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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1822-1829.)
© 1997 American Heart Association, Inc.

Articles

Hormonal Regulation of Lipoprotein(a) Levels: Effects of Estrogen Replacement Therapy on Lipoprotein(a) and Acute Phase Reactants in Postmenopausal Women

Catherine H. Tuck; Stephen Holleran; ; Lars Berglund

From the Departments of Medicine (C.H.T., L.B.) and Pediatrics (S.H.), Columbia University, New York, NY.

Correspondence to Lars Berglund, MD, PhD, Division of Preventive Medicine and Nutrition, Department of Medicine, Columbia University College of Physicians and Surgeons, P&S 9–510, 630 West 168th Street, New York, NY 10032. E-mail berglun{at}cudept.cis.columbia.edu

	Abstract

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Abstract Estrogen lowers lipoprotein(a) [Lp(a)] levels, but the mechanisms involved have not been clarified. To address the relationship between estrogenic effects on Lp(a) and serum lipids, and on other plasma proteins of hepatic origin, 15 healthy postmenopausal women participated in a randomized, double-blinded, placebo-controlled, crossover study with 4 weeks of oral conjugated estrogens (0.625 mg/d) and placebo, separated by a 6-week period. Lp(a) levels decreased during estrogen treatment in 14 of the 15 subjects (mean decrease, 23%; P<.001). In response to estrogen, apolipoprotein A-I (apoA-I), HDL cholesterol, and triglyceride levels increased by 12% (P=.001), 11% (P<.001), and 10% (P=.02), respectively. Apolipoprotein B (apoB) and LDL cholesterol levels decreased by 7% (P=.01) and 12% (P=.03), respectively. ApoB, LDL cholesterol, and Lp(a) levels fell within 1 week of treatment, whereas apoA-I and HDL cholesterol levels rose more slowly. Levels of acid ₁-glycoprotein (AAG) and haptoglobin (HPT), two hepatically derived acute phase proteins, also decreased during estrogen treatment by 18% (P<.001) and 25% (P=.002), respectively. Although the changes in AAG and HPT in response to estrogen were highly correlated (r=.67, P=.009), we were unable to detect a correlation between change in either acute phase protein and change in Lp(a) (r=-.14 and -.24, P=.64 and .41). The lack of correlation between the changes in two acute phase reactants and Lp(a) suggests different underlying mechanisms for the effects of estrogen on these liver-derived proteins.

Key Words: apolipoprotein(a) • hormone replacement therapy • conjugated equine estrogens

	Introduction

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Lp(a) is an LDL-like particle that consists of one apoprotein(a) [apo(a)] molecule covalently bound to an apoB molecule, surrounding a cholesterol-rich lipid core. Elevated plasma levels of Lp(a) have been shown to be an important independent risk factor for atherosclerosis in many retrospective case-control studies^1-4 and in most^5-8 but not all^9-11 prospective studies. Plasma Lp(a) levels are determined almost completely by genetic variation at the apo(a) locus.^12,13 About half of the genetic variation in Lp(a) levels from the apo(a) locus can be explained by an apo(a) size polymorphism; large alleles tend to be associated with low Lp(a) levels, and small alleles tend to be associated with high Lp(a) levels.¹² The factors that account for the remaining genetic variation from the apo(a) locus have not been identified conclusively. In an in vitro cell system, the promoter region, which consists of 1.3 kb of genomic DNA upstream from the transcription initiation site of apo(a), is able to drive transcription of a reporter gene¹⁴ and contains recognizable consensus sequences for nonspecific promoter elements, for liver-enriched transcription elements HNF-1, CEBP, and LF-A1, and for interleukin-6 (IL-6) binding.¹⁵ It has been shown that binding of the liver-enriched HNF-1 is an important positive regulatory factor for apo(a) gene expression and seems to confer liver-specific expression.¹⁶ Also, addition of IL-6 causes a 5-fold enhancement of reporter gene activity in transfected Hep G2 cells.¹⁴ Other potential regulatory sequences include a TTTTA repeat polymorphism in the apo(a) promoter that has been associated with lower than expected Lp(a) levels^17,18 and a C/T polymorphism that has been associated with decreased in vitro apo(a) translation.¹⁹ Finally, several liver-specific DNAse hypersensitive sites have been located far upstream from the apo(a) gene,^20,21 suggesting that other regulatory sites for apo(a) expression may be located in distant regions not yet studied in detail.

