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. Author manuscript; available in PMC: 2015 Feb 11.
Published in final edited form as: Andrology. 2014 Oct 1;2(6):967–976. doi: 10.1111/andr.285

Association of serum inorganic phosphate with sex steroid hormones and vitamin D in a nationally representative sample of men

W Wulaningsih 1, M Van Hemelrijck 1, K Michaelsson 2, N Kanarek 3,4, W G Nelson 4,5,6, J H Ix 7, E A Platz 4,5,8, S Rohrmann 9
PMCID: PMC4324600  NIHMSID: NIHMS660078  PMID: 25270590

SUMMARY

Defects in bone regulatory pathways have been linked to chronic diseases including cardiovascular disease and cancer. In men, a link between bone metabolism and gonadal hormones has been suggested. However, to date, there is lack of evidence on the association between serum inorganic phosphate (Pi) and sex steroid hormones. The objective of this study was to investigate the association between Pi, sex steroid hormones and a known Pi metabolic regulator, vitamin D, in men in the National Health and Nutrition Examination Survey III (NHANES III). From NHANES III, we selected 1412 men aged 20+ who participated in the morning session of Phase I (1988–1991) with serum measurements of Pi, sex hormones, and vitamin D. Multivariable linear regression was used to calculate crude and geometric mean Pi by total and estimated free testosterone and estradiol, sex hormone-binding globulin, androstanediol glucuronide (AAG), and vitamin D. Similar analyses were performed while stratifying by race/ethnicity and vitamin D levels. We found a lack of statistically significant difference in geometric means of Pi across quintiles of concentrations of sex hormones, indicating a tight regulation of Pi. However, Pi levels were inversely associated with calculated free testosterone in non-Hispanic black men, with geometric mean levels of Pi of 1.16 and 1.02 ng/mL for those in the lowest and highest quintiles of free testosterone, respectively (p-trend < 0.05). A similar but weaker pattern was seen between total testosterone and Pi. An inverse association was also seen between AAG and Pi in men with vitamin D concentration below the median (<24.2 ng/mL). No associations were observed among men with vitamin D levels at or above the median. Our findings suggest a weak link among sex hormones, vitamin D, and Pi in men. The observed effects of race/ethnicity and vitamin D indicate a complex association involving various regulators of Pi homeostasis.

Keywords: cross-sectional studies, gonadal steroid hormones, serum inorganic phosphate

INTRODUCTION

Inorganic phosphate (Pi) is well known for its role in bone mineralization, and physiological levels of Pi are a prerequisite for normal cellular function (Takeda et al., 2004). In addition to this, recent studies have suggested an active role of Pi in cellular growth and proliferation (Conrads et al., 2005; Chang et al., 2006), which indicates its potential implication in human diseases such as cancer. In animal models, a high Pi diet has been shown to increase lung and skin carcinogenesis, and interfere with normal brain growth (Jin et al., 2006, 2009; Camalier et al., 2010). The link between Pi and cancer is further corroborated by results from a population-based cohort study of 397 292 Swedish men and women: higher Pi levels were associated with increased risk of overall cancer in men, and conversely, with decreased cancer risk in women (Wulaningsih et al., 2013). Moreover, the inverse association was found to be driven by hormone-related cancers such as breast and endometrial cancers, suggesting the importance of sex-specific metabolic or endocrine factors.

Alongside calcium, Pi is a building block for hydroxyapatite in bone, and is regulated by metabolic and endocrine factors including vitamin D, parathyroid hormone (PTH), and fibroblast growth factor 23 (FGF-23) (Bansal, 1990; Takeda et al., 2004). However, skeletal homeostasis and serum Pi levels may also be affected by changes in the levels of sex steroid hormones, either directly or indirectly through the aforementioned bone regulators (Rossouw et al., 2002; Beral et al., 2004). In a study including healthy male adults, a significant increase in serum Pi levels was observed following a GnRH (gonadotropin-releasing hormone) analog-induced decrease of circulating testosterone and estradiol without any apparent changes in FGF-23 levels (Burnett-Bowie et al., 2007). On the other hand, similar gonadal suppression was shown to be related to an increased sensitivity of PTH in men (Leder et al., 2001; Lee et al., 2006). These findings indicate multiple metabolic pathways in which sex steroid hormones may affect Pi metabolism.

Despite the suggested link between sex steroid hormones and bone regulation, no clear association with Pi levels has been established. Given the importance of Pi in maintaining cellular function and its link to major chronic diseases including cardiovascular disease and cancer (Tonelli et al., 2005; Wulaningsih et al., 2013), understanding the relationship between Pi levels and sex steroid hormones may provide further insight into metabolic pathways involved in human diseases. Therefore, we performed a cross-sectional analysis to investigate the relation between serum Pi and concentrations of different sex steroid hormones while taking into account vitamin D levels in adult males using data from NHANES III, a nationally representative sample of non-institutionalized Americans.

METHODS

Study population

The National Center for Health Statistics (NCHS) conducted NHANES III between 1988 and 1994 (National Center for Health Statistics, 1994) and designed it as a multistage stratified, clustered probability sample of the non-institutionalized US population who was at least 2 months old. All subjects participated in an interview conducted at home and an extensive physical examination, which included a blood sample performed at a mobile examination center (National Center for Health Statistics, 1994). NHANES III was conducted in two phases (1988–1991 and 1991–1994) both of which are independent unbiased national estimates of health and nutrition characteristics when weighted. Within each phase, subjects were randomly assigned to participate in either the morning or afternoon/evening examination session. Of the 2205 men, who participated in the morning session of Phase I (1988–1991), we selected adult men who had serum measurements for total testosterone, total estradiol, sex hormone-binding globulin (SHBG), androstanediol glucuronide (AAG), Pi, albumin, creatinine, and vitamin D. Although data for men aged 17+ were available, different examination procedures were performed compared to those aged 20+ (National Center for Health Statistics, 1994), so that only the latter were selected for this study (n = 1412). Among these participants, 1098 men had measurements of percent body fat and were included in the final analysis with fully adjusted models.

Hormone measurements

Stored serum samples were assayed for sex steroid hormones at the Children’s Hospital Boston, MA. Testosterone, estradiol, and SHBG concentrations were measured with competitive electrochemiluminescence immunoassays on the 2010 Elecsys autoanalyzer (Roche Diagnostics, Indianapolis, IN, USA) in 2005. AAG, an indicator of the conversion of testosterone to dihydrotestosterone, was measured with an enzyme immunoassay (Diagnostic Systems Laboratories, Webster, TX, USA). Laboratory technicians were blinded to the participant characteristics. The detection limits of the assays were 0.02 ng/mL, 5 pg/mL, 0.33 ng/mL, and 3 nmol/L for testosterone, estradiol, AAG, and SHBG, respectively. The coefficients of variation for quality control specimens were as follows: testosterone 5.9 and 5.8% at 2.5 and 5.5 ng/mL, respectively; estradiol 2.5, 6.5, and 6.7% at 39.4, 102.7, and 474.1 pg/mL, respectively; AAG 9.5 and 5.0% at 2.9 and 10.1 ng/mL, respectively; and SHBG 5.3 and 5.9% at 5.3 and 16.6 nmol/L, respectively. Quality control samples with a mean estradiol concentration of 39.4 pg/nL were also assayed, which is in the range of typical male estradiol concentrations (interassay coefficient of variation: 2.5%) (Rohrmann et al., 2007). Free testosterone was estimated from total testosterone, SHBG, and albumin and free estradiol was estimated from total estradiol, SHBG, and albumin using mass action equations as described by Vermeulen et al. (1999) and Rinaldi et al. (2002). The full equations with an example are available in the website of International Society for the Study of the Aging Male (2014, www.issam.ch).

Exposure measurements

Information on age, race/ethnicity, cigarette smoking, alcohol consumption, and physical activity was collected during the interview. Race and ethnicity were combined into four racial/ethnic groups: non-Hispanic white, non-Hispanic black, Mexican American, and other. Participants were classified as never, former, and current smokers (<20, 20–39, ≥40 cigarettes per day) based on the self-reported smoking habits. Frequency of alcohol consumption was measured by a food frequency questionnaire and categorized by times per week. Vigorous physical activity was defined by the following activities: jogging or running; swimming or aerobics (for men ≥40 years); biking, dancing, gardening, and calisthenics (for men ≥65 years); and walking and lifting weights (for men ≥80 years). Percent body fat was estimated from anthropometric and bioelectrical impedance data using the equations of Chumlea et al. (2002).

