ORIGINAL

ARTICLE

Association between circulating sex steroids and vascular calcification in community dwelling men: The Framingham Heart Study 1,2,3

Thomas G. Travison, PhD; 3,4,5Christopher J. O’Donnell, MD, MPH, Shalender Bhasin,MD; 4,6Joseph M. Massaro,PhD; 3,7Udo Hoffmann,MD; 4,9 Ralph B. D’Agostino Sr.,PhD, 2,3Shehzad Basaria, MD 2,3

4,8

Ramachandr

1 Hebrew SeniorLife Institute for Aging Research, Roslindale, MA; 2Research Program on Men’s Health, Aging and Metabolism, Brigham and Women’s Hospital, Boston, MA; 3Harvard Medical School, Boston, MA; 4National Heart, Lung and Blood Institute’s Framingham Heart Study, Framingham, MA; 5 Cardiology Division, Department of Medicine, MA General Hospital, Boston, MA; 6Department of Biostatistics, Boston University School of Public Health; 7Department of Radiology, MA General Hospital, Boston, MA; 8Section of Preventive Medicine and Epidemiology, Boston University School of Medicine, Boston, MA; 9Department of Mathematics, Boston University, Boston, MA

Context: The relationship between sex steroids and atherosclerosis is poorly understood. Objective: To describe the association of serum total and calculated free testosterone (TT and cFT), estrone (E1), estradiol (E2), and sex hormone binding globulin (SHBG) to vascular calcification in adult men. Design: Observational study (The Framingham Heart Study; FHS). Analyses are cross-sectional. TT, E1 and E2 were measured by liquid chromatography-tandem mass spectrometry, SHBG by immunofluorometric assay, Estimates of association were obtained by Tobit regression, which acknowledges the influence of floor effects on outcomes. Setting: General community. Participants: N⫽1654 community-dwelling men from the Offspring and Third Generation cohorts of the FHS Main outcome measures: Coronary artery calcification (CAC), abdominal aortic calcification, and thoracic aortic calcification, measured by computed tomography. Results: Mean (standard deviation) age was 49 (10) years. Mean (SD) TT, cFT, and SHBG were 616 (224) ng/dl, 111 (45) pg/ml, and 46 (23) nmol/l, respectively. Mean (SD) E2 and E1 were 28 (10) and 39 (14) pg/ml. Vascular calcification at all sites was negatively associated with TT and cFT and positively associated with E2 and E1. A 100-ng/dl between-subject increase in TT was associated with a mean (95% CI) age-adjusted difference in CAC of -23% (-41%, -4%), p⫽0.02. After model adjustment for other cardiovascular risk factors, the estimated associations between testosterone and vascular calcification scores were statistically nonsignificant. Conclusions: Decreased circulating testosterone and estradiol levels are associated with an ageadjusted increase in CAC, but these associations appear to express relationships either attributable to or mediated by established cardiovascular risk factors.

ge-related declines in circulating testosterone concentrations have been associated with increased mor-

A

ISSN Print 0021-972X Printed in USA

bidity in aging men, (1– 6) but the association between endogenous testosterone and cardiovascular health in aging is controversial. The association of male sex with risk

ISSN Online 1945-7197

doi: 10.1210/jc.2015-4299

J Clin Endocrinol Metab

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Sex steroids and vascular calcification

