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ORIGINAL RESEARCH |
From the Department of Obstetrics and Gynecology, Kochi Medical School, Kochi, Japan.
Address reprint requests to: Akihiko Wakatsuki, MD Department of Obstetrics and Gynecology Kochi Medical School Oko-cho, Nankoku Kochi, 783-8505 Japan
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Methods: Twelve postmenopausal women were treated with conjugated equine estrogen, 0.625 mg daily, for 3 months. Plasma concentrations of total cholesterol, triglyceride, and high-density lipoprotein (HDL) cholesterol were measured before and after therapy. We also determined the amount of total, free, and esterified cholesterol, triglyceride, and apolipoprotein B in LDL. To evaluate lipid transfer reactions, plasma samples were incubated at 37C for 24 hours, and replacement of cholesteryl ester by triglyceride in LDL particles was analyzed. Cholesterol and triglyceride concentrations were measured enzymatically. Apolipoprotein B concentrations were determined by an immunoturbidimetric assay.
Results: Estrogen significantly reduced the plasma levels of total cholesterol and significantly increased those of triglyceride and HDL cholesterol. The ratio of cholesteryl ester to apolipoprotein B was reduced significantly, whereas the ratio of triglyceride to apolipoprotein B increased significantly after estrogen treatment. Both before and after estrogen treatment, incubation of plasma induced a significant increase in the ratio of LDL-triglyceride to apolipoprotein B with a concomitant decrease in the ratio of LDL–cholesteryl ester to apolipoprotein B. Incubation-induced changes in these ratios were significantly enhanced by estrogen therapy. The plasma concentration of triglyceride was correlated positively with incubation-induced changes in the ratio of LDL-triglyceride to apolipoprotein B (r = .83, P < .001) and correlated negatively with changes in the ratio of LDL–cholesteryl ester to apolipoprotein B (r = -.61, P < .01).
Conclusion: Estrogen-induced increase in the plasma level of triglyceride may enhance lipid transfer reactions, resulting in triglyceride-rich and cholesteryl ester-poor LDL particles.
Plasma levels of low-density lipoprotein (LDL) cholesterol increase after menopause,1 and women become more susceptible to coronary heart disease with age.2 We have demonstrated previously that a decrease in the plasma concentration of estrogen enhances the activity of lipoprotein lipase in postmenopausal and castrated women, which may lead to an increase in the plasma concentration of LDL.3 Arca et al4 have suggested that hypercholesterolemia in postmenopausal women is due to an impairment of the LDL receptor.
Low-density lipoprotein particles vary in size, density, and lipid composition,5 with various LDL subfractions differing in atherogenicity. Smaller, denser particles of LDL are associated with increased risk of atherosclerosis.6 Our previous findings demonstrated that the size of LDL particles decreases and the prevalence of LDL pattern B increases in women with natural or surgically induced menopause.7 Low-density lipoprotein pattern B, which represents particles with a diameter less than 25.5 nm, is associated with increased risk for coronary heart disease. Thus, an increased plasma level of LDL with a reduced size of LDL particles may be atherogenic in women with low plasma levels of estrogen.
Estrogen replacement therapy in postmenopausal women favorably affected lipid metabolism by reducing the plasma level of LDL cholesterol and increasing that of high-density lipoprotein (HDL) cholesterol.8 We also demonstrated that estrogen therapy reduced the size of LDL particles and increased the prevalence of LDL pattern B.9 Therefore, estrogen-induced small LDL particles may be atherogenic, and beneficial effects of estrogen might be offset partially by a concurrent decrease in LDL particle size.
Low-density lipoprotein particles are composed of free cholesterol, esterified cholesterol, triglyceride, phospholipid, and apolipoprotein B. The size of an LDL particle is influenced by its lipid constituents. In human plasma, the cholesteryl ester transfer protein catalyzes the net mass transfer of cholesteryl esters from LDL and HDL to very low-density lipoprotein, with a net mass transfer of triglycerides in the opposite direction, from very low-density lipoprotein to LDL and HDL.10 Therefore, lipid transfer reactions may be important in determining the composition of LDL and HDL and may play an important role in the development of atherosclerosis. According to our previous findings, the content of core lipids such as cholesteryl esters and triglycerides may be the main determinant of the size of LDL particles.11 In addition, we found that estrogen replacement may produce triglyceride-rich and cholesteryl ester–poor LDL particles that are small.11 Lipid transfer reactions may be involved in the mechanisms of small estrogen-induced LDL particles. However, the effect of estrogen on lipid transfer reactions has not been evaluated.
