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Serum C-Reactive Protein and Plasma Homocysteine Levels Are Associated With Hormone Therapy Use and Other Factors: a Population- Based Study of Middle-Aged Australian-Born Women

Posted on: Sunday, 18 September 2005, 03:00 CDT

ABSTRACT

Objective To investigate the associations between C-reactive protein (CRP), homocysteine levels, use of hormone therapy (HT) and other factors.

Methods A 12-year prospective study of 438 Australian-born women (Melbourne Women's Midlife Health Project), who at baseline were aged 45-55 years, had menstruated in the previous 3 months and were not taking HT. Fasting blood was collected in the 11th follow-up year for CRP, homocysteine, estradiol and follicle stimulating hormone (FSH) levels. Physical measurements and face-to-face interviews obtained information on health and lifestyle variables.

Results A total of 258 women (mean age 60 years) participated in the 11th follow-up year. Multiple regression analysis found that CRP levels were positively associated with body mass index (p < 0.001), HT use (p < 0.01), and negatively associated with statin use (p < 0.005) and exercising (p < 0.05). In postmenopausal women currently not using HT (n = 173) and after adjusting for body mass index, exercise and smoking, CRP was negatively associated with FSH levels (β= -0.32, p < 0.05). Homocysteine levels were positively associated with smoking (p < 0.001) and negatively associated with HT use (p < 0.05).

Conclusion In middle-aged Australian-born women, HT use was associated with increased CRP and decreased homocysteine levels. High CRP levels were also associated with high relative weight, low exercise levels and no statin medication.

Key words: C-REACTIVE PROTEIN, HOMOCYSTEINE, HORMONE THERAPY, WOMEN, STATINS, BODY MASS INDEX, EXERCISE

INTRODUCTION

Plasma concentration of C-reactive protein (CRP) is a sensitive marker of underlying systemic inflammation. In prior work, an increase in CRP levels has been associated with increased coronary heart disease (CHD) risk among healthy, middleaged men and women1- 3. Several studies indicate that oral postmenopausal hormone therapy (HT) leads to an increase in CRP levels4-6. In a nested case- control study of 608 women (mean age 69 years) followed for 3 years, CRP levels at baseline predicted vascular events among apparently healthy postmenopausal women. HT use did increase CRP; however, use or non-use of HT had less importance for cardiovascular risk than did baseline levels of CRP7. High measures of body weight and body fat have also been associated with higher CRP concentrations8-9 and a reduction in fat mass has been associated with a significant reduction in CRP levels10.

Statins have been shown to lower CRP levels in a community-based population11, as well as in patients with stable ischemic heart disease12. Aspirin therapy has also been reported to lower serum CRP levels13, although the results have been inconsistent12-14 and appear to depend on the initial levels of CRP13-15. The Melbourne Women's Midlife Health Project (MWMHP), an observational, prospective, population-based study of middle-aged Australian-born women, provided an opportunity to examine the association of CRP levels with physical variables, HT, statin and aspirin use.

Homocysteine has been reported as an independent risk factor for CHD and thrombosis16. Homocysteine interacts adversely with the endothelium and probably increases intima-media thickness17; homocysteine elevation may also be associated with increases in blood pressure18. Homocysteine levels can be lowered by folic acid and other nutritional supplements19. Levels of homocysteine rise after the menopause, suggesting an influence of sex steroids on the metabolism of homocysteine20. Homocysteine levels are reportedly lowered by both transdermal and oral estradiol administration over a 6-month period21.

As cardiovascular disease is the leading cause of death and morbidity in women of developed countries22-23, it would be useful to have a biomarker which was able to identify those at risk of CHD and to monitor treatments or interventions. The participants in the MWMHP were a relatively healthy group of middle-aged women in whom the predicted risk of a coronary event had previously been calculated24.

Table 1 Design of prospective study

The aim of this paper is to investigate, in the participants in the MWMHP, the associations between CRP, homocysteine levels, use of HT and other factors.

METHODS

The study was approved by the Human Research Ethics Committee of the University of Melbourne and the procedures followed were in accordance with the ethical standards of the National Health & Medical Research Council. All subjects provided written informed consent for their participation in the study.

