In AIDS Clinical Trials Group (ACTG) A5280, we reported that vitamin D (VitD) and calcium supplementation prevented bone loss that has been associated with antiretroviral therapy (ART) initiation with efavirenz/emtricitabine/tenofovir disoproxil fumarate (TDF) . After 48 weeks, percentage decline in bone mineral density (BMD) at total hip was smaller in the VitD/calcium arm compared with the placebo arm. Notably, no racial differences were apparent regarding this effect on BMD.
In the general population, black Americans have lower levels of 25-dihydroxyvitamin D [25-(OH)D] than white Americans, yet have higher bone mass and lower fracture rates . Studies from the general population have highlighted racial differences in the relationship between VitD and both parathyroid hormone (PTH) and BMD , suggesting that the threshold for VitD deficiency may differ between black and white individuals . One plausible explanation may be related to differences in expression of VitD-binding protein (VDBP). VDBP is the major serum transport protein for all VitD metabolites. VDBP has both a high capacity and affinity for VitD metabolites, binding 85–90% of total circulating 25-(OH)D. Bioavailable 25-(OH)D consists of albumin-bound 25-(OH)D (10–15% of total) and free 25-(OH)D (less than 1% of total) . Recently, Powe et al. reported that black Americans have lower serum VDBP concentrations as measured by a mAb assay than white Americans; therefore, despite a lower total 25-(OH)D level in black Americans, the calculated bioavailable concentration of 25-(OH)D is similar for black and white Americans. These data suggest that the commonly reported low VitD levels for black Americans may reflect the measurement of total rather than bioavailable 25-(OH)D concentrations ; however, this interpretation remains controversial as results differ with another VDBP assay [7–10]. In addition, two recent studies reported increases in both PTH and VDBP with initiation of TDF-containing ART [11,12], suggestive of an induced ‘functional’ VitD deficiency, but neither study calculated bioavailable 25-(OH)D or compared differences by race.
In this prespecified secondary analysis of A5280, we sought to clarify the complex relationship between VitD metabolism and race in HIV-infected participants receiving TDF-containing ART, by examining the effect of VitD/calcium supplementation on total 25-(OH)D, bioavailable 25-(OH)D, VDBP, PTH, bone turnover markers levels, and BMD by race in black and nonblack participants. We hypothesized that changes in bioavailable 25-(OH)D would correlate better with changes in calciotropic hormone levels and bone outcomes in black persons.
ACTG A5280 was a 48-week, randomized, double-blind, placebo-controlled study in which 165 HIV-infected participants, naïve to ART, with 25-(OH)D level at least 10 and less than 75 ng/ml (≥25 and <188 nmol/l), creatinine clearance at least 60 ml/min by Cockcroft–Gault, and serum calcium less than 10.5 mg/dl were randomized to receive 4000 IU cholecalciferol (VitD3) daily and 500 mg calcium carbonate twice daily or identically matching placebos (Tishcon Corporation, Westbury, New York, USA). Primary and secondary treatment outcomes of percentage change in total hip and lumbar spine BMD, and bone turnover markers, procollagen-1 N-terminal peptide (P1NP), and C-terminal telopeptide of type 1 collagen (CTX) from baseline to 48 weeks have been previously reported .
Serum samples were stored at −80°C until batched analysis at the Irving Institute Biomarkers Core at Columbia University Medical Center (New York City, New York, USA). We measured 25(OH)D2 and D3 by liquid chromatography–tandem mass spectrometry; intact PTH [radioimmunoassay (RIA); Scantibodies, Santee, California, USA; interassay coefficient of variation (CV), 6.8%]. We used the same monoclonal VDBP antibody assay use by Powe et al. (RIA; R&D Systems, Minneapolis, Minnesota, USA; interassay CV, 7.2%).
The analysis included all available data regardless of treatment change/discontinuation. Race groups in this report are defined as black and nonblack. The black group included all A5280 participants who self-identified as black or African-American (N = 52). The nonblack group included those self-identifying as White, Asian, or American Indian (N = 97). Participants with more than one race and with unknown race (N = 7) were excluded from the analyses with race. All 25-(OH)D VitD values below the lower limit of 1.25 ng/ml were imputed to 0 ng/ml and VDBP values above the upper limit of 500 μg/ml were imputed to 501 μg/ml. Bioavailable VitD was calculated using a formula that includes total 25-(OH)D, VDBP, and albumin levels from Powe et al.. The primary outcome measures of interest were defined by absolute change from baseline; post hoc secondary analyses outcomes defined as percentage change from baseline were also displayed graphically.