In keeping with the largely genetic control of Lp(a) levels, intraindividual Lp(a) levels are very stable over time.¹² However, a growing number of studies have shown that several hormones can significantly change Lp(a) levels in humans. Anabolic steroids given to women will lower Lp(a) levels substantially (60 to 80%).^22,23 In initial studies, large doses of orally and parenterally administered estrogens given to men with prostate cancer lowered Lp(a) levels by 50%.²⁴ Smaller replacement doses of estrogen given, either alone or in combination with progestins, long-term to postmenopausal women have subsequently been shown to lower Lp(a) levels significantly but by a smaller amount (~15 to 50%).^25-31 All of these studies have been long-term, and neither the timing of onset of the estrogen effect on Lp(a) nor its mechanism of action has been addressed in humans.

Apart from hormones, very few clinical conditions affect Lp(a) levels. However, some studies have suggested that Lp(a) levels may increase after stressful events, in the manner of the acute phase reactants,^32-34 although conflicting results have been obtained.³⁵ Estrogen therapy also influences the levels of several hepatically synthesized proteins,^36-38 and therefore we hypothesized that the response of Lp(a) to estrogen would be related to estrogen-induced changes in hepatically synthesized acute phase reactants.

In this study, we examined the effects of estrogen versus placebo on Lp(a) levels and measured the acute phase response in 15 postmenopausal women who were given oral conjugated estrogens and placebo for 4 weeks each in a randomized, double-blinded, crossover study. The changes in Lp(a) levels after estrogen were examined weekly in relation to changes in other lipid parameters and the acute phase reactants.

	Methods

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Subjects
Healthy women who had been postmenopausal for at least 1 year were recruited by advertisement within the medical center and by referral from medical clinics. Women were excluded from the study if they had any significant medical illness, including heart disease, diabetes, liver or kidney disease, or untreated thyroid disease. In addition, women with triglyceride levels >300 mg/dL or Lp(a) levels <2 mg/dL were excluded from the study. Of the 30 women who responded to the advertisements, 6 were excluded because they had Lp(a) levels <2 mg/dL, 2 were excluded because of medical illness, and 7 women dropped out before receiving any medication. Fifteen women (8 Caucasians, 4 African-Americans, 3 Hispanics) were thus enrolled in the study. Their clinical characteristics are given in Table 1. The average age was 55±6 years, the average time since menopause was 5.9±4.7 years, and the average body mass index was 26.7±6 kg/m². One woman had hypercholesterolemia treated with lovastatin; treatment was stopped 6 weeks before randomization, and she was maintained off medication during the trial. One woman had controlled hypothyroidism for many years on a stable dose of levothyroxine sodium, and one woman had depression treated with a stable dose of an antidepressant medication; both of those conditions and medications remained unchanged during the course of the study.

View this table: [in this window] [in a new window]   Table 1. Characteristics of the Subjects

Study Protocol
The study was designed as a randomized, double-blinded, placebo-controlled, crossover study and was approved by the Institutional Review Board. All subjects gave informed consent before enrollment in the study, and procedures for the study followed institutional guidelines. Eight subjects who had been receiving postmenopausal hormone replacement were advised to discontinue this medication 6 weeks before entry into the study. Four weeks before entry, each subject underwent a screening examination consisting of a complete history and physical examination. At that time, analysis of Lp(a), lipid profile, follicle-stimulating hormone (FSH), luteinizing hormone (LH), blood chemistry, complete blood count, and urinalysis were performed. Also at that visit, all subjects met with a dietitian and received instruction in the American Heart Association Step 1 diet. To reinforce the dietary guidelines, each subject was again contacted by the dietitian just before each treatment period, and all subjects were found to be compliant with the diet. At the start of the study, the subjects were randomly assigned to either 0.625 mg/d of conjugated equine estrogens (Premarin, Wyeth-Ayerst) or matching placebo for 4 weeks (Treatment Period 1). After the first treatment period, there was a 6-week washout period, followed by another 4-week treatment period with the corresponding regimen of either estrogen or placebo (Treatment Period 2). Fasting blood was drawn weekly in each treatment period for Lp(a), apoA-I, and apoB levels, lipid profile, and acute phase proteins. During the last week of each treatment period, additional blood samples were drawn for FSH/LH levels. Blood pressure and weight were recorded at each visit.

