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Original Article Relationship Between Serum Uric Acid and Bone Mineral Density in the General Population and in Rats with Experimental Hyperuricemia† Dihua Zhang, MD 1,2 *#, I. Alexandru Bobulescu, MD 1,2 *, Naim M. Maalouf, MD 1,2, Beverley Adams-Huet, MS 1,2,3, John Poindexter, BS 1,2, Sun Park, BS 2, Fuxin Wei 4, Christopher Chen, PhD 2,4, Orson W. Moe, MD 1,2,5, Khashayar Sakhaee, MD 1,2 1

Department of Internal Medicine, 2 The Charles and Jane Pak Center for Mineral Metabolism and Clinical Research, 3 Department of Clinical Sciences, 4 Department of Orthopaedic Surgery and 5 Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas, USA

* The first two authors (DZ and IAB) contributed equally to this work. #

DZ present address: Department of Nephrology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, People’s Republic of China. Address correspondence to: Khashayar Sakhaee, MD University of Texas Southwestern Medical Center The Charles and Jane Pak Center for Mineral Metabolism and Clinical Research 5323 Harry Hines Blvd Dallas, TX 75390-8885, USA Telephone: 214-648-9640; E-mail: [email protected]



This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/jbmr.2430]

Initial Date Submitted October 14, 2014; Date Revision Submitted November 24, 2014; Date Final Disposition Set December 8, 2014

Journal of Bone and Mineral Research This article is protected by copyright. All rights reserved DOI 10.1002/jbmr.2430

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Grant support: R01-DK081423 (to K. Sakhaee), R01-DK081423-06 (to K. Sakhaee & O.W. Moe), K01-DK090282 (to I.A. Bobulescu), P30-DK079328 (UT Southwestern O’Brien Kidney Research Core Center) Disclosures: During the studies, I.A. Bobulescu and Naim M. Maalouf received grant support for unrelated projects via the Investigator-Initiated Sponsored Research (IISR) mechanism from Takeda Pharmaceutical North America, and O.W. Moe received an investigator-initiated GRIP grant from Genzyme Corporation. There was no funding for this article, and no involvement from Takeda or any other commercial entity, directly or indirectly, in any aspect of the article. All other authors have nothing to disclose.

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Abstract

Higher serum uric acid concentrations have been associated with higher bone mineral

density in observational studies of older men and peri- or postmenopausal women, prompting speculation of a potential protective effect of uric acid on bone. Whether this relationship is present in the general population has not been examined and there is no data to support causality. We conducted a cross-sectional analysis of a probability sample of the US population. Demographic data, dietary intake, lifestyle risk factors and physical activity assessment data, serum biochemistry including serum uric acid, and bone mineral density were obtained from 6,759 National Health and Nutrition Examination Survey (NHANES; 2005-2010) participants over 30 years of age. In unadjusted analyses, higher serum uric acid levels were associated with higher bone mineral density at the femoral neck, total hip and lumbar spine in men, premenopausal women, and post-menopausal women not treated with estrogen. However, these associations were no longer statistically significant after adjustment for potential confounders, including age, body mass index, black race, alcohol consumption, estimated glomerular filtration rate (eGFR), serum alkaline phosphatase, and C-reactive protein (CRP). This is in contradistinction to some prevailing conclusions in the literature. To further examine the causal effect of higher serum uric acid on skeletal health, including biomechanical properties that are not measurable in humans, we used an established rat model of inducible mild hyperuricemia. There were no differences in bone mineral density, volume density, and biomechanical properties between hyperuricemic rats and normouricemic control animals. Taken together, our data do not support the hypothesis that higher serum uric acid has protective effects on bone health. This article is protected by copyright. All rights reserved