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of myocardial infarction (MI) and other cardiovascular disease events invites the suggestion that testosterone may promote and estrogen may protect against cardiac risk, (7) but in studies in men, the opposite has typically been observed (8, 9). The balance of epidemiologic data is suggestive of a protective role for testosterone in cardiovascular illnesses and deaths to which the illnesses contribute, (3, 10) though these findings are not universal (11) and exhibit some variation with the population under study (12). At the same time, a limited body of evidence suggests the potential for excess cardiovascular risk resulting from testosterone replacement therapy (TRT) in older men with multimorbidity (13–16). Coronary artery calcification (CAC) is a subclinical marker of atherosclerosis and strong independent predictor of risk of coronary heart disease (CHD) (CHD). CAC is consistently associated with established cardiovascular risk factors (17, 18). Recent results from the Multi-Ethnic Study of Atherosclerosis (MESA), for example, demonstrate that both the volume and progression of CAC are predictive of incident CHD and related events, (19, 20) findings similar to those observed elsewhere (18, 21–24). CAC predicts risk over and above conventional CHD risk factors, (25) and CAC may be especially predictive among asymptomatic subjects at low to intermediate risk of disease progression (22, 26). As markers of cardiovascular risk, abdominal aortic and descending thoracic aortic calcification (AAC and TAC) have generally received less attention than CAC. A recent meta-analysis, however, confirmed that AAC is a strong independent predictor of coronary events and cardiovascular mortality (27). TAC often tracks with CAC, (28, 29) but may be a stronger predictor of noncardiac and cerebrovascular events (30). Parental occurrence of premature cardiovascular disease predicts AAC and CAC in adult offspring in the Framingham Heart Study (FHS) and of CAC in MESA (31). There has been relatively little investigation of the direct association of endogenous sex hormones and vascular calcification in male population-based samples. In earlier studies with small sample sizes, there was an inverse relation between estradiol (E2) and generalized arterial calcification in men (32). These data are consistent with an hypothesis of a preventative influence of testosterone on atherosclerosis in male mice is transmitted via its aromatization to estradiol (33). In the Rotterdam study, reduced circulating testosterone was predictive of aortic calcification, (34) but AAC was related to neither total testosterone, E2 nor sex hormone-binding globulin (SHBG) in men enrolled in MESA (35). More recently, results from the

Multicenter AIDS Cohort Study (MACS) showed no association between calculated free Testosterone (cFT) and CAC in either HIV-infected or HIV-uninfected men, (36) while by contrast a study of nonobese Korean men demonstrated a negative association between bioavailable (ie, free ⫹ albumin bound) testosterone and coronary calcification among participants in whom some calcification was present (37). The role of estrone (E1) in male cardiometabolic health remains poorly understood; (6, 38 – 40) its relation to vascular calcification has not been explored. In this analysis, we utilized observational data from the Offspring cohort and Third Generation cohort of the FHS to determine whether total testosterone (TT), cFT, E1, E2, or SHBG exhibit cross-sectional association with CAC, AAC or TAC either alone or in the presence of conventional CHD risk factors. We measured testosterone, estrone, and estradiol using liquid chromatography tandem mass spectrometry (LC-MS/MS), widely considered the reference standard.

Copyright © 2016 by the Endocrine Society Received December 20, 2015. Accepted February 25, 2016.

Abbreviations:

Materials and Methods The FHS study design has been previously described (41). An original cohort sample of 5209 adult male and female residents of Framingham, MA was recruited in 1948. The FHS original cohort was predominantly white and of European ancestry. In 1971, children of the original cohort and their spouses were recruited as a second generation Offspring cohort. Recruitment of a Third Generation cohort, consisting of children of the Offspring cohort, was performed in 2002. All participants executed written informed consent approved by the institutional review board (IRB) at Boston University Medical Center. Analyses described here were based on data from men of the Offspring cohort with sex steroid measurements obtained at Examination 7, occurring between 1998 and 2001, and from men of the Third Generation cohort, who had sex steroid measurements between 2002 and 2005. Data on CAC, TAC, CAC and covariates were obtained concurrently.

Multidetector Computed Tomography (MDCT) Vascular calcification was measured using 8-slice MDCT (Lightspeed Ultra, GE, Milwaukee, WI) as previously described (42, 43). For the chest, 2.5 mm slices were acquired from the carina to the diaphragm during an inspiratory breath hold at 70% of the cardiac cycle (120 kVp, 320 mA). For the abdomen, 2.5 mm slices (120 kVp, 320 mA) were obtained of a 125 mm abdominal segment using the upper edge of the S1 vertebrae as the anatomic landmark of the lower field. The presence of calcium was determined using a workstation (Acquarius, Terarecon, San Mateo, Calif). Calcium was quantified using the Agatston method, adapted for use with MDCT; (42, 44) results are expressed here in Agatston Units (AU). Models contrasting any presence of calcification to none dichotomized (CAC, AAC, and TAC) by to compare those with 0 AU to those with ⬎ 0 AU. We

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also considered the definitive presence of calcification vs its absence dichotomized the measures at ⫾ 100 AU.

at least 60 years, who did not have prevalent diabetes or a history of cardiovascular disease.