Sample

The design of the MWMHP prospective study is outlined in Table 1. The MWMHP began in 1991 with population sampling by random telephone digital dialling and baseline interview of 2001 Australian-born women aged between 45 and 55 years and resident in Melbourne (71% response rate)25. Those women at baseline who had experienced menses in the prior 3 months, and who were not taking the oral contraceptive pill or HT, were invited to participate in a longitudinal study. Of those eligible, 56% accepted (n = 438). Compared to women who declined, these women more often reported better self-rated health, current employment, more than 12 years of education, exercising at least once a week and having had a Papanicolaou smear26. The retention rate by year 8 of follow-up was 88%. Women who dropped out were significantly less likely to be married/live with a partner, or to exercise at least once per week. Women were interviewed annually for the first 8 years of follow-up in their own homes and blood and physical measures were taken. In year 11 of follow-up, the women were invited to attend the Office for Gender and Health at the Royal Melbourne Hospital for cognitive function testing, blood collection and physical measurements. A total of 258 women (67%) accepted. Information on health status and medication use was obtained at the time of testing.

Blood and physical measures

Fasting morning blood samples were collected. Serum was obtained from all 258 women for CRP, estradiol and follicle stimulating hormone (FSH) assays. Blood was obtained from 205 of the cohort for plasma homocysteine estimations, as the procedure for this collection (using EDTA tubes and immediately centrifuging and collecting the plasma) was not initiated until after the start of the project.

FSH was measured using the Microparticle Enzyme Immunoassay (MEIA) technology (Abbott Diagnostics) and the inter-variation coefficient averaged 4.5%. Estradiol was measured by radioimmunoassay (RIA) following organic solvent extraction and Celite column partition chromatography. The intra- and intervariation coefficients for the estradiol RIA were 6.6% and 9.9%, respectively. CRP was measured with a high-sensitivity assay using a Dade Behring RxE Chemistry Analyser, with reagents and calibrators supplied by Dade Behring Diagnostics (Sydney, Australia). Between-assay coefficient of variations at CRP levels of 4.6 and 37mg/l were 5.6% and 3.0%, respectively. Homocysteine was measured using a commercial assay developed by Abbott Diagnostic Division for their IMx Analyzer. It was a fluorescence polarization assay (FPIA) where all components of the assay are fully automated and reagents are supplied in kit form. Between-assay coefficient of variations at homocysteine levels of 7.1 and 25.1 μmol/l were 5.0% and 3.0%, respectively.

Body height was measured to the nearest 0.1 cm with subjects in the erect position without shoes; body weight was measured to the nearest 0.1 kg with subjects wearing indoor clothes but no shoes. Body mass index (BMI) was calculated as weight in kg/(height in m)2. Three measures of lifestylerelated behavior were included:

(1) Exercise: recorded as frequency of engaging in exercise for fitness or recreational purposes, with seven answer options (never; less than once a month; a few times a month; once a week; 2-3 times per week; 4-6 times per week; every day);

(2) Current smoking (yes/no);

(3) Alcohol intake: recorded as number of alcoholic drinks consumed in the previous week (none; 1; 2; 3-6; 7; 8 or more).

Menopausal status was determined by menstrual history at the time of the interview. Women who reported at least 3 months but less than 12 months of amenorrhea were considered to be in late menopausal transition27. Women were considered postmenopausal if they had had amenorrhea for at least 12 months27. Women were classified as having a surgical menopause if they had experienced a hysterectomy with or without an oophorectomy. Current HT use was determined at interview and these women were classified separately.

Coronary heart disease risk

CHD risk was calculated using the Prospective Cardiovascular Munster (PROCAM) study's scoring system as described previously24, using eight CHD risk variables: age, low density lipoprotein (LDL) cholesterol, smoking, high density lipoprotein (HDL) cholesterol, systolic blood pressure, family history of premature (younger than 60 years) myocardial infarction, diabetes mellitus, and triglycrides. The methods used to measure these variables have been described previously" . The CHD risk calculated at year 8 of follow- up was used in t\his report.

Statistical analysis

Because of skewed distributions, CRP, homocysteine, estradiol and FSH measures were logtransformed before analysis. The log transformations were chosen because it was shown empirically that the residuals from fitted models were consistent with the normal distribution assumption. The means of the log-transformed values (geometric means) were calculated. There were two parts to this analysis:

(1) A cross-sectional analysis to determine variables associated with CRP or homocysteine levels at the 11th year of follow-up; and

(2) Analysis of associations of CRP and homocysteine with CHD risk variables collected 3 years previously.

