Deficiency of endogenous estrogens has been associated

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1 Menopause Is Associated With Reduced rotection From ostprandial Lipemia André. van Beek, Florianne C. de Ruijter-Heijstek, D. Willem Erkelens, Tjerk W.A. de Bruin Abstract Deficiency of endogenous estrogens has been associated with a higher incidence of coronary heart disease (CHD) in women. We investigated whether natural menopause is associated with reduced protection from postprandial lipemia, which represents a risk indicator of CHD. Twenty-three postmenopausal women (mean age, 50 1 [SD] years; body mass index, kg/m 2 ) and 21 premenopausal women matched for age and body mass index (age, 49 1 years; body mass index, kg/m 2 ) underwent an oral vitamin A fat-loading test. Vitamin A is a marker of the metabolism of chylomicrons and chylomicron remnants. All women were normolipidemic, were in good health, were nonsmokers, and used no medication. ostprandial lipids and vitamin A were measured at hourly intervals up to 12 hours. In postmenopausal women, plasma total cholesterol and LDL cholesterol concentrations were significantly higher. Fasting plasma triglyceride (TG) concentrations were mmol/l in postmenopausal women and mmol/l in premenopausal women ( NS). In the postprandial phase, postmenopausal women had higher plasma TG ( versus mmol L 1 h 1 ; 0.024) and vitamin A ( versus mg L 1 h 1 ; 0.001) responses. To correct for the possible confounding effect of fasting TG, 13 postmenopausal women were carefully matched with 19 premenopausal women. Although fasting TG levels were identical ( versus mmol/l), differences in postprandial vitamin A ( versus mg L 1 h 1 ; 0.006) and incremental TG (ie, after subtraction of baseline TG) ( versus mmol L 1 h 1 ; 0.023) persisted between postmenopausal and premenopausal women. Natural menopause is associated with aggravated postprandial lipemia in women matched for age and body mass index. Higher postprandial lipemia potentially explains the relation of TGs and CHD mortality risk in postmenopausal women. (Arterioscler Thromb Vasc Biol. 1999;19: ) Key Words: chylomicrons estrogens coronary heart disease risk factors lipids Deficiency of endogenous estrogens has been associated with a higher incidence of coronary heart disease (CHD). with an artificially early menopause by bilateral oophorectomy have an increased risk of myocardial infarction. 1 An early natural menopause poses an excess risk of CHD as well. 2 The decrease in risk in women who take estrogen replacement 3 is further evidence that the excess risk of CHD in women with a bilateral oophorectomy is a consequence of estrogen deficiency. How this effect is mediated remains unclear. One current hypothesis is that endogenous estrogen deficiency leads to an adverse lipid profile, which in turn results in an enhanced risk of CHD. It has consistently been shown that LDL cholesterol is raised after the menopause. 4,5 The results of the statin studies emphasize the importance of LDL as a risk factor in women. However, large prospective studies reveal that LDL cholesterol is not a very strong predictor of cardiovascular disease in women. 6,7 In contrast, triglycerides (TGs) are better predictors, 6 although their predictive power has been underestimated in epidemiological studies. 8 Exaggerated postprandial lipemia has invariably been observed in normolipidemic men with CHD 9 11 and in 1 study of normolipidemic women with CHD. 12 Moreover, the progression rate of CHD was related to the postprandial levels of chylomicron remnants. 