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The Journal of Clinical Endocrinology & Metabolism 87(10):4766 4770 Copyright 2002 by The Endocrine Society doi: 10.1210/jc.2002-020245

Effects of Hormone Replacement Therapy and Hepatic Lipase Polymorphism on Serum Lipid Profiles in Postmenopausal Japanese Women
YOSHIAKI SOMEKAWA, HIDENORI UMEKI, KIMIKO KOBAYASHI, SIGEO TOMURA, TAKESHI ASO, AND HIDEO HAMAGUCHI
Department of Obstetrics and Gynecology, Toride Kyodo General Hospital (Y.S., H.U.), Toride 3020022, Japan; Institute of Basic Medical Sciences (K.K.) and Institute of Community Medicine (S.T.), University of Tsukuba, Tsukuba 3050005, Japan; and Department of Comprehensive Reproductive Medicine, Regulation of Internal Environment and Reproductive, Systemic Organ Regulation, Graduate School, Tokyo Medical and Dental University (T.A.), Tokyo 1138519, Japan
The purpose of this study was to evaluate the relationships among hepatic lipase (HL) polymorphism, serum lipids, lipoproteins, and remnant-like particle cholesterol (RLP-C) and to determine the effects of hormone replacement therapy (HRT). We assessed the HL polymorphism in 209 postmenopausal Japanese women. Levels of serum total cholesterol, low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol, triglycerides, apolipoprotein (Apo) AI, Apo B, Apo E, Apo CII, Apo CIII, and RLP-C were measured before and after 3 months of HRT. The frequency of each genotype was 32% for 514 C/C, 41% for C/T, and 27% for T/T. Subjects with the C/T and T/T genotypes had higher levels of HDL cholesterol and Apo AI than those with the C/C genotype. Those with the T/T genotype had higher levels of RLP-C than those with the C/C or C/T genotype. Serum total cholesterol, LDL cholesterol, Apo B, Apo E, and Apo CII were decreased, and HDL cholesterol and Apo AI were increased significantly in all genotypes after 3 months of HRT. There were no differences in these changes with genotype. The HL polymorphism was associated with higher levels of HDL cholesterol, Apo AI, and RLP-C, and the HL gene variation may contribute to HL activity and affect serum lipoprotein metabolism. Effects of HRT on serum lipids, lipoproteins, and remnant lipoprotein metabolism were unaffected by the HL polymorphism. (J Clin Endocrinol Metab 87: 4766 4770, 2002)

IGH DENSITY LIPOPROTEIN cholesterol (HDL-C) is regarded as an antiatherogenic particle and is thought to be involved in the reverse transport of cholesterol from peripheral cells to hepatocytes (1). Remnant lipoproteins derived from very low density lipoproteins (VLDLs) and chylomicrons are atherogenic (2, 3). These triglyceriderich lipoproteins are of various sizes and density, which complicates their isolation. Nakajima et al. (4 6) recently developed a technique for isolating remnant-like lipoproteins as remnant-like particles (RLPs) using an immunoaffinity mixed gel containing antiapolipoprotein (anti-Apo) AI and anti-Apo B-100 monoclonal antibodies. The physical, chemical, and receptor-binding properties of RLP have been shown to resemble those of VLDL and chylomicron remnants (4). The serum levels of HDL-C and RLP cholesterol (RLP-C) are regulated by hepatic lipase (HL). HL is a lipolytic enzyme that hydrolyzes triglycerides and phospholipids in low density lipoprotein cholesterol (LDL-C) and HDL-C and contributes to determining lipoprotein particle size (2, 3). HL activity appears to be regulated by several factors, including 1) HL gene promoter polymorphism (7, 8), 2) intraabdominal fat (9), 3) sex steroid hormones (10, 11), and 4) age (12). HRT
Abbreviations: Apo, Apolipoprotein; BMI, body mass index; CAD, coronary artery disease; HDL-C, high density lipoprotein cholesterol; HL, hepatic lipase; HRT, hormone replacement therapy; LDL-C, low density lipoprotein cholesterol; MPA, medroxyprogesterone acetate; RLP-C, remnant-like particle cholesterol; TC, total cholesterol; TG, triglycerides; VLDL, very low density lipoprotein.

