1 Department of Obstetrics and Gynecology, 2 Department of Internal Medicine and 3 Department of Microbiology, Afyon Kocatepe University, Faculty of Medicine, Afyon, and 4 Department of Obstetrics and Gynecology, Ankara University, Faculty of Medicine, Ankara, Turkey
5 To whom correspondence should be addressed at: Istiklal Mah, Lale Sok, Yeni Candan Apt, A Blok, No. 3, Daire 5, 03200 Afyon, Turkey. e-mail: meyilmazer{at}hotmail.com
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Abstract |
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Key words: C3/C4/hormone replacement therapy/immunoglobulin G/immunoglobulin M
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Introduction |
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The complement system and immunoglobulins are the main components of humoral immunity. The activation of complement is known to be involved in a number of forms of cardiovascular disease. The results of some clinical studies have suggested that complement activation exacerbates myocardial defect following ischaemic injury (Gardinali et al., 1995), is involved in the generation of spontaneous atherosclerotic lesions (Seifert and Kazatchkine, 1988
), and may indeed be an initiating factor in lesion formation (Torzewski et al., 1996
). C3, the third complement component, is a cytokine and is produced by activated macrophages (Zimmer et al., 1982
), which are the cells mainly concerned with the development of atherosclerotic plaques (Libby, 1995
). Previous studies have indicated that serum C3 is a powerful indicator of the risk of myocardial infarction (Muscari et al., 1995
; 1998; 2000). Furthermore, there is some evidence suggesting that patients with established atherosclerosis have elevated levels of IgA, IgE, IgG and IgM (Criqui et al., 1987
; Muscari et al., 1988
). Moreover, elevated levels of IgA, IgE and IgG are predictive of myocardial infarction (Kovanen et al., 1998
).
In the present study, serum concentrations of complement (C3, C4), and immunoglobulin (IgM, IgG) were investigated in women treated with two different short-term HRT regimens for menopausal hormone deficiency symptoms, and in untreated women. To the best of the present authors knowledge, this is the first study to determine the impact of HRT on serum complement (C3, C4) and immunoglobulin (IgM, IgG) levels.
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Materials and methods |
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Women with a history of cardiovascular, rheumatismal, autoimmune and malignant diseases, with acute or chronic infection, and those with a clinically relevant abnormality in laboratory tests of haematological, renal and hepatic functions were excluded from participation. Patients with a body mass index (BMI) >35 kg/m2, caffeine consumption in excess of the equivalent of three cups of coffee per day, or who had been operated on within the past 6 months were also excluded. Additional exclusions included those women who had smoked cigarettes within the past year, had a medical history suggesting a possible contraindication to hormone use, a history of corticosteroid or any immunosuppressor drug use during the previous month. Those women with a plasma triglyceride level >200 mg/dl and total cholesterol level >240 mg/dl were also excluded.
In order to investigate the impact of short-term HRT use on the immune system, patients receiving oral or transdermal HRT for a duration of 39 months were involved in the study. For the analyses, 74 women receiving HRT were divided into two groups: group 1 (n = 18) received transdermal 17 estradiol (17
-E2; 50 µg/day) plus continuous medroxyprogesterone acetate (MPA; 2.5 mg/day), while group 2 (n = 56) received oral conjugated equine estrogen (CEE; 0.625 mg/day) plus continuous MPA (2.5 mg/day). The control group comprised 80 healthy post-menopausal women who had not taken any HRT regimen within the previous 12 months, with a serum FSH level >30 mIU/ml, a serum estradiol level <30 pg/ml, and had been amenorrhoeic for at least 12 months. Women who had used HRT or any other hormonal therapy within the previous 12 months were excluded from the control group.
Plasma complement (C3, C4), immunoglobulin (IgM, IgG) and hormone status were evaluated in all participants, and results were compared between control and study groups, as well as among women receiving different HRT regimens.
Blood samples were taken after a fasting period of 10 h in the study and control groups. All laboratory parameters were measured as soon as blood samples were taken. Serum C3, C4, IgG, IgM levels were measured using turbidimetry kits (Biosystem, USA) with an automatic analyser (SPACE, Schiapparelli Biosystem, Inc., USA). This analytical method was applied according to the manufacturers instructions. Normal ranges (provided by the manufacturers) for C3, C4, IgG and IgM were: 1.01.85 g/l, 0.20.45 g/l, 8.416.6 g/l and 0.52.2 g/l respectively. Daily calibrations were carried out for all measurements made during the study.
Statistical analysis
As many variables had a non-Gaussian distribution with positive skewness, statistical analysis was performed with non-parametric tests: Kruskal-Wallis and MannWhitney U-tests. The data were expressed as mean ± SD (range). Correlations between variables were calculated with Spearmans correlation coefficient. Statistical significance was set at P < 0.05. Data were collected in a Windows-based database (Microsoft Excel 2000) and analysed with the SPSS (Statistical Package for the Social Science, version 11.0) for Windows 98 (Microsoft Corp.).
