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Clinical Chemistry 48: 291-300, 2002;
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(Clinical Chemistry. 2002;48:291-300.)
© 2002 American Association for Clinical Chemistry, Inc.

Apolipoprotein E in Apolipoprotein B (apo B)- and Non-apo B-containing Lipoproteins in 3523 Participants in the Stanislas Cohort: Biological Variation and Genotype-specific Reference Limits

Françoise Schiele1, Monique Vincent-Viry1, Marjorie Starck1, Brigitte Beaud1, Geneviève Hennache2, Gérard Siest1, Sophie Visvikis1 and Bernard Herbeth1a

1 Centre de Médecine Préventive, INSERM U525, 2 Rue du Doyen Jacques Parisot, F54500 Vandoeuvre-lès-Nancy, France.

2 Sebia, 23 Rue Maximilien Robespierre, F92130 Issy-les-Moulineaux, France.

aAuthor for correspondence. Fax 33-3-83-44-87-21; e-mail Bernard.Herbeth{at}cmp.u-nancy.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Apolipoprotein (apo) E is a component of two major classes of plasma lipoproteins, apo B- (apo E-LpB) and non-apo B-containing (apo E-Lp-non-B) lipoproteins. The factors that affect total apo E in particles [lipoprotein E (LpE), apo E-Lp-non-B, and apo E-LpB], are incompletely characterized.

Methods: We studied the determinants of these lipoparticles in a sample population of presumably healthy individuals: 1784 children (age range, 8–18 years) and 1739 adults (age range, 19–50 years). Serum concentrations of LpE and apo E-Lp-non-B were measured by electroimmunoassays, and the concentration of apo E-LpB was calculated by a difference method.

Results: Serum LpE and apo E-Lp-non-B were higher in females than in males. Their concentrations decreased with age until 20–25 years and then increased in men but not in women. apo E-LpB concentrations increased up to 20–25 years and were similar in both sexes. Thereafter, adult men had higher values than women. Individuals carrying the {epsilon}2 allele had higher mean apo E-Lp-non-B concentrations and lower apo E-LpB concentrations than did individuals carrying the {epsilon}3 allele. Individuals with the {epsilon}4 allele showed an inverse profile compared with those with the {epsilon}2 allele. Age, gender, the common apo E polymorphism, puberty, serum lipid concentrations, and alcohol consumption were significantly associated with total LpE, apo E-Lp-non-B, and apo E-LpB concentrations. Reference limits were established according to age, gender, and the common apo E polymorphism.

Conclusions: Because measurements of LpE, apo E-Lp-non-B, and apo E-LpB concentrations may improve cardiovascular risk assessment, the proposed reference limits will aid interpretation of the results in clinical or therapeutic trials.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Serum apolipoprotein E (apo E) 1 associated with apo B or apo CIII is found in atherogenic lipoproteins such as LDL, intermediate-density lipoproteins, and VLDL. It is also, to a lesser extent, a constituent of nonatherogenic lipoproteins, i.e., HDL, in which apo E is associated mainly with apo AI, AII, CII, and CIII. apo E participates in the interaction between lipoproteins and cells and in the clearance of these lipoproteins through the receptor pathways (1). These mechanisms depend on both serum apo E concentration and a common apo E polymorphism (2). The common apo E polymorphism is manifested as three major isoforms (apo E2, apo E3, and apo E4) encoded by the {epsilon}2, {epsilon}3, and {epsilon}4 alleles, respectively. The presence of these alleles is related to changes in the concentrations of serum total cholesterol (TC), LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C), apo B, and apo E itself (3)(4). The presence of the {epsilon}4 allele is recognized as a risk factor in the etiology of coronary heart disease (CHD) (5)(6). Because apolipoproteins are known to play an important role in lipid metabolism, it has been suggested that lipoproteins be classified on the basis of their apolipoprotein composition (7). apo E-rich apo B-containing lipoproteins are associated with atherosclerosis and CHD (8)(9). This kind of particle is commonly found in residual particles produced during the catabolism of lipoproteins with high triglyceride (TG) concentrations. These data suggest that the concentration of apo E in lipoprotein particles containing apo B (apo E-LpB) could be more informative than the total serum apo E concentration. However, there is a lack of information regarding sources of biological variation. In addition, lipoprotein E (LpE), apo E-LpB, and apo E in particles without apo B (apo E-Lp-non-B) have been determined by various methods and calibrators, such as two-site ELISA with (10) or without (11) affinity chromatography and electroimmunoassay (8), which has led to a large variation in reported values.

