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Clinical Chemistry 44: 957-965, 1998;
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(Clinical Chemistry. 1998;44:957-965.)
© 1998 American Association for Clinical Chemistry, Inc.

Lipids and Lipoproteins

Biological variations and genetic reference values for apolipoprotein E serum concentrations: results from the STANISLAS cohort study

Monique Vincent-Viry1,3, Françoise Schiele1,3, René Gueguen2, Karin Bohnet1,3, Sophie Visvikis1,3, and Gérard Siest1,3,a

1 Laboratoire du Centre de Médecine Préventive and
2 Département statistiques, 2, avenue du Doyen Jacques Parisot, 54500 Vandoeuvre-lès-Nancy, France.

3 Université Henri Poincaré, 54000 Nancy, France.
a Address correspondence to this author at: Laboratoire du Centre de Médecine Préventive, 2, avenue du Doyen Jacques Parisot, 54500 Vandoeuvre-lès-Nancy, France. Fax 33 (0)3 83 44 87 21; e-mail Gerard.Siest{at}cmp.u-nancy.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Serum apolipoprotein (apo) E concentrations were determined by immunoturbidimetry in 4284 subjects from 4 to 71 years of age and belonging to 1003 nuclear families recruited for the STANISLAS cohort study between January 1994 and August 1995. Values for apo E ranged from 16 to 169 mg/L, with a geometric mean ± SD values of 46.6 ± 13.8 mg/L in the overall sample. The interindividual variability varied from 24.6% to 32.0% among family members. Females exhibited higher apo E values than males until the age of 17–26 years. Conversely, after the age of 26 years, serum apo E concentrations were higher in men than in women. Biological factors affecting serum apo E concentrations were described in fathers, mothers, sons, and daughters and explained up to 32.0% of the apo E variability in daughters and 19.0% in fathers. The main biological factors affecting apo E concentrations were the following: apo E polymorphism, waist-to-hip ratio, oral contraceptive intake, puberty, body mass index, age, and gender. Given the importance of apo E polymorphism in the regulation of apo E concentrations, we recommend the use of genetic-based reference values for the clinical interpretation of serum apo E concentrations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Apolipoprotein E (apo E)1 is a polymorphic protein existing in humans in three common isoforms: apo E2, apo E3, and apo E4, coded by three alleles {epsilon}2, {epsilon}3, and {epsilon}4. Apo E3 is the most common isoform, with a prevalence of ~77% in Caucasian populations. Apo E4 is the second most common isoform (~15%), and apo E2 is the rarest (~8%). Apo E polymorphism is functional and influences a variety of physiological and pathological processes. Its influence on serum concentrations of total cholesterol (TC) and LDL-cholesterol and the concentration of apo B is well known (1)(2)(3)(4)(5). Apo E polymorphism has also been implicated in the etiology of several diseases: cardiovascular disease, neurodegenerative diseases such as Alzheimer's disease, and many others (6).

In addition, this polymorphism strongly influences the apo E concentration (6), and there is growing evidence that variations of the apo E concentration have a direct influence on metabolic processes. Moreover, it has been suggested that both the apo E concentration and apo E genotype play an important role in lipoprotein metabolism (7)(8). Increased concentrations have been reported in patients with familial dysbetalipoproteinemia (9). Published physiological apo E concentrations vary between 30 and 250 mg/L (6). One of the main reasons for these discrepancies could be the variety of methods and calibrators used for apo E measurements. As in the example of apo AI and apo B, the use of a common reference material should improve the consistency of results. Another important reason for differences in apo E concentrations could be the sample population studied. Yet another reason for the discrepancies could be the effect of biological factors.