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Introduction

More than 10 million individuals in the United States are estimated to have osteoporosis, and

an additional 30 million have osteopenia (1). Uric acid is the final product of purine metabolism in humans and higher primates, and has been postulated to play a role in antioxidation (2),

although the relative importance of uric acid as antioxidant in vivo remains controversial (3). A number of recent studies have shown that higher serum uric acid levels associate with surrogate markers of better bone health, leading to speculation about a potential protective role of uric acid against bone loss (4-9). One proposed mechanism by which hyperuricemia could contribute to

higher BMD is via the potential anti-oxidant effects of uric acid, which in turn may inhibit osteoclastic bone resorption. However, this theory is highly speculative, and whether uric acid is indeed an antioxidant in vivo in humans remains controversial (3) In a cross-sectional study of men aged 70 or over, higher serum uric acid levels were associated with higher bone mineral density at various skeletal sites and a lower prevalence of non-vertebral fractures after adjusting for multiple covariates (4). Another recent observational study in peri- and postmenopausal women showed a similar positive correlation between serum uric acid and bone mineral density and in the rate of change in bone mineral density over time (6). Based on these associations, a model of a protective effect of uric acid on bone was postulated. Whether serum uric acid is independently associated with bone mineral density in the general population has not been evaluated. In the present study, we examined the relationship between serum uric acid and bone mineral density in a large database from the nationally representative National Health and Nutrition Examination Survey (NHANES) cohort. To examine the effect of uric acid on bone mineral density as well as bone biomechanical parameters under controlled conditions, we also conducted experiments in an established rodent model of inducible mild hyperuricemia and

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examined the bone.

Methods

Study population The National Health and Nutrition Examination Survey (NHANES) is a population-based

health examination survey that provides nationally representative cross-sectional data on the health status of the civilian, non-institutionalized US population. The design and operation of NHANES have been described on the Centers for Disease Control and Prevention (CDC) NHANES website (10), from which all data were downloaded. We studied the population from the NHANES 2005–2010 biennial surveys which specifically measured bone mineral density. A

total of 6,759 participants 30 years of age or older with available serum uric acid and bone mineral density data were included in this study, after exclusion of individuals with estimated

GFR < 60 ml/min/1.73m2, diagnosed diabetes, unknown menopausal status, as well as exclusion of those taking bisphosphonates, beta-blockers, corticosteroids, or allopurinol. Written informed consent was obtained from all adult participants in NHANES. Questionnaire information in NHANES provided the participants’ gender, age, and race/ethnicity.

Reproductive health variables The reproductive health questionnaire in NHANES was a complex, detailed set of questions

for women, including menstrual history, pregnancy history, lactation, oral contraceptive and hormone replacement therapy use and related conditions. Key variables of interest for this study included postmenopausal status and use of estrogen estrogen-containing agents. Post-menopausal

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status was defined as women who were ≥ 65 years old, or who had no menstrual cycles in the last 12 months, after excluding potential causes of physiological or pathological amenorrhea, including pregnancy and breastfeeding. For post-menopausal women, estrogen use was defined as a current user of estrogen or estrogen containing agents, such as estrogen pills/combo pills or estrogen patches/combo patches.

Co-morbidities/disease history and prescription medication use Co-morbidity and disease history were defined as self-reported physician diagnosis of

diabetes mellitus, liver diseases, thyroid diseases and malignant diseases. The prescription medication questionnaires provided personal interview data on use of prescription medications during a one-month period prior to the survey date.

Body measurements and dietary intake variables Key study variables of interest included height, weight, body mass index and body surface

area. Average daily nutrient intake estimation was based on 24-hour dietary recall. Data were collected by a trained dietary interviewer using the NHANES Dietary Data Collection system. Nutrient variables of interest included 24-hour intake of energy and protein.

Estimated glomerular filtration rate (eGFR) Estimated glomerular filtration rate (eGFR) was calculated from age, sex, race/ethnicity,

serum creatinine concentration, blood urea nitrogen, and albumin levels using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation (11). Body surface area calculated with Mosteller formula (12) was used to calculate the adjusted eGFR.