To date, most of the available data have been derived from case-control studies in the field of CHD (6)(8)(9). These studies focused mainly on the relationships between CHD, lipids, and lipoparticles or on the effect of lipid-lowering therapy, such as fluvastatin and cholestyramine (12). Factors affecting the biological variation of serum apo E-LpB and apo E-Lp-non-B concentrations have not been studied other than the influence of gender, menopausal status (13) and use of oral contraceptives in women (14), and the common apo E polymorphism in adult men (8). Because lipoprotein subspecies could play an important role in the development of CHD, our aim in the present study was to identify and quantify the effects of the main factors in biological variation that influence serum concentrations of LpE, apo E-LpB, and apo E-Lp-non-B. The factors studied included age, gender, puberty, morphometry, lifestyle, and the common apo E polymorphism. In addition, we established genetic reference limits for these particles in a large, selected sample population composed of healthy individuals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
sample population
The study population included apparently healthy individuals attending the Centre for Preventive Medicine at Vandoeuvre-lès-Nancy (France) for a periodic health examination and who had been recruited as part of the Stanislas Cohort (15). All gave written informed consent to participate in this cohort, which was approved by the local ethics committee of Nancy. The inclusion criteria were as follows: age, 8–50 years; body mass index (BMI) <40 kg/m2; {gamma}-glutamyltransferase (GGT) activity <300 U/L; alanine aminotransferase (ALT) activity <200 U/L; aspartate aminotransferase (AST) activity <200 U/L; serum TG concentration <10 mmol/L; and serum TC concentration <10 mmol/L. Individuals taking lipid-lowering drugs, oral contraceptives, or hormone replacement therapy and pregnant women were excluded. This sample population included 941 boys and 843 girls 8–18 years of age and 956 men and 783 women 19–50 years of age.

blood samples and data collection
Venous blood samples were collected by venipuncture after an overnight fast (15). Data collection included measurements of basic blood constituents, functional tests, a physical examination, and questionnaires on lifestyle, socio-professional characteristics, and medical history. Drug intake, notably lipid-lowering drugs, oral contraceptives, and hormone replacement therapies, was assessed by patient interview during the blood sampling. Alcohol and tobacco consumption were assessed by self-administered questionnaires. BMI was calculated according to the Quetelet formula: weight (kg)/[height (m)]2. The stages of puberty in girls and boys as defined by Marshall and Tanner (16) were scored by visual assessment of pubic hair and sexual maturation: stage 1 represents prepuberty, stage 2 represents early puberty, stages 3 and 4 represent mid-puberty, and stage 5 represents late puberty.

analytical methods
For measurements of serum TC, TG, HDL-C, apo AI, and apo B (15), the day-to-day imprecision (CV) was 1.7–8.0%. Serum LpE and apo E-Lp-non-B concentrations were measured by electroimmunoassay (Hydragel LpE reagent set; Sebia, Issy-les-Moulineaux, France) with agarose gel foils containing an anti-apo E polyclonal antibody, performed according to the manufacturer’s recommendations. Briefly, the apo E serum calibrator and samples from each patient (unaltered serum and serum preincubated with an anti-apo B antibody) were placed in gel wells. All classes of apo B-containing particles were precipitated by a goat anti-apo B polyclonal antibody (6). During electrophoresis, apo E migrated to form a complex with the anti-apo E antibody. The gel was then stained with violet acid and dried. After migration, the size (height) of the rockets was measured and compared with that of the calibrated serum calibrator to determine the concentrations of LpE (corresponding to the unaltered serum) and of apo E-Lp-non-B (treated serum) in the measured samples. The apo E-LpB concentration was calculated by the difference between the values for LpE and apo E-Lp-non-B. The total analytical variation (CV) between runs was <10%. DNA was extracted according to the method of Miller et al. (17). The apo E genotype was determined by PCR and subsequent digestion with the restriction enzyme HhaI (18).

