Contribution of Hepatic Lipase, Lipoprotein Lipase, and Cholesteryl Ester Transfer Protein to LDL and HDL Heterogeneity in Healthy Women
Hepatic lipase (HL) and cholesteryl ester transfer protein (CETP) have been independently associated with low density lipoprotein (LDL) and high density lipoprotein (HDL) size in different cohorts. These studies have been conducted mainly in men and in subjects with dyslipidemia. Ours is a comprehensive study of the proposed biochemical determinants (lipoprotein lipase, HL, CETP, and triglycerides) and genetic determinants (HL gene [LIPC] and Taq1B) of small dense LDL (sdLDL) and HDL subspecies in a large cohort of 120 normolipidemic, nondiabetic, premenopausal women. HL (P<0.001) and lipoprotein lipase activities (P=0.006) were independently associated with LDL buoyancy, whereas CETP (P=0.76) and triglycerides (P=0.06) were not. The women with more sdLDL had higher HL activity (P=0.007), lower HDL2 cholesterol (P<0.001), and lower frequency of the HL (LIPC) T allele (P=0.034) than did the women with buoyant LDL. The LIPC variant was associated with HL activity (P<0.001), HDL2 cholesterol (P=0.034), and LDL buoyancy (P=0.03), whereas the Taq1B polymorphism in the CETP gene was associated with CETP mass (P=0.002) and HDL3 cholesterol (P=0.039). These results suggest that HL activity and HL gene promoter polymorphism play a significant role in determining LDL and HDL heterogeneity in healthy women without hypertriglyceridemia. Thus, HL is an important determinant of sdLDL and HDL2 cholesterol in normal physiological states as well as in the pathogenesis of various disease processes.
Although ≈30% of the patients with premature coronary artery disease (CAD) have normal lipid values1,2⇓ by National Cholesterol Education Program guidelines,3 there has been increasing interest in lipoprotein particle size and composition as additional risk factors for atherosclerosis. Compared with large buoyant LDL particles, a predominance of small dense LDL (sdLDL) particles is associated with an increased risk of CAD.4–6⇓⇓ HDL2 particles, the larger and more buoyant subspecies of total HDL, are considered the more antiatherogenic HDL subspecies.7,8⇓ Certain constituents of lipoprotein metabolism (ie, lipoprotein lipase [LPL] activity, hepatic lipase [HL] activity, and cholesteryl ester transfer protein [CETP] and triglyceride [TG] levels) have been shown to contribute to the formation of sdLDL and sdHDL particles.
LPL hydrolyzes TGs in VLDL particles, forming smaller IDL particles.9 CETP facilitates the exchange of cholesteryl ester (CE) in LDL and HDL for TGs in VLDL.10 This transfer of TGs into LDL and HDL particles makes them more TG rich and, hence, a better substrate for HL activity. HL remodels LDL and HDL particles by hydrolysis of TGs and phospholipids, forming smaller denser particles.11,12⇓
There is a strong genetic influence on the level of HL and CETP activities, and genes that encode CETP and HL have been considered candidate genes for CAD. The HL gene (LIPC) promoter polymorphism (C-514T) has been consistently observed to be associated with plasma levels of HL activity,13,14⇓ LDL size and density,14 HDL2 cholesterol (HDL2-C),13,14⇓ and HDL cholesterol (HDL-C).15 The LIPC T allele occurs at a frequency of 0.21 in whites,15 0.53 in African Americans,16 and 0.47 in Japanese Americans.14 The Taq1B polymorphism in intron 1 of the CETP gene17 (allele frequency 0.40 in whites18) has been associated with plasma levels of CETP19 and HDL-C.18
Studies to date have focused on the influence of individual biochemical factors, eg, LPL, CETP, HL, and TG levels, and of genetic markers, eg, LIPC(−514) and CETP gene (Taq1B) polymorphisms, on LDL and HDL particle heterogeneity, mainly in the disease process and generally in men. In the present study, we report a unique and comprehensive study of all these proposed biochemical (LPL, CETP, HL, and TG) and genetic determinants of sdLDL and HDL subspecies in a single large cohort of normolipidemic premenopausal women. The present study was designed to investigate the primary determinants of LDL and HDL heterogeneity in healthy women. The purpose of the study was to identify the primary biochemical (LPL, CETP, HL, and TG) determinants of sdLDL and HDL subspecies and to establish the contributions of the HL [LIPC(−514)] and CETP gene (Taq1B) polymorphisms to this process.