The assessment of apo E concentration as a marker of cardiovascular risk requires the knowledge of its biological variations and of clearly defined reference limits and specific decision limits. The aims of this study were to identify the most important causes of biological variation in apo E concentrations, to determine serum apo E reference limits for the most frequent genotypes on a well-selected population sample belonging to the STANISLAS cohort study, and to estimate the reference values for the other apo E genotypes, using a translation factor and the {epsilon}3/{epsilon}3 genotype as a reference.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
sample population
Subjects were apparently healthy individuals attending the Centre for Preventive Medicine at Vandoeuvre-lès-Nancy (France) for a routine health examination that is repeated every five years. In this context, we recruited, between January 1994 and August 1995, 1003 nuclear families of Caucasian origin and including at least two children (4- to 26-years-old). This sample consisted of married subjects with their children, i.e., 4284 fasting individuals [1003 men, 1003 women, and 2278 children (1147 sons and 1131 daughters)]. This large familial sample called the "STANISLAS cohort study" will be followed for 10 years (10). All subjects gave their written informed consent for participating in this study, which was approved by the local ethics committee of Nancy. For practical reasons, the recruitment was limited to three or four families per day. This represented 10% to 13% of the subjects visiting the center per day. The health screening included multiple tests: 22 blood constituents, life-style descriptions, socio-professional environment questionnaires (questionnaires on the medical history and living conditions of the family), functional tests, and physical examinations.

The factors studied that could potentially affect the serum apo E concentration were apo E polymorphism, age, body mass index [BMI; calculated according to the Quetelet's formula, weight (kg)/height (m)], waist-to-hip ratio (WHR), alcohol and tobacco consumption, lipid-lowering drugs, oral contraceptive use, hormone replacement therapy, menopause, and puberty. For sexual maturation, the puberty variable was divided into three classes in males (before, during, and after puberty) and two classes in females (before and after menarche).

blood samples
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 Dickinson). Blood was centrifuged promptly at 1000g for 15 min at room temperature for serum separation and buffy coat preparation. The sera and buffy coat were frozen in liquid nitrogen until analysis or the extraction of DNA.

analytical methods
DNA extraction was performed according to the method of Miller et al. (11). Apo E genotype was determined by PCR amplification and subsequent digestion with the restriction enzyme HhaI as described by Hixson and Vernier (12). Aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2), {gamma}-glutamyltransferase (EC 2.3.2.2), TC, and triglycerides (TGs) were measured in fresh serum using established enzymatic methods (Merck) on an AU5000 automated analyzer (Olympus Merck). Serum lipoprotein(a), apo AI, and apo B were determined on a Behring automated nephelometer (Behring). Serum HDL-cholesterol and apo E were measured on a Cobas-Mira analyzer (Roche Diagnostics). Serum apo E concentrations were determined by immunoturbidimetry, using a kit from Daiichi (Apo E Auto"Daiichi," reference 114861) and according to the manufacturer's recommendations (13). Calibration curves were obtained by serial dilution of a serum calibrator (Daiichi High Level Standard, reference 125799; target value, 105 mg/L). The detection limit of the method was 6.3 mg/L with an upper limit of 100 mg/L. Sera were analyzed without pretreatment and diluted in double-distilled water when the apo E concentration exceeded 100 mg/L. Control sera (lyophilized 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 serum pools freshly prepared and stored at 4 °C; it varied from 2.8 to 3.4%. The day-to-day reproducibility was estimated to be 3.4% on a pooled serum (stored frozen at -20 °C). For the commercially available Daiichi control serum (stored at 4 °C), the reproducibility was 4.2% (one month) and 7.0% (12 months).

