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Andrology. 2014 November ; 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. Wulaningsih1, M. Van Hemelrijck1, K. Michaelsson2, N. Kanarek3,4, W. G. Nelson4,5,6, J.
H. Ix7, E. A. Platz4,5,8, and S. Rohrmann9

1Cancer Epidemiology Unit, Division of Cancer Studies, King’s College London, School of
Medicine, London, UK 2Department of Surgical Sciences, Uppsala University, Uppsala, Sweden
3Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public

Health, Baltimore, MD 4Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins,

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Baltimore, MD 5Department of Urology and the James Buchanan Brady Urological Institute,
Johns Hopkins School of Medicine, Baltimore, MD 6Departments of Oncology, Pathology,
Pharmacology and Molecular Sciences, Radiation Oncology and Molecular Radiation Sciences,
Johns Hopkins School of Medicine, Baltimore, MD 7Division of Nephrology and Hypertension,
Department of Medicine, University of California San Diego, San Diego, CA 8Department of
Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA 9Institute
of Social and Preventive Medicine, University of Zurich, Zurich, Switzerland

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
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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

Correspondence: Sabine Rohrmann, Institute of Social and Preventive Medicine, University of Zurich, Hirshengraben 84, CH-8001
Zurich, Switzerland. sabine.rohrmann@ifspm.uzh.ch.
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.
 Wulaningsih et al. Page 2

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
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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
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(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
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(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

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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
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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
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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
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(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

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full equations with an example are available in the website of International Society for the
Study of the Aging Male (2014, www.issam.ch).
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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
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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
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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

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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
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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
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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
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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.

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.

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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
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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).

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
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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).

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.
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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

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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).
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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.
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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
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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.

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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
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(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
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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/
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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

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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
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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
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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.
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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.

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

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Figure 1.
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.
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Table 1

Age-adjusted (standardized to the 2000 US Census age distribution) weighted characteristics of study
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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)
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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)
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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)

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Unweighted sample size Age-adjusted weighted mean/proportion (SE)

AAG (ng/L)
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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)

NHANES III, National Health and Nutrition Examination Survey III; eGFR, Estimated glomerular filtration rate; SHBG, sex hormone-binding
globulin; AAG, androstanediol glucuronide.
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Table 2

Geometric mean (95% CI) of Pi levels by quintiles of sex hormones and vitamin D in a nationally
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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)
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<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)
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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)

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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)

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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.
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Table 3

Geometric mean (95% CI) of Pi levels by quintiles of sex hormones and vitamin D in a nationally
NIH-PA Author Manuscript

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)
NIH-PA Author Manuscript

<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)
NIH-PA Author Manuscript

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)

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 Wulaningsih et al. Page 20

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)

NIH-PA Author Manuscript

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
NIH-PA Author Manuscript

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.
NIH-PA Author Manuscript

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 Wulaningsih et al. Page 21

Table 4

Geometric means (95% CI) of Pi levels by quintiles of sex hormones in a nationally representative sample of
NIH-PA Author Manuscript

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)
NIH-PA Author Manuscript

<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)
NIH-PA Author Manuscript

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)

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 Wulaningsih et al. Page 22

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)

NIH-PA Author Manuscript

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

NHANES III, National Health and Nutrition Examination Survey III; SHBG, sex hormone-binding globulin; AAG, androstanediol glucuronide; Pi,
inorganic phosphate.
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Andrology. Author manuscript; available in PMC 2015 February 11.