In both analyses, variables associated with CRP or homocysteine levels at a significance level of 0.1 were determined using linear regression and these were entered in multiple linear regression analyses. A 0.05 level of significance was adopted for retention in the models. SPSS software28 was used for all analyses.

RESULTS

Serum CRP levels were measured in 258 women from the MWMHP (mean age 59.8 years, standard deviation 2.5) during the 11th year of follow-up and plasma homocysteine levels were measured in 205 of these women. CRP levels for two women who had active inflammatory conditions at the time blood was collected were excluded from the analysis. Table 2 shows geometric means (95% confidence intervals) of CRP and homocysteine levels and physical, lifestyle characteristics and menopausal status of the cohort. Seventy-one percent of the HT users were taking oral estrogens plus progestins, 9% were taking oral estrogen only, and the remainder were using transdermal preparations. Thirty-four participants (13%) were taking statins, and 23 (9%) were on aspirin therapy, of whom half were also statin users.

C-reactive protein

Using data obtained in year 11 of follow-up only, multiple regression analysis found that CRP levels were positively associated with BMI (p < 0.001) and current HT use (p < 0.01), and negatively associated with exercising (p < 0.05) and statin use (p < 0.005) (Table 3). There was a trend for smoking to be positively associated with CRP levels (p = 0.07).

Table 2 Characteristics of cohort (n = 258). Data for homocysteine and C-reactive protein levels are expressed as geometric mean (95% confidence interval)

In postmenopausal women currently not using HT and after adjusting for BMI, exercise, smoking and statin use, CRP was negatively associated with FSH levels (β= -0.32, p < 0.05) and positively associated with estradiol levels (β = 0.14, p = 0.05). Women who took oral estrogen ( progestin) had higher CRP levels than transdermal estrogen users but the difference was only of borderline significance (p = 0.05). There was no difference in CRP levels between women taking oral estrogen plus progestin and oral estrogen only.

Using data obtained in year 8 of follow-up, CRP levels were positively correlated with CHD risk (calculated from the PROCAM scoring system, r = 0.2, p < 0.05). Of the eight variables used in the PROCAM score (age, LDL cholesterol, smoking, HDL cholesterol, systolic blood pressure, family history of premature myocardial infarction, diabetes mellitus and triglycerides), CRP was positively correlated with smoking (p < 0.05), systolic blood pressure (p < 0.001), and having diabetes (p < 0.01), and negatively associated with HDL cholesterol (p < 0.001); after adjusting for BMI and exercise, only HDL cholesterol and smoking remained in the model.

Homocysteine

Using data obtained in year 11 of follow-up, multiple regression analysis found that homocysteine levels were positively associated with current smoking (p < 0.005) and negatively associated with current HT use (p < 0.05) (Table 3). There was no significant correlation between homocysteine levels and the calculated CHD risk at year 8 of follow-up. The only component of the PROCAM score with which homocysteine was significantly and positively associated was smoking. In postmenopausal women not using HT, there was no significant association between homocysteine and estradiol and FSH levels.

Table 3 Results of cross-sectional analysis of measures from the 11th year of follow-up, with C-reactive protein and homocysteine as dependent variables. Regression coefficients (β) and p values

DISCUSSION

This paper confirms other reports7,29,30 that HT use, in particular orally, is associated with elevations in CRP. In randomized trials, increased cardiac events have been reported in the first few years following the initiation of oral estrogen and progestin therapy31,32 and the associated increase in CRP may directly or indirectly reflect the inflammatory processes associated with atheromatous plaque rupture. In one observational study7, although a positive association between HT and CRP was observed, the use or non-use of HT had less importance in terms of subsequent risk than baseline levels of CRP.

We also found that high CRP levels were associated with higher levels of endogenous estradiol and lower levels of FSH in postmenopausal women not using HT. The association of endogenous estradiol with increased CRP was only of borderline significance and the reason is unclear, especially as a comparison of pre- and postmenopausal women reported that menopausal status was not related to CRP levels9. Premenopausal women do have significantly higher estradiol levels than postmenopausal women33. The direct effects of hormonal changes on CRP may be complicated by changes in body fat and insulin sensitivity9.