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Results |
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Discussion |
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There is increasing evidence that post-menopausal HRT increases cardiovascular risk (Hemminki and McPherson, 1997; Rossouw et al., 2002
). In the Heart and Estrogen/progestin Replacement Study (HERS), HRT appeared to increase the risk of new cardiovascular events in the first year, despite favourable effects on lipids (Hulley et al., 1998
). Although the association between an increased cardiovascular risk and the administration of HRT has not yet been clarified, it was suggested that this might be mediated in part through an effect on the inflammatory system (Ross, 1999
).
C3 is a cytokine and an acute-phase reactant protein produced by activated macrophages (Zimmer et al., 1982), liver and adipose tissue (Alper et al., 1969
; Choy et al., 1992
). C3 and its fragments are recognized regulators of the humoral immune response and B-cell proliferation (Lenhardt and Melchers, 1988
). C3 complement is also heavily involved in the control of lipid and glucose metabolism through its fragment C3a-des-Arg (Baldo et al., 1993
). In addition, serum C3 levels are a potent indicator of the risk of myocardial infarction in men (Muscari et al., 1995
) and, together with C4 levels, have been found to be elevated in patients with severe angiographically assessed atherosclerosis who had already been affected by multiple ischaemic events (Muscari et al., 1988
). A significant prevalence of the fast allele of C3 has been demonstrated in atherosclerotic patients with respect to controls (Kristensen and Bruun Petersen, 1978
). Furthermore, increasing amounts of data indicate that serum C3 and C4 levels strongly correlate with several cardiovascular risk factors such as age, triglycerides, blood glucose and systolic blood pressure (Muscari et al., 1988
; 1995; 1998; 2000). Finally, complement activation is probably involved in the generation of spontaneous atherosclerotic lesions (Rus et al., 1986
; Niculescu et al., 1987
; Seifert and Kazatchkine, 1988
; Seifert et al., 1989
). Activated complement has cell-damaging and chemotactic properties (Marder et al., 1985
), and therefore might support the development of intimal lesion or monocyte recruitment at the site of atheroma formation (Muscari et al., 1995
).
From this perspective, the following two questions arise. First, might increased cardiovascular riskas was demonstrated to emerge during the early period of HRT use in the HERS trial (Hulley et al., 1998)be associated with the complement system? Second, in what way might the use of HRT affect the complement system? Limited data were available from the literature concerning the association of serum complement levels with HRT. However, HRT in the form of unopposed oral estrogen, or combined estrogen and progestin, increases serum C-reactive protein (CRP) concentrations in post-menopausal women (Cushman et al., 1999a
;b; Ridker et al., 1999
; Van Baal et al., 1999
; Walsh et al., 2000
; Luyer et al., 2001
; Garnero et al., 2002
). As a result of oral HRT use, hepatic synthesis of CRP increases (Van Baal et al., 1999
; Herrington et al., 2001
). Thus, serum levels of C3 which is an acute-phase reactant synthesized by the liver as CRP (Alper et al., 1969
)might also be raised following increased synthesis in liver due to the first-pass effect of oral HRT. Accordingly, no significant difference could be found between women receiving transdermal HRT and untreated women with respect to serum levels of C3 and C4. In transdermal HRT, hormones enter the systemic circulation without a hepatic first pass and, for this reason, serum C3 and C4 levels might not be affected.
The association of total immunoglobulins with cardiovascular disease remains the subject of debate. It has been proposed that serum IgA, IgG and IgE may be associated with cardiovascular disease in dyslipidemic men (Kovanen et al., 1998), but others (Muscari et al., 1988
; 1995; 1999) have suggested that the increase in IgM and IgG levels might be a consequence of the primary involvement of C3, as there was no independent association of IgM and IgG with atherosclerosis and myocardial infarction. In agreement with previous findings (Bukh et al., 1987
), in the present study no significant difference was found in IgG and IgM levels among women receiving oral or transdermal HRT, or in women not taking HRT.
The present study had certain limitations, the first being that the groups included only healthy women with a low risk of cardiovascular disease, and this may have affected the results. Second, the study was non-randomized, non-placebo-controlled and cross-sectional in design. Third, all of the participants enrolled had an intact uterus, and in order to protect the endometrium a combined HRT was used. The sole effect of progestins on the complement system or immunoglobulins is not known, but it may preclude the drawing of definitive conclusions regarding the effects of estrogen alone.
In summary, oral HRT use may cause significant changes in the serum levels of C3 and C4, which in turn may be associated with an increased risk of cardiovascular events during the early stages of treatment, as in the HERS trial. However, these are preliminary findings, and additional information must be derived from large, controlled studies before any clear-cut conclusions may be drawn.
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Submitted on January 6, 2003; resubmitted on February 27, 2003; accepted on April 2, 2003.