Apolipoprotein E and cholesteryl ester transfer protein polymorphisms in normal and preeclamptic pregnancies

European Journal of Obstetrics & Gynecology and Reproductive Biology 112 (2004) 9–15

Apolipoprotein E and cholesteryl ester transfer protein polymorphisms in normal and preeclamptic pregnancies Luı´s Beloa,b,*, Dairena Gaffneyc, Muriel Caslakec, Alice Santos-Silvaa,b, Luı´s Pereira-Leited, Alexandre Quintanilhab,e, Irene Rebeloa,b a

Department of Biochemistry, Faculty of Pharmacy, University of Porto, 4050-047 Porto, Portugal b Institute for Molecular and Cell Biology, University of Porto, 4150-180 Porto, Portugal c Department of Pathological Biochemistry, Glasgow Royal Infirmary, University N.H.S.Trust, G31 2ER Glasgow, UK d Department of Obstetrics and Gynaecology, Porto Medical School, Hospital of S.Joa˜o, 4200-319 Porto, Portugal e Institute of Biomedical Sciences of Abel Salazar, University of Porto, 4099-003 Porto, Portugal Received 9 September 2002; received in revised form 10 January 2003; accepted 26 March 2003

Abstract Objectives: To evaluate the association of apolipoprotein (apo) E polymorphism and a cholesteryl ester transfer protein (CETP) polymorphism (CETP/TaqIB) with preeclampsia and with lipid/lipoprotein profile in pregnancy. Materials and methods: A group of 144 normal pregnant women (67 in the third trimester) were compared with 51 cases of preeclampsia in the third trimester of gestation. Apo E and CETP genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism. Serum lipids, lipoproteins and apolipoproteins were evaluated using commercially available kits. LDL size was assessed by gradient gel electrophoresis. Results: No differences were found in the distribution of subjects with respect to genotypes, in the apo E and CETP polymorphisms, between control and pathologic groups. In the third trimester of gestation (both control and case groups considered), apo E polymorphism, but not CETP polymorphism, was associated with different lipid and lipoprotein levels. Patients carrying the E2 allele (E2þ) presented with significantly lower values of LDLcholesterol (LDLc) compared with carriers of E4 (E4þ) and E3/3 individuals. E2þ also presented with the highest triglyceride (TG) level, although this was not statistically significant. On the other hand, HDLcholesterol (HDLc) and apo A-I levels were significantly reduced in E4þ, compared with E3/3. Furthermore, E4þ presented with the highest total cholesterol and LDL and therefore LDLc/HDLc and apo B/apo A-I ratios were significantly higher in this group compared with the other two. Conclusions: Neither of our candidate genes showed association with preeclampsia. However, apo E genotype was associated with changes in lipid and lipoprotein profiles in pregnant women. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Apolipoprotein (apo) E polymorphism; Cholesteryl ester transfer protein (CETP) polymorphism; Lipids; Preeclampsia; Pregnancy

1. Introduction Preeclampsia, a characteristic multisystem disorder of pregnancy, presents a typical pattern of ‘‘acute atherosis’’ in the arteries of the uteroplacental bed and endothelial cell dysfunction is likely to have an important role in its pathophysiology [1]. Abnormal lipid profiles and species may promote oxidative stress and vascular dysfunction observed in this syndrome [2,3]. Preeclamptic women, compared with normal pregnant women, present with significantly higher triglyceride (TG) [4–7] and reduced HDLcholesterol *

Corresponding author. Tel.: þ351-22-2078906; fax: þ351-22-2003977. E-mail address: [email protected] (L. Belo).

(HDLc) and apolipoprotein (apo) A-I [4,7] concentrations. Moreover, LDL mean particle diameter is significantly decreased in preeclampsia relative to normal pregnancy [7]. Since preeclampsia has a higher incidence within families with a history of such a disorder [8–10], it is possible that some genes may be involved in its aetiology. However, and despite extensive research, the genetic basis of preeclampsia remains unclear [11]. Candidate genes associated with this disorder are apo E and cholesteryl ester transfer protein (CETP) genes. CETP is involved in reverse cholesterol transport and in remodelling lipoproteins. CETP effects a net exchange of HDL cholesteryl ester (CE) with the TG of triacylglycerolrich lipoproteins [12]. Subjects with the B1 allele of the CETP TaqIB polymorphism are more likely to have