Serum Pi and calcium were measured using a Hitachi 737 Analyzer (Boehringer Mannheim Diagnostics, Indianapolis, IN, USA) (Clase et al., 2007). 25-hydroxyvitamin D was measured using the Diasorin radioimmunoassay kit (Diasorin, Stillwater, MN, USA) on frozen serum from 1994 to 1995. Coefficients of variations from quality control samples ranged from 13 to 19%. The radioimmunoassay kit was calibrated using high-performance liquid chromatographically purified vitamin D every 6 months. Serum creatinine was measured using a Jaffe kinetic alkaline picrate method. To calculate the glomerular filtration rate (GFR), we estimated standardized serum creatinine based on Deming regression: standardized creatinine (mg/dL) = 0.960 × serum creatinine – 0.184 (National Center for Health Statistics, 2007; Selvin et al., 2007). Estimated GFR (eGFR) was then calculated according to the following formula from the Modification of Diet in Renal Disease Study: eGFR (mL/min/1.73 m2) = 175 × (standardized serum creatinine in mg/dL)−1.154 × (age)−0..203 × 1.212 (if black) (Levey et al., 2007). The protocols for the conduct of NHANES III were approved by the Institutional Review Board of the NCHS, Centers for Disease Control and Prevention. Written informed consent was obtained from all participants. The Institutional Review Boards at the Johns Hopkins Bloomberg School of Public Health and the NCHS, Centers for Disease Control and Prevention approved the assay of stored serum specimens for the Hormone Demonstration Program.

Statistical analysis

All analyses were conducted with SAS release 9.3 (SAS Institute, Cary, NC, USA) and SUDAAN 9.0 software (Research Triangle Park, NC, USA) as implemented in SAS 9.3. Phase I morning sampling weights for NHANES III were used to account for sampling variability and to adjust for differential probability of sample selection (National Center for Health Statistics, 1994). First, we calculated the age-adjusted means or percentages of baseline characteristics while adjusting for the age distribution of the US population according to the 2000 Census. Next, we observed the relation between Pi and sex hormones and vitamin D by displaying dose-dependent changes in Pi levels obtained from linear regression models using restricted cubic spline (RCS) function of sex hormone concentrations with four knots [0.05, 0.35, 0.65, 0.95 percentiles as previously described (Van Hemelrijck et al., 2013)]. The models were adjusted for potential confounders based on current literature, which included age (continuous) and race/ethnicity with further adjustment for percent body fat, vigorous physical activity (yes or no), cigarette smoking (never, former, current), alcohol intake (<2, 2–3, 4–6 times a week, or daily), history of diabetes, continuous levels of serum vitamin D, calcium, and standardized creatinine, as renal absorption is linked to both Pi and sex steroid hormones (Yi et al., 2009; Bergwitz & Juppner, 2010). This analysis was performed using the RCS_Reg SAS Macro created by Desquilbet & Mariotti, (2010).

To further assess how Pi is associated with sex hormones and vitamin D and to confirm findings observed with the splines, we calculated crude and adjusted geometric mean concentrations of serum Pi and their 95% confidence intervals (CI) by quintiles of sex hormones and vitamin D using multivariable linear regression. A test for trend using quintiles as an ordinal variable was performed to assess any statistically significant linear trend. Serum Pi levels were not normally distributed and were transformed using the natural logarithm. Test for interaction was conducted for quintiles of sex hormones and race/ethnicity, and with vitamin D levels. In addition, we performed stratified analyses based on race/ethnicity since it may be associated with Pi regulation (Gutierrez et al., 2011), and serum levels of vitamin D using its median value in the study population (</≥24.2 ng/mL) since vitamin D is a regulator of Pi metabolism and has been shown to be associated with the development of cancer (Blomberg Jensen, 2012).

In a sensitivity analysis, we performed an additional adjustment for comorbidity as both sex steroid hormones and Pi may be associated with other diseases such as cardiovascular and chronic lung diseases. The comorbidity was evaluated with a comorbidity coefficient similar to the Charlson Comorbidity Index, as used in other NHANES III-based analyses (Goldfarb-Rumyantzev et al., 2010). Each of the comorbidities available in the dataset contributed one point to the composite index with additional points given for older age. Finally, we also performed a sensitivity analysis in a subpopulation of men with eGFR ≥60 mL/min/1.73 m2 to exclude participants with chronic kidney disease (n = 960) (Lubwama et al., 2014).

RESULTS

Age-adjusted baseline characteristics of the study population are displayed in Table 1. When sampling weights were applied, the mean age of the participants was 46 years old and most (78%) were white Americans. The majority of men abstained from smoking (39%) and drank alcohol up to once a week (48%). Overall age-adjusted mean Pi was 1.03 mmol/L.

Table 1.

Age-adjusted (standardized to the 2000 US Census age distribution) weighted characteristics of study population, men, NHANES III 1988–1991

Unweighted sample size Age-adjusted weighted mean/proportion (SE)
Age (years)
 Mean (SE) 1412 45.74 (0.20)
Race–ethnicity (%)
 Non-Hispanic white 656 77.99 (3.19)
 Non-Hispanic black 353 9.47 (1.33)
 Mexican-American 346 4.82 (0.71)
 Other 57 7.72 (2.16)
Percent body fat (%)
 Mean (SE) 1098 25.24 (0.26)
Cigarette smoking (%)
 Never 483 38.51 (2.30)
 Former 484 36.23 (2.98)
 Current (<20 cigarettes per day) 205 13.68 (1.36)
 Current (20–40 cigarettes per day) 51 6.88 (1.57)
 Current (≥40 cigarettes per day) 36 4.70 (0.97)
Alcohol intake (%)
 Up to once a week 778 47.54 (3.24)
 2–3 times a week 210 17.38 (1.45)
 4–6 times a week 192 17.17 (1.48)
 Daily or more 232 17.91 (2.51)
Vigorous physical activity (%) 228 14.85 (1.33)
Diabetes (%) 100 3.75 (0.57)
Phosphate (mmol/L)
 Mean (SE) 1412 1.03 (0.01)
Calcium (mmol/L)
 Mean (SE) 1412 2.29 (0.01)
Vitamin D (ng/mL)
 Mean (SE) 1412 30.95 (0.67)
Creatinine (mg/dL)
 Mean (SE) 1412 1.17 (0.01)
eGFR (mg/dL)
 Mean (SE) 1412 93.08 (0.96)
Total testosterone (ng/L)
 Mean (SE) 1412 5.31 (0.07)
Total estradiol (pg/L)
 Mean (SE) 1412 36.95 (0.71)
SHBG (nmol/L)
 Mean (SE) 1412 39.53 (0.77)
AAG (ng/L)
 Mean (SE) 1412 14.02 (0.43)
Free testosterone (ng/L)
 Mean (SE) 1412 0.11 (0.001)
Free estradiol (pg/L)
 Mean (SE) 1412 0.95 (0.02)
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NHANES III, National Health and Nutrition Examination Survey III; eGFR, Estimated glomerular filtration rate; SHBG, sex hormone-binding globulin; AAG, androstanediol glucuronide.

Figure 1 displays the dose-dependent multivariable-adjusted associations between sex hormones and Pi levels. Overall, a decrease in Pi levels was observed with increasing levels of sex hormones. After reaching the nadir, Pi levels changed minimally with continued increasing levels of total and free testosterone, AAG, and free testosterone. Pi levels gradually shifted back to baseline value after reaching the nadir with increasing total and free estradiol, and SHBG, indicating a U-shape association. An inverse U-shape association was seen between Pi and vitamin D levels.

Figure 1.

Figure 1

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Dose–response association of inorganic phosphate (Pi) with sex hormones and vitamin D. Serum Pi was coded using an restricted cubic spline function with four knots arbitrarily located at the 0.05, 0.35, 0.65, and 0.95 percentile. Y-axis represents the adjusted changes of Pi levels based on the full linear regression models for any increase in concentrations of total testosterone, total estradiol, sex hormone-binding globulin (SHBG), androstanediol glucuronide (AAG), free testosterone, free estradiol, and vitamin D (not adjusted for vitamin D), compared to zero as the reference value.