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Serum chemistry Routine blood biochemistry profile included serum uric acid, total protein, blood urea

nitrogen, creatinine, total calcium, phosphorus and alkaline phosphatase were measured with a Beckman Synchron analyzer (Brea, CA). C-reactive protein (CRP) assays were performed on a Behring Nephelometer. In the NHANES 2005-2006 cycle only, serum PTH was measured by an electrochemiluminescence immunoassay on the Elecsys 1010 autoanalyzer (Roche Diagnostics, Indianapolis, IN) and serum 25 hydroxy vitamin D was measured using a Diasorin (formerly Incstar) 25-OH-D assay (Stillwater, MN).

Bone mineral density measurements The DXA examination was administered by trained and certified radiology technologists in

NHANES. Participants had their femur bone and lumbar spine (vertebrae L1 – L4) measured by

dual energy X-ray absorptiometry (DXA) with a Hologic QDR-4500A fan-beam densitometer (Hologic, Inc., Bedford, Massachusetts). Respectively, Hologic Discovery software, version 12.4, and Hologic software, APEX v3.0, were used to analyze femoral and spine scans acquired in 2005-2010. Total hip, femoral neck and L1-L4 spine bone mineral density were included in the data analyses.

Animal study All experimental procedures were performed according to protocols approved by the

Institutional Animal Care and Use Committee at UT Southwestern Medical Center. Eight 5-week old male rats (strain code 371, non-obese, non-diabetic) were purchased from Charles River

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Laboratories, Wilmington, MA. The rats were fed normal rodent chow for 1 week and then randomized to either rodent chow supplemented with 2% potassium oxonate as a uricase inhibitor to induce persistent hyperuricemia (13) or calorie- and nutrient-matched control chow (Test Diet, Richmond, IN) ad libitum. Sustained hyperuricemia was induced in rats from week 6 of life and lasted until sacrifice at 15-16 weeks. Serum uric acid and creatinine levels (Vitros Chemistry) were measured in serial fasting blood samples. Animals were sacrificed under anesthesia with Ketamine / Xylazine / Acepromazine (100 mg/kg / 10 mg/kg / 1 mg/kg, intraperitoneally), and both femurs of each animal were dissected, cleaned of soft tissue, and stored in 70% alcohol at -4°C.

Micro-CT of rat femurs Before scanning, femurs were positioned with gauze in the sample holder and allowed to

reach room temperature. The femurs was scanned at 12µm isotropic voxel size using an eXplore Locus SP (GE Healthcare, London, Ontario, Canada). Reconstructed volumes were processed using the Microview software (GE Healthcare) and analyzed for bone mineral density (BMD),

bone volume fraction (BV/TV) and trabecular morphometry including trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and trabecular number (Tb.N).

Biomechanical testing of rat femurs For biomechanical properties, femurs were rehydrated in PBS and preconditioned with 20

cycles of bending (0.2mm) load using a material testing system (Bose Electroforce model # 3230, Eden Prairie, MN). The sample was then loaded to failure by three-point bending (6mm and 6mm apart) under displacement control (0.06 mm/sec). Yield Load (N), Fracture Load (N)

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and Fracture Energy (mJoule), and Bending Stiffness (N/mm) from the linear region of displacement-load curve were determined (14).

Statistical analyses Due to the complex sampling design of NHANES, all analyses incorporate the study visit