Stratified Wilcoxon rank sum tests were used to test for differences in outcomes between the two treatment arms, stratified by the screening 25-(OH)D VitD stratum (10–20 ng/ml vs. 21–75 ng/ml). Exact Wilcoxon rank sum tests were used to test for differences in the two racial groups (black vs. nonblack both with respect to baseline characteristics as well as the primary and secondary outcomes of interest). Spearman correlations were used to evaluate associations between outcomes. Nonparametric smooth curves locally weighted scatterplot smoothing (LOESS) fits with 95% confidence limits were added to each scatter plot. All analyses by race were performed separately within treatment group. In the setting of relatively small subgroup sample sizes, inferences regarding between-race group differences in response to treatment are guided by both the magnitude of effect size as well as statistical significance.
All statistical tests are two-sided interpreted at the 5% nominal level of significance without adjustment for multiple testing. Analyses were conducted using SAS statistical software 9.4 (SAS Institute, Cary, North Carolina, USA).
Table 1 summarizes baseline demographic, immunologic, virologic, and other baseline parameters of the 149 black and nonblack populations. Overall, the median age was 32 years, 91% men, median CD4+ cell count 342 cells/μl with 19% having CD4+ cell count 200 cells/μl or less. No participants were coinfected with hepatitis B or C. The median estimated dietary calcium and VitD intake at entry were 859 mg and 129 IU, respectively; 17 and 21% of the participants reported calcium and VitD supplement use, respectively, within 30 days of study entry. The median BMD at the hip and at the spine were 1.06 g/cm3 and 1.13 g/cm3 (Z-scores 0.00 and −0.30), respectively.
Baseline and change in total 25-(OH)D, vitamin D-binding protein, bioavailable 25-(OH)D, and parathyroid hormone by treatment arm in all participants
At baseline, the distribution of levels of total 25-(OH)D [median (Q1, Q3) 28.4 (20.9, 38.5) vs. 26.4 (19.6, 33.0) ng/ml], bioavailable 25-(OH)D [2.1 (1.6, 3.8) vs. 2.1 (1.4, 3.3) ng/ml] and PTH (28.3 (24.5, 34.5) vs. 27.6 (22.1, 33.9) pg/ml) appeared to be similar between VitD/calcium and placebo arms. Total 25-(OH)D increased from baseline to week 48 in the VitD/calcium arm (median (Q1, Q3) +24.2 (+14.3, +35.8) ng/ml) but not in the placebo arm [+0.6 (−6.1, +4.3) ng/ml; P < 0.001]. Within arm increases in VDBP from baseline to 48 weeks were seen in both treatment groups (all P < 0.005), but a difference between the VitD/calcium and placebo arms was not apparent (P = 0.20). Bioavailable 25-(OH)D from baseline to 48 weeks increased significantly in the VitD/calcium arm [+1.5 (+0.5, +3.5) ng/ml, P < 0.001], but not in the placebo arm [−0.1 (−0.5, +0.4) ng/ml, P = 0.50; between arm P < 0.001]. In contrast, PTH increased from baseline to 48 weeks in the placebo arm [+5.2 (−0.7, +12.6) pg/ml, P < 0.001], but not in the VitD/calcium arm [+1.1 (−4.0, +6.1) pg/ml, P = 0.40; between arm P = 0.004].
Baseline and change in total 25-(OH)D, vitamin D-binding protein, bioavailable 25-(OH)D, and parathyroid hormone by race
At baseline, black participants had lower total 25-(OH)D [22.6 (15.8, 26.9) vs. 31.1 (23.1, 38.8) ng/ml, P < 0.001] and VDBP [125.6 (79.0, 264.0) vs. 289.6 (207.7, 393.2) μg/ml, P < 0.001], but higher bioavailable 25-(OH)D [2.9 (1.5, 5.2) vs. 2.0 (1.5, 3.0) ng/ml, P = 0.022] than nonblack participants (Table 2). Black participants also had slightly higher BMI than nonblack participants [25.3 (22.4, 30.1) vs. 24.2 (21.9, 26.8) kg/m2, P = 0.04]. Overall, a difference between the race groups in PTH levels was not apparent for any given 25-(OH)D level (Fig. 1a). However, the relationship between bioavailable VitD and PTH revealed a different pattern: black participants tended to have modestly higher levels of PTH compared with nonblack for any given level of bioavailable VitD at baseline (Fig. 1b). Furthermore, for a given level of PTH, black participants tended to have higher levels of BMD both at hip and spine compared with nonblack participants (Fig. 1c,d).