When looking at geometric means of Pi using both the age- and race-adjusted and fully adjusted models, mean Pi level did not statistically significantly differ across quintiles of sex hormones, although a weak decreasing Pi trend was seen with increasing AAG and free testosterone (Table 2). Further adjustment for comorbidity index and a sensitivity analysis in men with normal kidney function did not alter our findings (results not shown). When stratifying the analysis based on race/ethnicity, we found a statistically significant decreasing level of Pi with increasing free testosterone levels in non-Hispanic black men, suggesting an inverse association (p-trend = 0.01) (Table 3). However, no statistically significant interaction was observed between quintiles of sex hormones with race/ethnicity (results not shown).

Table 2.

Geometric mean (95% CI) of Pi levels by quintiles of sex hormones and vitamin D in a nationally representative sample of adult men in NHANES III 1988–1991. Full models were adjusted for age (continuous), race/ethnicity, % body fat (continuous), diabetes, cigarette smoking, alcohol intake, vigorous physical activity, and serum levels of vitamin D (continuous), calcium (continuous), and creatinine (continuous)

Quintiles of sex hormones Geometric mean (95% confidence intervals) of serum normalized Pi (mmol/L)
Age- and race-adjusted model (n = 1412) Fully adjusted modela (n = 1098)
Total testosterone (ng/mL)
 <3.59 1.03 (0.99–1.07) 1.04 (1.00–1.07)
 3.59–4.55 1.01 (0.99–1.04) 1.01 (0.98–1.04)
 4.55–5.58 1.03 (1.01–1.04) 1.03 (1.00–1.05)
 5.58–6.85 1.03 (1.01–1.06) 1.03 (1.01–1.05)
 ≥6.85 1.03 (1.01–1.05) 1.01 (0.98–1.04)
 p-value for trend 0.49 0.42
Total estradiol (pg/mL)
 <27.75 1.03 (1.00–1.05) 1.03 (1.00–1.06)
 27.75–32.98 1.01 (0.99–1.04) 1.01 (0.98–1.03)
 32.98–38.17 1.02 (1.00–1.04) 1.02 (1.00–1.04)
 38.17–45.71 1.05 (1.02–1.08) 1.03 (1.00–1.07)
 ≥45.71 1.03 (1.01–1.05) 1.02 (1.00–1.05)
 p-value for trend 0.44 0.67
SHBG (nmol/L)
 <24.91 1.02 (0.99–1.04) 1.02 (0.98–1.05)
 24.91–32.79 1.02 (0.99–1.06) 1.03 (0.99–1.06)
 32.79–42.32 1.01 (0.99–1.04) 1.02 (1.00–1.04)
 42.32–55.92 1.03 (1.01–1.06) 1.02 (1.00–1.04)
 ≥55.92 1.06 (1.03–1.09) 1.03 (1.00–1.06)
 p-value for trend 0.09 0.69
AAG (ng/mL)
 <6.57 1.04 (1.02–1.10) 1.05 (1.02–1.07)
 6.57–9.46 1.04 (1.01–1.07) 1.04 (1.00–1.08)
 9.46–12.57 1.03 (0.99–1.04) 1.02 (1.00–1.05)
 12.57–17.51 1.01 (0.99–1.04) 1.00 (0.98–1.02)
 ≥17.51 1.02 (1.00–1.05) 1.01 (0.99–1.04)
 p-value for trend 0.19 0.08
Free testosterone (ng/mL)
 <0.06 1.06 (1.02–1.10) 1.07 (1.02–1.12)
 0.06–0.09 1.02 (1.00–1.05) 1.02 (1.00–1.04)
 0.09–0.11 1.03 (1.00–1.05) 1.03 (1.01–1.06)
 0.11–0.14 1.02 (1.00–1.03) 1.00 (0.98–1.03)
 ≥0.14 1.04 (1.02–1.06) 1.02 (1.00–1.04)
 p-value for trend 0.75 0.17
Free estradiol (pg/mL)
 <0.68 1.03 (1.00–1.07) 1.02 (1.00–1.06)
 0.68–0.83 1.01 (0.98–1.06) 1.03 (1.00–1.05)
 0.83–0.98 1.02 (0.99–1.04) 1.01 (0.99–1.04)
 0.98–1.17 1.03 (1.01–1.06) 1.02 (1.00–1.05)
 ≥1.17 1.03 (1.00–1.05) 1.02 (1.00–1.04)
 p-value for trend 0.99 0.71
25-Hydroxy vitamin D (ng/mL)b
 <16.20 1.02 (0.97–1.07) 1.03 (0.97–1.09)
 16.20–21.50 1.01 (0.98–1.05) 1.02 (0.99–1.05)
 21.50–27.10 1.02 (0.99–1.05) 1.02 (0.99–1.05)
 27.10–33.90 1.03 (0.99–1.05) 1.03 (1.00–1.06)
 ≥33.90 1.03 (1.00–1.06) 1.02 (0.99–1.04)
 p-value for trend 0.24 0.30
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NHANES III, National Health and Nutrition Examination Survey III; SHBG, sex hormone-binding globulin; AAG, androstanediol glucuronide; Pi, inorganic phosphate.

a

Adjusted for age (continuous), race/ethnicity, % body fat (continuous), diabetes, cigarette smoking, alcohol intake, vigorous physical activity, and serum levels of vitamin D (continuous), calcium (continuous), and creatinine (continuous).

b

Not adjusted for vitamin D.

Table 3.

Geometric mean (95% CI) of Pi levels by quintiles of sex hormones and vitamin D in a nationally representative sample of adult men in NHANES III 1988–1991, stratified by race–ethnicity. All models were adjusted for age (continuous), % body fat (continuous), diabetes, cigarette smoking, alcohol intake, vigorous physical activity, and serum levels of vitamin D (continuous), calcium (continuous), and creatinine (continuous)

Quintiles of sex hormones Geometric mean (95% confidence intervals) of serum normalized Pi (mmol/L)
Non-Hispanic white (n = 498) Non-Hispanic black (n = 258) Mexican-American (n = 258)
Total testosterone (ng/mL)
 <3.59 1.03 (0.98–1.07) 1.10 (1.06–1.15) 1.07 (1.04–1.10)
 3.59–4.55 1.00 (0.97–1.04) 1.02 (0.97–1.07) 1.06 (1.04–1.09)
 4.55–5.58 1.02 (0.99–1.05) 1.08 (1.02–1.15) 1.06 (1.02–1.09)
 5.58–6.85 1.02 (1.00–1.04) 1.06 (1.02–1.10) 1.06 (1.02–1.10)
 ≥6.85 1.00 (0.96–1.03) 1.04 (0.99–1.09) 1.04 (0.99–1.10)
 p-value for trend 0.55 0.22 0.66
Total estradiol (pg/mL)
 <27.75 1.01 (0.99–1.05) 1.08 (1.04–1.13) 1.08 (1.05–1.11)
 27.75–32.98 0.99 (0.96–1.02) 1.05 (0.98–1.12) 1.08 (1.04–1.12)
 32.98–38.17 1.02 (1.00–1.04) 1.07 (1.00–1.15) 1.03 (1.00–1.07)
 38.17–45.71 1.02 (0.99–1.06) 1.05 (1.00–1.11) 1.07 (1.02–1.12)
 ≥45.71 1.02 (0.99–1.05) 1.05 (1.00–1.10) 1.00 (0.92–1.09)
 p-value for trend 0.59 0.45 0.10
SHBG (nmol/L)
 <24.91 1.00 (0.96–1.04) 1.07 (1.02–1.12) 1.05 (1.02–1.08)
 24.91–32.79 1.02 (0.98–1.06) 1.07 (1.02–1.11) 1.06 (1.02–1.09)
 32.79–42.32 1.01 (0.99–1.03) 1.05 (1.00–1.12) 1.05 (1.00–1.10)
 42.32–55.92 1.02 (0.99–1.04) 1.03 (0.97–1.10) 1.09 (1.06–1.13)
 ≥55.92 1.01 (0.98–1.05) 1.08 (1.01–1.14) 1.09 (1.03–1.16)
 p-value for trend 0.73 0.78 0.11
AAG (ng/mL)
 <6.57 1.03 (1.00–1.08) 1.09 (1.05–1.18) 1.04 (1.01–1.08)
 6.57–9.46 1.02 (0.99–1.08) 1.07 (1.03–1.12) 1.11 (1.06–1.16)
 9.46–12.57 1.01 (0.98–1.04) 1.08 (1.03–1.12) 1.04 (1.01–1.08)
 12.57–17.51 0.99 (0.97–1.01) 1.04 (0.98–1.10) 1.07 (1.04–1.11)
 ≥17.51 1.01 (0.98–1.04) 1.01 (0.95–1.08) 1.04 (0.99–1.09)
 p-value for trend 0.21 0.07 0.72
Free testosterone (ng/mL)
 <0.06 1.04 (0.99–1.10) 1.16 (1.02–1.16) 1.04 (1.00–1.08)
 0.06–0.09 1.01 (0.98–1.04) 1.07 (1.04–1.10) 1.06 (1.02–1.09)
 0.09–0.11 1.04 (1.01–1.06) 1.06 (1.01–1.10) 1.07 (1.04–1.10)
 0.11–0.14 0.99 (0.96–1.02) 1.06 (1.01–1.11) 1.07 (1.03–1.12)
 ≥0.14 1.01 (0.98–1.04) 1.02 (0.97–1.07) 1.04 (0.99–1.09)
 p-value for trend 0.37 0.01 0.81
Free estradiol (pg/mL)
 <0.68 1.02 (0.99–1.05) 1.09 (1.02–1.16) 1.07 (1.04–1.10)
 0.68–0.83 1.01 (0.99–1.03) 1.08 (1.02–1.14) 1.09 (1.06–1.13)
 0.83–0.98 1.00 (0.98–1.02) 1.04 (1.00–1.08) 1.06 (1.03–1.09)
 0.98–1.17 1.03 (0.99–1.06) 1.04 (0.98–1.10) 1.04 (1.00–1.09)
 ≥1.17 1.00 (0.98–1.04) 1.06 (1.01–1.11) 0.99 (0.90–1.09)
 p-value for trend 0.90 0.60 0.09
25-Hydroxy vitamin D (ng/mL)a
 <16.20 1.04 (0.92–1.18) 1.08 (0.99–1.17) 1.06 (0.97–1.18)
 16.20–21.50 0.99 (0.95–1.04) 1.08 (1.04–1.12) 1.10 (1.04–1.16)
 21.50–27.10 1.00 (0.96–1.03) 1.07 (0.97–1.14) 1.07 (1.03–1.11)
 27.10–33.90 1.03 (0.99–1.07) 1.05 (0.97–1.14) 1.02 90.97–1.07)
 ≥33.90 1.01 (0.99–1.04) 0.95 (0.84–1.09) 1.05 (1.00–1.10)
 p-value for trend 0.18 0.55 0.41
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NHANES III, National Health and Nutrition Examination Survey III; SHBG, sex hormone-binding globulin; AAG, androstanediol glucuronide; Pi, inorganic phosphate.