weights, primary sampling unit, and stratification design of the NHANES surveys. Our analyses were conducted in four subgroups, males, pre-menopausal females, post-menopausal women not treated with estrogen, and post-menopausal women on estrogen treatment. Results are reported as weighted means and 95% confidence intervals (CI) for continuous variables and as percentage and 95% CI for categorical variables. Statistical analyses were performed with SAS version 9.3 survey procedures (SAS Institute, Cary, NC) and utilize the entire NHANES 2005-2010 data set with the DOMAIN statement to obtain the variance estimates. A two-sided P value below 0.05 was considered statistically significant. The analysis aimed to investigate the relationship between serum uric acid and bone mineral density while controlling for different confounding factors. Potential confounding factors included demography data, lifestyle risk factors, dietary intake factors, body measurements, blood biochemistry, and co-morbidities. Using Proc SURVEYREG, regression analysis was performed to estimate unadjusted and multivariableadjusted weighted means for bone mineral density, which were subsequently compared across subgroup-specific quartiles of serum uric acid; tests for trends of unadjusted or adjusted bone mineral density means were conducted by using orthogonal linear contrasts. All multivariable models included age, body mass index (BMI), black race, alcohol consumption, eGFR, serum alkaline phosphatase, and c-reactive protein (CRP) since these variables were statistically significant in most models. Serum alkaline phosphatase and CRP were log transformed prior to

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regression analysis. In addition, the following independent variables were considered for inclusion in the regression models: smoking, total energy and protein dietary intake, serum calcium, PTH, and 25 hydroxy vitamin D. A similar regression analysis was conducted on a subgroup of age 65 years and older in men and post-menopausal women not treated with estrogen, and on a subgroup with eGFR ≥90 ml/min/1.73 m2. Student’s t test and ANOVA were used to analyze animal data, with a two-sided P value below 0.05 considered statistically

significant. This study had a power of 0.97 at a two-sided alpha of 0.05 to detect a linear trend in bone mineral density of 0.015 g/cm2 per serum uric acid quartile for males, premenopausal females, and postmenopausal females with estrogen replacement with standard deviation of 0.15 g/cm2. In the smaller subgroup of postmenopausal females with estrogen replacement, the power was 0.80 to detect a linear trend in bone mineral density of 0.03 g/cm2 per serum uric acid quartile.

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Results

Our human study dataset consisted of 6,759 NHANES participants, including 3,496 men and

3,263 women, the latter stratified by menopausal status and post-menopausal estrogen use.

Baseline demographic and clinical characteristics of the study population are presented in Table 1. Baseline serum biochemistry and bone mineral density are presented in Table 2.

Unadjusted association between serum uric acid and bone mineral density Bone mineral density measured at the total hip (Figure 1A), femoral neck (Figure 1B) and

lumbar spine (Figure 1C) increased significantly across quartiles of serum uric acid in men, premenopausal women, and post-menopausal women not treated with estrogen. Qualitatively similar trends were observed in post-menopausal women on estrogen treatment, but statistical significance was reached only for lumbar spine BMD.

Association between serum uric acid and bone mineral density after adjustment for confounding variables Multiple regression analysis was conducted to investigate the association between serum

uric acid and BMD after controlling for potential confounders. After adjustment for age, body

mass index, black race, alcohol consumption, estimated glomerular filtration rate (eGFR), serum alkaline phosphatase, and C-reactive protein (CRP), there were no differences in bone mineral density at the total hip (Figure 1D), femoral neck (Figure 1E) and lumbar spine (Figure 1F) across serum uric acid quartiles (Table S1) . Similar results were obtained in an older (age ≥ 65 years) subset of our NHANES 2005-2010 dataset (Supplemental Figure S1), as well as after

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exclusion of subjects on thiazide diuretics from the NHANES 2005-2010 dataset (Supplemental Figure S2), and after adjustment for PTH and 25 hydroxy vitamin D, only available in the NHANES 2005-2006 dataset (Supplemental Figure S3). A further sensitivity analysis, using an eGFR cut-off of 90 ml/min/1.73 m2 to completely exclude any potential interference of kidney function, also led to similar findings.

