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Lipolytic Enzyme Effect on Small Low-Density Lipoprotein Particles in Women Treated With Estrogen

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. E-mail: wakatuki{at}kochi-ms.ac.jp

	Abstract

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Objective: To test whether hydrolysis of low-density lipoprotein (LDL) triglyceride by lipolytic enzymes decreases the size of LDL particles in women treated with estrogen replacement.

Methods: Fifteen postmenopausal women received 0.625 mg conjugated equine estrogens daily for 3 months. Plasma concentrations of total cholesterol, triglyceride, and high-density lipoprotein (HDL) cholesterol were measured before and after therapy. We also assayed levels of total, free, and esterified cholesterol, triglyceride, and protein in LDL. Plasma samples were incubated at 37C for 24 hours and LDL fractions were isolated by ultracentrifugation. After LDL samples were further incubated with or without lipoprotein lipase (500, 700, and 1000 ng/mL) at 37C for 24 hours, LDL triglyceride, LDL protein, and the diameter of LDL particles were measured.

Results: Estrogen decreased total cholesterol and increased triglyceride and HDL cholesterol in plasma. Estrogen treatment decreased the ratio of cholesteryl ester/protein, whereas the ratio of triglyceride/protein increased. Estrogen decreased LDL particle diameter. Incubation of plasma increased the ratio of LDL triglyceride/protein from 0.40 ± 0.14 to 0.48 ± 0.15 (P < .05) and decreased the ratio of LDL cholesteryl ester/protein from 1.17 ± 0.25 to 1.09 ± 0.22 (P < .05), but LDL particle diameter did not change. Incubation of LDL with lipoprotein lipase reduced the LDL triglyceride/protein ratio, and decreased the diameter of LDL particles from 25.61 ± 0.87 nm to 24.89 ± 0.88 nm (500 ng/mL, P < .05), 24.62 ± 1.20 nm (700 ng/mL, P < .05), and 24.67 ± 1.19 nm (1000 ng/mL, P < .05).

Conclusion: In women treated with estrogen, hydrolysis of triglyceride in LDL particles might be accompanied by reduced particle size.

Estrogen replacement in postmenopausal women favorably affects lipid metabolism by reducing plasma concentration of low-density lipoprotein (LDL) cholesterol and increasing that of high-density lipoprotein (HDL) cholesterol.¹ An epidemiologic study indicated that long-term postmenopausal estrogen replacement therapy (ERT) significantly reduced mortality from coronary heart disease and other cardiovascular disease.² Contrasting findings from the Heart and Estrogen/Progestin Replacement Study indicated that hormone replacement therapy did not reduce the overall rate of coronary events in postmenopausal women with established coronary disease.³

Low-density lipoprotein particles vary in size, density, and lipid composition,⁴ with various LDL subfractions differing in atherogenicity. Smaller, denser LDL particles are associated with increased risk of atherosclerosis.⁵ According to a previous study, estrogen therapy reduced the size of LDL particles and increased the prevalence of LDL pattern B,^6,7 which represents particles with diameters less than 25.5 nm and is associated with increased coronary risk.^5,8 Beneficial effects of estrogen could be offset partially by a concomitant decrease in LDL particle size. Thus, whereas estrogen has an antiatherogenic effect, estrogen-induced small LDL particles might be atherogenic.

The size of LDL particles is influenced by their lipid constituents. In human plasma, the cholesteryl ester transfer protein catalyzes net mass transfer of cholesteryl esters from LDL and HDL to very low-density lipoprotein, with net mass transfer of triglycerides in the opposite direction from very low-density lipoprotein to LDL and HDL.⁹ Lipid transfer reactions might be important in determining the composition of LDL and HDL, so we analyzed the effects of estrogen on lipid transfer reactions in postmenopausal women and found that an estrogen-induced increase in plasma triglyceride concentration might stimulate lipid transfer reactions that result in triglyceride-rich, cholesteryl ester-poor LDL particles of small size.¹⁰ Enrichment of the LDL core with triglyceride does not promote formation of small LDL particles because the volume of triglyceride molecules might exceed that of cholesteryl ester molecules.^11,12 Other factors also might reduce the size of LDL particles.