Hormone Measurement

Statistical Analysis

Blood samples were drawn in the supine position in the early morning after an overnight fast. Sera were aliquoted and immediately stored at –70°C, remaining frozen until the time of assay. Serum TT levels were measured by LC-MS/MS as previously described (41). The functional sensitivity of the TT assay was 0.07 nmol/L and the interassay coefficient of variation was 15.8%, at 0.42 nmol/L, 10.6%, at 0.82 nmol/L, 7.9%, at 1.7 nmol/L, 7.7% at 8.4 nmol/L, 4.4% at 18.5 nmol/L, and 3.3% at 35.3 nmol/L, respectively. As part of the Centers for Disease Control’s (CDC) Testosterone Assay Harmonization Initiative, quality control (QC) samples provided by the CDC were run every three months; the bias in QC samples with testosterone concentrations in 3.5 to 35 nmol/L range was consistently less than 6.2%. Serum estradiol and estrone levels were measured simultaneously using LC-MS/MS after derivatization with dansyl chloride (45). The limit of quantitation for both hormones was 2 pg/mL. Interassay CVs for estrone were 4.5%, 7.7%, and 6.9% at estrone concentrations of 29.6, 285, and 773 pmol/L, respectively, and for estradiol 6.9%, 7.0%, and 4.8% at estradiol concentrations of 29.3, 283, and 756 pmol/L, respectively. SHBG was measured using a two-site directed immunofluorometric assay that had a sensitivity of 0.5 nM (Delphia-Wallac, Inc., Turku, Finland); cFT was estimated from total testosterone, SHBG, E1 and E2 using the law of mass action (46).

Analyses were performed using SAS v9.2 (SAS, Inc., Cary, NC) and R version 2.15.2 (R Foundation for Statistical Computing, Vienna, Austria). Exploratory analyses of association included smoothing via Generalized Additive Models (49). Owing to the large number of subjects with undetectable or nearundetectable levels of calcification, analyses of continuous CAC, AAC and TAC were performed using Tobit regression, which acknowledges this disproportionately large ‘spike’ at the floor of the sampling distribution of the outcome variable, while positing a linear association between covariates and the outcome above said floor. Analyses of presence/absence of calcification quantified association between covariates and these dichotomous outcomes via odds ratio (OR) estimates derived using logistic regression. For all models, measures of association between sex steroids and measures of calcification were adjusted first for age alone. We then fit a second set of multivariable-adjusted models including all covariates described above (including age). To provide for ease of interpretation, Tobit regression coefficients were scaled according to the median of the relevant outcome measurement (CAC, AAC, TAC) measured among subjects with detectable calcification. Thus the estimated slope parameter may be interpreted as roughly equivalent to crosssectional proportionate differences observed among ‘typical’ participants with calcification levels greater than zero. Sensitivity analyses considered the regressions described above for subjects who were less than and greater than 60 years of age, as well as a parallel set of analyses restricted to the ‘healthy older’ cohort described above.

Measurement of Covariates Standard anthropometric and risk factor measurements were obtained as previously described (47, 48) Age was determined at the date of the participant examination visit (at which blood was obtained). Body mass index (BMI) was computed from standardized measures of height and weight obtained during clinical study visits. Information regarding medication usage was collected. Glucose, high-density and low-density lipoprotein (LDL) (HDL and LDL) cholesterol, and triglycerides were obtained from measurement of fasting blood samples. Diastolic blood pressure (BP) was obtained in the seated position from the average of two measurements taken greater than five minutes apart in the left arm. The presence of type 2 diabetes mellitus (T2DM) was indicated by a fasting serum glucose measurement greater than 6.94 mmol/L and/or participant use of antidiabetic medications. Hypertension was diagnosed by the presence of a systolic BP greater than or equal to 140 mmHg, diastolic BP greater than or equal to 90 mmHg, or reported use of antihypertensive medication. Current smokers reported smoking at least one cigarette per day over the preceding year.

Analytic sample A total of 1773 participants were included in both the calcium and hormone sampling studies. Of these, 1657 (93%) had complete calcium and hormone data and did not report use of androgen or antiandrogen therapies. An additional three participants were excluded due to abnormally high circulating testosterone values (total testosterone ⬎ 2400 ng/dl or calculated free testosterone ⬎ 350 pg/ml). This left 1654 participants eligible for inclusion in analyses. We also performed sensitivity analyses that were restricted to ‘healthy older’ participants of age

