N Engl J Med. 2013 Nov 21;369(21):1991-2000. doi: 10.1056/NEJMoa1306357.
Powe CE1, Evans MK, Wenger J, Zonderman AB, Berg AH, Nalls M, Tamez H, Zhang D, Bhan I, Karumanchi SA, Powe NR, Thadhani R.
1From the Department of Medicine, Brigham and Women's Hospital (C.E.P.), Division of Nephrology, Massachusetts General Hospital (J.W., H.T., I.B., R.T.), Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School (A.H.B.), Division of Nephrology, Beth Israel Deaconess Medical Center (D.Z., S.A.K.), and Howard Hughes Medical Institute (D.Z., S.A.K.) - all in Boston; the Laboratory of Epidemiology and Population Sciences, National Institute on Aging, National Institutes of Health, Baltimore (M.K.E., A.B.Z.); the Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD (M.N.); and the Department of Medicine, San Francisco General Hospital and University of California, San Francisco, San Francisco (N.R.P.).
Low levels of total 25-hydroxyvitamin D are common among black Americans. Vitamin D-binding protein has not been considered in the assessment of vitamin D deficiency.
In the Healthy Aging in Neighborhoods of Diversity across the Life Span cohort of blacks and whites (2085 participants), we measured levels of total 25-hydroxyvitamin D, vitamin D-binding protein, and parathyroid hormone as well as bone mineral density (BMD). We genotyped study participants for two common polymorphisms in the vitamin D-binding protein gene (rs7041 and rs4588). We estimated levels of bioavailable 25-hydroxyvitamin D in homozygous participants.
Mean (±SE) levels of both total 25-hydroxyvitamin D and vitamin D-binding protein were lower in blacks than in whites (total 25-hydroxyvitamin D, 15.6±0.2 ng per milliliter vs. 25.8±0.4 ng per milliliter, P<0.001; vitamin D-binding protein, 168±3 μg per milliliter vs. 337±5 μg per milliliter, P<0.001). Genetic polymorphisms independently appeared to explain 79.4% and 9.9% of the variation in levels of vitamin D-binding protein and total 25-hydroxyvitamin D, respectively. BMD was higher in blacks than in whites (1.05±0.01 g per square centimeter vs. 0.94±0.01 g per square centimeter, P<0.001). Levels of parathyroid hormone increased with decreasing levels of total or bioavailable 25-hydroxyvitamin D (P<0.001 for both relationships), yet within each quintile of parathyroid hormone concentration, blacks had significantly lower levels of total 25-hydroxyvitamin D than whites. Among homozygous participants, blacks and whites had similar levels of bioavailable 25-hydroxyvitamin D overall (2.9±0.1 ng per milliliter and 3.1±0.1 ng per milliliter, respectively; P=0.71) and within quintiles of parathyroid hormone concentration.
Community-dwelling black Americans, as compared with whites, had low levels of total 25-hydroxyvitamin D and vitamin D-binding protein, resulting in similar concentrations of estimated bioavailable 25-hydroxyvitamin D. Racial differences in the prevalence of common genetic polymorphisms provide a likely explanation for this observation. (Funded by the National Institute on Aging and others.).
Vitamin D Binding Protein - Blacks and Whites
Vitamin D - Blacks and Whites
Vitamin D Binding Protein - Genes
Table of contents
St. Louis Veterans Affairs Medical Center, St. Louis, MO. sjweintraub at gmail.com
Current assays for vitamin D sufficiency measure total circulating 25-hydroxyvitamin D. However, Powe et al. (Nov. 21 issue)1 conclude that measurement of 25-hydroxyvitamin D that is not bound to the vitamin D–binding protein, which they refer to as bioavailable 25-hydroxyvitamin D, provides a better assessment of sufficiency. They report that the correlation between levels of bioavailable 25-hydroxyvitamin D and levels of parathyroid hormone is stronger than the correlation between levels of total circulating 25-hydroxyvitamin D and levels of parathyroid hormone when evaluated across races. However, they fail to reconcile their findings and the rationale underlying their conclusion with a critical role of the complex of 25-hydroxyvitamin D and vitamin D–binding protein.
This complex is taken up by renal proximal tubule epithelial cells through receptor-mediated endocytosis. The 25-hydroxyvitamin D component of the endocytosed complex then becomes the major precursor for circulating 1,25-dihydroxyvitamin D, the active steroid hormone form of vitamin D that is important in the regulation of parathyroid hormone levels.2,3 Thus, in the regulation of parathyroid hormone, the “bioavailable” form of 25-hydroxyvitamin D is, in fact, the form that is bound to the vitamin D–binding protein.