Effect of chronic hyperuricemia on bone properties in rats Oxonate-treated animals developed persistent mild hyperuricemia throughout the

duration of the experiment (Figure 2A), with no significant differences in body mass compared with control animals (Figure 2B). After 10 weeks of treatment, there were no significant

differences in bone mineral density (BMD) (Figure 2C) assessed by micro-CT (15) between hyperuricemic and control animals in cortical bone (326±27 vs. 336±37 mg/cm3, p=0.73) and trabecular bone (121±29 vs. 137±24 mg/cm3, p=0.43). Representative micro-CT images are shown in Figure S4. There were also no significant differences in bone volume fraction (BV/TV) (Figure 2D and Table S1) and measures of trabecular morphometry, including trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and trabecular number (Tb.N) (Table S2).

Using 3-point bending and fracture tests, we found no significant differences between groups in bone bending stiffness (biomechanical bending strength, Figure 2E), yield load (the load required to induce plastic deformation, Figure 2F), fracture load (the load required for bone fracture, Figure 2G) and fracture energy (the energy needed to induce bone fracture, Figure 2H).

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Discussion

The key finding from this large, cross-sectional study of 6,759 male and female NHANES

participants is that serum uric acid levels were not associated with bone mineral density at the total hip, femoral neck or spine after adjustment for age, BMI, eGFR and other confounders. To our knowledge, this is the first reported cross-sectional study to examine the association between serum uric acid and bone mineral density in the general population. In addition, induction of chronic mild hyperuricemia in a rodent model did not result in significant differences in bone mineral density, volume density, and biomechanical properties. High serum uric acid levels have been associated with a number of conditions including

cardiovascular disease, stroke (16, 17), hypertension, progressive renal disease (18, 19), diabetes mellitus (20), insulin resistance and the metabolic syndrome (21), but causality has been difficult to establish. In contrast with these potential deleterious effects, observational studies have suggested that uric acid might protect against bone loss. In 75 subjects with osteoporosis and 75 healthy controls, lower uric acid levels were found in osteoporotic subjects (22). In another

cross-sectional population-based study of 1,705 elderly men >70 years of age, high serum uric acid concentrations were associated with higher bone mineral density, lower bone resorption markers, and a lower prevalence of vertebral and non-vertebral fractures (4). In a longitudinal observational study, higher uric acid levels were associated with diminished bone loss at the lumbar spine, forearm, and total body in 356 peri-and postmenopausal women (6). In a second

cross-sectional study of 2,190 elderly persons, higher serum uric acid levels were associated with a lower prevalence of osteoporosis (23). Finally, in a third cross-sectional study of 7,502 healthy

postmenopausal women, higher serum uric acid concentrations were associated with higher bone

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mineral density at the lumbar spine and all proximal femoral sites after adjustment for multiple variables, as well as with lower bone turnover and lower prevalence of vertebral spine fragility fractures (5). Finally, using the Osteoporotic Fractures in Men (MrOS) database, higher serum uric acid levels were associated with significant reduction in incident non-spine fractures (9).

Study findings in context Our study did not find a positive association between serum uric acid and bone mineral

density in NHANES participants. Furthermore, chronic mild elevation of serum uric acid in an

established rodent model did not affect BMD and bone biomechanical properties. There may be a number of reasons for the apparent discrepancy between our findings and the results of prior observational studies in humans. Most importantly, the present study included subjects with a wide age range (30 to 85 years), rather than just peri- and postmenopausal women or elderly men. It is theoretically possible that uric acid has protective effects only in older individuals at higher risk for bone loss. However, this is not supported by our subgroup analysis in subjects ≥ 65 years of age (Supplemental Figure S1). Other potential causes for discrepancy between studies may be differences in the ethnic/racial makeup of the study populations, and differences in the number of potential confounders included in multivariable adjustment models. Serum uric acid levels in the highest quartile in our study was similar to uric acid levels in the highest quartiles in the previous studies (4, 9) (Supplemental Table 1).Finally, the predominance of

studies supporting a link between serum uric acid and bone may also be in part attributable to publication bias, with “negative” data being much more likely to be under-reported.