Results Characteristics of the Participants A total of 1654 men contributed to the analysis. A description of baseline characteristics and sex hormone levels is provided in Table 1. Mean (SD) total and free testosterone levels were well in the normal range at 617 (224) ng/dl and 111 (45) pg/ml, respectively. Total and free testosterone decreased, while estrone and estradiol increased (cross-sectionally) with age, as has been previously reported (41, 50). As expected, total and free testosterone were negatively associated with BMI and demonstrated positive association with HDL cholesterol; E1 and E2 demonstrated parallel associations, but these were not as strong. A description of the vascular calcification measures is given in Table 2. A substantial proportion of participants had zero scores for CAC and AAC; 80% of subjects had no detectable TAC and 89% had TAC ⬍ 100 AU. Among subjects with detectable calcification, the median CAC, AAC and TAC were 91, 392 and 131 AU, respectively. The Spearman correlation between CAC and AAC was

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Table 1. Description of baseline covariates and hormone levels, mean (SD) or n (%), n ⫽ 1654.

Age, years BMI, kg/ m2 Diastolic Blood Pressure, mmHg HDL-Chol, mg/dl Total Cholesterol, mg/dl Triglycerides, mg/dL Type-2 Diabetes Mellitus Prevalent Cardiovascular Disease Current Smoking Total Testosterone, ng/dl Free Testosterone, pg/ml Estrone, pg/ml Estradiol, pg/ml SHBG, nmol/ liter

CAC < 100 AU (n ⴝ 1229)

CAC > 100 AU (n ⴝ 425)

45 (8) 28 (4)

59 (10) 29 (5)

79 (9)

77 (10)

46 (12)

45 (14)

196 (34)

192 (35)

135 (96)

153 (112)

48 (4%)

63 (15%)

19 (2%)

65 (15%)

164 (13%)

53 (12%)

625 (216)

596 (246)

117 (45)

94 (42)

39 (14)

47 (17)

28 (10)

27 (9)

43 (21)

55 (27)

Abbreviations: SD ⫽ standard deviation; CAC ⫽ coronary artery calcium; AU ⫽ Agatston units; BMI ⫽ body mass index; HDL-Chol ⫽ HDL cholesterol; SHBG ⫽ sex hormone-blinding globulin.

0.41, whereas the correlation between CAC and TAC was 0.15. Total and Free Testosterone Analyses of association between sex steroids and calcification measures by Tobit regression is presented in Table 3. Regression estimates are scaled according to medians provided in the right most column of Table 2. For instance, among subjects with detectable CAC, the estimated mean absolute difference in CAC per 100 ng/dl cross-sectional increase in total testosterone is –21 AU with corresponding 95% confidence interval (CI) –38 AU to – 4 AU. Scaled to the median CAC among subjects with detectable calcification (91 AU), this translates to crosssectional mean (95% CI) trend of –23% (-41%, – 4%) the

CAC of the median participant per 100 ng/dl cross-sectional increase in total testosterone. Using this scaling method, the directionality, magnitude and statistical significance of age-adjusted associations between sex steroid levels and CAC is similar to those with AAC (Table 3). In age-adjusted analyses, there were statistically significant negative associations of TT and cFT with calcification at all sites, whereas there was a positive association of estrone with AAC and TAC. After adjustment for the other covariates, the associations between total and free testosterone and the calcification measures were reduced in magnitude and became statistically nonsignificant. In secondary analyses (results not shown), the associations between total or free testosterone and each of the measures of calcification became nonsignificant after adjustment for age, BMI and T2DM. Estradiol and Estrone In the multivariate model, E2 was negatively and significantly associated with CAC while estrone and was positively associated with TAC (Table 3). Estrone was likewise positively associated with AAC in age-adjusted models, but the magnitude of this effect was reduced substantially and nonsignificant once other factors were considered. The association between E2 and CAC was essentially unchanged in an alternative model controlling for SHBG in addition to the covariates listed in Table 3. Sensitivity Analysis Sensitivity analyses suggested that positive associations between estrone and AAC or TAC might be of lesser magnitude among younger as opposed to older subjects, though the evidence in favor of such age ⫻ hormone interactions was nonsignificant. Analyses restricted to the healthy older subsample were consistent with this trend; the directionality and significance of associations was similar to those displayed in Table 3, the magnitude of effects was of modestly greater magnitude. A similar pattern occurred with estradiol and CAC – estimates of negative association were somewhat greater in absolute magnitude when attention was restricted to the healthy older subset. By contrast to the pattern for testosterone, estradiol or estrone, there was no meaningful association of SHBG with vascular calcification even in models adjusting only for age. In logistic regression models, there was a negative ageadjusted association between testosterone and CAC (dichotomized at 100 AU) that became nonsignificant after adjustment for other cardiovascular risk factors (Figure 1). Similar patterns of association were observed for AAC and TAC. Results were similar in alternative models comparing those with CAC ⬍ 100 to those with CAC ⱖ 100.