Horizon Health Network, Fredericton, NB, Canada
yu.chen at horizonnb.ca
Powe et al. proposed that the most appropriate cross-racial marker of vitamin D status may be bioavailable 25-hydroxyvitamin D. This concept of “bioavailability” is analogous to other lipophilic hormones, such as testosterone, in that both free and albumin-bound ligands are available to exert biologic functions. However, the method used to determine bioavailable 25-hydroxyvitamin D is, in my view, questionable.
The authors adapted the Vermeulen equation, initially developed to calculate free and bioavailable testosterone.1 Estimated concentrations of bioavailable 25-hydroxyvitamin D in the study were compared with concentrations measured with the use of an analogue radioligand assay (Pearson's r=0.81 and 0.90). The assay is based on a competitive principle whereby a radiolabeled analogue competes with endogenous bioavailable 25-hydroxyvitamin D for a limited number of vitamin D–binding proteins immobilized on a solid surface. Unfortunately, analogue immunoassay methods for free and bioavailable testosterone are no longer recommended, because estimates by this approach reflect total hormone levels more closely than the free fraction.2-4
The authors should validate their estimation of free 25-hydroxyvitamin D, bioavailable 25-hydroxyvitamin D, or both with direct measurement by means of gas chromatography–mass spectrometry or liquid chromatography–mass spectrometry after ultrafiltration or equilibrium dialysis.
Katholieke Universiteit Leuven, Leuven, Belgium roger.bouillon at med.kuleuven.be
Kerry Jones, Ph.D.
Inez Schoenmakers, Ph.D. Medical Research Council Human Nutrition Research, Cambridge, United Kingdom
Powe et al. report that participants homozygous for the Gc1F phenotype of vitamin D–binding protein (>90% of black homozygotes), as compared with participants homozygous for the Gc1S or Gc2 phenotype (>90% of white homozygotes), have lower plasma levels of vitamin D–binding protein and similar levels of bioavailable 25-hydroxyvitamin D, despite lower levels of total 25-hydroxyvitamin D. However, the two monoclonal antibodies in the R&D Systems vitamin D–binding protein assay used in the study may not have equal affinity for all genotypes. As in the article by Powe et al., we found that plasma levels of vitamin D–binding protein measured with the R&D Systems assay were approximately 50% lower in Gambians (blacks only) than in participants from the United Kingdom (whites only) (unpublished data). However, in agreement with Winters et al.,1 we found similar vitamin D–binding protein concentrations in black (Congolese) and white (Belgian) men2 and in black Gambians (approximately 80% Gc1F homozygotes) and white participants from the United Kingdom (approximately 80% Gc1S or Gc2 homozygotes) (unpublished data) with an assay using polyclonal vitamin D–binding protein antibodies generated against mixed vitamin D–binding protein subtypes.3 Contrary to the results of Powe et al., serum vitamin D–binding protein is generally lower in Gc2 homozygotes than in persons with other Gc genotypes.4 Furthermore, differences in vitamin D–binding protein affinity owing to Gc genotype may be smaller than those used in the formula to calculate bioavailable 25-hydroxyvitamin D levels, because in-depth analysis under different conditions (buffers, pH, and temperature) showed no substantial differences.5 In our view, black Americans, as compared with white Americans, have lower levels of not only total 25-hydroxyvitamin D but also free or bioavailable 25-hydroxyvitamin D.
Medical University of South Carolina, Charleston, SC , hollisb at musc.edu
Daniel D. Bikle, M.D., Ph.D.
University of California, San Francisco, San Francisco, CA
_Powe et al. reported that black participants and white participants have different vitamin D requirements on the basis of the circulating concentration of vitamin D–binding protein. The authors used an assay for vitamin D–binding protein that is untested for the key measurement in their study. This assay is based on a primary monoclonal antibody produced against a single peptide fragment of the human vitamin D–binding protein. Standard vitamin D–binding protein assays are based on polyclonal antibodies against an intact vitamin D–binding protein.1-3 The problem is that the monoclonal-antibody assay gives lower values in blacks who have primarily the Gc1F variant of vitamin D–binding protein. The monoclonal antibody discriminates between the Gc1F and Gc1S variants to provide artificially low values for total vitamin D–binding protein in blacks, thus bringing the conclusion of this study into question. As shown in Fig. S4 in the Supplementary Appendix (available with the full text of the article at NEJM.org), “calculated” concentrations of bioavailable 25-hydroxyvitamin D correlate well with physically measured concentrations in participants homozygous for either the Gc1F or Gc1S variant, but the slopes of the relationships are very different. Specifically, for any given physically measured level of bioavailable 25-hydroxyvitamin D, the authors are overestimating bioavailable 25-hydroxyvitamin D by 2 to 2.5 times owing to underestimation of vitamin D–binding protein in blacks.