statistical analyses
Statistical analyses were performed with the BMDP® statistical software (19). Agreement of the genotype frequencies with the Hardy-Weinberg equilibrium was tested using a {chi}2 goodness-of-fit test. Distributions of all variables were tested for normality (20). Data for LpE, apo E-LpB, apo E-Lp-non-B, GGT, and TG concentrations were log10-transformed before statistical analysis. The LpE log-transformed values followed a gaussian distribution, whereas those for apo E-LpB and apo E-Lp-non-B trended toward gaussian shapes (skewness coefficients, -1.34 and -0.37 for log-transformed values vs 1.90 and 1.28 for untransformed values for apo E-LpB and apo E-Lp-non-B, respectively).

Comparisons between the age and sex groups were performed by ANOVA or the Student-Newman-Keul multiple range test. Multiple regression analysis with a stepwise procedure was used first to test relationships among LpE, apo E-LpB, and apo E-Lp-non-B concentrations and the factors known to be related with lipid metabolism: age; BMI; sexual maturation; menopause; smoking habits (current smokers: yes/no, and tobacco consumption in g/day); alcohol intake; TC, TG, apo AI, and apo B concentrations; and the apo E polymorphism. We then performed a multiple linear regression analysis including only factors significantly associated with LpE, apo E-LpB, and apo E-Lp-non-B concentrations in at least one of the four groups. Potential interactions between age-sex groups and apo E polymorphism and other predictors were evaluated by testing equality of slope in covariance analysis. Analyses were carried out separately for the four groups: children/adolescents and adults for both sexes.

The {epsilon}2 and {epsilon}4 allelic effects were estimated by a codominant additive model that included age, alcohol intake, Tanner stages, and TC, TG, apo AI, and apo B concentrations as covariates with two dummy variables coding, respectively, for the proportion of {epsilon}2 and {epsilon}4 alleles (0, 0.5, or 1) in each genotype. apo E values were log-transformed, and {epsilon}3/{epsilon}3 was chosen as the reference. The statistical significance was set at P <=0.05. Using a nonparametric method, we estimated the 2.5th, 5th, 50th, 95th, and 97.5th percentiles for the distribution of LpE, apo E-LpB, and apo E-Lp-non-B concentrations according to the following partition criteria: age, gender, and apo E polymorphism for the three most frequent apo E genotypes ({epsilon}3/{epsilon}2, {epsilon}3/{epsilon}3, and {epsilon}3/{epsilon}4).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
biological and sociodemographic data
Data on the sociodemographic characteristics and biochemical variables for the study groups are provided in Table 1 of the data supplement that accompanies the online version of this article (available at http://www.clinchem.org/content/vol48/issue2). The mean (SD) age was 12.9 (2.7) years for boys and 12.5 (2.6) years for girls. For the adults, the mean (SD) age was 38.6 (7.7) years for men and 38.6 (6.7) years for women. The mean values for BMI, alcohol and tobacco consumption, and all biological constituents (AST, ALT, GGT, TGs, TC, HDL-C, apo AI, apo B) were in accordance with the values found in apparently healthy individuals of the same age and sex. The four groups presented arithmetic means for LpE, apo E-Lp-non-B, and apo E-LpB concentrations that were significantly different (P <0.001). The mean LpE and apo E-Lp-non-B concentrations varied in a similarly increasing order: men < women < boys < girls. The mean apo E-LpB concentration was the highest in men, and the mean concentration in women was close to that observed in boys and girls. Allelic frequencies of the common apo E polymorphism were in Hardy-Weinberg equilibrium in children and adults [see Table 2 of the data supplement that accompanies the online version of this article (available at http://www.clinchem.org/content/vol48/issue2)]. The {epsilon}3 allelic frequency varied from 76.7% to 78.9%, that of {epsilon}4 from 11.6% to 14.6%, and that of {epsilon}2 from 8.7% to 9.9%.