13 We investigated whether the presence of natural menopause is associated with reduced protection from postprandial lipemia in normolipidemic women matched for age and body mass index. Methods Subjects were recruited by means of advertisements in local newspapers between May 1994 and May A questionnaire on health and menopausal status was sent to all responders. were defined as postmenopausal when they reported their last menses to have been at least 12 months previously and had elevated folliclestimulating hormone (FSH, 40 IU/L) levels at screening and test Received December 8, 1998; revision accepted March 24, From the Departments of Internal Medicine and Endocrinology, Laboratory of Lipid Metabolism, University Medical Center Utrecht, the Netherlands. Dr de Bruin is now at the Department of Medicine and Endocrinology, Maastricht University Medical School, Maastricht, Netherlands. resented in part at the Scientific Conference on Hormonal, Metabolic, and Cellular Influences on Cardiovascular Disease in of the American Heart Association, San Diego, Calif, October 19 21, 1995, and the 66th Congress of the European Atherosclerosis Society, Florence, Italy, July 13 17, Correspondence to André. van Beek, MD, hd, Departments of Internal Medicine and Endocrinology, F02.124, O Box , 3508 GA Utrecht, Netherlands. vanbeek.hopian@gironet.nl 1999 American Heart Association, Inc. Arterioscler Thromb Vasc Biol. is available at

2 2738 Arterioscler Thromb Vasc Biol. November 1999 TABLE 1. Characteristics of and No Age, y* 50 (47 52) 49 (47 52) Amenorrhea, y* 2.9 (1 7.5) Regular menses FSH, IU/L Luteinizing hormone, IU/L Estradiol, pmol/l Estrone, pmol/l Body mass index, kg/m Waist-to-hip ratio Total cholesterol, mmol/l TGs, mmol/l NS (0.078) HDL cholesterol, mmol/l NS LDL cholesterol, mmol/l Apolipoprotein A-1, g/l NS Apolipoprotein B, g/l NS (0.066) *Median and ranges. days. were considered premenopausal when they had had an unchanged and regular menstrual pattern during the previous 5 years, without typical climacteric complaints, and had compatible low FSH levels ( 20 IU/L). All healthy women, ranging in age from 47 to 52 years, were eligible for screening when they had a uterus and 2 ovaries in situ, used no medication currently or in the preceding year, and did not smoke. During a screening visit, the participant s medical history was taken with special attention to the menstrual cycle, and a physical examination was performed. At this visit, plasma cholesterol, TG, FSH, and estradiol levels were also determined. All postmenopausal and premenopausal women who were normolipidemic at screening (cholesterol 6.5 mmol/l and TG 2.0 mmol/l, representing the 75th percentile for Dutch women in this age group) and had body mass indices between 19 and 30 kg/m 2 were enrolled in the study. All participants gave their written informed consent before the study. The study was approved by the ethical committee of the Academic Hospital Utrecht. Oral Fat-Tolerance Test Cream consisting of 40% fat (wt/vol) with a ratio of polyunsaturated to saturated fatty acid of 0.06, 0.001% cholesterol (wt/vol), and 2.8% carbohydrates (wt/vol) was given as a single fat source in a dose of 50 g fat/m 2 body surface area. Vitamin A was added to the cream in a dose of IU aqueous retinyl palmitate (R)/50 g fat (125 ml cream). Vitamin A, a lipid-soluble vitamin, is incorporated in chylomicrons as R and is a marker for these intestinal lipoproteins. After an overnight fast of 12 hours, participants were admitted to the clinical research center at 8 AM. Blood samples were obtained before the test meal and hourly thereafter up to 12 hours in tubes containing EDTA. To prevent breakdown of R, all blood samples were protected from light. Water and decaffeinated coffee or tea, but no food, were allowed during the test. None of the subjects had diarrhea or other symptoms of malabsorption. The average cream fat load was 88 7 g in postmenopausal and 88 6 g in premenopausal women. Laboratory Assays and Measurements A rapid single-spin ultracentrifugation method was used at hourly intervals to separate lipoproteins into a fraction with a Svedberg flotation unit 1000 (Sf 1000) that contains chylomicrons, large chylomicron remnants, and large hepatic lipoproteins and a remaining infranatant fraction (Sf 1000) containing small chylomicron remnants and all other lipoproteins. 