is reported to attenuate the activity of HL, resulting in an increase in HDL2 and VLDL (13). Several studies indicated that the presence of the T allele at position 514 was associated with low HL activity and elevated HDL-C levels compared with those in normal subjects, and the frequency of the 514T allele is approximately 3 times higher in AfricanAmericans than in Caucasians (7, 8). We hypothesize that the 514T allele is associated with remnant metabolism and serum RLP-C level through the reduction of HL activity. The purpose of this study was to determine the relationships among the HL promoter polymorphism at position 514 (C to T), serum lipids, Apo, and RLP-C and the effects of hormone replacement therapy (HRT) on the concentrations of these lipids.
Subjects and Methods Subjects
We assessed the HL polymorphism in 209 postmenopausal Japanese women, aged 44 80 yr (mean age, 55.3 0.5 yr). In all cases, more than 6 months had elapsed since the last menstrual bleeding, the serum estradiol level was lower than 20 pg/ml (73.42 pmol/liter), and the serum FSH level was more than 50 IU/dl. Those who exercised regularly, smoked or drank heavily (consuming 30 cigarettes or 80 g alcohol/d for 5 yr), or were clinically diagnosed with liver disease, coronary artery disease, thromboembolic disease, diabetes, renal disease, or metabolic or other endocrine diseases that could influence lipid metabolism were excluded. None was receiving medications that could affect lipid metabolism. The study was approved by the institutional review of board of the hospital and was performed in accordance with the Declaration of Helsinki. Each subject gave her written informed

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consent for participation in the study before participation. Variables in the backgrounds of the patients, such as age, height, weight, body mass index (BMI), and age at menopause were measured.

were measured before and after 3 months of HRT. All patients in this study were advised to maintain a normal diet and not to change it throughout the duration of the study.

HL genotype
Venous blood samples were collected from 209 postmenopausal women. DNA was isolated from the leukocytes by the phenol extraction method. The PCR was performed on genomic DNA samples. The amplified fragments were digested with 1.5 U NlaIII restriction digestion (New England Biolabs, Inc., Beverly, MA), which can recognize a C to T substitution. The oligonucleotide primers for the 514 polymorphism detection were 5-TCACTTGGCAAGGGCATCTTTG-3 and 5-AGGTCGGGGTAGGTGGCTTCCA-3. PCR was performed with 35 cycles of 94 C for 30 sec, 55 C for 60 sec, and 72 C for 90 sec after a 2-min denaturation at 95 C. The fragments were resolved by electrophoresis in polyacrylamide gels and stained with ethidium bromide. The expected fragments were 226 and 48 bp for the T allele and 274 bp for the C allele. Genotypes were expressed as C/C (homozygous normal), C/T (heterozygous), and T/T, (homozygous mutant) (8).

Statistical analysis
The results are given as the mean se. Data analysis was performed using a StatView 5.0 software package (SAS Institute, Inc., Cary, NC). Baseline parameters were compared among the groups by the KruskalWallis test. One-way factorial ANOVA and Fishers protected least significant difference as post hoc tests were used for the evaluation of differences among the three groups. ANOVA for repeated measures was used to evaluate the significance of any changes in serum lipids, Apo, and RLP-C. Differences with P 0.05 were defined as statistically significant.