0301-2115/$ – see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0301-2115(03)00240-9

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increased CETP activity and lower HDLc levels [13]. This may be of importance in the genesis of atherosclerotic events, since there is a strong inverse relationship between HDLc concentration and such events [14]. Apo E also plays an important role in atherosclerosis, by modifying inflammatory responses, by facilitating cholesterol efflux from foam cells and by regulating hepatic uptake of remnant lipoproteins through the LDL receptor and LDL-receptor-related protein (or so-called ‘‘remnant’’ receptor) [15]. In the apo E polymorphism, subjects carrying the E2 (E2þ) and E4 (E4þ) alleles are associated, respectively, with lower and higher cholesterol levels compared to E3/3 individuals [16]. Concerning the effect of apo E allele on TG levels, contradictory results are found in literature. However, a meta-analysis reported that TG levels are higher in E2þ and in E3/4 than E3/3 subjects [16]. Moreover, apo E polymorphism also associates with plasma concentration of apo E, E2þ presenting the highest values [17]. Given apo E and CETP properties and the association of their polymorphisms with different lipid and/or lipoprotein profiles, it seems reasonable to assume that both may be associated with preeclampsia. Earlier reports have already focused on the association of apo E with this disorder, although conclusions remain controversial. Some authors noticed a higher frequency of the E2 and E4 alleles in women with preeclampsia [18,19]. However, these findings have not been confirmed [20,21]. The aim of our work was to clarify the potential association of apo E polymorphism with preeclampsia and to investigate whether CETP polymorphism might also be related with this disorder. We also intended to evaluate how both polymorphisms may influence lipids and lipoproteins in pregnant women.

2. Material and methods 2.1. Subjects The protocol used for all pregnant women was approved by the Committee on Ethics of the University Hospital S. Joa˜ o, Porto. Clinical data regarding the sample population was collected at the Obstetric Service of this Institution. Normal pregnancy was diagnosed on the basis of clinical and ultrasound findings. Healthy pregnant women had a normal course and outcome of pregnancy and did not receive any medication known to interfere with lipid metabolism. Diagnosis of preeclampsia was determined by gestational hypertension accompanied by proteinuria, oedema, or both. In agreement with the Committee on Terminology of the American College of Obstetricians and Gynecologists, gestational hypertension was defined as an increase by at least 30 mmHg systolic or 15 mmHg diastolic blood pressure compared with values obtained before 20 weeks of gestation, or a sustained blood pressure of at least 140/90 mmHg

after 20 weeks, if prior blood pressure was not known. Proteinuria was defined as the excretion of 300 mg of protein or greater in a 24 h urine collection specimen. This usually correlates with 30 mg/dl (1þ on dipstick testing) or greater in a random urine determination. Because proteinuria may be confounded with infection, routine cytobacteriologic exams and sequential urinary sediments were performed in all preeclamptic women to exclude this. Oedema was diagnosed as clinically evident swelling or as a rapid increase of weight. After evaluation of patients at emergency, women with no indication for immediate delivery were admitted and were considered on a case by case basis for administration of anti-hypertensive therapy and of low salt diet. All patients with significant obstetric disease other than preeclampsia or pregnancy-unrelated complications were eliminated from the study. Blood samples were collected in 144 normal pregnant women (primigravidas at different gestational ages) and in 51 cases of preeclampsia. All samples from preeclamptic women were obtained in the third trimester of gestation. In controls, 67 samples were obtained in the third trimester of gestation as well. 2.2. Procedures and assays 2.2.1. Blood samples Non-fasted blood samples were obtained and processed within 2 h of collection. Blood was obtained by venipuncture in EDTA containing tubes and in test tubes without anticoagulant. After separation by centrifugation, plasma and buffy-coat were taken from the EDTA containing tubes and serum from the tubes in which blood was allowed to coagulate. Aliquots were made and immediately stored at 70 8C until assayed. 2.2.2. DNA analysis Genomic DNA was extracted from white blood cells (buffy-coat) by proteinase K/salt precipitation method [22,23]. CETP genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) as described [24]. A fragment of 535 bp in the intron 1 of the CETP gene was amplified. Each amplification was performed in a thermal cycler (HYBAID—TouchDown) using 1 ml of DNA in a volume of 10 ml containing 1 PCR buffer [20 mM (NH4)2SO4, 75 mM Tris–HCl (pH 8.8), 0.01% (v/v) Tween, 1.5 mM MgCl2], 1 mM of each oligonucleotide primer (U: 50 -CACTAGCCCAGAGAGAGGAGTGCC-30 and L: 50 -CTGAGCCCAGCCGCACACTAAC-30 ), 0.2 mM dNTPs and 0.25 U of Taq polymerase (AB). The reaction mixture was heated 95 8C for 5 min for denaturation, and subjected to 31 cycles of amplification by primer annealing (60 8C for 1 min), extension (72 8C for 1 min), and denaturation (95 8C for 45 s), followed by a final extension at 72 8C for 8 min. The PCR products (10 ml) were subject to restriction enzyme analysis by digestion with 5 U