a

Not adjusted for vitamin D.

Next, a stratified analysis based on vitamin D levels was performed using the fully adjusted models. Among men with vitamin D levels greater than median (24.2 ng/mL), Pi levels did not statistically significantly differ across quintiles of sex hormones concentrations. However, among men with vitamin D levels less than or equal to the median, Pi levels decreased across increasing quintiles of AAG, with a geometric mean for Pi of 1.05 (95% CI: 1.00–1.10) mmol/L and 1.00 (95% CI: 0.97–1.03) mmol/L for those in the lowest and highest quintiles of AAG, respectively (p-trend = 0.04). Among men with lower vitamin D levels, U-shape patterns of Pi levels across quintiles of total and free estradiol were observed (mean Pi of 1.02, 0.99, and 1.05 mmol/L for those in the 1st, 3rd, and 5th quintiles of free estradiol, respectively; Table 4). No statistically significant interaction between quintiles of sex hormones with levels of vitamin D was found (results not shown).

Table 4.

Geometric means (95% CI) of Pi levels by quintiles of sex hormones in a nationally representative sample of adult men in NHANES III 1988–1991, stratified by vitamin D geometric mean (median) (Anderson et al., 2012). All models were adjusted for age (continuous), race/ethnicity, % body fat (continuous), diabetes, cigarette smoking, alcohol intake, vigorous physical activity, and serum levels of calcium (continuous), and creatinine (continuous)

Quintiles of sex hormones Geometric mean (95% confidence intervals) of serum normalized Pi (mmol/L)
Vitamin D < 24.2 ng/mL (n = 577) Vitamin D ≥ 24.2 ng/mL (n = 521)
Total testosterone (ng/mL)
 <3.59 1.02 (0.96–1.08) 1.04 (1.01–1.08)
 3.59–4.55 0.98 (0.94–1.03) 1.03 (1.00–1.07)
 4.55–5.58 1.02 (0.98–1.06) 1.03 (1.00–1.06)
 5.58–6.85 1.02 (0.99–1.06) 1.04 (1.00–1.07)
 ≥6.85 1.00 (0.95–1.06) 1.01 (0.98–1.04)
 p-value for trend 0.75 0.28
Total estradiol (pg/mL)
 <27.75 1.02 (0.97–1.07) 1.04 (1.00–1.07)
 27.75–32.98 0.99 (0.94–1.05) 1.01 (0.98–1.05)
 32.98–38.17 0.99 (0.95–1.03) 1.03 (1.01–1.05)
 38.17–45.71 1.00 (0.95–1.06) 1.04 (1.00–1.09)
 ≥45.71 1.05 (1.00–1.10) 1.00 (0.98–1.03)
 p-value for trend 0.30 0.65
SHBG (nmol/L)
 <24.91 1.01 (0.97–1.05) 1.02 (0.98–1.06)
 24.91–32.79 1.02 (0.98–1.06) 1.03 (0.99–1.07)
 32.79–42.32 1.00 (0.97–1.02) 1.03 (1.00–1.05)
 42.32–55.92 1.00 (0.96–1.04) 1.03 (1.01–1.05)
 ≥55.92 1.05 (0.99–1.12) 1.03 (1.01–1.05)
 p-value for trend 0.75 0.89
AAG (ng/mL)
 <6.57 1.05 (1.00–1.10) 1.04 (1.01–1.07)
 6.57–9.46 1.05 (1.00–1.11) 1.03 (0.99–1.07)
 9.46–12.57 0.99 (0.95–1.02) 1.04 (1.02–1.07)
 12.57–17.51 0.98 (0.95–1.02) 1.02 (0.99–1.04)
 ≥17.51 1.00 (0.97–1.03) 1.01 (0.98–1.05)
 p-value for trend 0.04 0.33
Free testosterone (ng/mL)
 <0.06 1.11 (1.05–1.17) 1.04 (0.98–1.10)
 0.06–0.09 1.01 (0.97–1.04) 1.03 (1.00–1.05)
 0.09–0.11 1.01 (0.96–1.05) 1.05 (1.03–1.08)
 0.11–0.14 0.97 (0.94–1.00) 1.01 (0.98–1.04)
 ≥0.14 1.02 (0.98–1.06) 1.01 (0.98–1.05)
 p-value for trend 0.10 0.34
Free estradiol (pg/mL)
 <0.68 1.03 (0.98–1.08) 1.03 (1.00–1.06)
 0.68–0.83 0.99 (0.95–1.03) 1.04 (1.01–1.07)
 0.83–0.98 0.98 (0.93–1.02) 1.03 (1.00–1.05)
 0.98–1.17 1.00 (0.98–1.03) 1.03 (0.99–1.06)
 ≥1.17 1.05 (1.01–1.09) 1.03 (0.97–1.04)
 p-value for trend 0.49 0.30
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NHANES III, National Health and Nutrition Examination Survey III; SHBG, sex hormone-binding globulin; AAG, androstanediol glucuronide; Pi, inorganic phosphate.

DISCUSSION

This study assessed the relationship between sex hormones overall and by vitamin D levels in non-institutionalized American men. We observed non-linear associations when plotting changes of Pi against increases in sex hormones, with patterns resembling U-shape for total and free estradiol and SHBG. When using quintiles, we found no association between sex hormones and Pi except for a statistically significant inverse trend between free testosterone and Pi in non-Hispanic black men. We also observed a similar inverse association between AAG and Pi and a U-shaped pattern between total and free estradiol and Pi in individuals with serum vitamin D <24.2 ng/mL.