statistics
Statistical analyses were performed using BMDP® statistical software (University of California, Los Angeles, CA) and using log-transformed values for serum apo E concentrations [Ln (apo E)]. We excluded 156 subjects with missing data and/or pathological values according to the following criteria: abnormal liver metabolism as defined by increased enzyme activities [alanine aminotransferase >200 U/L (5 subjects); aspartate aminotransferase >200 U/L (14 subjects); {gamma}-glutamyltransferase >300 U/L (1 subject)], abnormal lipid profiles [TGs >10 mmol/L (3 subjects), and TC >11 mmol/L (1 subject)] and pregnancy (one woman). All statistical analyses were conducted separately on fathers, mothers, sons, and daughters (14)(15). In the first step, unidimensional comparisons were performed by a one-way ANOVA to assess the degree of significance of the main biological factors on the serum apo E concentration. Significant factors were next introduced in a multiple regression analysis that was used to quantify the relationships between the apo E concentration and the biological factors. The significant factors served to define exclusion and partition criteria for selection of the reference sample population. The following variables were included in the regression: apo E polymorphism, age, BMI, WHR in parents only, puberty, oral contraceptive use, and the five genotype groups ({epsilon}2/{epsilon}2, {epsilon}2/{epsilon}3, {epsilon}2/{epsilon}4, {epsilon}3/{epsilon}4, and {epsilon}4/{epsilon}4); the {epsilon}3/{epsilon}3 genotype served as reference. Interaction testing involving genotypes with BMI, WHR, age, puberty, and oral contraceptive use was performed; however, the interaction effects increased by <1% the explained Ln (apo E) variability in each group, and the differences were not statistically significant (P >0.05). Finally, we estimated the 2.5th, 5th, 50th, 95th, and 97.5th percentiles of the apo E distribution in each reference group.

reference samples
Taking into account the results of the regression analyses, we excluded subjects with BMI values >30 for parents and >20 (5–11 years), >23 (11–14 years), >26 (14–17 years), and >29 (17–26 years) for children (16); we also excluded adults with WHR values >1 for fathers and >0.9 for mothers. The effect of oral contraceptive intake on the serum apo E concentration was adjusted by correcting values in women taking oral contraceptives by the regression coefficients obtained in the multiple regression analysis. Percentiles of apo E distribution were directly estimated in the {epsilon}3/{epsilon}3, {epsilon}3/{epsilon}2, and {epsilon}3/{epsilon}4 genotypes. Reference values for Ln (apo E) in genotypes other than {epsilon}3/{epsilon}3 were derived from those of the {epsilon}3/{epsilon}3 group by a translation coefficient estimated from the regression analysis. Reference values for Ln (apo E) were then converted to reference values of apo E by exponential transformation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
sample profile
Socio-demographic and biological characteristics of the total sample are presented in Table 1 . Most of the parents were of middle age, with an average age of 42.0 ± 5.0 years for fathers and 39.9 ± 4.8 years for mothers. Children were an average age of 13.6 ± 3.8 years for sons and 13.9 ± 4.1 for daughters. The concentrations of the biological variables, especially lipids and BMI, were almost within reference ranges, as shown by the means and SD values. Consumption of alcohol and tobacco was moderate. These life-style characteristics were in accordance with the familial recruitment of apparently healthy subjects. The frequency of E3, the most common isoform, reached 79% in the parental population and 78% in children. Isoforms E4 and E2 had a prevalence of 12% (E4) and 9% (E2) in parents and 13% (E4) and 9% (E2) in children. In this sample, the distribution of apo E genotypes was in Hardy-Weinberg equilibrium proportions (Table 1 ). These relative allele frequencies were similar to those seen in another Caucasian population (1).


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  		Table 1. Socio-demographic and biological characteristics of the total studied sample.

distribution of apo e values
In the overall sample (n = 4284 subjects), the serum concentration of apo E ranged from 16 to 169 mg/L. The distribution of apo E in each subgroup is shown in Fig. 1 , with mean and SD values as described in Table 1Up . All distributions were skewed, with skewness coefficients varying from 2.47 in fathers to 0.67 in daughters. A logarithmic transformation was therefore performed. In parents, the fathers had a mean apo E concentration greater than the mothers (49.7 ± 15.9 mg/L vs 44.7 ± 11.1 mg/L). In children, the means and SD values were 44.5 ± 11.1 mg/L in sons and 47.5 ± 11.7 mg/L in daughters. The total interindividual biological variation of serum apo E, including analytical variation, was ~26% in mothers, 25% in sons and daughters, and 32% in fathers.