One hundred twenty healthy premenopausal women, aged 42 to 55 years, were studied (Table 1). All had experienced menstrual flow in the previous 6 months. The women were not taking lipid-lowering medications, β-blockers, or estrogen. They had no lipid disorders or other medical conditions affecting lipid metabolism, including diabetes, liver disease, or pregnancy. Women with body mass index >40 kg/m2, with TG or LDL cholesterol (LDL-C) levels greater than the 95th percentile for age,20 or with fasting serum glucose >110 mg/dL were excluded from the study. There were 114 white and 6 nonwhite subjects. Four women used tobacco (all smoked <10 cigarettes/d). The Human Subjects Review Committee of the University of Washington approved the study protocol. Informed consent was obtained from all participants.
Blood was collected in 0.1% EDTA after a 12- to 16-hour fast for DNA isolation and lipoprotein measurements. A heparin bolus of 60 U/kg was given, and blood was collected after 10 minutes in lithium heparin tubes for the measurement of lipase activity.
Lipid and Hormone Determinations
Plasma total cholesterol, TG, HDL-C, HDL2-C, HDL3 cholesterol (HDL3-C), apoB, and Lp(a) levels were determined by standard methodologies at the Northwest Lipid Research Laboratory.21 LDL-C was calculated by the Friedewald formula: LDL-C=total cholesterol−HDL-C−(TG/5).22 HDL-C and HDL3-C were determined after plasma precipitation with dextran sulfate and magnesium chloride.23 Estradiol was measured by solid-phase chemoluminescent immunoassay.
Total lipolytic activity was measured in plasma after a heparin bolus, as previously described.24 Glycerol tri[1-14C]oleate (Amersham) and lecithin were incubated with postheparin plasma for 60 minutes at 37°C. LPL activity was calculated as the lipolytic activity removed from the plasma by the incubation with the specific 5D2 monoclonal antibody against LPL, and HL activity was determined as the activity remaining after incubation with the LPL antibody. The intra-assay coefficient of variation (CV) for HL activity was 2.7%, and the interassay CV was 10.4%.
Density Gradient Ultracentrifugation
A discontinuous salt density gradient was created by using a modification25 of a previous method.26 Samples were centrifuged at 65 000 rpm for 70 minutes (total ω2t=1.95×1011) at 10°C in a Beckman VTi 65.1 vertical rotor. The relative flotation rate (Rf), the LDL peak buoyancy, was obtained by dividing the fraction containing the LDL-C peak by the total number of fractions collected with a CV of 3.6%.
CETP Protein Mass Assay
Plasma CETP mass concentration was used to assess plasma CETP activity because they are strongly correlated.27 However, storage conditions and sample handling may influence the stability of CETP lipid transfer activity. CETP mass measurement was used in the present study to minimize the potential effect of storage and sample handling on the stability of the lipid transfer activity. CETP mass concentration was determined by a commercial sandwich ELISA immunoassay kit (Wako) by using 2 monoclonal antibodies. The intra-assay and interassay CVs were 3.1% and 10.5%, respectively. There was a correlation of 0.83 (n=42) between CETP mass, by ELISA, and CETP activity, determined by the CETP Diagnescent Kit (Diagnescent Technologies, Inc), which is a CETP activity assay kit commonly used for the determination of the lipid transfer activity.28,29⇓ CETP mass concentration values obtained by this kit were stable under various storage and sample-handling conditions.