Lipolytic enzymes such as lipoprotein lipase or hepatic triglyceride lipase hydrolyze triglyceride in lipoprotein particles. Hydrolysis of enriched triglyceride in LDL particles by such enzymes might be required to induce formation of LDL particles that are smaller than normal. The purpose of the present study was to test the hypothesis that hydrolysis of enriched LDL triglyceride by lipolytic enzymes reduced LDL particle size in postmenopausal women.

	Materials and Methods

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Between April 1, 1998, and September 30, 1998, we evaluated 15 postmenopausal Japanese women. The mean age was 54 years (range 48–64 years), and mean body mass index was 21.7 ± 1.9 kg/m² (range 18.1–25.8 kg/m²). No subjects smoked, used caffeine or alcohol, had histories of thyroid disease, liver disease, or diabetes mellitus, or were taking any medication known to influence lipoprotein metabolism. None of the women were taking ERT before this study. During a 6-month enrollment period, 15 subjects who satisfied inclusion criteria were registered sequentially. Written informed consent was obtained from each subject before admission. The study design was approved by the ethics committee of Kochi Medical School.

Each subject took 0.625 mg of conjugated equine estrogens orally every evening for 3 months. Endome-trial biopsies and blood samples were collected before and after treatment. During the 3 months of estrogen treatment, no subjects had exercise or dietary therapy, smoked, or used other sex steroids or phytoestrogen. Blood samples drawn between 8:00 AM and 10:00 AM after 12-hour fasts were centrifuged immediately at 1500g for 20 minutes at 4C to obtain plasma. Plasma concentrations of total cholesterol and triglyceride were measured by enzymatic methods as described.¹³ Plasma concentrations of HDL cholesterol were measured using the same method as for cholesterol after apolipoprotein B-containing lipoproteins had been precipitated with sodium phosphotungstate in the presence of magnesium chloride.¹³ The LDL fraction was isolated by ultracentrifugation according to the method of Havel et al,¹⁴ and concentrations of total cholesterol, free cholesterol, triglyceride, and protein in the LDL fraction were measured. The cholesteryl ester concentration of LDL was calculated as the difference between measured total cholesterol and the concentration of free cholesterol. The concentration of LDL protein was measured by the technique of Lowry et al.¹⁵

Low-density lipoprotein was subjected to gradient gel electrophoresis using 2–15% nondenaturing poly-acrylamide-agarose gels as described.¹² The gels were stained with Coomassie G-250 (Nacalai, Kyoto, Japan). The distribution profile of LDL subfractions was determined by densitometric scanning of the gels at 633 nm (Shimadzu, Kyoto, Japan). The apparent diameters of major LDL subfractions were measured by comparing results with a calibration curve constructed with ferritin, thyroglobulin, and latex beads.

Plasma samples before estrogen therapy were treated with 1.5 mmol/L iodoacetate to inhibit activity of lecithin cholesterol acyltransferase and maintained at 4C or incubated at 37C for 24 hours to analyze lipid transfer reactions and diameter of LDL particles.¹¹ The iodoacetate treatment was necessary because lecithin cholesterol acyltransferase esterifies free cholesterol in HDL. This esterified cholesterol is transferred to apolipoprotein B-containing lipoproteins by the cholesteryl ester transfer protein, which can influence lipid transfer reactions. Next, we isolated the LDL fraction by ultracentrifugation. Low-density lipoprotein particle diameter and concentrations of total cholesterol, free cholesterol, triglyceride, and protein in the LDL fraction were measured.

The LDL fraction isolated from the plasma samples incubated at 37C for 24 hours was further incubated with or without lipoprotein lipase (500–1000 ng/mL) at 37C for 24 hours.¹¹ Then the LDL fraction was reisolated, and concentrations of triglyceride and protein and the diameter of LDL particles were measured.

Estrogen-induced changes in lipid concentrations and differences in the ratio of LDL lipids/protein between 4C and 37C were analyzed by a paired Student t test when there was normal distribution or Wilcoxon rank-sum test when parameters were not normally distributed. The normality of measured parameters was evaluated by Shapiro-Wilk test. Effects of lipoprotein lipase on the ratio of LDL triglyceride/protein or the diameter of LDL particles were analyzed by repeated-measures analysis of variance. If analysis of variance showed a significant difference, Scheffé multiple comparison procedure was applied to determine which values differed. P < .05 was indicated statistical significance.