Results

Analysis of lipids and Apo

Fasting serum total cholesterol (TC), triglycerides (TG), HDL-C, LDL-C, Apo AI, Apo B, Apo CII, Apo CIII, and Apo E were measured to evaluate the risk of atherosclerosis. After an overnight fast, blood was collected from each patient for the estimation of lipids and lipoproteins. TC and TG levels were measured using enzymatic colorimetric methods (Ono Pharmaceutical, Osaka, Japan) and the enzymatic method (Shinotest, Tokyo, Japan). HDL-C levels were measured by a direct method (14) using commercially available kits (Kyowa Medix, Tokyo, Japan) on a Hitachi chemical analyzer (Tokyo, Japan). LDL-C levels were calculated with Friedewalds equation (15). Apo AI, Apo B, Apo CII, Apo CIII, and Apo E were measured by an immunoturbidimetric method using commercially available kits (Daiichi Pure Chemical, Tokyo, Japan). The intraassay coefficients of variation for TC, TG, HDL-C, Apo AI, Apo B, Apo CII, Apo CIII, and Apo E were 0.7%, 0.7%, 2.5%, 4.0%, and 2.3%, respectively. RLP-C levels were measured by the method described by Nakajima et al. (5). Remnant lipoproteins were isolated by application of fasting serum to immunoaffinity-mixed gel, which contained monoclonal antibodies to Apo AI and Apo B-100 (Japan Immunoresearch Laboratories, Takasaki, Japan). After a 2-h incubation at room temperature, cholesterol concentrations in the unbound fraction were measured by a sensitive cholesterol assay (6).

HRT
Of these 209 women, 172 women suffering from severe menopausal symptoms, diagnosed as osteoporosis according to the criteria [patients with low lumbar bone mineral density, 70% of young adult mean) or one or more nontraumatic vertebral fractures and lumbar bone mineral density less than 80% of young adult mean] proposed by the Japanese Society of Bone Metabolism in 1996 (16) or showing unfavorable lipid profiles [serum TC levels 220 mg/dl or LDL-C levels 140 mg/dl (17)] were treated by oral conjugated equine estrogen (0.625 mg/d) and medroxyprogesterone acetate (MPA) 2.5 mg/d continuously under the agreement to receive HRT. Levels of lipids, apolipoproteins, and RLP-C TABLE 1. Baseline clinical characteristics in three groups
Genotype C/C (n 67)

The frequency of the homozygous normal genotype (C/C) was 32%, that of the heterozygous genotype (C/T) was 41%, and that of the homozygous mutant genotype (T/T) was 27%. The allele frequency of the substitution was 47.6%, which was 3 times higher than that in Caucasian populations and consistent with the previous reports in Japan and Korea (18, 19). Background variables, such as age, height, weight, BMI, and age at menopause, did not exhibit significant differences among the genotypes (Table 1). Subjects with the C/T and T/T genotypes had higher levels of HDL-C, and those with the T/T genotype had higher levels of Apo AI than those having the C/C genotype, and those having the T/T genotype had higher levels of Apo CIII and RLP-C than those having the C/C genotype before HRT. There were no significant differences in serum levels of TC, TG, LDL-C, Apo B, Apo E, and Apo CII with genotype before HRT (Table 2). Serum TC, LDL-C, Apo B, Apo E, and Apo CII were decreased, and HDL-C and Apo AI were increased significantly in all genotypes after 3 months of HRT. There were no differences in the rates of change in serum TC, LDL-C, Apo B, Apo E, Apo CII, HDL-C, and Apo AI with the genotype. Serum levels of RLP-C were not changed in any genotype after 3 months of HRT (Table 2). Neither weight nor BMI was changed during this period.
Discussion

The presence of a C to T substitution at position 514 with respect to the transcription start site of the HL gene accounts for 20 30% of the variance in HL activity (8, 20). The 514T allele was shown to have a 30% lower promoter activity (21),

Genotype C/T (n 85)

Genotype T/T (n 57)

Pa

Age (yr) Height (m) Weight (kg) BMI Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Age of menopause (yr) Years since menopause (yr) Values are shown as means a One-way factorial ANOVA.
SE.

54 1 1.54 0.01 52.5 1.1 22.4 0.5 128.8 3.2 74.8 1.8 48.8 0.4 6.5 0.8

55 1 1.53 0.01 52.0 1.0 22.0 0.3 126.9 3.1 74.0 1.9 49.5 0.3 5.9 0.7

56 1 1.53 0.01 52.6 1.0 22.4 0.6 122.1 4.0 70.1 2.4 49.8 0.4 6.1 0.8

0.86 0.62 0.89 0.69 0.42 0.31 0.16 0.84

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Somekawa et al. Hepatic Lipase Polymorphism and Remnant Cholesterol

TABLE 2. Lipid, Apo, and RLP-C levels according to HL genotype before treatment
Genotype C/C C/T T/T Pa