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  		Figure 1. Distribution of values for apo E serum concentrations (mg/L) in fathers, mothers, sons, and daughters.

biological factors influencing apo e concentration
The ANOVA showed that alcohol and tobacco consumption, lipid-lowering drugs, hormonal replacement therapy, and menopause had no effect on serum apo E concentrations in this sample population (P >0.10). In contrast, age, BMI, WHR, puberty, and oral contraceptive intake had a significant influence on the serum apo E concentration (P <0.01 to P <0.001). Thus, these variables were included with apo E genotypes (using {epsilon}3/{epsilon}3 genotype as the reference) as explanatory variables in the multiple regression analysis. Table 2 shows the respective regression coefficients and SDs for each variable found to be significant (P <0.05). These biological indices explained 19–32% of the Ln (apo E) variability. In children, Ln (apo E) decreased with age only in males (P <0.05) and with puberty in both sexes (P <0.01). In adults 26- to 56-years-old, age did not affect the Ln (apo E) value. We decided therefore to present apo E reference values by age in children and without age stratification in adults.


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  		Table 2. Multiple regression analysis with regression coefficients (and SD) obtained for biological factors influencing apo E concentration.

Oral contraceptive use decreased the Ln (apo E) value in mothers and in daughters (P <0.001) with respective regression coefficients of -0.150 ± 0.017 and -0.191 ± 0.022. For the estimation of the apo E reference values, we adjusted Ln (apo E) values for oral contraceptive intake, using the previously determined regression coefficients. Because BMI and WHR significantly (P <0.01 to P <0.001) affected apo E concentrations, we excluded subjects with increased values (see Materials and Methods).

Polymorphism of apo E was the most important factor known to modify serum apo E concentrations. When the {epsilon}3/{epsilon}3 genotype was used for comparison, multiple regression analysis gave the mean deviation for each genotype in regard to the Ln (apo E) values observed in the {epsilon}3/{epsilon}3 group. As expected in fathers, for example, the most important positive deviation was observed for the {epsilon}2/{epsilon}2 group (0.878 ± 0.125). The deviations for the {epsilon}3/{epsilon}2 group and for the {epsilon}2/{epsilon}4 group were 0.211 ± 0.024 and 0.126 ± 0.059, respectively. For {epsilon}3/{epsilon}4 subjects (-0.034 ± 0.021) and for {epsilon}4/{epsilon}4 subjects (-0.038 ± 0.065), negative deviations were obtained.

We repeated the regression analysis with no more than one child of each sex from each family. No bias was observed in the age structure, the genotype proportions, means, and SDs for all of the variables tested, and the regression coefficients were not statistically different. Considering the small discrepancies between the two models of regression, we decided to present the first model, which included more subjects.

reference values of serum apo e concentration
The reference sample represented 3956 subjects, i.e., 92.3% of the overall sample. Among them, 2434 individuals carried the {epsilon}3/{epsilon}3 genotype and 1522 carried a different genotype. One hundred and seventy-two subjects were excluded because of increased BMI and/or WHR values. The characteristics of the reference sample are presented in Table 3 ; these characteristics were different from the overall sample for lipids, enzymes, and BMI values, with a less important dispersion. The reference values for serum apo E concentrations in the three most common genotype groups are summarized in Table 4 . The reference values for serum apo E concentrations in the three rare genotype groups are shown in Table 5 . These reference values were estimated from those of the {epsilon}3/{epsilon}3 genotype, using the regression coefficients listed in Table 2Up . Regarding age groups and median values, males exhibited lower apo E values than females until 14–26 years of age. In contrast, in adults older than 26 years, men had higher apo E values than women regardless of genotype. In both sexes, the lowest apo E concentration was found between the ages of 14 and 25. The effect of age, related to puberty in boys and girls, remained in girls even after the adjustment for oral contraceptive intake was made. For more information, we also examined apo E reference values between individuals 5–11, 11–14, 14–17, and 17–26 years of age in children carrying the {epsilon}3/{epsilon}3 genotype. When these values were compared with the reference values mentioned on Table 4 , we observed a decrease of apo E values estimated at the 50th percentile in children aged 11 to 25 years: 43.8 mg/L to 39.7 mg/L for boys and 48.9 mg/L to 45.2 mg/L for girls.


