load corresponding to a conventional French breakfast. In vitro studies have shown that insulin may accelerate the catabolism of LDL-choles-terol. The transfer of apo E from HDL to triglyceride rich lipoproteins demonstrated by Annuzi et al. [29] might explain in part this increase of apo E concentra-tion. The participants of this study were fasted for varying times before blood sampling, 4 h in Finland, at least 6 h in Northern Ireland, and 12 h in the other four centres. The comparison of apo E and triglyceride concentrations in Finnish subjects using covariance analysis (on log-transformed values and after adjustment for age, and apo E polymorphism) showed no statisti-cally significant difference regarding the fasting time (52, 2 – 6 and \6 h) on apo E concentration (P=0.44 in men and P=0.87 in women) and a statistically significant effect on triglyceride concentration in both sexes (PB0.001). It was reported that serum apolipo-protein AI and B concentrations decrease by 5 – 9%, respectively, in subjects who changed from standing to sitting or from standing to lying. These modifications occur rapidly within five min of altered posture, a time frequently equivalent to the duration of the blood sampling [30]. Posture is thus an important factor of pre-analytical variation. Nevertheless, in our study, the subjects from which the sample had been drawn in the sitting position (Finland, Northern Ireland) exhibited the lowest apo E values. However, it cannot be excluded that differences could have been greater if sitting subjects have be drawn in the supine position at well.
To minimise analytical variation, all apo E measure-ments were performed in one centre. Serum apo E concentration was measured by immunoturbidimetry using a commercial kit [16]. This automated method, which does not require any pre-treatment of the samples, is suitable for routine analyses and has an acceptable reproducibility (CVB4.5%).
One biological factor that significantly modifies the serum apo E concentration is apo E polymorphism. It has been reported that up to 30% of the total variability of serum apo E concentration can be attributed to its
common polymorphism:o2 has an increasing effect and o4 a decreasing effect on serum apo E concentration
[31 – 33]. In accordance with this, we found the highest apo E concentrations ino2 carriers and the lowest ino4
carriers in each region, independently of age and sex. We found no significant difference in apo E allele frequency by age and does not support an inverse association with age. This is probably in relation to the fact that the sample population was constituted by subjects aged B65 years. Aso4 is associated with CAD
or dementia, a decrease with age of the frequency ofo4
allele is generally described. However, AD frequency is age-dependent [34,35] and has been estimated to double every 5 years beyond age 65 years; from 1 to 4% at age 65 – 70 years to 22% or higher at age 85 – 90 years. The allele difference ino2 by gender is borderline significant
and could be due to a chance effect or a survival bias. However, it was only statistically significant in the Finnish cohort and not in other cohorts, which is an argument in favour of a chance effect. We observed significant difference in the distribution of the apo E alleles in the six participating centres. In Finland, theo4
allele is almost 2.5 times more frequent that in Greece (17.8 vs. 8.5%). This is in line with previous works that have reported a higher frequency of theo4 allele in the
North than in the South of Europe [36 – 40]. The decreas-ing North – South gradient in the frequency of the o4
allele and the less pronounced increasing gradient for the frequency of the o2 allele were verified in the present
study.
In addition to the effects caused by the genetic polymorphism of apo E, age and sex affect serum apo E concentration. Age effect differs between males and females. After the age of 50 years, females continue to increase their serum apo E concentration; this may be due to hormonal changes associated with the menopause. In men, aged more than 44 years, the levelling off of apo E concentration could be due to the preferential cardiovascular disease mortality in those with higher lipoprotein concentrations. This differential age-effect by gender may explain the discrepancies un-derlined by Vincent – Viry et al. [33]. This present study demonstrated that serum apo E concentration is higher in men than in women in the younger subjects, equiva-lent in middle-aged, and lower in older individuals. Such changes with age and sex are very similar to those observed for other plasma lipids and lipoproteins (total and LDL-cholesterol, triglycerides, apo AI and apo B) and are universal [24,41]. Therefore, differences in mean cholesterol or in mean triglyceride concentrations be-tween centres will not affect the age gender differences in apo E values even if apo E concentration is related to those of cholesterol and triglycerides.
E concentration is inverse to that reported for the o4
allele. It is not solely due to the influence of apo E polymorphism as it was observed within each genotypic group, even after adjustment for age. In this study, the variations in apo E concentration due to the geograph-ical area explain 5.7 and 12.0% of the total variability of serum apo E concentration, respectively in men and in women. These results suggest that environmental or genetic factors present in Mediterranean countries pro-mote a higher circulating concentration of apo E inde-pendent of the apo E genotype They underline the importance of taking into consideration the geographi-cal area in addition to the genetic polymorphism of apo E which contributes to explain 5.3% in men and 8.0% in women of the variability of the apo E concentration. In comparison age accounts for 0.5% in men and 6.2% in women.
It is well known that apo E polymorphism, particu-larlyo4 allele, is an important risk factor for CAD and
AD [1,5,6,36,39,42]. The frequency of this allele pre-sents large geographical variations, being more frequent in Northern European countries than in the Mediter-ranean ones and epidemiological studies have demon-strated a consistent positive relationship between the occurrence of CAD and o4 frequency [37,40,43].
Fur-thermore, serum apo E concentration is determined in part by the apo E polymorphism, thus the o2 allele is
associated with the higher apo E concentration; con-versely, theo4 allele is related to a lower apo E
concen-tration [4,31,32,44 – 46]. Several works give increasing evidence that serum apo E concentration, by itself, may influence the pathogenesis of common diseases such as CAD and AD [1,47 – 49]. However, at our knowledge no epidemiological data are available for CAD and those concerning AD are conflicting [8,9,50].