TC (mmol/liter) TG (mmol/liter) HDL-C (mmol/liter) LDL-C (mmol/liter) Apo AI ( mol/liter) Apo B ( mol/liter) Apo E ( mol/liter) Apo CII ( mol/liter) Apo CIII ( mol/liter) RLP-C (mmol/liter)

5.72 0.10 (5.28 0.08b) 1.41 0.09 (1.48 0.09) 1.42 0.04 (1.58 0.05b) 3.39 0.10 (3.02 0.10b) 50.7 0.9 (53.5 1.1d) 2.01 0.05 (1.90 0.06d) 1.78 0.25 (1.28 0.05b) 6.47 0.47 (5.52 0.47d) 11.8 0.35 (11.4 0.35) 0.15 0.02 (0.14 0.01)

5.67 0.10 (5.05 0.10b) 1.44 0.09 (1.39 0.08) 1.62 0.06 (1.74 0.05b) 3.39 0.10 (2.67 0.10b) 52.4 0.8 (57.4 1.1b) 1.89 0.05 (1.81 0.05d) 1.44 0.03 (1.14 0.03b) 5.64 0.35 (3.88 0.35b) 11.4 0.35 (11.4 0.35) 0.16 0.01 (0.14 0.01)

5.97 0.11 (5.32 0.10b) 1.54 0.10 (1.59 0.09) 1.65 0.06c (1.82 0.05b) 3.61 0.11 (2.77 0.09b) 55.4 1.1c (60.3 1.5b) 2.06 0.06 (1.87 0.06d) 1.69 0.08 (1.28 0.05b) 6.12 0.47 (4.70 0.47b) 12.9 0.47e (12.2 0.35) 0.18 0.02e (0.16 0.02)

0.11 0.60 0.006 0.11 0.005 0.06 0.17 0.39 0.01 0.05

Values are shown as means SE (after 3 months of HRT). a One-way factorial ANOVA. b P 0.001 by repeated ANOVA vs. baseline. c P 0.01 by Fishers protected least significant difference vs. C/C genotype. d P 0.01 by repeated ANOVA vs. baseline. e P 0.05 by Fishers protected least significant difference vs. C/C genotype.

which accounts for the increased HDL-C level compared with that in patients with a C/C genotype. Remnant lipoproteins are taken up by macrophages and cause foam cell formation (22). High levels of remnant lipoprotein cause endothelial vasomotor dysfunction in human coronary arteries and are independently associated with the presence of myocardial infarction (23). The RLP-C employed in this study has isolated Apo E-rich VLDL particles containing Apo B-100 together with chylomicron remnants containing Apo B-48, neither of which binds to the immunoaffinity gel (4 6). The removal of remnants is strongly associated with HL activity (24), and HL activity is sensitively regulated by sex hormones such as estrogen and progesterone (13, 25). It is more likely that the reduction of Apo E after HRT may reflect the increase in Apo B-E receptor (remnant receptor) activity, resulting in the reduction of remnant lipoprotein. The present findings raise two questions. The first is whether there is any difference among HRT and other lipidlowering therapies. Recently, Zambon et al. (26) reported that the response to lipid-lowering therapy with either lovastatin and colestipol or niacin and colestipol was different among subjects with different HL promoter genotypes. The T/T patients, who at baseline had lower HL activity and more buoyant, larger LDL particles, showed no change in HL activity or LDL buoyancy with therapy, whereas C/C patients exhibited a greater decrease in HL activity and a greater increase in LDL buoyancy with lipid-lowering therapy than carriers of the T allele. They proposed two concurrent and independent lipoprotein pathways accounting for drug-associated coronary artery disease (CAD): one leading to changes in LDL-C and Apo B levels and a new HLmediated pathway (26). We can propose similar pathways related to the effects of HRT on lipid metabolism: the general

