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From the Department of Internal Medicine, University of Texas, Dallas.
ABSTRACT
Objectives— Previous studies indicated that oral estrogen increased C-reactive protein by a first-pass hepatic effect. In this study, we determine whether the route of estrogen administration influences serum amyloid A (SAA), another acute-phase protein produced by the liver, and the SAA content of the high-density lipoprotein (HDL-SAA) in postmenopausal women.
Methods and Results— In 29 postmenopausal women without coronary heart disease, we conducted a randomized crossover placebo-controlled study to compare effects of transdermal versus oral estrogen on SAA and HDL-SAA. SAA, apolipoprotein A-I, HDL, and HDL-SAA were measured before and after 8 weeks of transdermal estradiol (100 μg per day), oral-conjugated estrogens (0.625 mg per day), or placebo. We found that oral estrogen significantly increased levels of SAA, HDL, and HDL-SAA, whereas transdermal estrogen reduced both SAA and HDL-SAA but had no effect on HDL in the same women.
Conclusions— Oral estrogen increased SAA and altered HDL composition to contain a higher level of SAA by a first-pass hepatic mechanism. Because elevated SAA levels predict adverse prognosis in healthy postmenopausal women, and elevated HDL-SAA levels have been shown to interfere with HDL function, the route of administration may be an important consideration in minimizing side effects of estrogen replacement therapy on cardiovascular outcomes.
In this study, we determine whether the route of estrogen administration influences serum amyloid A (SAA), another acute-phase protein produced by the liver, and the SAA content of the high-density lipoprotein (HDL-SAA) in postmenopausal women. Oral estrogen increased SAA and altered HDL composition to contain a higher level of SAA by a first-pass hepatic mechanism. Because elevated SAA levels predict adverse prognosis in healthy postmenopausal women, and elevated HDL-SAA levels have been shown to interfere with HDL function, the administration route may be an important consideration in minimizing side effects of estrogen replacement therapy on cardiovascular outcomes.
Key Words: hormones ? inflammation ? serum amyloid A ? menopause ? HDL
Introduction
The role of inflammation in the development of atherosclerosis is firmly established. Among markers of systemic inflammation, C-reactive protein (CRP) is one of the most important acute-phase proteins known to be independent predictors of adverse cardiovascular events in otherwise healthy women.1 Recent studies by our group and others indicated that oral estrogen replacement therapy (ERT) caused a sustained increase in CRP, which may explain the increased risks of cardiovascular events of ERT in clinical trials.2,3 This increase in CRP was avoided by transdermal estrogen, implicating a first-pass hepatic effect.
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Serum amyloid A (SAA) is another acute-phase protein that determines long-term cardiovascular prognosis in women.1 Like CRP, SAA is produced by the liver and has been shown to directly promote atherosclerosis and vascular inflammation.4 However, unlike CRP, SAA can form a complex with high-density lipoprotein (HDL) in the plasma, and the elevated SAA content of the HDL particle (HDL-SAA) has been shown to interfere with antiatherogenic, antioxidative, and anti-inflammatory HDL function.5 Although the influence of estrogen on CRP has been studied extensively, effects of estrogen administration on SAA are still unknown. The purpose of our present study is not only to determine whether the route of estrogen administration influences SAA levels but also HDL-SAA in postmenopausal women.
Methods
The study was approved by the institutional review board of the University of Texas Southwestern Medical Center at Dallas. After informed written consent was obtained, we performed studies in 29 postmenopausal women (19 normotensive, 10 hypertensive) to compare effects of oral versus transdermal ERT on SAA using a randomized, placebo-controlled, double-blind, crossover design. The rationale for using a crossover design is that the effect of multiple treatment can be assessed in a small group of subjects with the same power as that of a larger parallel study. Generally, the total number of subjects needed for a parallel trial is 4x larger than the total number of subjects needed for a 2-period crossover trial.6 Thus, a 2-group parallel trial that requires a total of 80 subjects (40 per group) would only need 20 subjects using a 2-period crossover design. In this regard, our 3-period crossover study of 29 subjects has the same power as a parallel trial of 58 subjects per each treatment group or total of >150 subjects.