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Table 2.

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Description of measures of vascular calcification. Mean (SD), AU

CAC AAC TAC

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201 (557) 845 (2101) 147 (788)

Median, AU

Participants with AU > 0

2 5 0

894 (54%) 912 (55%) 329 (20%)

Participants with AU > 100 425 (26%) 614 (37%) 179 (11%)

Median, AU, among participants with AU > 100 418 1128 404

Abbreviations: SD ⫽ standard deviation; AU ⫽ Agatston units; CAC ⫽ coronary artery calcium; AAC ⫽ abdominal aortic calcium; TAC ⫽ thoracic aortic calcium.

Discussion In a cross-sectional analysis of data from men in the Offspring cohort and the Third Generation cohort of the Framingham Heart Study, we observed age-adjusted associations between total or free testosterone and measures of coronary and aortic calcification, but these associations became attenuated and statistically nonsignificant after adjustment for conventional CHD risk factors, notably BMI and diabetes status. In models accounting for age alone, SHBG exhibited no association with any of the measures of calcification, estradiol was negatively associated with CAC, and estrone was positively associated with TAC. In the Tobit regression model, control for other factors was not sufficient to account for the relationship between circulating estrone levels and TAC, with a similar (though nonsignificant) trend in association with AAC. Age-related changes in sex steroids have been implicated in the development of illnesses of aging, including obesity, T2DM, osteoporosis, sarcopenia and mobility limitation and frailty. It remains unclear whether age-related declines in testosterone levels are a cause of, or rather tend to co-occur, with the constellation of illnesses and symptoms that are featured in male aging (51). While the phenotypic resemblance between androgen insufficiency in younger men and ‘typical’ male aging has long been noted, age-related trends in sex steroids appear to be blunted in men reporting excellent health, (52) and serum sex steroid levels are influenced both by health behaviors (notably smoking) as well as comorbidities to which they are also thought to contribute (notably changes in body composition (52, 53)). This suggests that the sex hormone alterations attending older age in men may have more to do with comorbidity than with aging per se. Our finding of an association between both testosterone and estradiol with vascular calcification in age-adjusted models, but not in multivariable-adjusted models – consistent with results from MESA and MACS (35, 36) – appear to accord with this interpretation. Our models suggest that the observed association between testosterone, E2 and calcification is either due to their joint correlation with conventional CHD risk factors, or alternatively that

the influence of sex steroids on cardiovascular calcification is transmitted along pathways those risk factors define. At the same time, the persistence of the association between estrone and TAC – in keeping with prior evidence of a role for estrone in the development of atherosclerosis (39, 40) and in cardiometabolic health more broadly, (50) is suggestive of a potential role for estrogens in the development of CHD in men somewhat reminiscent of that typically observed in women (54). Though it is seemingly counterintuitive that results obtained on estrone do not track those on estradiol, estrone is synthesized from androstenedione whereas estradiol is derived from testosterone and, the two hormones may exhibit differential activity in estrogen receptor subtypes, which in turn exhibit differential association with coronary atherosclerosis (55). This, along with the fact that estradiol (but not estrone) is a likely direct mediator of testosterone action on atherosclerotic risk, (33) lends some plausibility to the notion that estrone might express associations dissimilar from those observed for testosterone even as estradiol does not. These analyses have several strengths and also certain limitations. We measured testosterone, estradiol and estrone using LC-MS/MS, the method with the highest accuracy and sensitivity. The cohort included communitydwelling men over a wide age range from 19 to 89 years, in whom cardiovascular risk factors and disease have been well characterized. At the same time, the cross-sectional nature of the relationships renders assertions of causal association speculative. Free testosterone is calculated rather than directly measured, adding to the uncertainty in measurements. Finally, the FHS cohorts are largely homogenous in terms of ethnicity, geography, and to a lesser degree socioeconomic status, potentially inhibiting generalizability of results. This sample is also relatively young and healthy; and sensitivity analyses suggest that the effects observed might be somewhat stronger among older individuals, but we cannot speculate further. These results provide additional evidence for lack of association of either circulating testosterone or estradiol