14,15 Whole plasma and lipoprotein fractions were assayed for cholesterol and TGs with commercial enzymatic reagents. The concentration of R was measured by high-performance liquid chromatography. HDLs were prepared by the heparin-mncl 2 dextran sulfate precipitation method. LDLs were prepared by density gradient ultracentrifugation. Measurements of hormones were performed in baseline blood samples. FSH and luteinizing hormone were determined by ELISAs, estrone, and estradiol by competitive radioimmunoassays. 16 ApoE genotypes and postheparin lipolytic enzyme activities were measured as described. 16 Statistical Analysis Data are given as mean SD. ostprandial responses are expressed as TG or R area under the time curve (AUC) or under the incremental time curve ( AUC, ie, the increment after subtraction of baseline concentrations) from baseline to 8 hours postprandially. The unpaired t test was used to detect the significance of mean differences. To correct for the potentially confounding effect of baseline TGs, 13 postmenopausal women were matched to 19 premenopausal women with 1 single criterion: a difference in plasma TG 0.05 mmol/l. If 1 premenopausal woman met the matching criterion, all case subjects were selected and averaged out, resulting in a 1:1 matching. To determine the significance of mean differences in this subgroup analysis, a paired t test was used. Statistical calculations were performed with SSS 6.1 for Windows. Results Study Groups Baseline characteristics of the 2 groups are shown in Table 1. Both groups were of similar age and body composition. In women who had had a natural menopause, plasma cholesterol and LDL cholesterol concentrations were significantly increased, and plasma apob tended to be raised. Fasting plasma TGs were elevated in postmenopausal women, but this did not reach statistical significance. Although low TG values ( 0.5 mmol/l) were observed in both groups, 25% of the normolipidemic postmenopausal women had fasting TG concentrations 1.5 mmol/l, compared with 10% of the premenopausal women. Daily energy and fat intakes were not different for the 2 groups (data not shown). ApoE genotypes for the 2 groups were highly comparable: E2/E3 (postmenopausal, 0; premenopausal, 3), E2/E4 (2, 0), E3/E3 (15, 14), E3/E4 (5, 3) and E4/E4 (1, 1). Hepatic lipase activity was higher in

3 van Beek et al Natural Menopause and TG-Rich Lipoprotein Metabolism 2739 ostprandial TG and vitamin A responses for all postmenopausal (f) and premenopausal (E) women (A and B) and after matching for fasting TGs (C and D). postmenopausal women ( versus mu/ml; 0.001). LL activities were not different between the 2 groups ( and mu/ml for postmenopausal and premenopausal women, respectively). ostprandial Responses After an oral fat-tolerance test, time curves in postmenopausal women for TGs (Figure, panel A) and R (Figure, panel B) were higher than in premenopausal women. The mean (incremental) postprandial TG response (TG AUC and TG AUC) and chylomicron response (R AUC) were TABLE 2. ostprandial Responses in and significantly larger in postmenopausal women (Table 2). The statistically significant differences clearly appeared in Sf 1000 lipoprotein fractions with both TGs and R (ie, chylomicrons, large chylomicron remnants, and large hepatic TG-rich lipoproteins) but were also measured in Sf 1000 lipoproteins with R (ie, mainly small chylomicron remnants). All women in both groups had returned to fasting plasma TG concentrations after 8 hours after ingestion of the test meal, and TG levels continued to drop in the next 4 hours. This decrease was more exaggerated in postmenopausal women. The mean difference in plasma TGs between post- No ercent Increase* lasma SF Sf NS (0.064) Mean recovery, 91% lasma Vitamin A, mg L 1 h 1 lasma Sf Sf Mean recovery, 82% *Mean percent increase attributable to natural menopause. AUC 0 8 h indicates area under the incremental TG curve, ie, after subtraction of baseline TG concentrations.