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Strengths and Limitations To our knowledge, this is the first study showing a lack of association between serum uric

acid and BMD in a large population-based cohort. The key strengths of this study include the

nationally-representative cross-sectional data, strict inclusion and exclusion criteria, and thorough adjustment for confounding variables. In addition to our data in humans, we also studied the effect of chronic elevation of serum uric acid in tightly controlled conditions in rodents, with detailed measurements of BMD as well as bone biomechanical properties. This study also has limitations. While compromised bone quality contributes to fracture risk,

our NHANES data was limited to assessment of BMD, and lacks assessment of bone quality. However, in our animal model, estimates of bone strength were made, and indicate that hyperuricemia did not adversely impact bone strength. It also remains possible that uric acid has a beneficial effect on bone health in certain population subsets, but this effect was not captured by our study in the general population. For our animal study, we cannot exclude the possibility that hyperuricemia of longer duration, higher severity, or in animals with additional risk factors (e.g. senescence, post-menopausal state, or experimental induction of a high oxidative stress status) could yield different results. However, 9 weeks in rats (the duration of hyperuricemia in our experiment) is comparable with 6 human years (24) during which time serum uric acid levels were roughly doubled compared with those in control animals.

Conclusions Serum uric acid was not associated with bone mineral density in multivariable analyses after

adjustment for confounding variables in a large population-based cohort representative of the US adult population. Serum uric acid was also not associated with bone mineral density and bone

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biomechanical properties in a rodent model of chronic mild hyperuricemia. The association between serum uric acid and bone mineral density in specific population subsets at increased risk for bone loss requires further investigation.

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Acknowledgements

The authors would like to acknowledge funding by R01-DK081423 (to K. Sakhaee), R01DK081423-06 (to K. Sakhaee & O.W. Moe), K01-DK090282 (to I.A. Bobulescu), the Visiting

Scholars Program jointly sponsored by Sun Yat-Sen University First Affiliated Hospital and the University of Texas Southwestern Medical Center, the O’Brien Kidney Research Center (P30DK07938), and support from the Pak-Seldin Center of Human Metabolic Research in the Charles and Jane Pak Center for Mineral Metabolism and Clinical Research. Dr’s Zhang and Maalouf had major roles in data interpretation, intellectual content of the human study, and revision of final manuscript. Dr’s Bobulescu and Moe had a principle role in the conduct, intellectual content of the animal studies, and revision of final manuscript. Dr. Chen had a principle role in micro-CT analysis and revision of final manuscript.

Beverley Huet and John Poindexter

contributed to the study design, data analysis, data interpretation, and revision of final

manuscript. Dr. Sakhaee had an overall principle role in intellectual content, study design, data interpretation, and revision of final manuscript. Sun Park and Fuxin Wei had a major technical support role for the animal studies. All authors contributed to revision of the manuscript and approval of final version. Ms. Ashlei L. Johnson and Ms. Valeria Rodela for their assistance in the preparation of the manuscript.

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Table 1. Baseline demographic and clinical characteristics of the study population.

Men

Women, pre-

Women, post-

Women, post-

menopausal

menopausal,

menopausal,

not on estrogen

estrogen-treated

N, unweighted

3496

1741

1365

157

Age (years)

46.8 (46.2, 47.4)

40.7 (40.2, 41.1)

58 (57.4, 58.6)

54.8 (53, 56.5)

Body mass (kg)

86.3 (85.6, 87)

73.5 (72.4, 74.5)

72.9 (71.8, 74.1)

69.9 (67.7, 72.2)

Height (cm)

175.8

163.2

161.2

162

(175.4, 176.2)

(162.8, 163.6)

(160.8, 161.5)

(160.8, 163.2)

BMI (kg/m )

27.9 (27.7, 28.1)

27.6 (27.2, 28)

28.1 (27.6, 28.5)

26.6 (25.9, 27.2)

Hispanic (%)

