Cross-Sectional Associations of Serum Zinc with Bone Mineral Density in the NHANES 2011-2014 Cohort

Summary

This protocol aims to evaluate the association between serum zinc concentrations and total bone mineral density using nationally representative data from the NHANES 2011-2014 cohort.

Abstract

This cross-sectional study investigated the association between serum zinc concentrations and total bone mineral density (BMD) using data from 1,674 adults aged ≥ 20 years in the National Health and Nutrition Examination Survey (NHANES) 2011-2014. Participants with missing data on key variables were excluded, and analyses accounted for the NHANES complex survey design with 4-year fasting subsample weights (WTSAF4YR). Serum zinc levels were divided into quartiles, and multivariable linear regression models were applied to assess the relationship between serum zinc and total as well as site-specific BMD after adjustment for demographic, anthropometric, lifestyle, and biochemical confounders. Smooth curve fitting and threshold effect analyses based on generalized additive models (GAMs) were used to explore potential nonlinear associations. Serum zinc was positively associated with BMD after adjustment for covariates, with a nonlinear threshold effect observed at approximately 13.20 µmol/L, beyond which the positive association became more pronounced. These findings provide new insight into zinc's role in skeletal health, highlighting that maintaining serum zinc above this level may contribute to optimal bone density and inform strategies for osteoporosis prevention and nutritional assessment.

Introduction

Osteoporosis represents a major public health concern worldwide, affecting an estimated 18.3% of the global population^1,2. Epidemiological data indicate that this condition does not impact all demographic groups equally, with women experiencing a markedly higher burden compared to men, particularly after menopause due to hormonal changes that accelerate bone loss^1,2. Bone mineral density (BMD) serves as a fundamental indicator of skeletal health and is widely recognized as a crucial determinant of bone strength³. Clinically, it is considered one of the most reliable predictors of an individual's susceptibility to fractures³. Individuals with higher BMD generally possess stronger, more resilient bones, which confer protection against bone fragility and the occurrence of fractures^4,5. Conversely, reduced BMD is associated with compromised bone integrity, placing individuals at elevated risk for conditions such as osteopenia and osteoporosis, as well as fracture-related morbidity^4,5.

BMD decline is a multifactorial and biologically intricate process influenced by a constellation of factors, including aging, hormonal milieu, lifestyle behaviors, nutritional status, and underlying pathological conditions⁶. With advancing age, the rate of osteoblastic bone formation progressively diminishes, whereas osteoclastic bone resorption becomes relatively enhanced, resulting in a net loss of bone mass over time⁷. Among the biological determinants, hormonal alterations, particularly the marked reduction in circulating estrogen following menopause, constitute one of the principal drivers of rapid bone demineralization⁸. Nutritional inadequacies, such as insufficient intake of calcium, vitamin D, protein, and essential trace elements like magnesium and zinc, further compromise skeletal integrity by impairing bone mineralization and matrix formation^3,8. Lifestyle-related factors, including physical inactivity, cigarette smoking, excessive alcohol consumption, and chronic psychosocial stress, exacerbate bone resorption and accelerate bone loss⁹. Additionally, prolonged administration of glucocorticoids, anticonvulsants, or aromatase inhibitors, as well as chronic systemic disorders such as diabetes mellitus, renal insufficiency, and gastrointestinal diseases, can perturb calcium homeostasis and disrupt normal bone remodeling, collectively contributing to the progressive reduction in BMD and heightened susceptibility to osteoporosis³.

Zinc, as a vital trace element, is integral to the regulation of bone metabolism and the preservation of skeletal integrity¹⁰. Accumulating evidence indicates a positive correlation between zinc status and BMD, although the magnitude and consistency of this relationship are influenced by baseline zinc levels, existing bone health, and demographic characteristics of the study population¹¹. From a mechanistic perspective, zinc facilitates osteogenesis by enhancing osteoblast differentiation, promoting collagen matrix synthesis, and activating critical enzymes such as alkaline phosphatase, which is essential for proper bone mineralization¹⁰. Concurrently, zinc exerts inhibitory effects on osteoclast-mediated bone resorption, thereby contributing to the homeostatic balance between bone formation and degradation^10,12. Observational and interventional studies have shown that higher circulating zinc concentrations are generally associated with increased BMD, whereas zinc insufficiency correlates with elevated osteoporosis risk¹².

In the present study, we leveraged data from the National Health and Nutrition Examination Survey (NHANES) 2011-2014, which provides a large, nationally representative cohort of U.S. adults with comprehensive biochemical, demographic, and lifestyle data. Compared with previous investigations, this approach offers several methodological advantages, including an expanded sample size that enhances statistical power and the ability to detect subtle associations, as well as the implementation of threshold modeling to explore potential nonlinear relationships between serum zinc concentrations and BMD. By systematically examining zinc-BMD associations across different quartiles and adjusting for relevant confounders, our study provides a more nuanced understanding of how zinc status may influence skeletal health. Importantly, the findings derived from this nationally representative dataset can inform clinical decision-making and public health strategies, enabling clinicians and researchers to better assess the potential role of zinc in bone health maintenance and osteoporosis prevention.

