Associations of serum vitamins levels with bone mineral density in the different race-ethnicities US adults

This study was conducted under the National Health and Nutrition Examination Survey. The purpose of this study was to examine the associations of serum vitamins levels with BMD at hip. Given both serum vitamins and BMD at hip were collected only in the third National Health and Nutrition Examination Survey (NHANES III), this study only used the data of the NHANES III. The NHANES III is designed to assess the health and nutritional status of residents in the US, and focused on oversampling many groups within the U.S. population aged ≥2 months. These oversampled groups included children aged 2 months to 5 years, persons over age 60, Mexican-American persons, and non-Hispanic black persons. The NHANES III interview includes demographic, socioeconomic, dietary, and health-related questions. The examination component consists of medical, dental, and physiological measurements, as well as laboratory tests administered by highly trained medical personnel. The detailed description of the NHANES III was published elsewhere [17]. This study was approved by the Institutional Review Board of the National Center for Health Statistics NHANES. All participants gave their written informed consent.

The included criteria were as follows: who were with complete data of interesting variables, such as BMD, serum vitamins, and other covariates, such as physical index, health behaviors, and medical histories; and who aged ≥18 years. The excluded criteria were as follows: who were with missing data in the interesting variables; or who had the history of bone diseases, such as arthritis and bone tumor. The flowchart is shown in Fig. 1.

All participants were interviewed to collect data, including demographic-socioeconomic index and health-related questions. Meanwhile, fasting blood samples were collected to assay the levels of serum vitamins.

Serum samples from participants aged ≥18 years who were interviewed during phase II of this survey (1991–1994) in the NHANES III surplus sera project were used to assay serum vitamins concentrations including vitamins A, B9, B12, C, and E using the Quantaphase II Radioassay Kit (Bio-Rad) in the NHANES Laboratory of the Centers for Disease Control and Prevention. The coefficients of variation for vitamins A, B9, B12, C, and E in the NHANES III were 6, 7, 6, 4, and 5%, respectively. Furthermore, the intra-assay and inter-assay coefficients of variation were 5.2 and 2.5%, respectively. Details of the detection method are accessible at the NHANES website [18]. Dual-energy X-ray absorptiometry by using Hologic QDR 4500A fan-beam densitometers was employed to measure BMD at hip, including femur neck and the total hip BMD. The coefficients of variation were 3.2 and 2.4%, respectively. Details on the DXA examination protocol have been published elsewhere [13].

Height and weight were measured according to the standardized protocol, and used to calculate body mass index. All participants were divided into the White and Black as race, Mexican-American and Not Hispanic as ethnicity, or Non-Hispanic white, Non-Hispanic black, and Mexican-American as race-ethnicity. Urbanization classification based USDA Rural/Urban continuum codes was as follows: central counties or fringe counties of metro areas with ≥1 million population were considered as urban region; all other areas were considered as rural region. Health behaviors, such as smoking status, alcohol consumption, and physical activity, as well as medical histories, such as history of hypertension, history of diabetes, and history of fracture, were collected using a valid questionnaire.

Since all continuous variables were normal distribution, means and standard deviations were used to express the distributions of continuous variables. Categorical variables were described by frequencies and percentages. Linear regressions were employed to examine the associations of serum vitamins levels with BMD at different sites and obtain the regression coefficients and 95% confidential intervals (CIs). Furthermore, linear regressions were stratified by race, ethnicity, age, and sex. In order to correct the confounding effects of covariates, age, sex, body mass index, smoking, alcohol consumption, physical activity, years of school completed, living regions, race, ethnicity, history of hypertension, history of diabetes, history of fracture, and sample design weights were adjusted. All analyses were conducted using SAS 9.4 (SAS Institute Inc., Cary, NC, USA.). A two-tailed P ≤ 0.05 indicated the statistical significance.

There were 6023 participants included in the final analysis. The mean of age was 48.62 years. The averages of serum vitamins levels were 1.98 μmol/L for vitamin A, 15.67 nmol/L for folate, 358.75 pmol/L for vitamin B12, 41.25 mmol/L for vitamin C, and 26.60 μmol/L for vitamin E. And the averages of BMD were 0.83 g/cm² and 0.95 g/cm² at femur neck and the total hip, respectively. There were 2686 males, accounting for 44.60%. All characteristics are shown in Table 1.