14.5 (11.7, 17.5)

14 (11.2, 16.9)

9.4 (6.6, 12.2)

5.3 (3.3, 7.4)

Non-Hispanic White (%)

69.8 (65.8, 73.7)

70 (65.5, 74.4)

75.2 (70.5, 80)

83.6 (77.4, 89.9)

Non-Hispanic Black (%)

9.1 (7.6, 10.6)

9.9 (7.9, 11.9)

10.1 (7.8, 12.4)

6.1 (2, 10.2)

Other race/ethnicity (%)

6.6 (5, 8.1)

6.1 (4.5, 7.7)

5.2 (3.1, 7.3)

4.9 (0.2, 9.7)

Smoking history (%)

53.3 (50.2, 56.3)

39.4 (36.8, 41.9)

45.9 (42.8, 49)

46.0 (39.3, 52.6)

Alcohol intake (g/day)

19.3 (17.5, 21.2)

8.3 (7.3, 9.3)

6.1 (5, 7.3)

8 (4.4, 11.6)

Caffeine intake (mg/day)

240 (227, 253)

187 (173, 202)

204 (190, 219)

197 (157, 237)

Dietary energy intake

2681 (2625, 2737)

1900 (1857, 1944)

1704 (1653, 1755)

1798 (1678, 1917)

102.2 (99.5, 104.9)

72.1 (70.2, 73.9)

65.4 (63.2, 67.6)

67.4 (61.8, 73)

Liver disease (%)

4.4 (3.5, 5.3)

2.6 (1.8, 3.5)

3.3 (1.9, 4.7)

2.3 (0, 4.9)

Thyroid disease (%)

2.6 (1.8, 3.4)

9.7 (7.9, 11.6)

18.5 (15.6, 21.4)

27.6 (19.4, 35.8)

Malignancy (%)

5.6 (4.6, 6.7)

4.6 (3.4, 5.7)

13.2 (10.8, 15.6)

17.1 (9.7, 24.6)

On diuretics (%)

4.0 (3.0, 4.9)

2.9 (2.2, 3.7)

10.5 (8.4, 12.5)

13.2 (7.3, 19.1)

2

(kcal/day) Dietary protein intake (g/day)

Weighted means, proportions, and 95% CIs are provided unless otherwise specified. Abbreviations: BMI, body mass index

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Table 2. Baseline serum biochemistry and bone mineral density. Men

Women, pre-

Women, post-

Women, post-

menopausal

menopausal,

menopausal,

not on estrogen

estrogen-treated

Reference Range

N, unweighted

3496

1741

1365

157

Total protein (g/dL)

7.16 (7.13, 7.19)

7.08 (7.05, 7.11)

7.07 (7.04, 7.11)

6.99 (6.91, 7.07)

6.4-7.7

Uric acid (mg/dL)

5.99 (5.93, 6.04)

4.47 (4.41, 4.54)

4.82 (4.74, 4.91)

4.66 (4.49, 4.83)

Men: 3.6-8.4 Women: 2.9-7.5

PTH (pg/mL)

39.5 (38.1, 40.9)

42.1 (40.2, 44.0)

44.1 (42.0, 46.2)

41 (36.2, 45.7)

18-74

25-OH-D (ng/mL)

23.6 (22.4, 24.8)

23.3 (22.1, 24.6)

23.4 (22.2, 24.6)

26.1 (24.3, 27.9)

10-55

Phosphorus (mg/dL)

3.67 (3.64, 3.69)

3.74 (3.71, 3.77)

3.94 (3.9, 3.98)

3.87 (3.76, 3.99)

2.6-4.4

Calcium (mg/dL)

9.46 (9.44, 9.49)

9.34 (9.31, 9.37)

9.51 (9.48, 9.54)

9.45 (9.4, 9.51)

8.5-10.5

Alkaline phosphatase

67.8 (66.8, 68.8)

60.5 (59.3, 61.7)