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  		Table 1. Predictors of LpE, apo E-LpB, and apo E-Lp-non-B concentrations, using multiple regression analysis.


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  		Table 2. Reference limits1 for serum LpE, apo E-LpB, and apo E-Lp-non-B concentrations (mg/L) by genotype, age, and gender.

distribution of apo E lipoparticles in serum
Serum LpE concentrations were 4–180 mg/L, and those of apo E-Lp-non-B and apo E-LpB were 4–170 and 1–119 mg/L, respectively. Shown in Fig. 1 are histograms of the serum concentrations of LpE, apo E-Lp-non-B, and apo E-LpB in all four groups. The distributions of the LpE, apo E-Lp-non-B, and apo E-LpB concentrations were asymmetric and skewed toward increased values. The LpE concentration range was 12–156 mg/L in adults and 4–180 mg/L in children/adolescents. Values for apo E-LpB showed an inverse pattern. In addition, the values for apo E-Lp-B were very close to those for apo E-Lp-non-B in men, whereas in the three other groups, the apo E-LpB concentrations were clearly shifted toward lower values compared with apo E-Lp-non-B concentrations.



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  		Figure 1. Distribution of the LpE ({diamondsuit}), apo E-Lp-non-B ({blacksquare}), and apo E-LpB ({blacktriangleup}) concentrations in men (A), women (B), boys (C), and girls (D).

factors affecting variability of apo E in particles
Among the different potential predictors, only the common apo E polymorphism, age, lipid concentrations (TC, TGs, apo AI, and apo B), puberty (Tanner stages), and alcohol consumption were significantly associated with LpE, apo E-Lp-non-B, and apo E-LpB concentrations in at least one of the four groups (Table 1Up ). BMI, tobacco consumption in both sexes, and menopausal status were not significantly related to LpE, apo E-Lp-non-B, and apo E-LpB concentrations. In boys and girls, puberty, age, lipids, and the common apo E polymorphism accounted for 36.5% and 42.3%, 22.1% and 19.3%, and 52.2% and 51.8% of the variance in LpE, apo E-LpB, and apo E-Lp-non-B concentrations, respectively (Table 1AUp ). The portions of variance explained by age, alcohol consumption, lipids, and the common apo E polymorphism were 31.2% and 35.4%, 32.3% and 16.9%, and 40.4% and 48.2% for LpE, apo E-LpB, and apo E-Lp-non-B concentrations, respectively, in men and women 19–50 years of age (Table 1BUp ).

Significant sex interactions were observed for various factors of biological variation. In children/adolescents, significant differences between regression coefficients were observed according to sex (P <0.05). These differences concerned predictors such as age, {epsilon}4 allele, and cholesterol and apo B concentrations for the serum LpE concentration, and age, cholesterol concentration, and Tanner stages (1 and 4, in particular) for the apo E-Lp-non-B concentration. In adults, for the LpE concentration, significant sex interactions (P <0.05) were observed for age, alcohol intake, and cholesterol, apo B, and TG concentrations; for the apo E-LpB concentration, significant interactions were observed for {epsilon}2 and {epsilon}4 apo E alleles, alcohol intake, and TG concentrations; and for apo E-Lp-non-B concentration, significant interactions were observed for alcohol intake and cholesterol, apo B, and TG concentrations.

