BACKGROUND: Vitamin D deficiency is associated with poor bone health and other adverse health outcomes; however, the associations are greatly attenuated in black vs white individuals. One possible explanation for this attenuation is different concentrations of bioavailable vitamin D metabolites in plasma, which are estimated with 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 with 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 cohort and compared the results with a widely used monoclonal immunoassay. RESULTS: With 10μL of serum or plasma, the lower limit of quantification for the assay (μ20% CV) was 71 μg/mL. The assay was linear from 62 to 434 μg/mL, with total imprecision of 7.3-9.0% CV at approximately 250 μg/mL. Significant hemolysis interfered with quantification. The identification of isoforms was 97% concordant with genotyping (< coefficient). Method comparison with immunoassay revealed significant isoform-specific effects in the immunoassay. Mean concentrations (SD) of VDBG by mass spectrometry were 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 can provide additional information beyond immunoassay. Counter to prior observations by immunoassay, VDBG concentrations did not vary by race.
Bibliographical noteFunding Information:
The Atherosclerosis Risk in Communities Study is carried out as a collaborative study supported by National Heart, Lung, and Blood Institute contracts (HHSN268201100005C, HHSN268201100006C, HHSN268201100007C, HHSN 268201100008C, HHSN268201100009C, HHSN268201100010C, HHSN268201100011C, and HHSN268201100012C). The University of Washington has received grant (Waters, Inc.) and instrumentation (Waters, Inc., and Thermo, Inc.) support from mass spectrometry companies. R&D Systems provided immunoassay kits. C.M. Henderson, NIH grant HL007028; P.L. Lutsey,NIHgrants HL103706 and HL103706-S1, nonsponsored McKnight Land-Grant Professorship funds; E. Selvin, NIH grant DK089174; A.N. Hoofnagle, NIH grants CA160034 and DK035816. The authors thank R&D Systems for providing the immunoassay kits used in this study and Hannah Pflaum for technical assistance.
© 2015 American Association for Clinical Chemistry.