Table 2 displays the associations of serum vitamins levels with BMD. Serum folate level was positively associated with BMD at femur neck and the total hip (P = 0.035 and 0.010; β = 0.00024 and 0.00023; and 95% CI: 0.00002–0.00051 and 0.00005–0.00051, respectively). Similarly, serum vitamins A and C levels were significantly associated with BMD at femur neck and the total hip (vitamin A: P = 0.010 and 0.006; β = 0.00749 and 0.00837; and 95% CI: 0.00176–0.01322 and 0.00240–0.01433, respectively) and (vitamin C: all P <  0.001; β = 0.00026 and 0.00025; and 95% CI: 0.00012–0.00039 and 0.00011–0.00039, respectively). No significant associations of serum vitamins B12 and E levels with BMD were observed.

The results stratified by race are presented in Table 3. The associations of serum vitamins B12 and E levels with BMD were consistent with those of the total population. However, there were positive associations of serum folate level with BMD at femur neck and the total hip in the Black participants (P = 0.013 and 0.018; β = 0.00086 and 0.00082; and 95% CI: 0.00018–0.00154 and 0.00014–0.00151, respectively) but not in the White participants. Significant associations of serum vitamin A level with BMD were observed at femur neck in the White participants (P = 0.012) and at the total region in the Black participants (P = 0.024). Serum vitamin C was associated with BMD at femur neck both in the White and Black populations (P = 0.011 and 0.017, respectively), and at total region only in the White population (P = 0.007).

As stratified by ethnicity, both in the Mexican-American and Not Hispanic participants, there were no significant associations of serum vitamins B12 and E levels with BMD, which were comparable with the total results. There were positive associations of serum folate, vitamin A, and vitamin C levels with BMD at femur neck (P = 0.047, 0.001, and 0.001, respectively) and the total hip (P = 0.039, 0.001, and <  0.001, respectively) in the Not Hispanic participants but not in Mexican-American participants (Table 4).

The results stratified by age are shown in Table 5. Significant associations of serum vitamin C level with BMD at femur neck and the total hip were observed both in the 20–49 years and 50–90 years groups (P = 0.003, 0.019, 0.013, and 0.025, respectively). There were positive associations of serum folate level with BMD at femur neck and the total hip only in the 50–90 years group (P = 0.022 and 0.041; β = 0.00018 and 0.00013; and 95% CI: 0.00007–0.00028 and 0.00004–0.00023, respectively). Similarly, serum vitamin A level was significantly associated with BMD in the 50–90 years group at femur neck (P = 0.043; β = 0.00834; and 95% CI: 0.00026–0.01642) and at the total region (P = 0.042; β = 0.00913; and 95% CI: 0.00032–0.01793). In line with the total results, there were no significant associations of serum vitamins B12 and E levels with BMD both in the 20–49 years and 50–90 years groups.

Table 6 presents the associations of serum vitamins levels with BMD at femur neck and the total hip stratified by sex. In males, there were positively associations of serum folate, vitamin A and C levels with BMD at both femur neck (P = 0.014, 0.004, and 0.046; β = 0.00040, 0.01365, and 0.00022; and 95% CI: 0.00010–0.00089, 0.00431–0.02298, and 0.00001–0.00044, respectively) and total region (P = 0.010, < 0.001, and 0.016; β = 0.00042, 0.01709, and 0.00028; and 95% CI: 0.00018–0.00092, 0.00761–0.02657, and 0.00005–0.00050, respectively). However, in females, only serum vitamin C was associated with BMD at femur neck and total region (P = 0.001 and 0.002; β = 0.00029, and 0.00028; and 95% CI: 0.00012–0.00045 and 0.00010–0.00045, respectively).

There were some strengths in this study. First, this study comprehensively evaluated the associations of serum vitamins levels with BMD at hip in the US adults. Furthermore, some major covariates were adjusted in this study, such as race/ ethnicity, history of fracture, BMI and sample design weights. Therefore, this study would provide accurate evidence on the associations of serum vitamins levels with BMD. Second, differing from other studies to use dietary vitamins intake or supplement, serum vitamins levels were used in this study. Given the complicated relationship between dietary vitamins intake and serum level, serum vitamins levels seemed to better reflect the biological availability and activity of vitamins. Third, this study further examined the modified effect of race and ethnicity on the associations of serum vitamins levels with BMD. As a result, it would provide a better understanding on the important role of race-ethnicity in the associations of serum vitamins levels with BMD.

However, there were some limitations to be stated. First, this study was based on the NHANES III, in which the study population were restricted to the US subjects. Furthermore, the population of this study were relatively young. Therefore, it should be cautious when generalizing the results to older populations or populations outside the USA. Second, this study was a cross-sectional study, which was poor to examine the causal relationship between serum vitamins levels and BMD. A well designed cohort study should be conducted to confirm the findings of this study in the future. Third, since the complicated backgrounds of different races or ethnicities, the mechanisms of race/ ethnicity modifying the associations of serum vitamins levels with BMD failed to be fully explained in this study.