Analytical Procedures
Plasma total cholesterol and triglyceride levels were determined using standard enzymatic techniques. HDL cholesterol was determined after precipitation of apoB-containing lipoproteins with phosphotungstic acid.³⁹ LDL cholesterol levels were calculated using the formula of Friedewald et al.⁴⁰ In addition, LDL cholesterol levels were corrected for the cholesterol carried in Lp(a) by subtracting the Lp(a) level multiplied by 0.3.⁴¹ Serum levels of apoA-I and apoB, C-reactive protein (CRP), HPT, and AAG were determined by immunoturbidimetric procedures on a Beckman Array 360 nephelometer using commercially available reagents. The interassay coefficients of variation for these measurements were 3.9% for apoA-I, 3.4% for apoB, 5.6% for HPT, and 3.6% for AAG. Non-Lp(a) apoB levels were estimated by subtracting from total apoB levels the Lp(a) level multiplied by 0.184.⁴² Lp(a) levels were analyzed using an immunonephelometric procedure on a Beckman Array 360 nephelometer. Lp(a) antibody was obtained from DAKO Chemicals, and Lp(a) standards, standardized against the Northwest Lipid Research Laboratory, were purchased from International Enzymes. In our hands, this assay had interassay coefficients of variation of 4% and 8% at Lp(a) levels of 48 mg/dL and 8 mg/dL, respectively. FSH/LH levels were assayed using a kit (Diagnostic Products Corp.) according to the manufacturer's instructions. HDL subfractions, HDL₂ and HDL₃, were determined in plasma by sequential precipitation using heparin-manganese and dextran sulfate as described by Gidez et al.⁴³ ApoE genotypes were determined essentially as described by Hixson and Vernier.⁴⁴

Apo(a) Phenotyping
Apo(a) phenotyping was performed using Western blotting essentially as described by Kamboh et al.⁴⁵ Briefly, plasma samples were separated on a 2% submarine agarose gel for 15 hours at 100 V at 4°C. The proteins were then blotted onto nitrocellulose using an electroblotter for 3 hours in the cold. The nitrocellulose membrane was blocked using powdered skim milk and then incubated with a primary antibody against Lp(a) (INCStar). The apo(a) bands were visualized with the ECL Amersham technique on Kodak X-OMAT films using a second, labeled antibody. The results were related to standards with multiple defined apo(a) isoforms from Immuno AG (Innsbruck, Austria), taking into account that kringle size number is related in an inverse logarithmic fashion to isoform mobility on agarose gel electrophoresis.⁴⁶ Additional apo(a) isoform standards with defined molecular weights were also kindly provided by Dr. Neal Azrolan (Wyeth-Ayerst).

Statistics
As the distribution of values for Lp(a) and triglycerides were skewed, square root or logarithmic transformation of these data was done before statistical tests were performed. The skewness of the Lp(a) levels in the estrogen and placebo groups before square root transformation was .45 and .15, and the skewness in the estrogen and placebo groups after square root transformation was -.02 and -.26; the considerable improvement in skewness after transformation for the estrogen group argues for transforming the raw data. Similarly, the skewness of the triglyceride levels in the estrogen and placebo groups before logarithmic transformation was .6 and .6; after transformation, the corresponding skewness values were .1 and -.1. Comparisons between groups were made using paired t tests (two-tailed). A P value of .05 was considered significant for the primary outcome variable, Lp(a), and a smaller P value of .01 was considered significant for secondary outcome variables, because multiple comparisons were being made. Correlations were made by calculating Pearson's coefficient of correlation. Data are expressed as mean±SD except for Figs 1 and 4, where data are expressed as mean±SE.

View larger version (12K): [in this window] [in a new window]   Figure 1. Time course of Lp(a) response to estrogen and placebo. Lines represent the mean±SE of the Lp(a) levels of all 15 subjects during each week of the placebo and estrogen phase.

View larger version (25K): [in this window] [in a new window]   Figure 4. Time course of the lipid, lipoprotein, and acute phase protein response to estrogen versus placebo. Each line represents the mean±SE of the measurements for all subjects during each week of the corresponding treatment phase. Values for LDL cholesterol and apoB were adjusted for Lp(a) cholesterol and apoB, as described.41-42

	Results

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There were no adverse effects related to estrogen therapy, and pill counts indicated compliance with the medications. Baseline gonadotropin levels were high, consistent with each subject's postmenopausal status. As expected, FSH and LH levels were suppressed during the estrogen treatment compared with placebo, further confirming that the patients were compliant with taking the medication (Table 1). Weight and blood pressure remained stable during the course of the study. All subjects reported only occasional alcohol use, and none changed exercise habits during the course of the study.

