Measurement by a Novel LC-MS/MS Methodology Reveals Similar Serum Concentrations of Vitamin D–Binding Protein in Blacks and Whites

Abstract

Background Vitamin D deficiency is associated with poor bone health and other adverse health outcomes. However, the associations are greatly attenuated in blacks compared with whites. One possible explanation for this attenuation could be different concentrations of bioavailable vitamin D metabolites in plasma, which are estimated using equations that include the total concentration of vitamin D binding globulin (VDBG) and haplotype-specific dissociation constants. Methods We developed a method to quantify VDBG using LC-MS/MS that could also identify the haplotypes/isoforms of VDBG present. We validated the method according to recent recommendations for publications of biomarker studies. We determined serum VDBG concentrations in samples from the Atherosclerosis Risk in Communities (ARIC) cohort and compared the results with a widely-used monoclonal immunoassay. Results An assay with a lower limit of quantification (<20% CV) of 71 µg/mL was developed using 10 µL of serum or plasma. The assay was linear from 64 to 434 µg/mL with total imprecision of 7.3–9.0% CV at ~250 µg/mL. Significant hemolysis interfered with quantification. The identification of isoforms was 97% concordant with genotyping (kappa coefficient). Method comparison with immunoassay revealed significant isoform-specific effects in the immunoassay. The concentration (SD) of VDBG by mass spectrometry was similar in whites and blacks [262 (25) vs. 266 (35) µg/mL, respectively; p=0.43]. Conclusions Validated mass spectrometric methods for the quantification of proteins in human samples are sensitive and specific and can provide additional information beyond immunoassay. Counter to prior observations by immunoassay, VDBG levels did not vary by race.

Assessment of vitamin D status – a changing landscape

“…One allele copy of rs7041 reduced DBP by 189 μg/mL whereas one allele copy of rs4588 increased DBP by approximately 50 μg/mL. However, the monoclonal immunoassay from R&D Systems used in this study appears to be sensitive to DBP polymorphisms [110]. The aforementioned polymorphisms result in protein variants with different affinities to the monoclonal antibodies used in R&D Systems assay.…”

“…As a response to the limitations of 25-OHD, it has been speculated that other surrogate markers of vitamin D metabolism might better reflect pathophysiology and predict clinical outcome [109,110]. Some of these markers are illustrated in Figure 1.…”

“…The aforementioned polymorphisms result in protein variants with different affinities to the monoclonal antibodies used in R&D Systems assay. In a more recent study from Henderson et al DBP was measured with a LC-MS/MS method demonstrated to reliably detect the common DBP variants [110]. With this method, comparable DBP concentrations were measured in Blacks and Whites.…”

Vitamin D and Cancer Risk and Mortality: State of the Science, Gaps, and Challenges

“…The current literature also suggests that there may be other important factors that interact with vitamin D status, such as menopausal status in breast cancer (17), sex in colorectal cancer (140), and circulating vitamin D binding protein in prostate cancer (141). Relevant to the latter observation, studies have begun to pay more attention to the measurement of 25(OH)D by examining the role of free versus total vitamin D in cancer etiology, with free 25(OH)D appearing to be more important for some cancer sites and total 25(OH)D appearing to be more important for others (12,80,(141)(142)(143) (145). Attention to these dimensions in future studies will be critical for the conduct of informative and reproducible research in the field.…”

The Power of Mass Spectroscopy as Arbiter for Immunoassays

“…DBP is a glycoprotein with either 3 (GC1 isoforms) or 2 carbohydrate chains on some (2%-20%) of the circulating DBP molecules (4 ); it is a multifunctional protein probably belonging to the oldest member of the albumin gene family (1,2 ). It has a very high affinity for actin and helps to depolymerize filamentous actin into globular actin when released in the bloodstream after cell damage .…”