L. Belo et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 112 (2004) 9–15

of the restriction endonuclease TaqI at 65 8C for 3 h, in the buffer recommended by the manufacturer (React 2, Gibco BRL, Paisley Scotland). After separation by electrophoresis, on 1.5% agarose gel in TAE, the digested DNA fragments were stained with ethidium bromide and visualized by UV illumination. The sizes of TaqI fragments were estimated by comparison with known size markers (GeneRulerTM 100 bp DNA ladder, MBI Fermentas). Apo E genotyping was also performed by PCR-RFLP using the method of Hixson and Vernier [25], with some modifications. A 244 bp fragment located in the exon 4 of the apo E gene was amplified using oligonucleotide primers that flank positions 112 and 158 in the referred exon (F4: 50 -ACAGAATTCGCCCCGGCCTGGTACAC-30 and F6: 50 -TAAGCTTGGCACGGCTGTCCAAGGA-30 ). The PCR reaction was carried out in a thermal cycler (HYBAID—TouchDown) using 1 ml of DNA in a volume of 20 ml containing 1 PCR Buffer (HotStarTaq Polymerase Buffer, with a final concentration of 2.0 mM MgCl2), 1 mM of each primer, 10% (v/v) DMSO, 0.2 mM dNTPs, and 0.5 U of HotStarTaq DNA Polymerase. The PCR conditions were 95 8C for 15 min, and subsequently 31 cycles at 95 8C for 45 s, 60 8C for 1 min, and 72 8C for 2 min, and finally at 72 8C for 8 min. The PCR products (20 ml) were digested with 10U of HhaI in the recommended buffer (React 2, Gibco BRL), for 3 h at 37 8C. Each reaction mixture was loaded onto an 8% polyacrylamide gel in TBE and electrophoresed. The gel was stained with ethidium bromide and DNA fragments were visualised by UV illumination. The sizes of HhaI fragments were estimated by comparison with known size markers (10 bp DNA ladder, Invitrogen life technologies). 2.2.3. Serum analysis Serum lipids, lipoproteins and apolipoproteins analysis were performed in an auto-analyser (Cobas Mira S, Roche) using commercially available kits. Serum total cholesterol (Chol) and TG concentrations were determined by enzymatic colorimetric tests (CHOD-PAP and GPO-PAP methods, Roche, respectively). HDLcholesterol (HDLc) and LDLcholesterol (LDLc) levels were measured using enzymatic colorimetric tests after selective separation of HDL and LDL fractions (Direct HDL-cholesterol and Direct LDL-cholesterol, Roche). Apo A-I and apo B levels in serum were evaluated by immunoturbidimetric assays (uni-kit apolipoprotein A-I and B specific antiserums, Roche). 2.2.4. Plasma analysis LDL at d¼ 1:0191:063 g/ml was isolated from plasma. Electrophoresis was carried out, as described elsewhere [26]. We used 2–16% polyacrylamide gels (Alamo Gels, San Antonio, Texas) to determine LDL mean particle diameter (LDL-MPD). The gels were standardised against three samples prepared by density gradient ultracentrifugation with different LDL peak particle diameters (LDL I, II