Among participants receiving VitD/calcium, increases in total 25-(OH)D and bioavailable 25-(OH)D levels from week 0 to 48 were observed in both blacks and nonblack groups, but differences between-race groups were only apparent for bioavailable 25-(OH)D (P < 0.001, Table 2). Similarly, percentage increase from baseline in bioavailable 25-(OH)D over 48 weeks was also greater in black compared with nonblack participants (Fig. 2). Although an increase in levels of VDBP from baseline was apparent among nonblack participants, this was not apparent in black participants; a difference between the two racial groups was not evident (P = 0.098). Changes in PTH from baseline were not apparent in either race group (Table 2).
Among participants who received placebo, no changes in 25-(OH)D or bioavailable 25-(OH)D were apparent from week 0 to 48 for either race group. Although an increase in levels of VDBP from baseline was apparent among black participants, this was not apparent in nonblack participants; a difference between the two racial groups was not evident (P = 0.43). In contrast, an increase in levels of PTH was apparent in both race groups; the magnitude of this increase did not differ between groups (P = 0.49; Table 2).
Bone mineral density, procollagen-1 N-terminal peptide, and C-terminal telopeptide of type 1 collagen by treatment arm and race
At baseline, BMD was higher in the black than the nonblack group at the spine and hip, but Z-scores did not differ between groups (Table 1). No differences between black and nonblack groups in change in BMD were apparent in either the VitD/calcium or placebo arms (Table 3). Similarly, there were no between-race differences in bone turnover marker response (P1NP and CTX) in either the VitD/calcium and placebo arms (Table 3).
Examination of the relationships between 48-week increases in total or bioavailable 25-(OH)D and increases in hip or spine BMD or decreases in P1NP and CTX among those who received VitD/calcium revealed weak associations among nonblack participants (r = 0.306 to r = 0.431, P < 0.05) that were not apparent among blacks (all r < |0.40|, all P > 0.15). Visual examination of these data in scatterplots with LOESS fits (supplementary Figure 1, http://links.lww.com/QAD/B150) highlight greater variability in total or bioavailable 25-(OH)D changes among blacks compared with nonblacks that contribute to this finding. Overall, the associations between change in bone outcomes and changes in bioavailable 25-(OH)D vs. total 25-(OH)D appear consistent.
With VitD/calcium supplementation, total and bioavailable 25-(OH)D levels increased over 48 weeks with beneficial effects on BMD in both black and nonblack participants initiating ART. Despite having a total 25-(OH)D in the insufficient range (median <30 ng/ml), black HIV-infected individuals had higher bioavailable 25-(OH)D and similar PTH values at baseline, and achieved greater increases in bioavailable 25-(OH)D with VitD/calcium supplementation compared with nonblack participants. Although changes in both total and bioavailable 25-(OH)D levels appeared to be weakly associated with bone outcomes among nonblack participants, associations were not apparent for either measure among black participants. These data fail to confirm that bioavailable VitD is a more useful measure of VitD status compared to total 25-(OH)D in HIV-infected blacks initiating ART and receiving VitD and calcium supplementation.
The importance of measuring VitD levels in the setting of HIV infection has been hotly debated, particularly given the ‘epidemic’ of hypovitaminosis D reported among HIV-infected persons (and the general population) despite unclear benefit beyond established BMD protection [13,14]. The well characterized racial differences in total 25-(OH)D have created consternation among clinicians given the fact that black Americans tend to have higher BMD despite lower 25-(OH)D levels than whites. Furthermore, PTH levels have been reported lower for black Americans at any given 25-(OH)D level suggesting that either total VitD measurement may not reflect the active VitD metabolites or an alternative set point at which PTH expression increases . The recognition that much of the measured VitD is actually bound to VDBP provides the opportunity to explain these discrepancies by estimating free or bioavailable VitD after measurement of VDBP levels.
Unfortunately, the measurement of VDBP has not been standardized. Recent studies suggest that the mAb assay for VDBP (R&D Systems), used by Powe et al. and in our study, detects a lower concentration of VDBP in black Americans because of a lower affinity for VDBP GC1f isoform (commonly expressed in people of African origin), and a higher affinity for the GC12 isoform (commonly expressed in white individuals) [8–10]. In contrast to findings reported with the mAb assay, studies using an alternative polyclonal Ab assay [7–10], or liquid chromatography–tandem mass spectrometry to directly measure VDBP [7,15] have not found VDBP to be lower or bioavailable 25-(OH)D higher in black individuals. Currently, there is no consensus regarding which assay to use, or whether bioavailable 25-(OH)D is a better predictor of VitD status or clinical endpoints than total 25-(OH)D [5,16,17].