Lp(a) levels began to decrease at 1 week after estrogen therapy compared with placebo (Fig 1). Because the response was stable after 3 weeks, we combined results from weeks 3 and 4 in each treatment period for comparison. Lp(a) levels were lower in 14 of 15 patients during the estrogen treatment period compared with placebo (Fig 2). The mean decrease during estrogen treatment was 12.9 mg/dL or 23% (P=.0002) (Table 2). As shown in another study,³¹ the absolute amount of Lp(a) lowering was inversely correlated with the initial Lp(a) level (r=-.6, P=.018). However, when viewed in terms of percentage drop, there was no difference in Lp(a) lowering to estrogen when those subjects with Lp(a) levels <30 mg/dL were compared with those with Lp(a) levels >40 mg/dL (26% decrease in each group). Our sample consisted of subjects from three different ethnic groups. Although the small number of patients in our study precludes comparing the response of Lp(a) to estrogen between ethnic groups, it is evident from the figure that nearly all subjects in this racially mixed group responded to estrogen to about the same relative degree.

View larger version (18K): [in this window] [in a new window]   Figure 2. The responses of Lp(a) to estrogen versus placebo in individual subjects. Each point represents the mean Lp(a) level during weeks 3 and 4 of the corresponding treatment phase. African-American (), Hispanic (x), Caucasian ().

View this table: [in this window] [in a new window]   Table 2. Lipid and Lipoprotein Responses to Estrogen Versus Placebo

A Western blot representing all the subjects' apo(a) isoforms arranged by isoform size is shown in Fig 3. Twelve of the 15 women clearly had two visible bands on Western blot; three subjects had only one band visible. The apo(a) sizes ranged from approximately 14 to 35 kringle-4 U, or about 430 to 850 kDa. As seen in Fig 3, there was a general linear relationship between baseline Lp(a) levels and apo(a) isoform size, with a few exceptions. The r value for dominant isoform size versus Lp(a) level was -.79. We could not detect any major isoform-dependent response to estrogen; however, the number of subjects with only large or only small apo(a) isoforms was small.

View larger version (82K): [in this window] [in a new window]   Figure 3. Western blot of apo(a) isoforms for each subject. Lanes 1, 7, 13, 19, and 21 represent apo(a) size controls. The baseline Lp(a) level for each subject is shown, as well as the ethnicity of each subject [Caucasian (C), Hispanic (H), African-American (AA)]. It can be seen that three subjects (lanes 9, 11, and 20) had only a single apo(a) allele visible, and that the range of apo(a) sizes was from 14 to 35 kringle-4 U (K4 U), or about 430 to 850 kDa.

In response to estrogen treatment, total cholesterol levels were not different compared with placebo (Table 2). However, as expected, LDL cholesterol levels decreased by 12%, and HDL levels increased significantly, by 11%. The increase in serum HDL levels was accounted for completely by changes in the HDL₂ subfraction. The increase in serum HDL was paralleled by a significant increase in apoA-I levels of 12%. The decrease in LDL cholesterol levels was not statistically significant. In addition, some of this nonsignificant drop in LDL may have been accounted for by a drop in Lp(a) cholesterol rather than LDL cholesterol, because the difference between LDL levels corrected for Lp(a) cholesterol was smaller than the difference between uncorrected LDL levels (Table 2). Similarly, unadjusted apoB levels decreased significantly, by 7%, but after adjustment for Lp(a) apoB, the decrease in apoB levels after estrogen was no longer significant (P=.05). Serum triglycerides increased by about 10%, but this change did not reach statistical significance.