Previous studies have shown that sex steroid hormones, especially estrogen, play important roles in bone conservation (Riggs et al., 2002), and therefore may interfere with Pi metabolism. The osteoprotective role of estrogen is particularly evident in women (Cauley et al., 2003; Robbins et al., 2013). In men, estrogen is also strongly linked to decreased bone loss, although a similar but weaker association is shown for testosterone (Gennari et al., 2003; Amin et al., 2006; Araujo et al., 2008; Khosla et al., 2008). There are several ways in which sex steroid hormones may influence the dynamic of bone turnover. Directly, both estrogen and testosterone were suggested to modulate gene expression leading to shortened osteoclasts lifespan while prolonging that of osteoblasts in a gender- and site-specific fashion (Kawano et al., 2003; Michael et al., 2005; Nakamura et al., 2007; Imai et al., 2009; Wang & Stern, 2011; Yang et al., 2013). Through bone regulators, sex steroid hormones may also indirectly interfere with bone metabolism, as shown by inhibition of PTH-stimulated formation of osteoclasts following in vitro estradiol treatment (Liu et al., 2002).

In the context of diseases, the association between sex steroid hormones and Pi may be important given a number of similar molecular pathways in bone regulation and development of diseases such as cardiovascular disease and cancer. For instance, PTH is known to enhance the expression of receptor activator of NF-κB ligand (RANKL), a cellular surface protein important in both osteoclastogenesis and cancer progression (Blair et al., 2006; Huang et al., 2006; Odero-Marah et al., 2008). In cardiovascular disease, higher RANKL expression has also been linked to left ventricular dysfunction in heart failure (Ueland et al., 2005). Interestingly, upregulation of RANKL has been reported in deficiency of estrogen and androgen receptor in animal studies, further emphasizing the role of sex steroid hormones (Liu et al., 2002; Michael et al., 2005). Another common link between bone metabolism, cancer, and cardiovascular disease is β-catenin, a component of the Wnt pathway, which is also involved in carcinogenesis and regulation of heart muscle (Miller et al., 1999; Klaus & Birchmeier, 2008; Mill & George, 2012). The evidence suggests the potential importance of the link between Pi and sex hormones in bone remodeling and disease pathogenesis.

Despite the effects of sex steroid hormone on bone turnover, and the role of Pi as a major bone constituent, the link between sex steroid hormones and Pi regulation has not been explicitly described in the literature. Serum Pi is conventionally known to be positively regulated by vitamin D and negatively regulated by PTH (Bergwitz & Juppner, 2010). The importance of Pi-calcium homeostasis is reflected by a feedback mechanism triggered by abnormal levels of Pi and calcium on vitamin D and PTH secretion. Adding to this system are the recently found Pi regulators, FGF-23 and Klotho, an anti-aging hormone (Renkema et al., 2008). FGF-23 induces hypophosphatemia through renal phosphate wasting, while Klotho forms heterodimers with FGF receptors, producing a specific receptor for FGF-23 (Goetz & Mohammadi, 2013).

Sex steroid hormones may affect Pi levels through modulation of Pi regulators as levels of PTH and vitamin D are influenced by estrogen (Liu et al., 2002; Uebelhart et al., 2009; Wang & Stern, 2011). Also, sex steroid hormones indirectly favor osteoblasts (Yang et al., 2013), which produce FGF-23, and treatment with estrogen results in higher FGF-23 mRNA and serum levels in vitro and in vivo (Carrillo-Lopez et al., 2009). Estrogen may also affect Pi homeostasis through its effects on calcium, as it was reported to increase renal absorption of calcium in a vitamin D-independent manner (Renkema et al., 2008). Moreover, estrogen is suggested to directly regulate Pi levels through PTH-independent downregulation of a major Pi-dependent cotransporter NaPi-IIa in the renal proximal tubule (Faroqui et al., 2008). Interestingly, a counter-regulation of sex steroid hormones by bone regulators may also occur, as shown by a decreased estrogen biosynthesis and insufficient gonadal functions in animal models lacking vitamin D receptor (Kinuta et al., 2000; Jensen, 2014). However, little has been reported regarded the link between serum sex steroid hormones and Pi in men in observational studies.

In 1346 men aged 65 or older in The Osteoporotic Fractures in Men (MrOs) Study which majorly (~90%) consisted of Caucasians, a clear inverse relation of Pi with both total and bioavailable estradiol and testosterone was reported, with a decrease of 0.05 mg/dL serum Pi (95% CI: −0.09 to −0.02; p < 0.01) for a 10 pg/mL increase in total estradiol and a decrease of 0.09 mg/dL (95% CI: −0.13 to −0.04; p < 0.001) for a 200 ng/dL increase in total testosterone (Meng et al., 2010). Correspondingly, an increase in Pi levels was observed in men whom GnRH analog was administered, with concomitant estrogen and testosterone deficiency (Burnett-Bowie et al., 2007). In this study, we found the association between Pi and sex hormones to be non-linear, suggesting a feedback mechanism following low levels of Pi. These associations seem to be weaker than those reported in the MrOs US Study (Meng et al., 2010). However, our study population had younger mean age, and differences in findings may be accounted by age-related kidney function and morbidities despite attempts to adjust for these factors in the analyses. Further detailed studies with methods enabling threshold estimation are needed to confirm the observed non-linearity of the association between Pi and sex hormones.

We observed a statistically significant inverse relationship between Pi and calculated free testosterone in non-Hispanic black men. A suggested effect of race/ethnicity on Pi regulators may explain a stronger association in this particular subgroup. Circulating vitamin D, the positive regulator of Pi, is generally lower in black Americans compared to whites (Looker et al., 2011; Powe et al., 2013) which is mainly caused by a decreased synthesis in more heavily pigmented skin (Libon et al., 2013). Interestingly, a different association between vitamin D and PTH has also been reported in non-Hispanic blacks compared to white and Mexican-American individuals (Gutierrez et al., 2011). In the two latter groups, levels of vitamin D were inversely associated with those of PTH, while in blacks an inverse association was only seen when vitamin D levels were <26 ng/mL. This may indicate a lack of PTH-negative regulation and possibly greater Pi wasting in blacks with adequate vitamin D, or a different threshold of normal Pi in respect of races. Levels of sex hormones have also been reported to vary among races/ethnicities, with higher levels of estradiol in American black compared to white and Mexican-American men (Rohrmann et al., 2007). It is therefore possible that higher levels of sex steroid hormones and lower Pi threshold in black men result in a more pronounced inverse association between free testosterone and Pi levels, However, no statistically significant interaction between sex steroid hormones and race/ethnicity was observed in this study and thus confirmation in future studies is needed to exclude a possibility of chance findings.

Despite lack of marked effect modification, associations between AAG, a testosterone metabolite, and Pi differed across vitamin D levels. Vitamin D increases Pi and calcium absorption and negatively regulates PTH and FGF-23, with a net effect of increased Pi and calcium serum concentrations (Erben et al., 2002; Bergwitz & Juppner, 2010). Our findings support a negative feedback of vitamin D following high Pi levels, which may be caused by the action of PTH and FGF-23 in inhibiting CYP27B1, a renal enzyme synthesizing 1,25(OH)2D3, the active metabolite of vitamin D (Anderson et al., 2012). A lower expression of this enzyme was also found in men with hypogonadism, accompanied by low vitamin D and PTH levels (Foresta et al., 2011), which is relevant to the findings of low levels of sex steroid hormones in men with vitamin D deficiency (Araujo et al., 2008). Moreover, when the association between vitamin D and testosterone in men was plotted, vitamin D levels were shown to increase the following higher testosterone levels until a certain point, then decrease into a plateau (Nimptsch et al., 2012). Such association may allow effect modification to occur interchangeably between vitamin D and androgen markers as observed in this study. Also, since vitamin D levels are lower in non-Hispanic blacks (Looker et al., 2011), the observed effect modification by vitamin D, although weak, may reflect race-specific genetic polymorphisms in genes regulating Pi and sex hormones.

This strength of this study is its generalizability following the use of nationally representative data of the US population. We were able to adjust for many potential confounding factors and examine effect modifications by vitamin D levels. A limitation of this cross-sectional study is that it relied on a single measurement at one point in time so that it may be prone to measurement error and within-person variation. Furthermore, our study used immunoassays instead of mass spectrometry to evaluate serum levels of sex steroid hormones and 25-hydroxyvitamin D. Since estradiol measurements with immunoassay were reported to be less well correlated with the mass spectrometry compared to testosterone (Cauley et al., 2003), there is a possibility that this may affect the strength of the observed associations in our study. However, similar patterns of association between sex steroid hormones with BMD was observed in both immunoassay and mass spectrometry (Cauley et al., 2003), suggesting the relevance of both methods in assessing sex steroid hormones and bone metabolism. Spurious correlations may be of concern when performing multiple comparisons as shown in our study (Rothman, 1990). However, we planned our analyses based on prior evidence and our results are explicable by suggested biological pathways and findings from other studies. Another limitation is that we were unable to account for the effects of various Pi and calcium compounds. However, we performed a sensitivity analysis excluding men with reduced kidney function since kidney failure has been linked to an increased ectopic formation of these compounds (O’neill, 2007). Since we only have information on sex hormones in male participants of the NHANES III study, we were unable to assess the gender specificity of the association between Pi, sex hormones, and vitamin D. Finally, the information on PTH and FGF-23 as other Pi regulators was unavailable in this study.