Age, puberty, and gender.
Shown in Fig. 2 are the geometric means for serum LpE, apo E-Lp-non-B, and apo E-LpB concentrations according to sex and age classes (8–9, 10–11, 12–13, 14–15, and 16–17 years for children and adolescents; 18–29, 30–34, 35–39, 40–44, and 45–50 years for adults). From 10 to 25 years, serum LpE and apo E-Lp-non-B concentrations decreased significantly (global ANOVA, P <=0.001), whereas that of apo E-LpB increased between 10 and 29 years (P <=0.001 in males; P <=0.05 in females) in both sexes. Using Student-Newman-Keul multiple range tests, we found that among children <18 years of age, only girls 10–13 years had significantly higher serum LpE and apo E-Lp-non-B concentrations than in the other age groups (P <=0.05). In boys, LpE and apo E-Lp-non-B concentrations were not significantly different between 8 and 15 years and between 8 and 13 years, respectively, whereas the concentrations in the other age groups were significantly different from each other. The apo E-LpB concentration did not differ between 8 and 13 years in boys and between 14 and 29 years in young adults even if the concentration in the group 8–13 years of age was significantly lower than that observed in the group 14–29 years of age (P <=0.05). Among adults 30–50 years of age, the Student-Newman-Keul multiple range test failed to find a significant difference in lipoparticle concentrations among the age classes. Nevertheless, age (Table 1BUp ) was significantly associated with LpE concentration (P <0.001) in women and with apo E-Lp-non-B concentration in both sexes (P <0.01 in men and <0.001 in women). The effect of puberty was studied by use of the Tanner stage (1 to 4; Table 1AUp ) and was significantly associated with LpE and apo E-Lp-non-B concentrations in boys (P <0.05) and in girls (P <0.001 for LpE and <0.01 for apo E-Lp-non-B).



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  		Figure 2. Geometric means of LpE, apo E-Lp-non-B, and apo E-LpB concentrations according to age and sex.

{diamondsuit}, apo E in males; {diamond}, apo E in females; {blacksquare} apo E-LpB in males; {square}, apo E-LpB in females; •, apo E-Lp-non-B in males; {circ}, apo E-Lp-non-B in females. Numbers of males per age group: 8–9 years, n = 115; 10–11 years, n = 189; 12–13 years, n = 232; 14–15 years, n = 199; 16–17 years, n = 162; 18–29 years, n = 163; 30–34 years, n = 37; 35–39 years, n = 254; 40–44 years, n = 374; 45–50 years, n = 172. Numbers of females per age group: 8–9 years, n = 128; 10–11 years, n = 185; 12–13 years, n = 207; 14–15 years, n = 196; 16–17 years, n = 107; 18–29 years, n = 81; 30–34 years, n = 61; 35–39 years, n = 288; 40–44 years, n = 231; 45–50 years, n = 142.

Between 8 and 17 years, girls had significantly higher serum concentrations of LpE and apo E-Lp-non-B than did boys (global ANOVA, P <=0.001), whereas apo E-LpB concentrations did not significantly differ between sexes. In middle-aged adults (30-50 years), LpE and apo E-Lp-non-B concentrations were significantly higher in women than in men (P <=0.001), whereas apo E-LpB concentrations were lower in women (P <=0.001).

The percentage of apo E-Lp-B in LpE lipoparticles is shown in Fig. 3 . Age-related effects were different between males and females. In men, the increase in apo E-LpB concentration, without a corresponding decrease in apo E-Lp-non-B, produced an increase in the proportion of apo E Lp-B with age until 42 years. This increase was particularly marked between 12.5 and 23.5 years: from ~27% to 45%. On the other hand, in women, the percentage of apo E Lp-B in LpE lipoparticles exhibited slight variations with age. The age-related variations in LpE concentration reflected essentially those of apo E-Lp-non-B in children/adolescents and in women, in whom apo E-Lp-non-B represented approximately two-thirds of the LpE content.



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  		Figure 3. Mean percentage of apo E-LpB in LpE lipoparticles according to age and sex.

{blacktriangleup}, males; {blacksquare}, females.

Lipids and lifestyle factors.
In all participants, LpE, apo E-LpB, and apo E-Lp-non-B concentrations were significantly associated with the concentrations of lipids and apolipoproteins.

In boys and girls (Table 1AUp ), the serum concentrations of LpE and apo E-Lp-non-B were positively associated with TC (P <0.001) and negatively associated with apo B (P <0.001). Moreover, LpE was also positively associated with apo A1 (P <0.05 in boys; P <0.01 in girls) and not significantly associated with TGs, whereas the apo E-Lp-non-B concentration was negatively associated with TGs (P <0.001) and not significantly associated with apo A1. The serum apo E LpB concentration was negatively associated with TC in boys (P <0.001) and in girls (P <0.01), positively associated with TG concentration in both sexes (P <0.001), and showed significant relationships with apo B only in boys (P <0.001) and with apo A1 (P <0.05) in both sexes.