CRP levels were undetectable in all but one subject, which was expected because the subjects were healthy women. One subject showed a rise in CRP during the placebo treatment period, at a time when she developed an upper respiratory infection (data not shown). Exclusion of her data did not change the overall results of the study, and her results were therefore included in all computations. Thirteen of 15 subjects showed a decrease in HPT in response to estrogen, and all subjects showed a decrease in AAG in response to estrogen. Mean decreases for HPT and AAG were 25% (P=.002) and 18% (P=.0006), respectively (Table 3). The time course of the response was similar to that observed with Lp(a) (Fig 4), but the magnitude of the decrease in HPT and AAG did not correlate with the decrease in Lp(a) (r=-.24, P=.41, confidence limits -.65 to .37; and r=-.14, P=.64, confidence limits -.62 to .4, respectively). This was true even when Lp(a) was measured as percentage change rather than absolute change (P=.6 for AAG; P=.8 for HPT). It should be noted that our study sample size was small and the confidence limits for the r values were wide. However, in contrast to the lack of correlation between the acute phase reactants and Lp(a), there was a strong and significant correlation between the responses in HPT and AAG, with a correlation coefficient of r=.67 (P=.009), despite the small size of our study.

View this table: [in this window] [in a new window]   Table 3. Acute Phase Protein Response Compared With Lp(a) Response to Estrogen Versus Placebo

The changes over time in the lipid, apolipoprotein, and acute phase proteins are shown in Fig 4. For LDL cholesterol and apoB levels, adjustments for Lp(a) content are given. There was a rapid reduction in LDL cholesterol and apoB levels in response to estrogen, but these changes did not reach statistical significance. In contrast, the response of HDL cholesterol and apoA-I was more gradual during the first 3 to 4 weeks. Accordingly, the total cholesterol levels appeared initially to decrease at 1 to 2 weeks, but by 3 to 4 weeks the levels were unchanged compared with placebo. There was no correlation between changes in any of the lipid or lipoprotein parameters and changes in Lp(a).

	Discussion

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In the present randomized, placebo-controlled, crossover, short-term estrogen study, we have established that the Lp(a)-lowering response to estrogen replacement therapy is of rapid onset. A decrease in Lp(a) levels was seen after 1 week of treatment, and this decrease in Lp(a) plasma levels seemed to stabilize during the last 2 weeks of the 4-week treatment period. The magnitude of the Lp(a) decrease, 23%, was similar to that observed in long-term studies in postmenopausal women. Estrogen treatment also significantly increased HDL cholesterol levels, confirming previous studies. Estrogen treatment caused a nonsignificant drop in LDL cholesterol, which may have been partly because of a drop in Lp(a) cholesterol, because corrected LDL cholesterol levels were more similar between treatment groups than were noncorrected LDL cholesterol levels. Similarly, apoB levels did not change significantly between treatment groups once they were adjusted for the apoB present in Lp(a).

The effects of estrogen on other lipoproteins also had a rapid onset, although the patterns were somewhat different. Both LDL cholesterol and apoB levels dropped rapidly during the first week of treatment and did not decrease further during the study. This is in agreement with previous studies, where administration of estrogens resulted in a rapid increase of LDL clearance.⁴⁷ In contrast, the onset of the effects on HDL cholesterol and apoA-I levels was slower, and plasma levels increased gradually during the 4-week treatment period. Although these results have to be interpreted with caution, because of the relatively small number of subjects in the present study, they nevertheless suggest that the temporal effects of estrogen treatment on the various plasma lipoprotein fractions differ, indicating that multiple mechanisms of action may be present.

Thus far, the underlying mechanisms for the Lp(a)-lowering effects of estrogen have not been clarified. Although there is a well-established increase in LDL receptor activity during estrogen administration,^47,48 this mechanism does not seem to affect Lp(a) levels.^49,50 It has previously been demonstrated that oral administration of estrogens results in an increased secretion of VLDL.⁵¹ Currently, there are indications that apo(a) under postprandial conditions, with increased synthesis and secretion of triglyceride-rich lipoproteins from the liver, may associate to an increased extent with VLDL.^52,53 However, also under these conditions, overall VLDL-associated apo(a) levels have been relatively low. Although redistribution of apo(a) between different plasma lipoproteins may occur during estrogen treatment, it is unlikely that this would, to a major extent, explain the observed decrease in plasma Lp(a) levels.