CONCLUSION

We found weak non-linear associations between levels of Pi with sex steroid hormones in non-institutionalized US male adults. Although no statistically significant effect modification by race/ethnicity or vitamin D was observed, an inverse relation was found between Pi levels with free testosterone in non-Hispanic black men, and with androgen-derived metabolite concentrations in those with lower vitamin D levels. Our findings further emphasize the complex regulation of bone metabolism involving sex steroid hormones in addition to conventional Pi regulators, i.e., vitamin D, PTH, and FGF-23. As abnormal Pi levels have been linked to the pathogenesis of cancer and other chronic diseases, further study assessing sex steroid hormones and Pi should include Pi regulators as well as accounting for sex and racial differences.

Acknowledgments

This is the 29th study from the Hormone Demonstration Program, which is supported by the Maryland Cigarette Restitution Fund at Johns Hopkins.

Footnotes

CONFLICT OF INTERESTS

None declared.

AUTHOR CONTRIBUTIONS

WW, MVH, KM, EAP, and SR interpreted analysis results; WW, MVH, KM, NK, WGN, JHI, EAP, and SR have edited and reviewed the manuscript; WW, MVH, and SR conceived and designed the experiments; WW and SR performed the experiments; WW and SR analyzed the data; NK, WGN, JHI, and EAP contributed reagents/materials/analysis tools; WW wrote the manuscript.