In the setting of HIV infection, two studies have reported an increase in both PTH and VDBP, using the mAb assay, with initiation of TDF-containing ART [11,12], but neither study calculated bioavailable 25-(OH)D nor compared differences by race. Hsieh et al. reported a 30% increase in median VDBP levels from 154 (91, 257.4) to 198.3 (119.6, 351.9) μg/ml in Chinese individuals after 48 weeks of efavirenz/emtricitabine/TDF. Similarly, Havens et al. reported an increase in VDBP using the mAb assay with TDF-containing ART that corresponded to a decrease in free 1,25(OH)2D, suggesting a functional VitD deficiency induced by TDF. Within the placebo arm of our study, modest (14%) increases in VDBP levels were apparent only among black participants and did not translate to a decrease in bioavailable 25-(OH)D. In contrast, within the VitD/calcium arm, an increase in VDBP from baseline was observed in the nonblack group only, but increases in bioavailable 25-(OH)D were apparent in both black and nonblack groups. Notably, the increase in bioavailable 25-(OH)D within the VitD/calcium arm, was greater for black than nonblack groups. Whether this fact relates back to the specific mAb assay used or an actual difference in VDBP for these different groups cannot be determined based on available data. As all participants received TDF, our data also cannot completely address the functional VitD deficiency associated with TDF exposure reported by Havens et al. However, the observed increases in PTH in both race groups in the placebo but not the VitD/calcium supplementation arm are suggestive of a similar process.
Although having total 25-(OH)D levels in the insufficient range (<30 ng/ml), black participants had higher bioavailable 25-(OH)D at baseline, similar PTH levels, and greater increases in bioavailable 25-(OH)D with VitD/calcium supplementation than nonblack participants. These data suggest that total 25-(OH)D levels do not uniformly indicate VitD deficiency. Previous studies have evaluated the relationship between total serum 25-(OH)D levels and PTH by race; in these studies, the inflection point in a LOESS curve, which represents the 25-(OH)D level above which PTH levels remain constant, occurs at a lower 25-(OH)D level in black compared with white individuals . One can extrapolate that VitD sufficiency has a lower 25-(OH)D set point for black than white individuals and the accepted threshold for VitD deficiency [<20 ng/ml 25-(OH)D] may not have the same physiological implications . Our LOESS curves for total or bioavailable 25-(OH)D vs. PTH did not reveal a clear inflection point in either blacks or nonblack participants (Fig. 1); however, overall, it appeared that for the same level of bioavailable 25-(OH)D at baseline, black participants tend to have a modestly, although not significantly, higher level of PTH at baseline compared with nonblack participants.
We recognize that this study has limited power to confidently estimate the relationship between-race, VDBP expression, and bioavailable 25-(OH)D. Our analyses were limited to within subgroup analyses and were underpowered to formally assess effect modification via tests for interaction. To this end, we are conservative with our inferences, paying attention to effect size differences and not solely relying on subgroup tests of statistical significance. Another limitation of this secondary analysis is that we estimated bioavailable 25-(OH)D using a single assay, the mAb test for VDBP, and did not compare results to the polyclonal antibody assay or directly measured bioavailable 25-(OH)D. Given the current controversies regarding the VDBP assays and the appropriate mechanism to measure bioavailable VitD, additional research is needed to establish best practices for determining these clinical parameters and their utility in bone health and beyond.
In conclusion, with vitamin/calcium supplementation during ART initiation, we observed similar increases in total 25-(OH)D, but greater increases in calculated bioavailable 25-(OH)D in black compared with nonblack participants when using a monoclonal assay to measure VDBP. VitD/calcium supplementation prevented BMD loss at both the spine and hip in both black and nonblack participants. However, the benefits of supplementation was not consistently reflected in outcomes of bioavailable 25-(OH)D by race. Given the current uncertainties with available VDBP assays, calculating bioavailable 25-(OH)D does not appear to provide benefit beyond measuring total 25-(OH)D for assessment of VitD status in black HIV-infected persons.
We wish to gratefully acknowledge all of the study sites and study participants who have devoted their time and effort to this research endeavor.
Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number UM1 AI068634, UM1 AI068636, UM1 AI106701, R01 AI095089 and supported by National Institute of Mental Health (NIMH), National Institute of Dental and Craniofacial Research (NIDCR). This publication was also supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number UL1TR001873. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health, Bristol-Myers Squibb and Gilead funded the DXA scans and ancillary laboratory testing. Study medications were provided by Bristol-Myers Squibb, Gilead Sciences, and Tischcon Corporation.
Conflicts of interest
There are no conflicts of interest.
The work was presented at the Conference on Retroviruses and Opportunistic Infections 2016 (22–25 February 2016, Abstract #700) in Boston, Massachusetts, USA.
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