It is well known that plasma Lp(a) levels are largely determined by apo(a) genetic factors,¹² indicating that a reduced apo(a) synthesis might contribute to the estrogen-induced lowering of Lp(a). Studies in transgenic mice also indicate that estrogen reduces apo(a) synthesis.⁵⁴ We have recently demonstrated that exogenous parenteral administration of estrogens in large doses did not affect Lp(a) levels despite drastic changes in serum hormone levels.⁵⁵ This indicates that a high liver uptake of orally administered estrogens might be associated with the Lp(a) changes and focuses interest on hepatic mechanisms. Studies have also shown no effect of transdermally administered estrogen on HDL and VLDL metabolism, further underscoring an impact of high hepatic extraction during the "first pass" of orally administered estrogen.^51,56 Oral administration of estrogen elicits responses in a number of hepatically derived plasma proteins,^36-38 but it is not known whether this is related to the decrease in Lp(a). Of particular interest are results from several studies indicating that Lp(a) levels may change significantly after stressful events such as surgery and myocardial infarction.^32-34 This might suggest a link between Lp(a) and effects on hepatically derived acute phase proteins. However, Lp(a) levels have been found to be stable during acute phase reactions in another study.³⁵ Because apo(a) is known to have IL-6 response elements in its promoter but not an estrogen response element,¹⁵ we hypothesized that estrogen might regulate Lp(a) indirectly, perhaps via cytokines such as IL-6. As an example of one such cytokine-mediated estrogen effect, studies of osteoclast cells in culture have shown that estrogen affects those cells only indirectly, by modulating IL-6 levels.⁵⁷ As cytokines also regulate hepatic acute phase reactant responses, it might be expected that the changes in Lp(a) would correlate with changes in the acute phase proteins. In the present study, the response in Lp(a) levels was compared with estrogen-induced changes in levels of HPT and AAG. These two proteins are key liver-derived acute phase proteins with different functions during the acute phase response. Their synthesis is affected by different interleukins, mainly IL-6 and IL-1.⁵⁸ The response in both AAG and HPT was rapid, with a significant decrease observed after 4 weeks of estrogen treatment. Furthermore, the decreases in these two plasma proteins were highly correlated (r=.67, P=.009). Interestingly, no significant correlation was observed between the decrease in Lp(a) levels and the decrease in HPT or AAG levels, suggesting that different mechanisms may be responsible for the changes in the levels of Lp(a) on one hand and the two acute phase proteins on the other. However, our sample size was small and heterogeneous, and we acknowledge that a larger sample size might be necessary to establish definitively whether there is a correlation between Lp(a) and the acute phase reactants. Also, we only tested a limited number of acute phase reactants, and there may have been correlations present with other acute phase proteins. Because the mechanisms involved in the acute phase response are complex, with considerable cross-talk at the molecular level,⁵⁹ further studies addressing the possible impact of interleukins on Lp(a) are therefore warranted.

Our subjects represented a variety of ethnic groups and a wide range of apo(a) isoform sizes. Although the numbers in specific subgroups were too small to make meaningful comparisons between them, it seems likely that the response to estrogen was independent of these variables, as a similar relative decrease in Lp(a) in response to estrogen was seen in nearly all of the subjects.

In conclusion, we have, in the present study, established that the response in Lp(a) to estrogen replacement therapy is of rapid onset and has a different temporal sequence than estrogen-induced changes in total cholesterol, HDL cholesterol, apoB, or apoA-I levels. Furthermore, the observed changes in Lp(a) were not correlated to estrogen-induced changes in HPT or AAG levels, although we may have had too few subjects to detect a significant correlation. In addition, no obvious isoform-induced modulation of the Lp(a) response was observed, and the response was seen in all ethnic groups studied. Further studies are warranted to elucidate the mechanisms behind the estrogen-induced changes in plasma Lp(a) levels.

	Selected Abbreviations and Acronyms

 

AAG = 1-glycoprotein apo = apolipoprotein CRP = C-reactive protein FSH = follicle-stimulating hormone HPT = haptoglobin IL-6 = interleukin-6 LH = luteinizing hormone

	Acknowledgments

 
The assistance of the nursing staff and the resources of the Irving Center for Clinical Research at Columbia University are gratefully acknowledged. Jeffrey Jones and Nelson Fontanez provided expert technical assistance. Wahida Karmally, Maudene Nelson, and Riska Platt provided expert nutritional counseling. We also gratefully acknowledge Wyeth-Ayerst Corporation for kindly providing the estrogen and placebo. This study was supported by a grant-in-aid from the American Heart Association, New York City Affiliate, by funds from Columbia University, and by a grant from the National Institutes of Health, National Center for Research Resources (RR-00645). C.H.T. is a GCRC-funded Clinical Associate Physician, and L.B. is a Florence Irving Associate Professor of Medicine and an Established Scientist of the American Heart Association, New York City Affiliate.

Received September 20, 1996; accepted January 3, 1997.

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