CRP was independently positively associated with BMI and negatively associated with exercise. The strong association of CRP with relative body weight is in agreement with other studies8,9 and, although exercise is associated with lower relative body weights, exercise appears to be having an effect on CRP levels independent of its effect on weight. Manns and colleagues34 have reported that the association between higher physical activity and lower serum CRP levels is dependent on the lower body fat of the more active woman. Future research needs to look at whether reductions in body fat have an effect on CRP levels, particularly in women taking HT.

In our cohort, women taking statins had lower levels of CRP independent of BMI, exercise, smoking and HT use. This is in agreement with other studies in which statins lowered CRP levels in a randomized trial of a community-based sample11 and also in patients with stable ischemic heart disease12. The results of our observational study provide further evidence that statins may have an anti-inflammatory effect in addition to lipid-lowering effects.

A limitation of this study is that our data on CHD risk factors were measured at least 3 years prior to the CRP measure and we have only three reports of coronary events23. So, we cannot make any conclusions as to whether CRP may provide a strong risk prediction for cardiovascular disease. However, it is of interest that these prospectively measured CRP levels were associated with a calculated risk of CHD, in particular with low HDL cholesterol levels. High HDL cholesterol levels in women reduce CHD risk.

Homocysteine levels in our participants were not significantly associated with the calculated risk of CHD nor were they associated with other correlates of CHD risk - high relative weight and low physical activity. Hyperhomocysteinemia has been implicated as an independent risk factor for cardiovascular disease, with levels greater than 12 μmol/1 being considered clinically significant35. As only 13% of our cohort was in this category, it may explain the lack of association with calculated CHD risk.

Smoking was associated with high homocysteine levels and, after adjusting for smoking, current HT users had significantly lower homocysteine levels than non-users, in agreement with other studies36,37. There was no association between homocysteine levels and endogenous hormone levels in postmenopausal women not using HT. Plasma homocysteine levels have been shown to be significantly lower in premenopausal women than in postmenopausal women20, suggesting that estrogen does modulate plasma homocysteine levels although the mechanisms are largely unknown38.

There are other limitations in this investigation. The main one, and the major weakness of this study, is that only one blood sample was taken for CRP and homocysteine measures and it is possible that these markers may vary over time. Bogaty and colleagues39 have reported fluctuations in CRP levels in patients with stable ischemic heart disease. Other researchers40 have reported variability in repeated measures over time from healthy volunteers. Another limitation is that, due to the racial mix of the Australian population, our Australian-born, communitybased sample consists only of Caucasian women and so we cannot comment on any race/ ethnicity effects that may apply to the variables studied.

CONCLUSION

In this population-based study of middle-aged Australian born women, HT use was associated with increased CRP levels and decreased homocysteine levels. High CRP levels were associated with high relative weight and low exercise, both of which are predictive of CHD risk. Further longterm follow-up of this cohort is required to see if CRP levels are predictors of acute events. Future research is needed to establish whether interventions such as weight loss, increased physical activity and statin use can significantly modify CRP levels and influence CHD outcomes in our population.

ACKNOWLEDGEMENTS

We thank the Department of Biochemistry at the Monash Medical Centre for the FSH, homocysteine and C-reactive protein assays and Frank Stanczyk PhD, Professor of Research, Departments of Obstetrics and Gynecology, and preventative medicine, University of Southern California, Keck School of Medicine, Los Angeles, CA for the estradiol assays.

Conflict of interest J. R. Guthrie, M. S. Clarke and H. G. Burger do no\t have any potential conflicts of interest. L. Dennerstein has been a consultant to Eli Lilly Ltd, Procter and Gamble, and Pfizer drug companies.

Source of funding This study was supported by grants from the Victorian Health Promotion Foundation, the National Health and Medical Research Council of Australia, the Alzheimer's Association and the University of Melbourne. Prince Henry's Institute of Medical Research received grants from Organon Pty Ltd for the blood assays.

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J. R. Guthrie, M. S. Clark, L. Dennerstein and H. G. Burger*

Office for Gender and Health, Department of Psychiatry, The University of Melbourne, Victoria; *Prince Henry's Institute of Medical Research, Clayton, Victoria, Australia

Correspondence: Dr J. R. Guthrie, Office for Gender and Health, Department of Psychiatry, The University of Melbourne, 4th Floor, 766 Elizabeth Street, Melbourne, VIC 3010, Australia

Received 24-01-05

Revised 13-04-05

Accepted 14-04-05

Copyright CRC Press Sep 2005

Source: Climacteric

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