In adults (Table 1BUp ), LpE and apo E-Lp-non-B concentrations showed positive relationships with TC and apo A1 (P <0.001) and negative relationships with apo B for the both sexes (P <0.01 to <0.001). In addition, the LpE concentration was positively related to TGs only in males (P <0.001), whereas the apo E-Lp-non-B concentration was negatively related to TGs both in males and females (P <0.001). The apo E-LpB concentration was strongly positively associated with the TG concentration in both sexes, with regression coefficients being higher in males than in females (P <0.001). apo E-LpB was also negatively associated with TC and positively associated with apo B in both sexes (P <0.05 to <0.001). We failed to find a relationship between apo E-LpB and apo A1 in adults.

Alcohol consumption was negatively related to LpE, apo E-LpB, and apo E-Lp-non-B (P <0.01, <0.001, and <0.05, respectively) concentrations in women only (Table 1BUp ).

Common apo E polymorphism.
The common apo E polymorphism was a significant predictor of LpE, apo E-Lp-non-B, and apo E-LpB concentrations in children/adolescents (Table 1AUp ) and in adults (Table 1BUp ). Compared with the {epsilon}3/{epsilon}3 genotype, individuals carrying the {epsilon}2 allele showed significantly higher LpE and apo E-Lp-non-B concentrations (P <=0.001) and a lower apo E-LpB concentration (P <=0.001). Conversely, the {epsilon}4 allele was significantly associated with lower concentrations of LpE and apo E-Lp-non-B (P <=0.001) in children/adolescents and adults of both sexes, whereas the apo E-LpB concentration was significantly higher in boys and men (P <0.05) and not significantly higher in girls and women. The mean allelic effects of {epsilon}2 and {epsilon}4 on the three lipoparticle concentrations are shown in Fig. 4 . The results were obtained with a codominant additive model for the log-transformed apo E values in comparison with {epsilon}3/{epsilon}3 genotype. {epsilon}2 and {epsilon}4 allelic frequencies were coded as 0, 0.5, and 1. The mean allelic effect of {epsilon}2 was significantly different among the four groups for LpE concentration (P <=0.001), as was that of {epsilon}4 for LpE (P <=0.001) and apo E-Lp-non-B concentration (P <=0.05) as given by the test for equality of slope, with men exhibiting the lowest allelic effects.



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  		Figure 4. Mean effects of {epsilon}2 ({blacksquare}) and {epsilon}4 () allelic frequencies on LpE, apo E-LpB, and apo E-Lp-non-B concentrations according to age and sex.

Values were log-transformed and adjusted for age, Tanner stages, alcohol consumption, and cholesterol, TG, apo AI, and apo B concentrations; the {epsilon}3 allele was used as reference. {epsilon}2 and {epsilon}4 allelic frequencies were coded as 0, 0.5, or 1.