Protocol

The National Center for Health Statistics (NCHS) Research Ethics Review Board (ERB) approved the NHANES data collection protocols. Since the NHANES datasets are publicly accessible, researchers are exempt from obtaining additional approval from their own institutional review boards (IRBs).

Study design and characteristics of the study population

In the present cross-sectional investigation, we sought to examine the potential independent association between serum zinc concentrations and BMD utilizing data derived from the 2011-2014 cycles of the NHANES. NHANES, which is administered by the National Center for Health Statistics (NCHS), employs a carefully designed stratified, multistage probability sampling methodology to generate a nationally representative dataset of the civilian, non-institutionalized population of the United States. Each survey cycle systematically collects extensive information encompassing demographic characteristics, lifestyle factors, dietary intake, clinical parameters, and anthropometric measurements through a combination of standardized interviews, structured questionnaires, and physical examinations. Comprehensive protocols and methodological details for each survey component are publicly accessible on the official NHANES website. For the purposes of this analysis, we focused on adults aged 20 years and older and excluded individuals with missing or incomplete data on key anthropometric measures or other essential covariates required for the study models. The process of participant selection, along with specific inclusion and exclusion criteria, is visually summarized in Figure 1 to provide a clear overview of the analytic sample.

Assessment indicators

Assessment of Anthropometric Measures

Trained and certified health technicians performed the collection of anthropometric measurements, including body weight and standing height, at dedicated NHANES mobile examination centers, following rigorously standardized protocols established by the NCHS. Body weight was recorded to the nearest 0.1 kg using a calibrated digital scale, ensuring consistency and accuracy across all measurements. Standing height was measured to the nearest 0.1 centimeter employing a fixed stadiometer, with participants instructed to maintain an upright posture and heels together for precision. Subsequently, body mass index (BMI), a widely recognized indicator of general adiposity, was computed for each participant by dividing the measured weight in kilograms by the square of the height in meters (kg/m²).

Zinc status in serum

Venous blood specimens were obtained from study participants at the designated mobile examination centers (MECs) by certified phlebotomists following standardized NHANES procedures. Collected samples were promptly processed and transported under controlled conditions by trained personnel to the National Center for Environmental Health (NCEH) laboratory for subsequent biochemical analysis. After initial centrifugation, serum aliquots were kept at °C during transit to preserve sample integrity and were then stored at ultra-low temperatures of either -20 °C or -70 °C until the time of analysis. Quantification of serum zinc concentrations was performed using inductively coupled plasma dynamic reaction cell mass spectrometry (ICP-DRC-MS), a highly sensitive and precise analytical method. Comprehensive details regarding the laboratory protocols, quality control measures, and analytical specifications for serum zinc assessment are documented in the NHANES laboratory data file (LBDZN), which can be accessed on the official NHANES website, with further methodological description provided at: https://wwwn.cdc.gov/nchs/nhanes/2011-2012/PBCD_G.htm.

BMD

All participants who met the inclusion criteria for this study underwent dual-energy X-ray absorptiometry (DXA) scans to obtain precise measurements of BMD. The scans were conducted by certified radiologic technologists using a fan-beam densitometer, with data acquisition and analysis performed through Hologic APEX software (version 4.0) to ensure accuracy and consistency. The standardized DXA examination protocols, including participant positioning, calibration procedures, and quality control measures, are fully detailed on the official NHANES website, with the corresponding link provided in the Table of Materials. Certain individuals were excluded from undergoing DXA assessment based on specific safety and technical criteria, including pregnancy, administration of contrast agents within the preceding seven days, body weight exceeding 450 pounds, multiple prior hip replacements, bilateral hip fractures, or the presence of hip prostheses, in order to maintain the validity and reliability of BMD measurements.

Covariates

To rigorously assess the association between serum zinc concentrations and BMD, it was essential to account for potential confounding variables and the complex interactions among multiple covariates. Demographic factors included participants' age (≥20 years), sex (male or female), race/ethnicity (categorized as Mexican American, non-Hispanic White, non-Hispanic Black, other Hispanic, and other races), educational level (less than high school, high school or equivalent, and above high school), and the ratio of family income to the federal poverty threshold. Anthropometric parameters, including BMI, were collected during standardized physical examinations. Biochemical measures, specifically serum total calcium and serum phosphorus concentrations (both in mmol/L), were also incorporated as key covariates due to their critical roles in bone metabolism and mineralization. In addition, lifestyle and health-related characteristics were assessed through structured questionnaires, capturing self-reported participation in vigorous physical activity (yes/no), history of diabetes (yes/no), hypertension status (yes/no), and alcohol consumption. Alcohol intake was quantified as the average number of alcoholic beverages consumed per day over the preceding 12 months¹³. Participants' smoking status was determined based on their cumulative lifetime cigarette consumption. Individuals who had smoked fewer than 100 cigarettes throughout their lifetime were classified as non-smokers, whereas those who reported having smoked 100 or more cigarettes were categorized as smoker¹⁴.