The average age of subjects, duration of menopause, and body mass index were 56±2 years, 11.4±1.7 years, and 27.2±0.8 kg/m2, respectively. Twelve women reported previous hormone use before enrollment in the study, but none had received hormonal therapy for at least 4 weeks before the study. This duration has been shown to be adequate in allowing CRP to return to baseline in our previous study.2 All postmenopausal women had no history of diabetes mellitus or coronary heart disease and received the following 3 regimens in random order: (1) transdermal estradiol (Climara; Berlex) 100 mcg per day plus oral placebo for 8 weeks, (2) 0.625 mg of oral-conjugated estrogens (Premarin; Wyeth-Ayerst) plus placebo patch for 8 weeks, and (3) placebo patch plus oral placebo for 8 weeks. Serum and plasma samples were obtained at baseline and after each treatment period for measurement of estradiol, insulin-like growth factor I (IGF-I), total SAA, HDL, HDL-SAA, and apolipoprotein A-I (apoA-I) levels. IGF-I levels were measured by radioimmunoassay after acid-ethanol extraction (Nichols Institute Diagnostics). CRP was measured with a highly sensitive latex-enhanced immunonephelometric assay (Dade Behring). Both interassay and intra-assay coefficients of variation were <5%. Total SAA and HDL-SAA levels were measured by ELISA using reagents from Biosource. HDL-SAA was measured in the supernatant, after precipitation of other lipoproteins with dextran sulfate and magnesium chloride.7 The mean coefficient of variation of 3 lots of pooled serum samples assayed as 10 replicates were 6.2% and 9.2% for serum and HDL-SAA, respectively.
Statistical Method
All analyses were performed using SAS version 8.2 (SAS Institute). Nonparametric tests were used for analysis of SAA and HDL-SAA because these data were skewed. The 4 phases were compared with the Friedman test, and where the interaction was significant at the 0.05 level, pairwise comparisons were made using the Wilcoxon signed rank test. SAA and HDL-SAA data are expressed as median and interquartile range (from the 25th to the 75th percentile). Repeated-measures ANOVA models were used to compare estradiol, IGF-I, HDL, and apoA-I levels during the 4 phases of study. Where the ANOVA was significant at the 0.05 level, pairwise contrasts were made using the least-square means derived from these models. Carryover effect was also tested using linear models incorporating period and the period times treatment interaction. Models that examined specific contrasts were developed, and a factor for the treatment of the previous period was evaluated in those models. No statistically significant effects for period or carryover were found (all P>0.30). Estradiol, HDL, IGF-I, and apoA-I levels are expressed as mean±SEM.
Results
We found that oral and transdermal estrogen caused a similar increase in estradiol levels. As expected, HDL and apoA-I levels increased by 10% with oral ERT but were unaffected by transdermal ERT or placebo in the same women (P<0.01 versus baseline and placebo). We also found that, as described in previous studies,2,8 CRP increased with oral estrogen (from 2.9±0.56 to 5.4±1.05 μg/mL; mean±SE; P<0.01) but was unaffected by transdermal estrogen (2.8±0.53) or placebo (3.0±0.62), whereas IGF-I levels reduced significantly with oral estrogen but was unaffected by transdermal estrogen and placebo (Table). Despite elevated HDL levels, oral ERT significantly increased total SAA and HDL-SAA levels by 20% and 10%, respectively. In contrast, transdermal ERT significantly reduced total SAA and HDL-SAA levels by 25%, whereas placebo had no effects in the same women (Figure and Table). Overall, serum levels of total SAA and HDL-SAA during transdermal ERT were 35% lower than those during oral ERT (P<0.01; Figure and Table).
Effects of Oral Versus Transdermal Estrogen Administration on Total SAA and SAA-HDL
Total SAA levels (left) and HDL-SAA (right) levels at baseline after 8 weeks of oral estrogen, placebo, and transdermal estrogen. Data are median and interquartile range (from 25th to 75th percentile). P<0.01 vs baseline; P<0.01 vs placebo; P<0.01 vs transdermal estrogen; ||P=0.01 vs baseline; ?P=0.01 versus placebo.
Discussion
Previous studies in postmenopausal women indicated that estrogen increased acute-phase protein CRP by a first-pass hepatic effect,2,3 which may explain increased cardiovascular events during oral ERT.9 Our present study extends previous observations in that estrogen not only increased another acute-phase protein, SAA, but also altered HDL composition to contain higher levels of SAA by a first-pass hepatic mechanism. However, our present study differs from previous studies in an important way. Although transdermal estrogen has no effect on CRP, the major new finding is that it paradoxically reduces SAA and HDL-SAA levels in the same postmenopausal women.