74.8 (73.5, 76.1)

65.6 (62.3, 69.0)

36-113

0.31 (0.28, 0.35)

0.39 (0.36, 0.43)

0.40 (0.36, 0.45)

0.44 (0.31, 0.57)

0-1

BUN (mg/dL)

12.9 (12.7, 13.1)

10.8 (10.6, 11.0)

12.6 (12.3, 12.9)

12.3 (11.6, 13.0)

6-23

Creatinine (mg/dL)

0.95 (0.94, 0.95)

0.75 (0.74, 0.75)

0.76 (0.75, 0.77)

0.77 (0.75, 0.79)

Men: 0.7-1.3

(U/L)

C-reactive protein (mg/dL)

2

Women: 0.6-1.1

eGFR (ml/min/1.73m )

81.9 (81.2, 82.7)

97.4 (96.1, 98.7)

85.4 (84.0, 86.8)

87.0 (8.04, 90.0)

Total femur BMD

1.024 (1.017, 1.031)

0.957 (0.949, 0.965)

0.854 (0.845, 0.864)

0.894 (0.874, 0.913)

(gm/cm²)

[n=3428]

[n=1683]

[n=1283]

[n=152]

Femoral neck BMD

0.860 (0.853, 0.866)

0.844 (0.837, 0.851)

0.735 (0.726, 0.744)

0.766 (0.748, 0.783)

(gm/cm²)

[n=3428]

[n=1683]

[n=1283]

[n=152]

L1-L4 spine BMD

1.047 (1.039, 1.056)

1.067 (1.061, 1.074)

0.95 (0.938, 0.963)

1.0 (0.975, 1.025)

(gm/cm²)

[n=2863]

[n=1600]

[n=1068]

[n=122]

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Weighted means, proportions, and 95% CIs are provided unless otherwise specified. Abbreviations and definitions: BUN, blood urea nitrogen; eGFR, estimated glomerular filtration rate; PTH, parathyroid hormone; 25-OH-D, 25 hydroxy vitamin D ; BMD, bone mineral density. Conversion factors to SI units are as follows: Total protein, g/dL x 10.0 = g/L; Uric acid, mg/dL x 0.059 = mmol/L; PTH, pg/mL x 0.1061 = pmol/L; 25-OH-D, ng/mL x 2.496 = nmol/L; Phosphorus, mg/dL x 0.3229 = mmol/L; Calcium, mg/dL x 0.2495 = mmol/L; C-reactive protein, mg/dL x 10 = mg/L; BUN, mg/dL x 0.3570 = mmol/L; Creatinine, mg/dL x 88.4 = μmol/L.

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Figure Legends

Figure 1: Bone mineral density (BMD) per quartile of serum uric acid. A-C) Unadjusted BMD. D-F) BMD adjusted for age, body mass index, black race, alcohol consumption, estimated glomerular filtration rate (eGFR), serum alkaline phosphatase, and C-reactive protein (CRP). Results are presented as the survey weighted least squares means and 95% confidence intervals from regression analysis. P-values are from the test for linear trend across serum uric acid quartiles.

Figure 2. Effect of chronic mild hyperuricemia on bone properties in rats. A) Serum uric acid. B) Body mass. C) Bone mineral density (BMD). D) Bone volume fraction (BV/TV).

E) bending stiffness (biomechanical bending strength). F) Yield load (the load required to induce plastic deformation). G) Fracture load (the load required for bone fracture). H) Fracture energy (the energy needed to induce bone fracture). Results shown are mean and standard deviation (N=4 per condition).

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Figure 1

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Figure 2

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Figure S1

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Figure S2

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Figure S3

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Figure S4

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Relationship between serum uric Acid and bone mineral density in the general population and in rats with experimental hyperuricemia.

Higher serum uric acid concentrations have been associated with higher bone mineral density (BMD) in observational studies of older men and perimenopa...
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