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and III) which had previously been calibrated against markers: Seradyn latex (38 nm), thyroglobulin (17.00 nm) and ferritin (12.20 nm). The gels were scanned by laser densitometry (Bio-Rad Multi-AnalystTM/PC Version 1.1). LDL-MPD was calculated to give the mean diameter across the entire LDL profile. To achieve this, each peak of the LDL profile was sliced into ten portions and the peak area under the curve (volume) was calculated. For each portion, the particle size was calculated using the known reference sizes of LDL I, II and III. Then, the frequency for each particle was calculated (size  volume). Finally, the sum of frequencies divided by the sum of volumes gave the mean particle diameter (MPD). 2.3. Statistical analysis For the polymorphisms examined, the distribution of subjects with respect to genotypes was analysed using chi-squared (w2) test and Fisher’s exact test. Multiple comparisons between groups were performed by one-way ANOVA supplemented with Tukey’s HSD post hoc test. Preeclamptic and control patients were compared using Student’s unpaired t-test or Mann–Whitney U-test. Adjustment of statistical differences for confounding factors was performed using ANCOVA. Significance was accepted at P <0.05.

3. Results The entire control group (n ¼ 144) consisted of different normal pregnant women at different gestational ages, 67 of whom were in the third trimester of gestation. The mean age of the entire control group (26:5  4:6 years) did not differ from the preeclamptic group (28:0  5:5 years). In the polymorphisms examined, no differences were found in the distribution of subjects with respect to genotypes between both groups (Table 1). For comparison of lipids and lipoproteins we only considered the control subgroup obtained in the third trimester Table 1 Frequency of apo E and CETP genotypes in the study groups of pregnant women Polymorphism

Genotype

Normal (n ¼ 144)

Preeclamptic (n ¼ 51)

P-value

Apo E

E2/2 E2/3 E2/4 E3/3 E3/4 E4/4

1 11 2 104 24 2

0 4 0 40 6 1

(0%) (7.8%) (0%) (78.4%) (11.8%) (2.0%)

NS

CETP

B1/1 B1/2 B2/2

13 (25.5%) 29 (56.9%) 9 (17.6%)

NS

NS, not significant.

(0.7%) (7.6%) (1.4%) (72.2%) (16.7%) (1.4%)

56 (38.9%) 69 (47.9%) 19 (13.2%)

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Table 2 Comparison of normal and preeclamptic women in the third trimester of pregnancy Normal (n ¼ 67) Clinical characteristics Age (years) Gestational age at sampling (weeks) Gestational age at delivery (weeks) Maternal weight at sampling (kg) Uric acid (mg/dl)

26.2 34.0 38.7 73.9 4.0

Blood pressure (mmHg) Systolic Diastolic Cases presenting proteinuria (þ) Cases with 1þ Cases with 2þ Cases with 3þ Cases with 4þ TG (mg/dl) Apo A-I (mg/dl) HDLc (mg/dl) LDL-MPD (nm) LDLc:Apo B

    

Preeclamptic (n ¼ 51)

4.8 2.8 1.2 9.9 1.1

28.0 34.4 35.8 80.1 6.5

119  11 62  9 0 (0%) – – – – 185.8  38.4 279.5  70.6 61.8  14.2 26.34  0.52 0.80  0.12

Genotype E 2/2 E 2/3 E 2/4 E 3/3 E 3/4 E 4/4 B 1/1 B 1/2 B 2/2

1 5 1 46 13 1 24 35 8

    

P-value

5.5 3.6 2.9a 14.8 1.8

NS NS <0.001 <0.05 <0.001

152  12 92  9 45 (88.2%) 17 (33.3%) 11 (21.6%) 10 (19.6%) 7 (13.7%) 238.8  85.6 237.5  63.9 54.2  14.5 26.05  0.59 0.73  0.13

(1.5%) (7.5%) (1.5%) (68.7%) (19.4%) (1.5%) (35.8%) (52.2%) (11.9%)

0 4 0 40 6 1 13 29 9

<0.001 <0.001 <0.001

<0.001 <0.01 <0.01 <0.01 <0.01

(0%) (7.8%) (0%) (78.4%) (11.8%) (2.0%) (25.5%) (56.9%) (17.6%)

NS

NS

Values are given as mean  S:D., unless otherwise indicated. NS, not significant. a n ¼ 49 (two cases of foetal mortality).

of pregnancy; that subgroup was representative, in terms of genotype distribution, of the entire control sample (Table 2). Table 2 also summarises the most significant differences that we found in clinical data and lipid profile, between both groups. Preeclamptic women presented with significantly

higher mean weight, uric acid level and blood pressure compared to control pregnant women. Forty-five preeclamptic women (88.2%) presented with proteinuria (1þ) and 42 (82.4%) presented with oedema. Mean gestational age at delivery was significantly lower for the preeclamptic