	Results

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Histologic examination of endometrial biopsy specimens showed no hyperplasia before or after treatment. Body mass index did not change significantly during the 3 months of estrogen treatment. Estrogen treatment significantly reduced plasma concentrations of total cholesterol and significantly increased plasma concentrations of triglyceride and HDL cholesterol. The LDL cholesteryl ester/protein ratio was significantly reduced, whereas the LDL triglyceride/protein ratio was significantly increased by estrogen treatment. Estrogen treatment significantly decreased the diameter of LDL particles (Table 1).

	Discussion

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To evaluate whether increased lipid exchange in LDL particles mediated by estrogen-induced enhanced lipid transfer reactions reduces the size of LDL particles, plasma samples collected before estrogen therapy were incubated at 37C for 24 hours. Similarly to estrogen therapy, incubation led to significant decreases in the ratio of LDL cholesteryl ester/protein with concomitant increases in the ratio of LDL triglyceride/protein. The ratios indicated that incubation of plasma enhanced replacement of cholesteryl ester by triglyceride through lipid transfer reactions in LDL particles, resulting in triglyceride-rich and cholesteryl ester-poor LDL particles. According to our previous findings,¹⁰ concentrations of core lipids such as triglyceride and cholesterol ester might be a major determinant of the size of LDL particles. However, the size of LDL particles did not change significantly, indicating that exchange of LDL core lipids by stimulated lipid transfer reactions might not affect the size of LDL particles. The enrichment of the LDL core with triglyceride did not promote the formation of small LDL particles because the volume of the triglyceride molecules might exceed that of the cholesterol ester molecules.^11,12 Therefore, additional factors might be needed to reduce the size of LDL particles.

Lipolytic enzymes such as lipoprotein lipase or hepatic triglyceride lipase are involved in hydrolysis of triglyceride. Lipoprotein lipase catalyzes the hydrolysis of the triglyceride in very low-density lipoprotein to form intermediate-density lipoprotein. Hepatic triglyceride lipase catalyzes the hydrolysis of triglyceride in intermediate-density lipoprotein to produce LDL, and converts HDL2 to HDL3. Hydrolysis of LDL core triglyceride by these enzymes might be accompanied by a concomitant reduction in the size of LDL particles.

To investigate the effects of lipolytic enzymes on the hydrolysis of LDL triglyceride and the size of LDL particles, LDL samples from the plasma incubated at 37C for 24 hours were further incubated with or without a lipolytic enzyme.¹¹ We used lipoprotein lipase from bovine milk, which has structural and functional similarities to human lipoprotein lipase. Concentrations of lipoprotein lipase used in this study were chosen to be equivalent to physiologic activity in healthy post-menopausal women.^1,16 Although hepatic triglyceride lipase is responsible for hydrolysis of LDL triglyceride, we used lipoprotein lipase instead, given that it is commercially available. Both enzymes hydrolyze the triglyceride in LDL despite differences in the substrate specificity.^11,17

Low-density lipoprotein fractions isolated by sequential ultracentrifugation in the present study were free from contamination by plasma proteins. Therefore the concentration of LDL protein might be equivalent to the concentration of LDL apolipoprotein B. Each LDL particle contains one molecule of apolipoprotein B, so the LDL triglyceride/protein ratio indicates the concentrations of triglyceride in each LDL particle. In the present study, incubation of LDL samples with lipoprotein lipase decreased the LDL triglyceride/protein ratio, indicating that lipoprotein lipase might stimulate hydrolysis of LDL triglyceride and reduce the concentration of triglyceride in LDL particles. The decreased concentration of LDL triglyceride also was accompanied by a concomitant reduction in the size of LDL particles. Based on those findings, estrogen-induced hypertriglyceridemia might enhance lipid transfer reactions initially, resulting in triglyceride-rich and cholesteryl ester-poor LDL particles. Next, hydrolysis of the enriched triglyceride by lipolytic enzymes might induce formation of small LDL particles. However, according to our previous study, estrogen therapy significantly inhibited lipolytic enzyme activity by 30%.¹ The concentration of lipoprotein lipase in that study, 700 ng/mL, was equivalent to the average activity of lipoprotein lipase in postmenopausal women.¹⁶ In the present experiments, lipoprotein lipase at 500 ng/mL also significantly reduced the ratio of LDL triglyceride/protein and decreased the size of LDL particles. That shows that estrogen-induced reduction in lipolytic enzyme activity is unlikely to completely inhibit hydrolysis of LDL triglyceride to result in production of small LDL particles.