Weintraub notes that our findings may not reconcile with previous data suggesting that vitamin D–binding protein–bound 25-hydroxyvitamin D is the primary precursor for 1,25-dihydroxyvitamin D. Megalin is a vitamin D–binding protein receptor and takes up vitamin D–binding protein–bound 25-hydroxyvitamin D in the proximal renal tubules. Megalin knockout mice are susceptible to bone disease and signs of vitamin D deficiency.1 In contrast, vitamin D–binding protein knockout mice do not have signs of deficiency on a vitamin D–replete diet, a finding that indicates that non–vitamin D–binding protein–bound 25-hydroxyvitamin D may be sufficient.2 Megalin also binds albumin; thus, albumin-bound 25-hydroxyvitamin D (bioavailable 25-hydroxyvitamin D) may be a relevant analyte converted to 1,25-dihydroxyvitamin D.
Chen writes that we should validate the assay used for direct measurement of bioavailable vitamin D because previous studies have found problems with analogue immunoassay methods for directly measuring free testosterone. These issues may not be applicable to our assay method, because our assay is different in its calibrator and affinity-binding approach. We agree that validation of direct assays for bioavailable 25-hydroxyvitamin D would strengthen our findings.
Bouillon et al. and Hollis and Bikle raise concerns regarding the use of the monoclonal antibody–based vitamin D–binding protein assay, because it may have differential immunoreactivity against variant vitamin D–binding proteins. We were unable to directly address this issue, because genotype-specific standards were not available. Previous research using polyclonal immunoassays did not reveal racial differences in levels of circulating vitamin D–binding protein.3 However, polyclonal antibodies raised against vitamin D–binding protein may cross-react with other proteins.4 Consistent with this observation, when we spiked serum with pooled vitamin D–binding protein, we recovered 91 to 103% of the added vitamin D–binding protein using the R&D Systems kit (monoclonal format) but recovered 171 to 176% using the ALPCO Diagnostics kit (polyclonal format). Furthermore, vitamin D–binding protein levels determined with the use of the polyclonal assay did not correlate with our directly measured results (Fig. S6 in the Supplementary Appendix).
Hollis and Bikle suggest that an underestimation of vitamin D–binding protein concentrations in blacks could explain the different slopes observed for Gc1S homozygotes and Gc1F homozygotes (Fig. S4 in the Supplementary Appendix). In our direct assay, vitamin D–binding protein enriched in Gc1S was used as a calibrator and adsorbed affinity-binding reagent. This assay format may result in asymmetric competition for binding between the Gc1S vitamin D–binding protein adsorbed to the plate and Gc1F vitamin D–binding protein in participants' serum. Buffer conditions also may have differentially influenced the binding affinities of the vitamin D–binding protein variants and thus altered slopes. To compare absolute levels of bioavailable 25-hydroxyvitamin D in participants of differing genotypes, genotype-specific calibrators are needed. Importantly, the significant association that we observed between total 25-hydroxyvitamin D levels and vitamin D–binding protein genetic polymorphisms was independent of vitamin D–binding protein levels.
Camille E. Powe, M.D.
Brigham and Women's Hospital, Boston, MA
S. Ananth Karumanchi, M.D. Beth Israel Deaconess Medical Center, Boston, MA
Ravi Thadhani, M.D., M.P.H. Massachusetts General Hospital, Boston, MA
Download the PDF from VitaminDWiki
Measurement by a Novel LC-MS/MS Methodology Reveals Similar Serum Concentrations of Vitamin D–Binding Protein in Blacks and Whites
Clark M. Henderson1, Pamela L. Lutsey2, Jeffrey R. Misialek2, Thomas J. Laha1, Elizabeth Selvin3, John H. Eckfeldt4 and Andrew N. Hoofnagle1,5,*
1 Department of Laboratory Medicine and
5 Department of Medicine, University of Washington, Seattle, WA;
2 Division of Epidemiology and Community Health,
4 Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN;
3 Department of Epidemiology and the Welch Center for Prevention, Epidemiology and Clinical Research, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD.
↵* Address correspondence to this author at: Box 357110, University of Washington, Seattle, WA 98115. Fax 206-598-6189. E-mail ahoof at u.washington.edu.
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 10 μL of serum or plasma, the lower limit of quantification for the assay (<20% 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 (κ 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.