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  		Table 3. Socio-demographic and biological characteristics of the reference sample population (all genotypes).


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  		Table 4. Reference values1 for serum apo E concentrations (mg/L) by genotype, age, and sex.


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  		Table 5. Reference values1 for serum apo E concentrations (mg/L) by age and sex for the less frequent genotypes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The measurement of serum apo E concentrations is, at this moment, rarely used in clinical chemistry, in contrast to the measurement of apo AI and apo B. The effect of apo E polymorphism on the concentration of apo E and on the concentrations of other lipids, such as cholesterol and TGs, is well documented (1)(2)(3)(4)(5)(6)(17)(18). However, no reference values that include the polymorphism effect and biological factors influencing apo E concentrations have been established. Here we report a detailed description and quantification of apo E biological variations, and we provide genetic reference intervals for the apo E concentration in Caucasian individuals belonging to a large population-based study. The population sample was selected a posteriori, according to IFCC recommendations (19)(20) and taking into account the main biological factors that have been shown to affect the apo E concentration. Serum apo E concentrations were measured by immunoturbidimetry using a kit from Daiichi Pure Chemicals (13). This method, which can be automated, does not require any pretreatment of the samples and is suitable for routine analyses with an acceptable reproducibility (CV <4.5%).

The total interindividual variation of serum apo E concentrations in the overall sample population studied is considerable (25–32%) but similar to that reported for other apolipoproteins (21)(22)(23).

Biological factors that significantly modified serum apo E concentrations were, in decreasing order of importance, apo E genetic polymorphism (mainly {epsilon}2/{epsilon}2 genotype), WHR in adults, oral contraceptive intake, puberty in both sexes, BMI, and age in sons.

Age and sex significantly influence serum apo E concentrations. We found lower apo E values in boys than in girls. However, in adults over 26 years of age, the apo E concentration was higher in men than in women. This interaction of age and sex on apo E concentrations is similar to that observed for apo B, with lower values in males than in females until 25–35 years of age and higher apo B concentrations in older males (21). These data could explain the different results published in the literature, i.e., higher apo E concentrations in women (9)(24), in men (25), or no difference (26). The decrease of apo E values with age in children and the lack of age variation in adults are in agreement with other studies (27)(28).

In addition to the age effect, puberty significantly decreases serum apo E concentrations in both sexes. This independent impact of puberty on apo E concentrations has not been reported previously. This finding could be compared in girls with the diminishing effect of estrogens or the use of oral contraceptives on serum apo E concentrations as described by others (17)(29)(30)(31). In the overall sample studied, the decrease in apo E concentrations in women taking oral contraceptives leads to a shift of the apo E distribution towards lower values in mothers and in daughters.

Alcohol and tobacco consumption did not significantly modify apo E concentrations. However, the consumption of alcohol and tobacco was relatively moderate in this group. It has been reported that alcohol abuse increases apo E concentrations (32)(33), although moderate drinking (34) and smoking do not seem to influence the concentration of apo E (27)(33).

Increased BMI and WHR values were associated with increased apo E concentrations, in agreement with others (17)(35). BMIs and/or WHRs were positively correlated with TGs, LDL-cholesterol, and apo B and negatively correlated with HDL-cholesterol and apo AI (17)(35). Relationships between body mass, TGs, and some hepatic enzymes, especially alanine aminotransferase and {gamma}-glutamyltransferase, were described some years ago (36)(37). {gamma}-Glutamyltransferase activity is often increased in non-insulin-dependent diabetics. This observation could be related in part to the modified lipid metabolism in patients with insulin-resistant syndrome (unpublished results).