DNA Isolation and Analysis
DNA was extracted from leukocytes in 10 mL freshly drawn blood by the method of Miller et al.30 The LIPC(−514) and the Taq1B polymorphism in intron 1 of the CETP gene were determined by polymerase chain reaction amplification as described previously.31
Body fat composition was measured by CT scan (GE Highspeed Advantage) and DEXA scan (Hologic QDR 1500). The CT image was analyzed for cross-sectional area of fat, as described previously,32 by a single observer blinded to the conditions of the study. Total body fat (percentage) was measured by DEXA scan with an interassay CV of 1.6%, 1.3%, and 1.3% for fat mass, lean mass, and percent body fat, respectively.
Statistical analyses were performed by using SigmaStat, version 2.0 (Jandel Scientific). Comparisons of Taq1B genotypes were performed by using 1-way ANOVA and Kruskal-Wallis ANOVA on ranks. Comparisons of cohort separated by HL genotype and cohort separated by LDL-Rf were performed by using unpaired t tests and Mann-Whitney rank sum tests. The relationships of LDL-Rf, HDL-C, HDL2-C, and HDL3-C with HL, LPL, TG, and CETP were performed by using forward stepwise regression. Comparisons of the frequency of LIPC and Taq1B genotypes in the cohort separated by LDL-Rf were performed by using the χ2 test.
CETP Taq1B Genotype Is Associated With CETP Mass and HDL3-C
The women were separated by Taq1B genotype and compared by ANOVA (Table 2). The women with the B2 allele had significantly lower CETP mass (P=0.002) and significantly higher HDL3-C (P=0.039). There was no significant difference in HDL2-C among the different Taq1B genotypes, but there was a trend toward higher HDL-C (P=0.059) with the B2 allele. LDL buoyancy (LDL-Rf, P=0.58) was not significantly different between the 3 genotypes.
LIPC Genotype Is Associated With HL Activity, HDL2-C, and LDL Density
The women were separated by the LIPC genotype (presence or absence of HL −514T allele, Table 3). The carriers of the T allele (CT and TT combined) had significantly lower HL activity than did those with the CC genotype (179.7±75.7 versus 252.0±93.2 nmol/mL per minute, P<0.001). This lower HL activity with the T allele was associated with significantly higher HDL2-C (P=0.034) and significantly more buoyant LDL-Rf (P=0.03). There were no significant differences in intra-abdominal fat (IAF, P=0.97), TG (P=0.95), estradiol (P=0.22), or LPL (P=0.52), which are all known to affect LDL density, which could have accounted for the significantly more buoyant LDL particles in the carriers of the T allele.
Multiple Regression Analyses
Stepwise regression analyses (Table 4) revealed that HL (P<0.001) and LPL (P=0.006) were related to LDL-Rf (dependent variable) but that CETP (P=0.76) and TG (P=0.06) were not. These factors accounted for 26% of the variance in LDL-Rf (r2=0.26). In a similar model (Table 4), LPL (P<0.001), TG (P<0.001), and HL (P=0.027) were all independently related to HDL-C, but CETP (P=0.16) was not (r2=0.49). Also, in Table 4, TG (P<0.001), HL (P=0.003), and LPL (P<0.001) all contributed independently to variance in HDL2-C, but CETP did not (P=0.56). These 3 variables accounted for 46% of the variance in HDL2-C (r2=0.46). HDL3-C (Table 4) was independently related to TG (P=0.007) and LPL (P<0.001) but not to HL (P=0.29) or CETP (P=0.12). These factors accounted for 40% of the variance in HDL3-C (r2=0.40).
Determinants of LDL Buoyancy
The cohort was separated in half by LDL buoyancy (LDL-Rf). The women (n=60) with the relatively more dense LDL buoyancy (Table 5) had a mean LDL-Rf of 0.297±0.022 versus the women (n=60) with the more buoyant LDL-Rf (0.343±0.012, P<0.001). The women with the more dense LDL particles had significantly higher HL activity (P=0.007) and significantly lower LPL activity (P=0.012). Although there was no significant difference in total HDL-C (P=0.08) or HDL3-C (P=0.58), the women with more dense LDL particles had significantly lower HDL2-C (P<0.001). There was a significant difference in LIPC genotype distribution between the 2 groups: the women with the large buoyant LDL particles were significantly more likely to be carriers of the T allele (P=0.034), which most likely contributes to the lower HL activity in this group. There were no differences in Taq1B genotype frequency, CETP mass, TG, IAF, total percent body fat, or estradiol levels (all P>0.05), which could have accounted for the difference in LDL buoyancy.