According to the findings of the Heart and Estrogen/Progestin Replacement Study, hormone replacement therapy had beneficial effects on plasma concentrations of LDL cholesterol and HDL cholesterol, but the plasma triglyceride concentration was increased,³ as in our results. That estrogen-induced increase in plasma triglyceride can be atherogenic by a decrease in LDL particle size, and might offset partially the beneficial effects of estrogen. Interventional studies are needed to investigate whether a reduction in plasma triglyceride concentrations during hormone replacement therapy can reduce the risk of cardiac events in postmenopausal women with established coronary disease.

	Footnotes

 
PII S0029-7844(00)01185-6

Received July 17, 2000. Received in revised form November 28, 2000. Accepted November 29, 2000.

	References

Top Abstract Materials and Methods Results Discussion References

 
1. Wakatsuki A, Sagara Y. Effects of continuous medroxyprogester-one acetate on lipoprotein metabolism in postmenopausal women receiving estrogen. Maturitas 1996;25:35–44.[Medline]

2. Ettinger B, Friedman GD, Bush T, Quesenberry CP Jr. Reduced mortality associated with long-term postmenopausal estrogen study. Obstet Gynecol 1996;87:6–12.[Abstract]

3. Hulley S, Grady D, Bush T, Furberg C, Herrington D, Riggs B, et al. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. Heart and Estrogen/Progestin Replacement Study (HERS) Research Group. JAMA 1998;280:605–13.[Abstract/Free Full Text]

4. Shen MM, Krauss RM, Lindgren FT, Forte TM. Heterogeneity of serum low density lipoproteins in normal human subjects. J Lipid Res 1981;22:236–44.[Abstract]

5. Austin MA, Breslow JL, Hennekens CH, Buring JE, Willett WC, Krauss RM. Low-density lipoprotein subclass patterns and the risk of myocardial infarction. JAMA 1988;260:1917–21.[Abstract]

6. Wakatsuki A, Ikenoue N, Sagara Y. Effect of estrogen on the size of low-density lipoprotein particles in postmenopausal women. Obstet Gynecol 1997;90:22–5.[Abstract]

7. Wakatsuki A, Ikenoue N, Sagara Y. Estrogen-induced small low-density lipoprotein particles in postmenopausal women. Obstet Gynecol 1998;91:234–40.[Abstract]

8. Austin MA, King MC, Vranizan KM, Newman B, Krauss RM. Inheritance of low-density lipoprotein subclass patterns: Results of complex segregation analysis. Am J Hum Genet 1988;43:838–46.[Medline]

9. Tall AR. Plasma cholesteryl ester transfer protein. J Lipid Res 1993;34:1255–74.[Medline]

10. Wakatsuki A, Ikenoue N, Okatani Y, Izumiya C. Lipid transfer reactions and lipid composition of low-density lipoprotein particles in postmenopausal women receiving estrogen. Obstet Gynecol 1999;94:492–7.[Abstract/Free Full Text]

11. Lagrost L, Gambert P, Lallemant C. Combined effects of lipid transfers and lipolysis on gradient gel patterns of human plasma LDL. Arterioscler Thromb 1994;14:1327–36.[Abstract/Free Full Text]

12. Gambert P, Bouzenard-Gambert C, Athias A, Farnier M, Lallemant C. Human low density lipoprotein subfractions separated by gradient gel electrophoresis: Composition, distribution, and alterations induced by cholesteryl ester transfer protein. J Lipid Res 1990;31:1199–210.[Abstract]

13. Allain CC, Poon LS, Chan CG, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem 1974;20:470–5.[Abstract]

14. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human plasma. J Clin Invest 1955;34:1346–53.

15. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75.[Free Full Text]

16. Ikeda Y, Takagi A, Ohkaru Y, Nogi K, Iwanaga T, Kurooka S, et al. A sandwich-enzyme immunoassay for the quantification of lipoprotein lipase and hepatic triglyceride lipase in human postheparin plasma using monoclonal antibodies to the corresponding enzymes. J Lipid Res 1990;31:1911–24.[Abstract]

17. Homma Y, Nakaya N, Nakamura H, Goto Y. Increase in the density of lighter low density lipoprotein by hepatic triglyceride lipase. Artery 1985;13:19–31.[Medline]

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