To investigate the effect of estrogen on lipid transfer reactions, we treated postmenopausal women with conjugated equine estrogen. Plasma samples before and after estrogen therapy were incubated, and changes in lipid distribution of LDL were measured.
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Materials and Methods |
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Each subject received 0.625 mg of conjugated equine estrogen orally daily in the evening for 3 months. Endometrial biopsies and blood samples were obtained before and after treatment. Blood samples were drawn between 8:00 AM and 10:00 AM following a 12-hour fast and were centrifuged immediately at 1500 xg for 20 minutes at 4C to obtain plasma.
Plasma levels of total cholesterol and triglyceride were measured by enzymatic methods as described previously.12 The plasma levels of HDL cholesterol were determined using the same method for cholesterol after apolipoprotein B–containing lipoproteins had been precipitated with sodium phosphotungstate in the presence of magnesium chloride.12 The concentration of LDL–apolipoprotein B was measured by a turbidimetric immunoassay.13 Low-density lipoprotein was fractionated subsequently by ultracentrifugation according to the method of Havel et al.14 The concentrations of total cholesterol, free cholesterol, and triglyceride in the LDL fraction were determined using enzymatic assays.14 The cholesteryl ester concentration of LDL was calculated as the difference between measured total cholesterol and the concentration of free cholesterol.
Lecithin cholesterol acyltransferase esterifies free cholesterol in HDL. Esterified cholesterol in HDL is transferred to apolipoprotein B–containing lipoproteins by the cholesteryl ester transfer protein. Lecithin cholesterol acyltransferase–induced cholesterol esterification in HDL may influence lipid transfer reactions between lipoproteins. Therefore, the plasma samples were supplemented with 1.5 mmol/L iodoacetate to inhibit the activity of lecithin cholesterol acyltransferase and were maintained at 4C or incubated at 37C for 24 hours to analyze lipid transfer reactions.15 Subsequently, the LDL fraction was isolated and the concentrations of total cholesterol, free cholesterol, triglyceride, and apolipoprotein B were measured.
Data are expressed as mean ± standard error (SE). Changes in lipid levels induced by estrogen therapy were analyzed by a Student paired t test. Differences in lipid levels of LDL between 4C and 37C incubation or differences in incubation-induced changes in LDL lipid levels between before and after estrogen therapy were analyzed by Student unpaired t test. Regression lines were determined by the least squares method. A level of P < .05 was accepted as statistically significant.
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Concentrations of free cholesterol, esterified cholesterol, and apolipoprotein B in the LDL were all decreased significantly by estrogen treatment. Triglyceride content in the LDL increased significantly after treatment. The LDL–cholesteryl ester/apolipoprotein B ratio was significantly reduced, whereas the LDL-triglyceride/apolipoprotein B ratio was significantly increased. The LDL–free cholesterol/apolipoprotein B ratio was unaffected by estrogen treatment (Table 1
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) and correlated negatively with the change in the ratio of LDL–cholesteryl ester/apolipoprotein B (Y = -364.4x + 94.8, r = -.61, P < .01) (Figure 2).
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Discussion |
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McNamara et al19 have suggested that the plasma level of triglyceride is the single most important factor to affect the size of the LDL particles, and that variations in the plasma concentration of triglyceride affect the size of the LDL particle. Several studies18–22 have demonstrated that estrogen reduced the size of LDL particles in association with a significant increase in plasma triglyceride levels. In the present study, the plasma level of triglyceride was increased after estrogen treatment, consistent with our previous report.9 We have found previously that estrogen-induced increase in the plasma level of triglyceride may reduce the size of LDL particles.9
Estrogen stimulates the synthesis of LDL receptors and lowers the plasma level of LDL cholesterol.23 Because each LDL particle has one molecule of apolipoprotein B, a decrease in the plasma level of LDL–apolipoprotein B in our study indicates that estrogen most probably reduces the plasma concentration of LDL particles by removing them by way of LDL receptors. To estimate lipid levels in individual LDL particles, we calculated the ratio of lipids to apolipoprotein B because each LDL particle has one molecule of apolipoprotein B. The ratio of LDL– cholesteryl ester/apolipoprotein B was decreased and the ratio of triglyceride/apolipoprotein B was increased by estrogen treatment, but the ratio of LDL–free cholesterol/ apolipoprotein B did not change. This indicates that estrogen therapy induces triglyceride-rich and cholesteryl ester–poor LDL particles, which are reported to be associated with small LDL particles.6
According to Musliner et al,24 increased plasma triglyceride concentrations enhance the replacement of cholesteryl ester by triglyceride in LDL particles by way of lipid transfer reactions. When the plasma triglyceride level is lowered, the composition of LDL can be normalized as a result of a reduced rate of lipids transfer between very low-density lipoprotein and the LDL and HDL fractions.25,26 We demonstrated previously that plasma triglyceride levels were correlated positively with the ratio of LDL-triglyceride/apolipoprotein B, and negatively with the ratio of LDL–cholesteryl ester/apolipoprotein B. On the basis of these findings, we speculated that an estrogen-induced increase in the plasma level of triglyceride may affect lipid transfer reactions to produce triglyceride-rich and cholesteryl ester–poor LDL particles.