Statistical analysis

All statistical analyses for this study were performed using EmpowerStats software (version 4.1). Analyses were conducted using the NHANES complex survey design, accounting for sampling weights, stratification, and clustering to produce nationally representative estimates. Specifically, the 4-year fasting subsample weight (WTSAF4YR), stratification variable (SDMVSTRA), and primary sampling unit (SDMVPSU) were applied in all analyses. The demographic, clinical, and biochemical characteristics of the study population were first summarized and compared across quartiles of serum zinc concentrations to provide an overview of population distributions. For continuous variables, group differences were assessed using linear regression models, whereas categorical variables were compared using chi-square tests to identify statistically significant variations between quartiles.

To evaluate the relationship between serum zinc levels and BMD, multivariable linear regression models were employed. Three progressively adjusted models were constructed to account for potential confounding factors. Model 1 represented the unadjusted association. Model 2 controlled for basic demographic covariates, including age, sex, and race/ethnicity. Model 3 further incorporated additional potential confounders such as educational attainment, diabetes status, engagement in vigorous physical activity, smoking status, hypertension, serum total calcium, serum phosphorus, and the ratio of family income to the federal poverty threshold. Furthermore, to investigate potential nonlinear relationships between serum zinc concentrations and BMD, smooth curve fitting analyses were conducted. All statistical tests were two-sided, and a p-value of less than 0.05 was considered indicative of statistical significance.

Results

Descriptive characteristics and baseline profile of the participants included in the study

Since serum zinc concentrations were measured in a fasting subsample, the corresponding 4-year fasting subsample weight (WTSAF4YR) was applied in accordance with NHANES analytic guidelines. Because data from two survey cycles (2011-2012 and 2013-2014) were combined, the 4-year weight was derived by dividing each participant's 2-year fasting subsample weight (WTSAF2YR) by two, following t...

Discussion

In this cross-sectional study of 1,674 adults from NHANES 2011-2014, higher serum zinc concentrations were positively associated with total and lumbar spine BMD, with the strongest effects observed in the highest zinc quartile. Multivariable analyses confirmed these associations after adjusting for demographic, lifestyle, anthropometric, and biochemical covariates, and subgroup analyses suggested a more pronounced effect in men. Site-specific results indicated that trabecular-rich sites like the lumbar spine were more re...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
APEX DXA Analysis Software	Hologic Inc.	Version 4.0	Used for DXA data acquisition and analysis.
EmpowerStats (for statistical analysis, regression, and threshold effect modeling)	X&Y Solutions, Boston, MA, USA	Version 4.1	Statistical software used for multivariable linear regression, smooth curve fitting, and threshold effect analysis. RRID if available.
Hologic QDR-4500A fan-beam densitometer	Hologic Inc., Bedford, MA, USA	https://www.hologic.com/file/403701/download?token=9xR5yyeg	Used for DXA scans to measure bone mineral density in NHANES participants.
ICP-DRC-MS (Inductively Coupled Plasma Dynamic Reaction Cell Mass Spectrometry)	NHANES Laboratory, CDC	https://wwwn.cdc.gov/nchs/data/nhanes/public/2017/labmethods/UM?J?MET?508.pdf	Used for quantification of serum zinc concentrations with high sensitivity and precision.
NHANES 2011–2014 Demographics & Socioeconomic dataset	CDC NHANES	DEMO	Publicly available dataset with demographic, socioeconomic, and lifestyle variables.
NHANES 2011–2014 DXA Bone Mineral Density dataset	CDC NHANES	DXXSPN	Publicly available dataset containing total and site-specific BMD measurements via DXA.
NHANES 2011–2014 Serum Zinc dataset	CDC NHANES	LBDZN	Publicly available dataset containing serum zinc measurements for U.S. adults.
NHANES 2011–2016 public datasets (LBDZN, DXXSPN, DEMO)	CDC NHANES	https://wwwn.cdc.gov/nchs/nhanes/tutorials/datasets.aspx	National Health and Nutrition Examination Survey datasets for serum zinc, DXA BMD, and demographic/socioeconomic data. Repository: https://www.cdc.gov/nchs/nhanes/
NHANES Official Website	National Center for Health Statistics (NCHS), CDC	https://www.cdc.gov/nchs/nhanes/	Official DXA examination protocol details are available at: https://www.cdc.gov/nchs/
QDR-4500A Fan-Beam Densitometer	Hologic Inc. (Bedford, MA, USA)	QDR-4500A	Used for DXA scans to obtain BMD measurements.