Table 3 Lipids and lipoproteins, according to apo E distribution, in pregnant women (normal and preeclamptic) at the third trimester of gestation Apo E E 2þ (n ¼ 10) TG (mg/dl) Chol (mg/dl) Apo B (mg/dl) Apo A-I (mg/dl) HDLc (mg/dl) LDLc (mg/dl) LDLc/HDLc Apo B/Apo A-I LDL-MPD (nm) LDLc:Apo B

222.8 225.4 101.2 208.4 54.5 101.8 2.10 0.52 26.43 0.99

         

94.9 70.5 34.0 64.8 18.6 41.4b 1.33 0.23 0.61 0.14c

E 3/3 (n ¼ 86) 207.6 281.2 130.0 206.8 60.8 145.1 2.39 0.61 26.18 1.09

         

61.5 83.1 42.9 45.0 13.5 54.7 0.99 0.20 0.53 0.16

E 4þ (n ¼ 21) 208.6 294.6 137.1 178.1 52.2 159.4 3.05 0.76 26.27 1.17

         

83.1 46.9 25.7 42.5a 15.7a 32.7 1.00b 0.20b 0.69 0.16

E2þ and E4þ represent carriers of alleles 2 (E2/2 and E2/3) and 4 (3/4 and E4/4), respectively; the E2/4 subject was not included in analysis; values are given as mean  S.D. a P < 0:05 vs. E3/3. b P < 0:05 vs. other two groups. c P < 0:05 vs. E4þ.

L. Belo et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 112 (2004) 9–15

group. Regarding lipid profile, preeclamptic women exhibited significantly higher mean serum TG concentration and lower HDLc and apo A-I levels compared with healthy pregnant women. LDL-MPD and LDLc/apo B ratio were also significantly reduced in the pathologic group. All these results remained significant after adjustment for weight. In the third trimester of gestation (both control and case groups considered), apo E polymorphism, but not CETP polymorphism, was associated with different lipid and lipoprotein status. To assess correlation of lipids and lipoproteins with the apo E polymorphism, we divided subjects in three groups: E2 carriers (E2/2 and E2/3), E3/3 individuals and E4 carriers (E3/4 and E4/4). The E2/4 subject was not included in analysis. The results obtained in E2 carriers (E2þ) and E4 carriers (E4þ), were mainly due to the contribution of E2/3 and E3/4 genotypes, respectively. E2þ presented with significantly lower values of LDLc compared with E4þ and E3/3 individuals and lower LDLc/apoB ratio compared with E4þ (Table 3). E2þ also presented with the highest TG level, though without statistical significance. On the other hand, HDLc and apo A-I levels were significantly reduced in E4þ, compared with E3/3 (Table 3). Furthermore, E4þ presented with the highest Chol and LDLc and therefore LDLc/HDLc and apo B/apo A-I ratios were significantly higher in this group compared with the other two. No differences in LDL size (LDL-MPD) were found between the different apo E genotypes. All statistical significant results remained significant after being adjusted for case/controls. After adjustment for weight, age and gestational age at sampling significance was lost for LDLc/HDLc ratio.

4. Discussion Samples were collected on a non-fasting basis because preeclamptic cases were generally admitted on an emergency basis and therefore standardised blood sampling after an overnight fast was not feasible. However, postprandial serum lipid and lipoprotein levels seem to have no substantial short-term effects, except for TG concentration [27]. We have previously demonstrated (see resume in Table 2) that preeclampsia is associated with a combination of certain risk factors (increased TG concentration, reduced LDL-MPD and reduced HDLc and apo A-I levels) that may contribute to endothelial dysfunction and to the process of ‘‘acute atherosis’’ in the arteries of the uteroplacental bed, observed in this syndrome [7]. Oedema was considered, by some classifications, and for a long time, as being part of the diagnostic criteria of preeclampsia. However, there is now universal agreement that it should not be considered in the diagnosis of preeclampsia, which is primarily defined as gestational hypertension plus proteinuria [28]. We did not remove oedema from the diagnostic criteria of preeclampsia since it was part of the classification adopted by clinicians at the beginning of