4 2740 Arterioscler Thromb Vasc Biol. November 1999 TABLE 3. ostprandial Responses in and Matched for Fasting TG Concentrations No Fasting TGs, mmol/l ercent Increase* lasma Sf NS Sf NS Mean recovery, 91% lasma Vitamin A, mg L 1 h 1 lasma Sf Sf NS Mean recovery, 78% *Mean percent increase attributable to natural menopause. AUC 0 8 h indicates area under the incremental TG curve, ie, after subtraction of baseline TG concentrations. menopausal and premenopausal women at baseline and at 8, 10, and 12 hours after the fat load was 30%, 15%, 7%, and 4%, respectively. To correct for the possible confounding effects of fasting TGs, postmenopausal and premenopausal women were precisely matched for baseline TGs (Table 3). All matched pairs had fasting TG levels 1.2 mmol/l, and differences in fasting TG in matched pairs were not exceeding 0.05 mmol/l. Time curves for TGs and R (figure, panels C and D, respectively) showed again that postmenopausal women had higher postprandial lipemia than premenopausal women. Despite identical fasting TGs, the mean (and incremental) plasma TG responses (TG AUC and TG AUC) were significantly higher in postmenopausal women. The chylomicron response (R AUC) was also higher after the menopause. This difference was caused by the Sf 1000 lipoprotein fraction, which contained mainly the chylomicrons and large chylomicron remnants. Discussion The present results showed differences in postprandial TG and chylomicron response between postmenopausal and premenopausal women with similar age, body mass index, daily energy and fat intake, apoe genotype, LL activity, and HDL concentrations even after careful matching for fasting TGs. The main differences were found in the vitamin A concentrations in the Sf 1000 fraction, which contains chylomicrons and large chylomicron remnants. We studied nonsmoking women of similar age and body mass index, because smoking, obesity, and age influence both lipid metabolism and the occurrence of menopause. 17 In theory, the selection of age-matched groups might be a problem, because the fact that menopause was reached at a different chronological age could be an indication of underlying differences between the groups that also relate to lipid metabolism. 18 We believe that this is not the case, because no data exist in the literature to support such an idea. We studied 2 groups of healthy women within a narrow age limit who were at the lower and higher ends of the normal distribution for age at menopause. It is unlikely that underlying differences, rather than menopause per se, have blurred the study results. Age at menopause is determined primarily by the number of ovarian follicles. 19 Menopausal changes in fasting plasma lipids in our study were highly comparable to those found by others in longitudinal 5,20 or cross-sectional 4,21 studies, with the exception of TGs. We found that fasting TGs were 30% higher in postmenopausal women. Other studies found no significant increase 5 or higher levels after the menopause, with values ranging from 12% 4 to 18%. 21 This variability in findings can be explained in 2 ways. First, different definitions of menopausal status were used in the literature, and therefore, variable groups, with conceivably varying differences in plasma lipids, were identified. It is likely that changes in lipids are not instantaneous but rather gradual, expressing ongoing decline of ovarian function. 20 Second, the distinct possibility should be recognized that regulation of plasma TGs in premenopausal women is different from that in postmenopausal women. Ninety percent of the premenopausal women had plasma TG levels 1.5 mmol/l at baseline, in contrast to only 75% of the postmenopausal women, whereas very low fasting TG concentrations ( 0.5 mmol/l) were observed in both groups. Thus, after the menopause, there is a larger population-based variation in fasting TGs. This was also observed after an oral fat load, showing higher postchallenge peak TG levels and lower compensatory trough levels. Thus, concomitant with the loss

5 van Beek et al Natural Menopause and TG-Rich Lipoprotein Metabolism 2741 of endogenous estrogens, the tight regulation of plasma TGs is lost. The postprandial differences in TG and vitamin A in the Sf 1000 fractions suggest a difference in the metabolism of chylomicrons and large chylomicron remnants, because fat intake and postheparin lipoprotein lipase activity were similar. This can be explained in several ways. First, higher concentrations of hepatic TG-rich lipoproteins may have resulted in enhanced competition at the level of the common lipolytic pathway. 22 However, after correction for endogenous TGs in the fasting state, these differences persisted. Second, there may have been a different composition of chylomicrons, eg, reduced size or different apoprotein composition. Furthermore, the deficiency of endogenous estrogens may lead to a decreased chylomicron clearance capacity. This is supported by a report on the effects of estrogen replacement on chylomicron metabolism in postmenopausal women. 16 In conclusion, substantial and significant differences in postprandial lipid metabolism were found in women who differed only in their menopausal status and therefore in their endogenous estrogen production. The loss of protection from postprandial lipemia associated with the postmenopausal state potentially explains the lipid contribution to the increased risk of coronary artery disease and has the potential to be therapeutically corrected by nutritional or pharmaceutical interventions. Acknowledgments This work was made possible by the Dutch Heart Foundation (grant NHS ). Dr de Bruin is a recipient of the ioneer grant of the Dutch Organization of Fundamental Research (NWO). The authors wish to thank H.E. Westerveld for the study design and grant application. References 1. Rosenberg L, Hennekens CH, Rosner B, Belanger C, Rothman KJ, Speizer FE. 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