To investigate the implications of lipid transfer in modulating lipid distribution in LDL particles, plasma was incubated at 37C for 24 hours, and replacement of cholesteryl ester by triglyceride in LDL particles was analyzed.15 Incubation led to significant decreases in the ratio of LDL–cholesteryl ester/apolipoprotein B with concomitant increases in the ratio of LDL-triglyceride/apolipoprotein B. This indicates that incubation of the plasma enhances replacement of cholesteryl ester by triglyceride through lipid transfer reactions in LDL particles. Incubation-induced changes in the ratio of LDL–cholesteryl ester/apolipoprotein B and the ratio of LDL-triglyceride/apolipoprotein B both were increased by estrogen treatment. The plasma level of triglyceride showed a negative correlation with the incubation-induced changes in the ratio of LDL–cholesteryl ester/apolipoprotein B, and a positive correlation with the changes in the ratio of LDL-triglyceride/apolipoprotein B, indicating that estrogen-induced hypertriglyceridemia may enhance lipid transfer reactions to result in triglyceride-rich and cholesteryl ester–poor LDL particles. However, enrichment of the LDL core with triglyceride does not promote formation of small LDL particles.15,27 The triglyceride-rich LDL particles tend to enlarge because the volume of the triglyceride molecules may exceed that of the cholesteryl ester molecules.28 Therefore, additional factors may be involved in the reduction of the size of the LDL particle. Lypolytic enzymes such as lipoprotein lipase and hepatic triglyceride lipase are involved in the hydrolysis of triglyceride. Lipoprotein lipase catalyzes hydrolysis of the triglyceride in very low-density lipoprotein to form intermediate-density lipoprotein. Hepatic triglyceride lipase catalyzes hydrolysis of the triglyceride in intermediate-density lipoprotein to produce LDL and converts HDL2 to HDL3. Despite differences in substrate specificity, both enzymes hydrolyze the triglyceride in LDL. According to Homma et al,29 hydrolysis of LDL core triglyceride by these enzymes is accompanied by a concomitant reduction in the size of LDL particles. Accordingly, hydrolysis of the enriched triglyceride in LDL particles by lipolytic enzymes may be required to induce the formation of LDL particles that are smaller than normal. According to our previous findings, estrogen therapy did not affect the activity of lipoprotein lipase, but the activity of hepatic triglyceride lipase was inhibited significantly (a 30% reduction).8 However, estrogen-induced reduction in the activity of hepatic triglyceride lipase is unlikely to inhibit completely hydrolysis of LDL-triglyceride. Additional investigations of the effect of estrogen on the process of triglyceride hydrolysis in LDL particles are required to clarify the mechanisms of the change in LDL particle size.
Long-term postmenopausal estrogen replacement therapy significantly reduces mortality from coronary heart disease and from cardiovascular disease.30 The anti-atherogenic effect of estrogen may be related to a decrease in the number of LDL particles. Furthermore, estrogen stimulates nitric oxide production in the endothelial cells31,32 and inhibits the migration and proliferation of vascular smooth muscle cells.33 These favorable effects of estrogen may lead to a reduction in the incidence of coronary heart disease. However, these beneficial effects could be offset partially by a concurrent decrease in LDL particle size. Studies are needed to investigate whether or not a reduction in the plasma level of triglyceride during estrogen replacement therapy further reduces the incidence of coronary heart disease in postmenopausal women.
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Footnotes |
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Received November 23, 1998. Received in revised form March 2, 1999. Accepted March 11, 1999.
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2. Castelli WP. Epidemiology of coronary heart disease: The Framingham Study. Am J Med 1984;76:4–12.[Medline]
3. Wakatsuki A, Sagara Y. Lipoprotein metabolism in postmenopausal and oophorectomized women. Obstet Gynecol 1995;85: 523–8.[Abstract]
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