The relationship of HL activity and LDL-Rf was examined in the CC and CT/TT genotypes by linear regression (Figure). As HL activity increased, LDL-Rf decreased, with a correlation coefficient of 0.43 (P<0.001) in the CC genotype and 0.39 (P=0.006) in the CT/TT genotypes. Even though the women with the CT/TT genotypes had lower HL activity and more buoyant LDL particles than did the women with the CC genotype, they fell on the same regression line (r=0.45, P<0.001 for entire cohort). Adjustment of LDL-Rf for IAF did not change these relationships (data not shown).
The predominance of sdLDL particles5,6⇓ and low HDL2-C8,33⇓ have been associated with an increased risk of CAD. In the present study, we investigated the role of HL, CETP, LPL, and plasma TG levels as determinants of LDL and HDL size heterogeneity in healthy women. Our data suggest that HL activity is the strongest contributor to LDL and HDL size distribution, whereas CETP has a small contribution to lipoprotein heterogeneity in this cohort of normotriglyceridemic women.
Effects of HL on ApoB-Containing Lipoproteins
Compared with CETP, LPL, and plasma TG levels, HL activity was the most important determinant of LDL buoyancy in normal women. Examination using multiple regression revealed that HL and LPL activities were the strongest independent contributors to LDL buoyancy assessed by LDL-Rf (Table 4). In a study of 15 women, Watson et al34 reported that HL, but neither CETP nor LPL, was a strong independent predictor of LDL1 and LDL3 concentrations. Segregation of our cohort into 2 groups of buoyant and dense LDL (Table 5) showed a significantly elevated HL activity in the dense LDL group (low LDL-Rf). However, LPL activity was higher in the women with large buoyant LDL particles, and this was most likely because the higher LPL led to more rapid lipolysis of VLDL with shorter VLDL residence time and, hence, less exchange of VLDL-TG for LDL-CE and HDL-CE.
The HL gene promoter genotype appeared to contribute the most to this higher HL activity seen in the women with more dense LDL particles (Table 5). There was a significant difference in the frequency of the T allele of the HL promoter polymorphism, thus explaining why the women with more dense LDL particles had higher HL activity. The women with more dense LDL particles had higher HL activity and were more likely to carry the −514C allele. Two reports have recently shown that the T allele is less transcriptionally active than is the C allele35,36⇓ and provide an explanation for the association of this allele with lower HL activity.
HL activity is known to be positively associated with IAF, independent of body mass index,37 and IAF contributes to a significant amount of the variance in HL activity.38 We have shown previously that as IAF increases, HL activity increases, but the maximum HL activity appears to be determined by the HL genotype.39 Women in this cohort generally have low amounts of visceral adiposity compared with a similar healthy male cohort.38 Despite the relatively low IAF and low HL activity (Tables 4 and 5⇑), LDL buoyancy in these women was determined primarily by the variance in HL activity (Figure). This observation suggests that that the HL promoter genotype may play a greater role in determining HL activity when IAF content is low than when IAF levels are elevated by central obesity. Lower IAF may also mitigate the contribution of CETP to the metabolism of sdLDL.
Role of CETP in ApoB-Containing Lipoprotein Metabolism
There was no relation of LDL buoyancy to CETP protein levels (Table 4) or the Taq1B genotype (Table 2) in these normal women. This is consistent with findings from the Framingham study; ie, the CETP Taq1B polymorphism is not an important determinant of LDL size and density in women.40 Several groups have been unable to demonstrate an independent effect of CETP on LDL subfraction concentration in normotriglyceridemic41,42⇓ and normolipidemic42,43⇓ subjects, whereas Talmud et al44 have shown a significant association of the Taq1B B2 allele with LDL size. This inconsistency may be due to the fact that the latter study44 included women with TG levels ≤400 mg/dL. CETP is thought to facilitate the generation of sdLDL through an indirect mechanism of increased rate of TG transfer from VLDL in exchange for CE in LDL and HDL.45 Thus, we subsequently examined the role of TG in LDL and HDL particle heterogeneity.
