Bioavailable 25-Hydroxyvitamin D Measurement

Bioavailable 25-hydroxyvitamin D was defined as circulating 25-hydroxyvitamin D not bound to vitamin D–binding protein, which is analogous to the definition of bioavailable testosterone.^{28 (link)} Concentrations of bioavailable 25-hydroxyvitamin D were calculated in 1025 homozygotes, for whom we could use a single genotype-specific binding affinity constant on the basis of the presence of a single vitamin D–binding protein variant (see the Methods section in the Supplementary Appendix).^{22 (link)} Calculated concentrations of bioavailable 25-hydroxyvitamin D were validated by direct measurement in a subgroup of homozygous participants with the use of a competitive radioligand-binding assay (see the Methods section and Fig. S2 and S3 in the Supplementary Appendix). Measured and calculated 25-hydroxyvitamin D concentrations were correlated (Pearson's r = 0.81 in 32 Gc1F homozygotes and 0.90 in 13 Gc1S homozygotes; P<0.001 for both relationships) (Fig. S4 in the Supplementary Appendix).