reference limits for apo E in particles
Because the most important variables for these lipoproteins were apo E polymorphism, age, and gender, they were used as partition criteria to produce genetic reference limits. Because of the low frequencies of the {epsilon}2/{epsilon}2, {epsilon}4/{epsilon}4, and {epsilon}4/{epsilon}2 genotypes in this sample population, we estimated the reference limits for LpE (Table 2AUp ), apo E-LpB (Table 2BUp ), and apo E-Lp-non-B (Table 2CUp ) for only the three most frequent apo E genotypes ({epsilon}3/{epsilon}2, {epsilon}3/{epsilon}3, and {epsilon}3/{epsilon}4). The reference limits for LpE and apo E-Lp-non-B varied in the same way. According to age and median values, men exhibited lower concentrations than women for these two lipoparticles regardless of apo E genotype. For apo E-LpB concentrations, the differences between men and women were less pronounced than those observed for the LpE and apo E-Lp-non-B lipoparticles, whereas variations attributable to age were more important in men than in women regardless of the apo E genotype. The influence of the common apo E polymorphism on the concentrations of the three lipoparticles was clear. Serum LpE and apo E-Lp-non-B concentrations varied in the following decreasing order: {epsilon}3/{epsilon}2 < {epsilon}3/{epsilon}3 < {epsilon}3/{epsilon}4. On the other hand, the apo E-LpB concentration showed an inverse increasing order: {epsilon}3/{epsilon}2 > {epsilon}3/{epsilon}3 > {epsilon}3/{epsilon}4.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The importance of apo E and its common polymorphism in CHD is now well documented. However, the common apo E polymorphism is insufficient to explain the development of CHD, and it has been suggested that the serum concentration of apo E itself could play an independent role in this process (5). Nevertheless, the mechanism remains unclear, and there is growing evidence that lipoprotein subspecies, particularly lipoparticles containing or not containing apo B, could play a role in the development of atherosclerosis and the occurrence of myocardial infarction or CHD (2)(6)(9). In addition, several studies have pointed out the importance of these lipoprotein subspecies and of the common apo E polymorphism in surveys of individuals taking lipid-lowering drugs (12) or oral contraceptives (14). Clinical validation of these apo E lipoparticles requires knowledge of the factors involved their biological variation and the establishment of reference limits. The present work deals with these objectives for LpE, apo E-LpB, and apo E-Lp-non-B, using a large sample population from the Stanislas Cohort study (15).

In this study, the main biological factors significantly modifying the LpE, apo E-LpB, and apo E-Lp-non-B concentrations were age, gender, and apo E polymorphism. The effect of age differed between apo E-LpB and apo E-Lp-non-B. This sex-differential pattern was demonstrated by the increase with age of the proportion of apo E-LpB, especially in adult men, in whom the percentage of apo E-LpB was nearly one-half of the total LpE content. The age and gender effects on apo E-LpB concentration and its proportion in LpE lipoparticles were similar to those observed for other lipids, such as TC or TGs. These age and gender effects are probably explained in part by the presence of circulating sex steroid hormones. The influence of puberty, which has been described previously by Vincent-Viry et al. (3) in relation to apo E concentration, was observed in this study for LpE, apo E-LpB, and apo E-Lp-non-B concentrations. In children and adolescents, puberty was associated with a decrease in apo E-Lp-non-B and LpE concentrations and with a slight increase in apo E-LpB concentration. In boys, the effect of puberty could be related to the increase in testosterone, which was demonstrated to modify the lipid profile (21). In females, the change in apo E lipoparticles could be associated with the modification in estrogen concentrations during life or with hormone treatments (puberty, oral contraceptive intake or hormone replacement therapy, and menopause). Recently, we showed that oral contraceptives decreased the concentrations of apo E and apo E-Lp-non-B and increased apo E-LpB, producing a redistribution of apo E from Lp-non-B toward LpB (14). For this reason, we did not study the effect of oral contraceptives and excluded treated women from the sample population. Li et al. (13) found that the concentration of apo E-LpB was higher in postmenopausal women than in premenopausal women. In our sample population, menopausal women were excluded because of the small number (n = 14).

Lifestyle factors were less associated with the serum concentrations of apo E lipoparticles. Alcohol consumption was negatively associated with apo E-LpB only in adult women, which is in agreement with the known beneficial effect of moderate intake of alcohol on the lipid profile and the low alcohol consumption of the women in our sample population.