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Sex steroids and vascular calcification

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Table 3. Tobit regression: weighted proportionate difference in calcification per difference in sex steroid levels, scaled to median calcification among those with detectable calcium.a Multivariable-Adjusteda

Age-Adjusted % Difference (95% CI) CAC Total Testosterone, 100 ng/dl Free Testosterone, 10 ng/ dl SHBG, 10 nmol/ liter Estrone, pg/ml Estradiol, pg/ml AAC Total Testosterone, 100 ng/dl Free Testosterone, 10 ng/ dl SHBG, 10 nmol/ liter Estrone, pg/ml Estradiol, pg/ml TAC Total Testosterone, 100 ng/dl Free Testosterone, 10 ng/ dl SHBG, 10 nmol/ liter Estrone, pg/ml Estradiol, pg/ml a

pvalue

% Difference (95% CI)

pvalue

⫺23 (-41, ⫺4)

0.02

⫺0.8 (-20, 19)

0.94

⫺12 (-22, ⫺2)

0.02

⫺6 (-16, 4)

0.25

⫺5 (-24, 13)

0.58

12 (-7, 31)

0.21

2 (-1, 4)

0.25

⫺0.7 (-4, 2)

0.65

⫺4 (-8, 1)

0.09

⫺5 (-9, ⫺1)

0.01

⫺21 (-36, ⫺7)

0.004

⫺0.4 (-15, 14)

0.95

⫺13 (-21, ⫺5)

0.002

⫺6 (-14, 2)

0.13

⫺0.3 (-15, 14)

0.97

13 (-1, 28)

0.08

4 (2, 6)

⬍0.001

2 (-0.2, 4)

0.09

⫺0.3 (-4, 3)

0.86

⫺0.6 (-4, 3)

0.69

⫺45 (-89, ⫺0.8)

0.046

⫺16 (-61, 29)

0.47

⫺25 (-53, 3)

0.08

⫺12 (-39, 15)

0.38

⫺11 (-53, 30)

0.59

8 (-34, 50)

0.70

15 (9, 21)

⬍0.001

11 (5, 17)

⬍0.001

⫺3 (-14, 7)

0.55

0.2 (-9, 10)

0.96

See Table 2

b

Adjusted for age, BMI, Type 2 Diabetes Mellitus, HDL-Chol, total cholesterol, triglycerides, smoking, diastolic blood pressure, prevalent CVD

Abbreviations: CI ⫽ confidence interval; AU ⫽ Agatston units; CAC ⫽ coronary artery calcium; AAC ⫽ abdominal aortic calcium; TAC ⫽ thoracic aortic calcium; SHBG ⫽ sex hormone-blinding globulin; BMI ⫽ body mass index; HDL-Chol ⫽ HDL cholesterol.

with atherosclerosis in aging men independent of conventional CHD risk factors, but our data suggest a potential

independent role for estrone. Prospective studies are

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needed to confirm or rule out this putative association in adult men. 2.

Acknowledgments Address all correspondence and requests for reprints to: Thomas G. Travison, PhD, Senior Scientist, Hebrew SeniorLife Institute for Aging Research, 1200 Centre St., Boston, MA 02 131, t: 617.971.5386, f: 617.971.5309, [email protected]. Disclosures: The authors have nothing to disclose This work was supported by primarily by NIH grants 1RO1AG31206 and 5R01DK092938 to SB and RSV. Additional support was provided by the Boston Claude D. Pepper Older Americans Independence Center grant 5P30AG031679 from the National Institute on Aging and by a grant from the CDC Foundation. The Framingham Heart Study is supported by the National Heart, Lung and Blood Institute contract N01-HC-25 195.

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Figure 1. Logistic regression: cross-sectional association sex hormones and the presence of detectable CAC. In unadjusted or age-adjusted models, total circulating testosterone concentrations exhibit a statistically significant negative association with CAC (first three confidence intervals, left to right, respectively). After adjustment for conventional risk factors, however, testosterone and CAC are near-independent (final interval, with point estimate indistinguishable with the null value). Control for age and other factors induced similar effects for free testosterone, estradiol and estrone.

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Circulating Sex Steroids and Vascular Calcification in Community-Dwelling Men: The Framingham Heart Study.

The relationship between sex steroids and atherosclerosis is poorly understood...
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