References

  1. Amin S, Zhang Y, Felson DT, Sawin CT, Hannan MT, Wilson PW, Kiel DP. Estradiol, testosterone, and the risk for hip fractures in elderly men from the Framingham study. Am J Med. 2006;119:426–433. doi: 10.1016/j.amjmed.2005.10.048. [DOI] [PubMed] [Google Scholar]
  2. Anderson PH, Turner AG, Morris HA. Vitamin D actions to regulate calcium and skeletal homeostasis. Clin Biochem. 2012;45:880–886. doi: 10.1016/j.clinbiochem.2012.02.020. [DOI] [PubMed] [Google Scholar]
  3. Araujo AB, Travison TG, Leder BZ, Mckinlay JB. Correlations between serum testosterone, estradiol, and sex hormone-binding globulin and bone mineral density in a diverse sample of men. J Clin Endocrinol Metab. 2008;93:2135–2141. doi: 10.1210/jc.2007-1469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bansal VK. Serum inorganic phosphorus. In: Walker HK, Hall WD, Hurst JW, editors. Clinical Methods: The History, Physical, and Laboratory Examinations. 3. Chapter 198. Butterworths; Boston, MA: 1990. Available from: http://www.ncbi.nlm.nih.gov/books/NBK201/ [PubMed] [Google Scholar]
  5. Beral V, Banks E, Reeves G. Million Women Study Collaborators (2004) Effects of estrogen-only treatment in postmenopausal women. JAMA. 292:684. doi: 10.1001/jama.292.6.684-a. author reply 685–686. [DOI] [PubMed] [Google Scholar]
  6. Bergwitz C, Juppner H. Regulation of phosphate homeostasis by PTH, vitamin D, and FGF23. Annu Rev Med. 2010;61:91–104. doi: 10.1146/annurev.med.051308.111339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Blair JM, Zhou H, Seibel MJ, Dunstan CR. Mechanisms of disease: roles of OPG, RANKL and RANK in the pathophysiology of skeletal metastasis. Nat Clin Pract Oncol. 2006;3:41–49. doi: 10.1038/ncponc0381. [DOI] [PubMed] [Google Scholar]
  8. Blomberg Jensen M. Vitamin D metabolism, sex hormones and male reproductive function. Reproduction. 2012;144:647. doi: 10.1530/REP-12-0064. [DOI] [PubMed] [Google Scholar]
  9. Burnett-Bowie SM, Mendoza N, Leder BZ. Effects of gonadal steroid withdrawal on serum phosphate and FGF-23 levels in men. Bone. 2007;40:913–918. doi: 10.1016/j.bone.2006.10.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Camalier CE, Young MR, Bobe G, Perella CM, Colburn NH, Beck GR., Jr Elevated phosphate activates N-ras and promotes cell transformation and skin tumorigenesis. Cancer Prev Res (Phila) 2010;3:359–370. doi: 10.1158/1940-6207.CAPR-09-0068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Carrillo-Lopez N, Roman-Garcia P, Rodriguez-Rebollar A, Fernandez-Martin JL, Naves-Diaz M, Cannata-Andia JB. Indirect regulation of PTH by estrogens may require FGF23. J Am Soc Nephrol. 2009;20:2009–2017. doi: 10.1681/ASN.2008121258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cauley JA, Robbins J, Chen Z, Cummings SR, Jackson RD, Lacroix AZ, Leboff M, Lewis CE, Mcgowan J, Neuner J, Pettinger M, Stefanick ML, Wactawski-Wende J, Watts NB Women’s Health Initiative Investigators. Effects of estrogen plus progestin on risk of fracture and bone mineral density: the women’s health initiative randomized trial. JAMA. 2003;290:1729–1738. doi: 10.1001/jama.290.13.1729. [DOI] [PubMed] [Google Scholar]
  13. Chang SH, Yu KN, Lee YS, An GH, Beck GR, Jr, Colburn NH, Lee KH, Cho MH. Elevated inorganic phosphate stimulates Akt-ERK1/2-Mnk1 signaling in human lung cells. Am J Respir Cell Mol Biol. 2006;35:528–539. doi: 10.1165/rcmb.2005-0477OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chumlea WC, Guo SS, Kuczmarski RJ, Flegal KM, Johnson CL, Heymsfield SB, Lukaski HC, Friedl K, Hubbard VS. Body composition estimates from NHANES III bioelectrical impedance data. Int J Obes Relat Metab Disord. 2002;26:1596–1609. doi: 10.1038/sj.ijo.0802167. [DOI] [PubMed] [Google Scholar]
  15. Clase CM, Kiberd BA, Garg AX. Relationship between glomerular filtration rate and the prevalence of metabolic abnormalities: results from the Third National Health and Nutrition Examination Survey (NHANES III) Nephron Clin Pract. 2007;105:c178–c184. doi: 10.1159/000100489. [DOI] [PubMed] [Google Scholar]
  16. Conrads KA, Yi M, Simpson KA, Lucas DA, Camalier CE, Yu LR, Veenstra TD, Stephens RM, Conrads TP, Beck GR., Jr A combined proteome and microarray investigation of inorganic phosphate-induced pre-osteoblast cells. Mol Cell Proteomics. 2005;4:1284–1296. doi: 10.1074/mcp.M500082-MCP200. [DOI] [PubMed] [Google Scholar]
  17. Desquilbet L, Mariotti F. Dose-response analyses using restricted cubic spline functions in public health research. Stat Med. 2010;29:1037–1057. doi: 10.1002/sim.3841. [DOI] [PubMed] [Google Scholar]
  18. Erben RG, Soegiarto DW, Weber K, Zeitz U, Lieberherr M, Gniadecki R, Moller G, Adamski J, Balling R. Deletion of deoxyribonucleic acid binding domain of the vitamin D receptor abrogates genomic and nongenomic functions of vitamin D. Mol Endocrinol. 2002;16:1524–1537. doi: 10.1210/mend.16.7.0866. [DOI] [PubMed] [Google Scholar]
  19. Faroqui S, Levi M, Soleimani M, Amlal H. Estrogen downregulates the proximal tubule type IIa sodium phosphate cotransporter causing phosphate wasting and hypophosphatemia. Kidney Int. 2008;73:1141–1150. doi: 10.1038/ki.2008.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Foresta C, Strapazzon G, De Toni L, Perilli L, Di Mambro A, Muciaccia B, Sartori L, Selice R. Bone mineral density and testicular failure: evidence for a role of vitamin D 25-hydroxylase in human testis. J Clin Endocrinol Metab. 2011;96:E646–E652. doi: 10.1210/jc.2010-1628. [DOI] [PubMed] [Google Scholar]
  21. Gennari L, Merlotti D, Martini G, Gonnelli S, Franci B, Campagna S, Lucani B, Dal Canto N, Valenti R, Gennari C, Nuti R. Longitudinal association between sex hormone levels, bone loss, and bone turnover in elderly men. J Clin Endocrinol Metab. 2003;88:5327–5333. doi: 10.1210/jc.2003-030736. [DOI] [PubMed] [Google Scholar]
  22. Goetz R, Mohammadi M. Exploring mechanisms of FGF signalling through the lens of structural biology. Nat Rev Mol Cell Biol. 2013;14:166–180. doi: 10.1038/nrm3528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Goldfarb-Rumyantzev AS, Rout P, Sandhu GS, Khattak M, Tang H, Barenbaum A. Association between social adaptability index and survival of patients with chronic kidney disease. Nephrol Dial Transplant. 2010;25:3672–3681. doi: 10.1093/ndt/gfq177. [DOI] [PubMed] [Google Scholar]
  24. Gutierrez OM, Farwell WR, Kermah D, Taylor EN. Racial differences in the relationship between vitamin D, bone mineral density, and parathyroid hormone in the National Health and Nutrition Examination Survey. Osteoporos Int. 2011;22:1745–1753. doi: 10.1007/s00198-010-1383-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Huang H, Ryu J, Ha J, Chang EJ, Kim HJ, Kim HM, Kitamura T, Lee ZH, Kim HH. Osteoclast differentiation requires TAK1 and MKK6 for NFATc1 induction and Nf-kappaB transactivation by RANKL. Cell Death Differ. 2006;13:1879–1891. doi: 10.1038/sj.cdd.4401882. [DOI] [PubMed] [Google Scholar]
  26. Imai Y, Youn MY, Kondoh S, Nakamura T, Kouzmenko A, Matsumoto T, Takada I, Takaoka K, Kato S. Estrogens maintain bone mass by regulating expression of genes controlling function and life span in mature osteoclasts. Ann N Y Acad Sci. 2009;1173(Suppl 1):E31–E39. doi: 10.1111/j.1749-6632.2009.04954.x. [DOI] [PubMed] [Google Scholar]
  27. International Society for the Study of the Aging Male. Free Testosterone Calculator. 2014 Available at: http://www.issam.ch/freetesto.htm.
  28. Jensen MB. Vitamin D and male reproduction. Nat Rev Endocrinol. 2014;10:175–186. doi: 10.1038/nrendo.2013.262. [DOI] [PubMed] [Google Scholar]
  29. Jin H, Hwang SK, Yu K, Anderson HK, Lee YS, Lee KH, Prats AC, Morello D, Beck GR, Jr, Cho MH. A high inorganic phosphate diet perturbs brain growth, alters Akt-ERK signaling, and results in changes in cap-dependent translation. Toxicol Sci. 2006;90:221–229. doi: 10.1093/toxsci/kfj066. [DOI] [PubMed] [Google Scholar]
  30. Jin H, Xu CX, Lim HT, Park SJ, Shin JY, Chung YS, Park SC, Chang SH, Youn HJ, Lee KH, Lee YS, Ha YC, Chae CH, Beck GR, Jr, Cho MH. High dietary inorganic phosphate increases lung tumorigenesis and alters Akt signaling. Am J Respir Crit Care Med. 2009;179:59–68. doi: 10.1164/rccm.200802-306OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kawano H, Sato T, Yamada T, Matsumoto T, Sekine K, Watanabe T, Nakamura T, Fukuda T, Yoshimura K, Yoshizawa T, Aihara K, Yamamoto Y, Nakamichi Y, Metzger D, Chambon P, Nakamura K, Kawaguchi H, Kato S. Suppressive function of androgen receptor in bone resorption. Proc Natl Acad Sci USA. 2003;100:9416–9421. doi: 10.1073/pnas.1533500100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Khosla S, Amin S, Singh RJ, Atkinson EJ, Melton LJ, 3rd, Riggs BL. Comparison of sex steroid measurements in men by immunoassay versus mass spectroscopy and relationships with cortical and trabecular volumetric bone mineral density. Osteoporos Int. 2008;19:1465–1471. doi: 10.1007/s00198-008-0591-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kinuta K, Tanaka H, Moriwake T, Aya K, Kato S, Seino Y. Vitamin D is an important factor in estrogen biosynthesis of both female and male gonads. Endocrinology. 2000;141:1317–1324. doi: 10.1210/endo.141.4.7403. [DOI] [PubMed] [Google Scholar]
  34. Klaus A, Birchmeier W. Wnt signalling and its impact on development and cancer. Nat Rev Cancer. 2008;8:387–398. doi: 10.1038/nrc2389. [DOI] [PubMed] [Google Scholar]
  35. Leder BZ, Smith MR, Fallon MA, Lee ML, Finkelstein JS. Effects of gonadal steroid suppression on skeletal sensitivity to parathyroid hormone in men. J Clin Endocrinol Metab. 2001;86:511–516. doi: 10.1210/jcem.86.2.7177. [DOI] [PubMed] [Google Scholar]