pathway that includes Apo AI, Apo B, Apo E, VLDL, LDL-C, and HDL-C and the HL-mediated pathway. However, these pathways are not perfectly independent; the RLP-C level is regulated by Apo B-E receptor (remnant receptor) activity as well as the HL-mediated pathway (Fig. 1). A reduction was observed in TC and LDL-C as well as Apo B and Apo E levels, whereas a consistent increase was shown in HDL-C and Apo AI levels after HRT treatment, similar to the response to the other lipid-lowering therapy used by Zambon et al. (26). Neither C/C patients nor carriers of the T allele show a different change in RLP-C values after HRT treatment, whereas T/T patients had higher levels of RLP-C than C/C patients before HRT. However, Zambon et al. (26) have shown differences in the changes in HL activity with genotype. One possible explanation for the discrepancy between the two therapies is related to the effects on HL activities of these therapies. Tilly-Kiesi et al. (27) reported that oral 17-estradiol (2 mg/d) combined with norethisterone acetate (1 mg/d) decreased HL activity, but transdermal 17-estradiol (50 g/d) combined with MPA (10 mg/d) did not affect HL activity. HL activity is decreased by estrogen and conversely increased by progesterone (13, 25). We employed HRT with 0.625 mg conjugated equine estrogen and 2.5 mg MPA/d in our study. The effect of estrogen might surpass the antagonistic effect of MPA and reduce HL activity, resulting in attenuation of the differences in HL activity of HRT among the three genotypes. The second point concerns the association of HL polymorphism with the risk of CAD. Increased HL activity is associated with small, dense LDL particles and lower levels of the antiatherogenic large HDL particles, and patients with small, dense LDL have a 3-fold increased risk of premature CAD (28). Conversely, lowered HL activities seem to be associated with an increased atherosclerotic risk (29). The

Somekawa et al. Hepatic Lipase Polymorphism and Remnant Cholesterol

J Clin Endocrinol Metab, October 2002, 87(10):4766 4770 4769

FIG. 1. Two pathways related to the effects of HRT on lipid metabolism: the general pathway containing Apo AI, Apo B, Apo E, VLDL, and LDL-C, and the HL-mediated pathway containing HDL-C and remnant lipoprotein.

prevalence of alleles with the C to T substitution has been reported to be higher in CAD patients than in normal subjects (30). No significant difference in allele frequency between the two groups was found (31), even in TT patients with elevated HDL-C. RLP-C, which is elevated in TT patients, may play a key role in resolving this contradiction. Other lipid-lowering therapies, such as statin, which reduces LDL-C and increases HDL-C, have been reported to be effective for the primary and secondary prevention of CAD (32, 33). However, HRT has been reported to be ineffective for the prevention of secondary CAD (34, 35), although it reduced LDL-C, Apo B, and homocysteine while increasing HDL-C and Apo AI (34). The main reason for this discrepancy may be related to the increased risk of thromboembolism (35) or the proinflammatory effects (36) of HRT, but the negative effect of HRT on RLP-C may be partially responsible for the failure in reducing the risk of CAD. Effects of HL polymorphism on LDL-C and Apo B are inconsistent; one study reported significantly higher LDL-C levels and borderline higher Apo B levels in T/T patients than in those with the C/C genotype (26), whereas in other studies there were no differences in LDL-C and Apo B levels with genotype (20, 31) as in this study. The mechanism by which LDL-C and Apo B are reduced in T/T patients is not clear, and the effects of HL on LDL-C metabolism may be limited to LDL size and buoyancy through the HL-related removal of phospholipids and free cholesterol from LDL (37). Both LDL-C and Apo B levels are independent risk factors for CAD and may not be closely associated with HL activity. In summary, reduced HL activity in the T/T genotype is associated with higher HDL-cholesterol, Apo AI, and RLP-C levels. The effects of HRT on serum lipids, lipoproteins, and RLP-C are unaffected by the HL polymorphism.
Acknowledgments
We gratefully thank the patients who agreed to participate in this study, and Hiroshi Hirasawa and Kazuyoshi Hosoya for their assistance with the laboratory procedures.

Received February 15, 2002. Accepted July 9, 2002. Address all correspondence and requests for reprints to: Yoshiaki Somekawa, M.D., Toride Kyodo General Hospital, Hongo 2-1-1 Toride, Ibaraki 3020022, Japan. E-mail: pv2t-sigi @asahi-net.or.jp.

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