Mechanisms by which first-pass hepatic metabolism of oral ERT increases SAA are unknown. One possibility is that oral ERT increased levels of interleukin 6 (IL-6), the main proinflammatory cytokine known to stimulate hepatic production of CRP and SAA. However, this is unlikely because our previous study indicates that IL-6 and other proinflammatory cytokines were unaffected by either oral or transdermal ERT.2 Mechanism by which transdermal ERT reduced SAA and HDL-SAA is also unknown but may be related to anti-inflammatory effects of estrogen because subcutaneous estradiol administration has been shown to reduce leukocyte infiltration into injured vessels in animals.10 We speculate that during transdermal ERT, physiological levels of estrogen in portal circulation may exert anti-inflammatory effect by reducing hepatic SAA production at the same level of IL-6. In contrast, with oral ERT, supraphysiological concentration of estrogen may paradoxically promote SAA production despite the same levels of proinflammatory cytokines. Because only oral-conjugated estrogens was tested as oral estrogen in our study, we consider the possibility that SAA may not increase with other oral preparations that do not share the same estrogenic constituents as oral-conjugated estrogens. However, this is unlikely because previous studies indicated that oral estrogen has been shown to consistently increase acute phase protein CRP regardless of specific preparations (ie, oral-conjugated estrogens or oral 17?-estradiol, or estradiol valerate).11,12
Our observation may have important clinical implication for several reasons. First, elevated SAA levels are independently associated with adverse cardiovascular events in women,1 possibly by their direct proinflammatory and proatherogenic effects. The purified SAA particle was also shown to enhance cholesterol uptake in aortic smooth muscle cells in rabbits4 and induce monocyte chemotaxis and monocytic release of proinflammatory cytokines in mice.13 Second, elevated levels of SAA within HDL particles can interfere with antiatherogenic, antioxidative, and anti-inflammatory function of HDL. SAA-enriched HDL particles preferentially bind to macrophage rather than hepatocytes.14 As a result, cholesterol in HDL is less available to be metabolized by the liver or excreted in the bile. Compared with native HDL, SAA-enriched HDL has a lower capacity to generate cholesterol efflux from macrophage and reduced ability to protect LDL from oxidation as well as prevent LDL-induced monocyte chemotaxis.5 A prospective observational study in humans indicated that the increase in HDL-SAA after acute injury changed the anti-inflammatory property of HDL to directionally opposite that of proinflammatory lipoprotein, promoting expression of adhesion molecules and monocyte chemotaxis in the human endothelium cells.15 Precise mechanisms by which SAA interfere with HDL function during acute inflammation is not known but may be related to displacement of paraoxonase enzyme or other HDL-associated enzymes essential for anti-inflammatory and antioxidative function of HDL.16,17
Oral ERT is thought to have an advantage over transdermal ERT with regard to a more favorable lipid profile, particularly higher HDL levels. Despite this favorable effect of oral ERT on HDL, large randomized studies failed to demonstrate that oral ERT had any impact on progression or regression of coronary18 or carotid19,20 atherosclerosis and even increased risks of stroke and myocardial infarction.9 We speculate that the increased SAA and HDL-SAA during oral ERT negate the beneficial effects of increase in HDL levels. In this regard, reduced HDL-SAA during transdermal ERT may augment HDL function even if HDL levels were unchanged. Furthermore, recent studies have also indicated that oral ERT increased risk of dementia.21 SAA is a precursor of amyloid fibril,22,23 which is found to deposit in the brain of patients with Alzheimer’s disease, promoting chronic neuronal inflammation and neuronal loss.24 Whether chronic elevation in SAA during oral ERT will lead to deposition of amyloid fibrils in brain tissue and contribute to development of dementia also remains unknown. Large prospective studies are needed to determine whether the anti-inflammatory effect of transdermal ERT will constitute an effective strategy for preventing atherosclerosis without increasing risk of dementia after menopause.
Acknowledgments
The study is funded by grants to W.V. from the National Institutes of Health (NIH; K23RR16321), Donald W. Reynolds Cardiovascular Clinical Research Center, and a United States Public Health Service grant (M01-RR00633) through NIH–National Center for Research Resources, to I.J. from NIH (K24AT00596). We gratefully acknowledge Beverley A. Huet, MS, for statistical assistance.
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