  36. Lee H, Finkelstein JS, Miller M, Comeaux SJ, Cohen RI, Leder BZ. Effects of selective testosterone and estradiol withdrawal on skeletal sensitivity to parathyroid hormone in men. J Clin Endocrinol Metab. 2006;91:1069–1075. doi: 10.1210/jc.2005-2495. [DOI] [PubMed] [Google Scholar]
  37. Levey AS, Coresh J, Greene T, Marsh J, Stevens LA, Kusek JW, Van Lente F Chronic Kidney Disease Epidemiology Collaboration. Expressing the Modification of Diet in Renal Disease Study equation for estimating glomerular filtration rate with standardized serum creatinine values. Clin Chem. 2007;53:766–772. doi: 10.1373/clinchem.2006.077180. [DOI] [PubMed] [Google Scholar]
  38. Libon F, Cavalier E, Nikkels AF. Skin color is relevant to vitamin D synthesis. Dermatology. 2013;227:250–254. doi: 10.1159/000354750. [DOI] [PubMed] [Google Scholar]
  39. Liu BY, Wu PW, Bringhurst FR, Wang JT. Estrogen inhibition of PTH-stimulated osteoclast formation and attachment in vitro: involvement of both PKA and PKC. Endocrinology. 2002;143:627–635. doi: 10.1210/endo.143.2.8614. [DOI] [PubMed] [Google Scholar]
  40. Looker AC, Johnson CL, Lacher DA, Pfeiffer CM, Schleicher RL, Sempos CT. NCHS data brief. 59. National Center for Health Statistics; Hyattsville, MD: 2011. Vitamin D Status: United States, 2001–2006. [PubMed] [Google Scholar]
  41. Lubwama R, Nguyen A, Modi A, Diana C, Miller PD. Prevalence of renal impairment among osteoporotic women in the USA, NHANES 2005–2008: is treatment with bisphosphonates an option? Osteoporos Int. 2014;25:1607–1615. doi: 10.1007/s00198-014-2645-1. [DOI] [PubMed] [Google Scholar]
  42. Meng J, Ohlsson C, Laughlin GA, Chonchol M, Wassel CL, Ljunggren O, Karlsson MK, Mellstrom D, Orwoll ES, Barrett-Connor E, Ix JH. Associations of estradiol and testosterone with serum phosphorus in older men: the osteoporotic fractures in men study. Kidney Int. 2010;78:415–422. doi: 10.1038/ki.2010.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Michael H, Harkonen PL, Vaananen HK, Hentunen TA. Estrogen and testosterone use different cellular pathways to inhibit osteoclastogenesis and bone resorption. J Bone Miner Res. 2005;20:2224–2232. doi: 10.1359/JBMR.050803. [DOI] [PubMed] [Google Scholar]
  44. Mill C, George SJ. Wnt signalling in smooth muscle cells and its role in cardiovascular disorders. Cardiovasc Res. 2012;95:233–240. doi: 10.1093/cvr/cvs141. [DOI] [PubMed] [Google Scholar]
  45. Miller JR, Hocking AM, Brown JD, Moon RT. Mechanism and function of signal transduction by the Wnt/beta-catenin and Wnt/Ca2+ pathways. Oncogene. 1999;18:7860–7872. doi: 10.1038/sj.onc.1203245. [DOI] [PubMed] [Google Scholar]
  46. Nakamura T, Imai Y, Matsumoto T, Sato S, Takeuchi K, Igarashi K, Harada Y, Azuma Y, Krust A, Yamamoto Y, Nishina H, Takeda S, Takayanagi H, Metzger D, Kanno J, Takaoka K, Martin TJ, Chambon P, Kato S. Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts. Cell. 2007;130:811–823. doi: 10.1016/j.cell.2007.07.025. [DOI] [PubMed] [Google Scholar]
  47. National Center for Health Statistics. Note for Correction of Serum Creatinine for NHANES III, NHANES 1999–2000, 2001–2002 and 2003–2004. 2007 Available at: http://www.cdc.gov/nchs/data/nhanes/nhanes_03_04/general_%20note_for_serum_creatinine.pdf.
  48. National Center for Health Statistics. Plan and operation of the Third National Health and Nutrition Examination Survey, 1988–94. Series 1: programs and collection procedures. Vital Health Stat 1. 1994;32:1–407. [PubMed] [Google Scholar]
  49. Nimptsch K, Platz EA, Willett WC, Giovannucci E. Association between plasma 25-OH vitamin D and testosterone levels in men. Clin Endocrinol (Oxf) 2012;77:106–112. doi: 10.1111/j.1365-2265.2012.04332.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Odero-Marah VA, Wang R, Chu G, Zayzafoon M, Xu J, Shi C, Marshall FF, Zhau HE, Chung LW. Receptor activator of NF-kappaB ligand (RANKL) expression is associated with epithelial to mesenchymal transition in human prostate cancer cells. Cell Res. 2008;18:858–870. doi: 10.1038/cr.2008.84. [DOI] [PubMed] [Google Scholar]
  51. O’neill WC. The fallacy of the calcium-phosphorus product. Kidney Int. 2007;72:792–796. doi: 10.1038/sj.ki.5002412. [DOI] [PubMed] [Google Scholar]
  52. Powe CE, Evans MK, Wenger J, Zonderman AB, Berg AH, Nalls M, Tamez H, Zhang D, Bhan I, Karumanchi SA, Powe NR, Thadhani R. Vitamin D-binding protein and vitamin D status of black Americans and white Americans. N Engl J Med. 2013;369:1991–2000. doi: 10.1056/NEJMoa1306357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Renkema KY, Alexander RT, Bindels RJ, Hoenderop JG. Calcium and phosphate homeostasis: concerted interplay of new regulators. Ann Med. 2008;40:82–91. doi: 10.1080/07853890701689645. [DOI] [PubMed] [Google Scholar]
  54. Riggs BL, Khosla S, Melton LJ., 3rd Sex steroids and the construction and conservation of the adult skeleton. Endocr Rev. 2002;23:279–302. doi: 10.1210/edrv.23.3.0465. [DOI] [PubMed] [Google Scholar]
  55. Rinaldi S, Geay A, Dechaud H, Biessy C, Zeleniuch-Jacquotte A, Akhmedkhanov A, Shore RE, Riboli E, Toniolo P, Kaaks R. Validity of free testosterone and free estradiol determinations in serum samples from postmenopausal women by theoretical calculations. Cancer Epidemiol Biomarkers Prev. 2002;11(10 Pt 1):1065–1071. [PubMed] [Google Scholar]
  56. Robbins JA, Aragaki A, Crandall CJ, Manson JE, Carbone L, Jackson R, Lewis CE, Johnson KC, Sarto G, Stefanick ML, Wactawski-Wende J. Women’s health initiative clinical trials: interaction of calcium and vitamin D with hormone therapy. Menopause. 2013;21:116–123. doi: 10.1097/GME.0b013e3182963901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Rohrmann S, Nelson WG, Rifai N, Brown TR, Dobs A, Kanarek N, Yager JD, Platz EA. Serum estrogen, but not testosterone, levels differ between black and white men in a nationally representative sample of Americans. J Clin Endocrinol Metab. 2007;92:2519–2525. doi: 10.1210/jc.2007-0028. [DOI] [PubMed] [Google Scholar]
  58. Rossouw JE, Anderson GL, Prentice RL, Lacroix AZ, Kooperberg C, Stefanick ML, Jackson RD, Beresford SA, Howard BV, Johnson KC, Kotchen JM, Ockene J Group Writing for the Women’s Initiative Health Investigators. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the women’s health initiative randomized controlled trial. JAMA. 2002;288:321–333. doi: 10.1001/jama.288.3.321. [DOI] [PubMed] [Google Scholar]
  59. Rothman KJ. No adjustments are needed for multiple comparisons. Epidemiology. 1990;1:43–46. [PubMed] [Google Scholar]
  60. Selvin E, Manzi J, Stevens LA, Van Lente F, Lacher DA, Levey AS, Coresh J. Calibration of serum creatinine in the National Health and Nutrition Examination Surveys (NHANES) 1988–1994, 1999–2004. Am J Kidney Dis. 2007;50:918–926. doi: 10.1053/j.ajkd.2007.08.020. [DOI] [PubMed] [Google Scholar]
  61. Takeda E, Yamamoto H, Nashiki K, Sato T, Arai H, Taketani Y. Inorganic phosphate homeostasis and the role of dietary phosphorus. J Cell Mol Med. 2004;8:191–200. doi: 10.1111/j.1582-4934.2004.tb00274.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Tonelli M, Sacks F, Pfeffer M, Gao Z, Curhan G Cholesterol & Recurrent Events Trial Investigators. Relation between serum phosphate level and cardiovascular event rate in people with coronary disease. Circulation. 2005;112:2627–2633. doi: 10.1161/CIRCULATIONAHA.105.553198. [DOI] [PubMed] [Google Scholar]
  63. Uebelhart B, Herrmann F, Rizzoli R. Effects of the serm raloxifene on calcium and phosphate metabolism in healthy middle-aged men. Clin Cases Miner Bone Metab. 2009;6:163–168. [PMC free article] [PubMed] [Google Scholar]
  64. Ueland T, Yndestad A, Oie E, Florholmen G, Halvorsen B, Froland SS, Simonsen S, Christensen G, Gullestad L, Aukrust P. Dysregulated osteoprotegerin/rank ligand/rank axis in clinical and experimental heart failure. Circulation. 2005;111:2461–2468. doi: 10.1161/01.CIR.0000165119.62099.14. [DOI] [PubMed] [Google Scholar]
  65. Van Hemelrijck M, Michaelsson K, Linseisen J, Rohrmann S. Calcium intake and serum concentration in relation to risk of cardiovascular death in NHANES III. PLoS ONE. 2013;8:e61037. doi: 10.1371/journal.pone.0061037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Vermeulen A, Verdonck L, Kaufman JM. A critical evaluation of simple methods for the estimation of free testosterone in serum. J Clin Endocrinol Metab. 1999;84:3666–3672. doi: 10.1210/jcem.84.10.6079. [DOI] [PubMed] [Google Scholar]
  67. Wang J, Stern PH. Sex-specific effects of estrogen and androgen on gene expression in human monocyte-derived osteoclasts. J Cell Biochem. 2011;112:3714–3721. doi: 10.1002/jcb.23297. [DOI] [PubMed] [Google Scholar]
  68. Wulaningsih W, Michaelsson K, Garmo H, Hammar N, Jungner I, Walldius G, Holmberg L, Van Hemelrijck M. Inorganic phosphate and the risk of cancer in the Swedish AMORIS study. BMC Cancer. 2013;13:257. doi: 10.1186/1471-2407-13-257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Yang YH, Chen K, Li B, Chen JW, Zheng XF, Wang YR, Jiang SD, Jiang LS. Estradiol inhibits osteoblast apoptosis via promotion of autophagy through the ER-ERK-mTOR pathway. Apoptosis. 2013;18:1363–1375. doi: 10.1007/s10495-013-0867-x. [DOI] [PubMed] [Google Scholar]
  70. Yi S, Selvin E, Rohrmann S, Basaria S, Menke A, Rifai N, Guallar E, Platz EA, Astor B. Endogenous sex steroid hormones and measures of chronic kidney disease (CKD) in a nationally representative sample of men. Clin Endocrinol (Oxf) 2009;71:246–252. doi: 10.1111/j.1365-2265.2008.03455.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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