The apo E genotype is the most important factor affecting its serum concentrations. Between 6% and 20% of the total variability of the apo E concentration has been attributed to its polymorphism by several authors (2)(5)(27)(38)(39). Results from a recent study conducted in the same cohort provided evidence that apo E variability is determined by its genetic polymorphism and clearly demonstrated the nonadditive effects of {epsilon}2 and {epsilon}4 alleles (17). As expected, the highest apo E concentrations are found in the {epsilon}2/{epsilon}2 genotype and the lowest in the {epsilon}4/{epsilon}4 genotype (see Table 2Up ).

Mean deviations estimated by multiple regression analysis on Ln (apo E) are in agreement with those described in previous studies [for review see (6)]. The great impact of polymorphism on apo E concentrations justifies the selection of a homogeneous sample regarding its genotype for the establishment of apo E reference values. Because the {epsilon}3/{epsilon}3 genotype occurs most frequently, we chose to define apo E reference values using {epsilon}3/{epsilon}3 subjects as the baseline to estimate reference values in the three less frequent genotypes by applying a translation coefficient to Ln (apo E). In addition, this genotype is not reported to be associated with pathological states.

Apo E polymorphism not only influences apo E concentration but also cholesterol and apo B concentrations, which are cardiovascular risk factors. The concentration of apo E has been shown to modulate lipid metabolism (8), and it was suggested that the apo E concentration, in addition to polymorphism, might become a risk factor for cardiovascular disease (6)(27). The use of reference intervals, the knowledge of the main factors causing variation, and case-control studies to assess relationships between apo E concentrations and pathological states will help to determine the relevance of apo E in clinical chemistry. An apo E concentration in a nongenotyped individual can first be compared with apo {epsilon}3/{epsilon}3 reference limits. If it is outside the 95% reference interval, a genotype could be determined. An apo E concentration in a genotyped individual should, of course, be compared with its corresponding genotype reference interval. However, the question remains open about the predictive value of an apo E concentration outside the reference limits in each genotype.

In conclusion, we have shown that the serum apo E concentration is affected by its polymorphism and by most of the current biological factors of variation: age, gender, BMI, WHR, puberty, and oral contraceptive use. The most important sources of variation in the studied population were the {epsilon}2 allele and WHR. Age was the least important. The large number of subjects and the amount of information collected in the STANISLAS cohort study allowed us to quantify the different observed variations. For the first time, we produced genetically derived reference values for serum apo E concentrations. We therefore recommend, to interpret results of apo E measurements, taking into account all possible source of variability. Consequently, the use of reference limits according to the apo E genotype is essential for the interpretation of its concentration.


   Acknowledgments
 
We are grateful to the staff of the Centre for Preventive Medicine of Vandoeuvre-lès-Nancy, France, for their contributions in recruitment and the collection of data of the STANISLAS cohort. Special thanks to M.J. Longis for her help in the statistical analyses and to W. Ruff (Howard University, Washington, DC) for his assistance with the translation. We are also indebted to the families of the STANISLAS survey, who made this study possible. K. Bohnet was the recipient of a grant of the Gottlieb Daimler-und Karl Benz-Stiftung, Ladenburg, Germany. This work was supported by the European Commission (Contract No.: CT 961543) and by Daiichi Pure Chemicals; the STANISLAS cohort study was supported by Beckman Instruments, Biomérieux, Johnson & Johnson, and Merck.


   Footnotes
 
Presented in part at the 70th Scientific Sessions, American Heart Association, November 9–12, 1997, Orlando, Florida.

1 Nonstandard abbreviations: apo, apolipoprotein; TC, total cholesterol; STANISLAS: Suivi Temporaire Annuel Non Invasif de la Santé des Lorrains Assurés Sociaux; BMI, body mass index; WHR, waist-to-hip ratio; TG, triglyceride; and Ln (apo E), log-transformed apo E value.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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