There have been several reports suggesting that plasma TG levels strongly influence the role of CETP in lipid metabolism, specifically LDL heterogeneity.46,47⇓ This has led to the hypothesis that CETP activity can be rate limiting only in the presence of hypertriglyceridemia.48 The present cohort had normal TG levels, and this may explain why CETP was not associated with LDL-Rf. It may be that when IAF and TGs increase with menopause,49 CETP plays a more important role in determining LDL particle size in women.
Effects of HL and CETP on HDL
Comparisons of HL and CETP gene polymorphisms as well as plasma HL activity and CETP mass levels suggest that HL and CETP independently contribute to different subspecies of HDL particles. The HL gene is associated with HDL2-C (P=0.034) and total HDL-C (P=0.058) levels, whereas CETP is primarily associated with HDL3-C (P=0.039) and total HDL-C (P=0.059) levels. Although there has been no comparison of this magnitude, previous work on the individual gene polymorphisms or protein products supports our findings.14,18,50⇓⇓ Whereas there was no association between the Taq1B polymorphism and HDL2-C in the present cohort, Ordovas et al40 have shown that the Taq1B B2 allele is associated with significantly higher HDL2-C as well as HDL3-C in a large cohort of men and women. This paradoxical finding may be attributed to differences in plasma TG levels in these 2 different cohorts. Alternatively, for the present group of women, the ability to detect small differences in HDL2-C levels may be underpowered.
The present study may be limited by the fact that the cohort was healthy and had no clinical evidence of CAD. But these nondiabetic women had normal IAF and normal TG levels, which would have reduced the confounding effects of diabetes, obesity, and hypertriglyceridemia on lipid metabolism. The associations seen in this cohort cannot verify the true causal relationships of the biochemical or genetic factors that contribute to LDL and HDL size heterogeneity because interventional prospective studies are needed to verify these relationships.
In summary, the present work represents a comprehensive comparison of previously proposed biochemical factors and genetic markers to determine the relative contribution of each parameter to the metabolism of sdLDL and HDL particle heterogeneity in a large group of normolipidemic, nondiabetic, premenopausal women. We have shown that HL activity is the strongest contributor to plasma LDL and HDL size heterogeneity. The contribution of CETP to particle size heterogeneity was small and only limited to HDL3-C in this female cohort. The HL gene variant was associated with variance in HL activity, HDL2-C, and LDL buoyancy, whereas the Taq1B gene polymorphism was associated with variance in CETP mass and HDL3-C. The present results suggest that HL activity and the HL gene polymorphism may have a significant role in determining LDL and HDL heterogeneity in healthy women without hypertriglyceridemia. Thus, HL is an important determinant of sdLDL and HDL2-C in normal physiology as well as in the pathogenesis of various disease processes. The role of HL activity in contributing to the profile of the metabolic syndrome (sdLDL and reduced HDL2-C) supports the proatherogenic role of elevated HL activity and suggests that HL may be an effective therapeutic target in modifying LDL and HDL size heterogeneity and the related CAD risk.
This study was supported by National Institutes of Health (NIH) grants HL-30086, HL-64322, and NR-04141; an NIH K23 grant (RR-16067 to Dr Carr); and a grant from Bristol Myers Squibb Foundation. Dr Murdoch was supported by an NIH training grant (T32 DK-07247), and Dr Ayyobi was supported by an American Diabetes Association mentor-based fellowship grant. These studies were performed with the support of the University of Washington Clinical Nutrition Research Unit (DK-35816) and General Clinical Research Center (M01-RR-00037). We thank the volunteers for their generosity in participating in this study, Alegria Aquino-Albers and Steve Hashimoto for their technical expertise, and Linda Floyd for recruitment.
Received January 14, 2002; revision accepted February 6, 2002.
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