Assessing vitamin D status: time for a rethink?

Vitamin D status is universally assessed by measuring circulating total 25-hydroxyvitamin D [25(OH)D], the sum of 25(OH)D3 and 25(OH)D2. However, despite the widespread acceptance of this biomarker of vitamin D status, studies have suggested that measurement of 25(OH)D alone may not be sufficient to understand the relationship between vitamin D exposure and certain health outcomes. As a result, attention has turned to other aspects of vitamin D biology. Approximately 90%–95% of 25(OH)D is tightly bound to a specific globulin, vitamin D binding protein (VDBP), which is structurally related to serum albumin. Of the remaining 25(OH)D, about 1% is unbound and the rest loosely bound to serum albumin. A current hypothesis is that the free and albumin-bound moieties are responsible for delivering 25(OH)D to the cell, with the VDBP-bound 25(OH)D acting as a reservoir of the metabolite. In humans, common genetic polymorphisms produce 3 major circulating variants of the VDBP (Gc1F, Gc2, and Gc1S), which differ in their affinity for 25(OH)D. The prevalence of these polymorphs has been shown to vary across different ethnicities and populations, with the Gc1F variant generally more common among individuals of African ancestry. In a recent study reported in the New England Journal of Medicine (1 ), Powe et al. investigated the prevalence of the different VDBP phenotypes (Gc1F, Gc2, and Gc1S) in a cohort of black and white Americans and examined the relationship with 25(OH)D concentrations as well as other parameters. Unlike previous investigations, this study included measurements of the concentration of serum VDBP. In this work, black Americans were found to have a preponderance of the high-affinity Gc1F phenotype, with homozygotes having about half the concentration of total VDBP found in whites, in whom the Gc1S variant predominated. The mean concentration of total 25(OH)D was also lower in blacks than in whites. An additional aspect of the study by Powe et al. was the estimation of “bioavailable” 25(OH)D for the homozygotes, using the measured concentrations of 25(OH)D and VDBP and literature values for their binding affinities to albumin or specific variants of VDBP. Bioavailable 25(OH)D was also measured in a subset of these samples using a competitive radioligand-binding assay. Based on their results, the authors suggest that the low VDBP concentrations found in black Americans result in concentrations of bioavailable 25(OH)D that are similar to those found in whites (calculated values 2.9 and 3.1 ng/mL, respectively). This might explain why black Americans can have apparently good bone health in the presence of low total 25(OH)D concentrations. If true, these findings could have profound implications for the interpretation of total 25(OH)D results. Current guidelines, including those recently published by the US Institutes of Medicine, do not distinguish between ethnic groups. Should the results of the study by Powe et al. be confirmed, these guidelines will have to be revisited and a new approach for assessing vitamin D status will need to be developed, possibly including the measurement of VDBP or a direct estimate of bioavailable 25(OH)D. The Powe et al. study does have some limitations, and further research is clearly warranted. The VDBP assay used in this work (R&D Systems) is intended only for research purposes and has not been validated to the standard required for diagnostic use. Worryingly, results of the R&D assay showed no significant correlation with those given by an alternative VDBP assay (Alpco Diagnostics) that was also investigated in the Powe et al. study. Bioavailable 25(OH)D calculated using the VDBP concentration (R&D kit) and previously published affinity constants for Gc1F and Gc1S did correlate with values obtained with a radioligand binding assay. Although these results are encouraging, no procedure currently exists to evaluate the accuracy of these methods. Furthermore, standardization of such assays is currently problematic because the available calibration materials tend to contain either a single 1 Imperial College Healthcare NHS Trust, Oncology/Endocrine Laboratory, Charing Cross Hospital, London, UK; 2 Biomolecular Measurement Division, NIST, Gaithersburg, MD * Address correspondence to this author at: Imperial College Healthcare NHS Trust, Oncology/Endocrine Laboratory Charing Cross Hospital, Fulham Palace Rd., London W6 8RF, UK. Fax 44-(0)20-8846-7007; e-mail b.carter1@ which.net. Disclaimer: Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by NIST, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose. Received January 30, 2014; accepted February 4, 2014. Previously published online at DOI: 10.1373/clinchem.2013.219386 3 Nonstandard abbreviations: 25(OH)D, 25-hydroxyvitamin D; VDBP, vitamin D binding protein; LC-MS/MS, liquid chromatography–tandem mass spectrometry. Clinical Chemistry 60:6 809–811 (2014) Perspective