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the study. Even though, we performed statistical analysis without the few cases (six, 11.8%) that presented with hypertension and oedema (but not with proteinuria) and we realised that those cases had no marked effect in the statistical analysis (data not shown) and, therefore, in our conclusions. The abnormal lipid profile that we found in preeclampsia may have a genetic explanation and/or contribution. Indeed, it is true that preeclampsia runs at higher rates in families with a history of such disorder [8–10]. Most data suggest that maternal genotype is the major factor responsible for the susceptibility, but some studies have ascribed an influence to the foetus, and thus the contribution of the father [29,30]. By analysis of maternal DNA, we show that none of our candidate polymorphisms (on apo E and CETP genes) seem to be associated with this disorder. We believe that expanding the study to a larger sample population would not change the conclusions, since no indication of a trend towards a difference was observed. Some authors have noticed a higher frequency of the E2 and E4 alleles in women with preeclampsia and suggested that these alleles may be risk factors for preeclampsia [18,19]. Our results differ from those but are in agreement with recent studies in South African [20] and Finnish populations [21]. Given the lack of consistency from the conclusions of different studies, it appears unlikely that apo E plays a pivotal role in the development of preeclampsia. We present the first study investigating any association between CETP polymorphism and preeclampsia. In nonpregnant individuals, TaqIB CETP polymorphism is associated with altered CETP activity [13] or mass [24,31,32] and with HDLc [13,24,31–35] and apo A-I levels [24,32,33]. Non-pregnant subjects with the B1 allele are more likely to have increased CETP activity and lower HDLc levels [13]. However, the effects of the CETP gene on plasma CETP and HDLc are possibly independent [24,31,32]. In the present study, we did not find a higher frequency of B1B1 individuals in the preeclamptic group to explain lower HDLc and/ or apo A-I concentrations. Furthermore, this study demonstrates that apo E, but not CETP, polymorphism is associated with changes in lipid and lipoprotein profiles in pregnant women. The results observed in carriers of E2 (E2þ) and E4 (E4þ) alleles were mainly due to the contribution of E2/3 and E3/4 subjects, respectively. E2þ presented with the lowest LDLc levels, compared with the E3/3 genotype and E4þ, in agreement with previous reports in non-pregnant [17] and pregnant [36] individuals. E2þ also presented with the highest TG levels, though without statistical significance. On the other hand, HDLc and apo A-I levels were significantly reduced in E4þ, compared with E3/3 genotype. A previous study in pregnant women found no differences in HDLc between women with different apo E genotypes [36]. However, a metaanalysis reported HDLc to be lower in E3/4 than in E3/3 non-pregnant subjects [16]. Furthermore, in this study, E4þ presented with the highest Chol and LDLc levels

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L. Belo et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 112 (2004) 9–15

and therefore LDLc/HDLc and apo B/apo A-I ratios were significantly higher in this group compared with the other two. Because the association of apo E isoforms with lipids and lipoproteins were obtained in the third trimester of the combined groups (cases and controls) and since both groups differ statistically in the lipid profile, that could be a major confounder in the association that we found. However, all the results remained significant after being adjusted for case/ controls. Moreover, after adjustment for environmental factors, such as weight, age and gestational age at sampling, significance was lost only for the LDLc/HDLc ratio. The relatively low number of subjects involved may explain, in part, the lack of association of CETP genotypes with HDLc and/or apo A-I, and also the lack of significance of some parameters with apo E isoforms. In addition, the strength of the relation between polymorphisms in the apo E and CETP genes and lipid profiles may be affected by unadjusted environmental factors. Indeed, the effect of the apo E polymorphism in determining LDLc concentration is affected by alcohol consumption [37]. Also, the strength of the association between CETP polymorphism and HDLc levels was reduced by obesity and smoking [34] and enhanced by alcohol intake [24]. In another CETP polymorphism, hypertriglyceridaemia also enhanced such association [38]. Moreover, CETP activity can be modulated without presenting variation in CETP mass by the metabolic state and especially by levels of TG [31]. It seems that regulation of CETP activity depends on TG levels, with a positive relationship with net CE transfer evident only in the presence of high TG [39]. In normal pregnancy, and especially in preeclampsia, levels of TG are likely to be a major determinant of net CE transfer and thus HDLc concentration. In conclusion, none of the polymorphisms examined explained the different lipid and lipoprotein profile found in preeclampsia. However, we demonstrated that apo E, but not CETP genotype influences lipid and lipoprotein levels in pregnant women. Further studies, by enlarging the sample population, are warranted to strength our findings.

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