This large multinational study confirms the well-known North – South decreasing gradient of the o4
al-lelic frequency and the effect of apo E genotype, age and sex on serum apo E concentration. Moreover, it evidences a positive North – South gradient in apo E concentration. The contribution of this last gradient to the pathogenesis of CAD and AD independently from theo4 apo E genotype remains to be explained. Parts II
and III of the ApoEurope project will give more infor-mation about this question. For this purpose it was of great importance to perform a detailed quantitation of serum apo E concentrations and to determine the distri-bution of the apo E alleles in six different European populations in order to provide a basis for the use of apo E measurement in clinical chemistry.
Acknowledgements
We are grateful to all the participants for their contribution in recruitment and the collection of data
of the ApoEurope Project. Special thanks to all subjects who made this study possible. This work was supported by the Re´gion Lorraine, Daiichi Pure Chemicals and, by the European Community: contract no. BMH4-1543 (DG 126SSMA) and contract S.M.&T: MAT1-CT94-0046.
Appendix A
Concerted action contract N° BMH4-CT96-1543 (DG 12-SSMA) between The European Community represented by the commission of the European Com-munities (‘the Commission’) represented by the Direc-tor General for DG XII Science, Research and Development, or its authorised representative, and
Universite´ Henri Poincare´ Nancy I, (‘UNANCY.CM’) established in France (‘the Contrac-tor’), represented by its authorised representative, have agreed to a concerted action called ‘Apolipoprotein E : Health and disease marker in Europe’.
The Contractor: Universite´ Henri Poincare´ (Nancy I), Centre du Me´dicament, Nancy, France: Professor Siest Ge´rard and Dr Visvikis Sophia.
Members of the Steering Committee: Professor G. Siest and Dr S. Visvikis, Nancy as coordinators, and Professor A. Evans, Belfast; G. De Backer, Gent; Pro-fessor J. Shepherd, Glasgow.
The ApoEurope group, collaborating centres, and their associated investigators:
Belgium: Department of Public Health, University of Gent, Gent: Professor Guy De Backer *, Dr Dirk De Bacquer.
Croatia: Clinical Institute of Laboratory Diagnosis, Zagreb University School of Medicine and Clinical Hospital Centre, Zagreb: Professor Rukavina Ana Staljvenic*, Dr Jadranka Sertic, Dr Tomislav Babic and Dr Branka Ribaric.
Finland: National Public Health Institute, Depart-ment of Biochemistry, Helsinki: Professor Christian Ehnholm*, Veikko Salomaa; National Public Health Institute, Department of Epidemiology and Health Pro-motion, Helsinki: Professor Jaakko Tuomilehto; Re-search Institute of Public Health, University of Kuopio: Professor Jukka T. Salonen.
France: Centre de Me´decine Pre´ventive, Vandoeuvre-les-Nancy: Professor Ge´rard Siest*, Dr Sophia Visvi-kis*, Dr Franc¸oise Schiele, Dr Monique Vincent-Viry, Dr Bernard Herbeth, Dr Catherine Sass, Dr Aksam Merched, Dominique Aguillon, Mickael Maurice and the laboratory staff of the Centre;
Germany: Medizinische Klinik, Universita¨ts-Krankenhaus Eppendorf, Hamburg: Dr Ulrike Beisiegel*;
Central laboratory, University Hospital, Freiburg: Dr Winfried Ma¨rz.
Greece: The University of Crete, School of Medicine, Department of social Medicine, Heraklion: Professor Anthony Kafatos*, Dr Joanna Moschandreas.
Italy: Clinical Chemistry, Legnano Hospital, Leg-nano-Milano: Dr Paolo Brambilla*;
IRCCS Ospedale San Raffaele, Clinical Molecular Biology Laboratory, Milano: Dr Maurizio Ferrari, Dr ssa Stefania Stenirri; Department of Neurology, Mi-lano: Dr Massimo Franceschi;
di Ricerca per le Malattie Cronico Degenerative, Universita` di Milano, Ospedale di Monza: Dr Marco Ferrari.
Macedonia: Clinic of Neurology, Faculty of Medicine, Skopje: Dr Angel Mitrevski*.
Portugal: Unidade de Quimica Clinica, Instituto Na-cional de Saude, Lisbon: Dr Maria do Carmo Mar-tins*, Maria Odete Rodrigues, Maria Isabel Albergaria, Maria Liseta Alpendre.
Spain: Programa Cronicat, Institute of Health Stud-ies, Barcelona: Dr Susanna Sans*, Paluzie Guillem, Balan˜a Lluisa and In˜aki Pe´rez;
Unitat Recerca Lipids i Arteriosclerosi, Facultat de Medecina, Hospital de Sant Joan, Universitat Rovira i Virgili, Reus: Dr Luis Masana, Dr Josep Ribalta, Dr Pilar Dra Martinez, Dr Jose Maria Olive.
United Kingdom: Department of Epidemiology and Public Health, The Queen’s University of Belfast, Belfast, Northern Ireland: Professor Alun Evans*, Dr John Yannell; Department of Medicine, The Queen’s University of Belfast, Belfast, Northern Ireland: Dr D McMaster;
Department of Pathological Biochemistry, Royal Infirmary, University of Glasgow, Glasgow, Great Britain: Professor James Shepherd.
Yugoslavia: Faculty of Pharmacy, Department of Medical Biochemistry, Belgrade: Prof Marina Stojanov*.
*Principal investigator.
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