Another important factor affecting LpE, apo E-Lp-non-B, and apo E-LpB concentrations was the common apo E polymorphism. In this study, results regarding the influence of the common apo E polymorphism on the three lipoparticle concentrations are in agreement with those observed by Luc et al. (6) in healthy men 25–64 years of age. Our study showed that {epsilon}2 and {epsilon}4 allelic effects were also present in females and children/adolescents. However, these effects varied according to age and gender and were more pronounced in adult men and in girls for LpE concentration and in adult men for apo E-Lp-non-B concentration. The quantitative effect of the {epsilon}2 allele was greater than that of the {epsilon}4 allele regardless of the study group and was attributable mainly to variations in the apo E-Lp-non-B concentration. These results are in accordance with the known preference of apo E4 for apo B-containing lipoproteins and the preferential association of the apo E2 isoform with HDL particles. As hypothesized by others (6), the distribution of apo E isoforms among lipoproteins depends on several factors, such as affinity for the receptors and the different types of lipoproteins, formation of complexes with other apolipoproteins, and the presence of apo E dimers. The apo E concentration in nonatherogenic apo E-Lp-non-B particles significantly decreased from the {epsilon}2 to the {epsilon}3 and to the {epsilon}4 allele, which is opposite to the increasing risk of CHD: {epsilon}4 > {epsilon}3 > {epsilon}2 (5)(6)(8)(22)(23)(24). Moreover, it has also been reported that the common apo E polymorphism is associated with modifications in the lipid profile, in particular the {epsilon}4 allele is associated with higher concentrations of TC, LDL-C, and apo B (13)(25)(26) and with smaller LDL size (27). The {epsilon}2 allele is associated with an inverse pattern.

For TG concentrations, both alleles are associated with higher serum concentrations (25). Individuals carrying the {epsilon}4 allele present a higher risk for CHD and also have an increased apo E-LpB concentration (24) and an increased proportion of apo E-LpB (11). In our study, the apo E-Lp-non-B concentration was negatively associated with TG concentration, whereas apo E-LpB was positively and strongly associated in adults and in children/adolescents with TG concentration, which is already known to be an independent risk factor for CHD.

Generally speaking, the apo E-Lp-non-B concentration is often associated with a nonatherogenic lipid profile. It is also well documented that age, male gender, and hormonal status associated with an altered lipid profile maximize the risk of CHD. In particular, it has been demonstrated that patients with CHD have higher concentrations of apo E-LpB and LDL-C (9)(28) and that all apo B-containing lipoproteins are increased in postmenopausal women (13). Here we report age- and sex-related modifications of the proportion of apo E-LpB in LpE particles. The lipid profile and lipoprotein subspecies associated with the effects of age, gender, and common apo E polymorphism could lead to an increased risk for the development of CHD. Thus, age- and gender-specific genetic reference limits for LpE, apo E-LpB, and apo E-Lp-non-B concentrations are essential for interpretation of the results in clinical or therapeutic trials. In practice, the concentrations of the three lipoparticles could first be measured, and then the values could be compared with the genetic reference limits obtained for the {epsilon}3/{epsilon}3 genotype. Individuals with values outside the 95% reference interval could be genotyped for apo E. Thus, clinical interpretation will be more precise in the evaluation of cardiovascular risk or in therapeutic monitoring.

In conclusion, the clinical value of lipoparticle determinations needs to be validated by large clinical studies because the current published data are encouraging in the field of cardiovascular risk. We provide genetic reference limits for LpE, apo E-LpB, and apo E-Lp-non-B particles stratified by age, gender, and apo E polymorphism for the three most frequent genotypes, which may help this effort to progress.


   Acknowledgments
 
We are grateful to the staff of the Centre for Preventive Medicine at Vandoeuvre-lès-Nancy (France) for their contributions in recruitment of participants and collection of data for the cohort. We are also indebted to the families of the Stanislas survey, who made this study possible. We express our gratitude to the public institutions, such as the University of Nancy, INSERM, and the Region Lorraine, and to the private companies (Roche and Bayer), who supported this work. We also thank Sebia for the kind gift of reagents.


   Footnotes
 
1 Nonstandard abbreviations: apo, apolipoprotein; TC, total cholesterol; LDL-C and HDL-C, LDL- and HDL-cholesterol, respectively; CHD, coronary heart disease; TG, triglyceride; LpE, lipoprotein E; apo E-LpB, apo E in lipoprotein particles containing apo B; apo E-Lp-non-B, apo E in lipoprotein particles not containing apo B; BMI, body mass index; GGT, {gamma}-glutamyltransferase; ALT, alanine aminotransferase; and AST, aspartate aminotransferase.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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