Laboratory measures
Vitamin D is highly lipophilic, and in plasma is almost entirely protein-bound. VDBP is the principal carrier of both 25(OH)D and 1,25(OH)2D (85-90% of total): a smaller amount of each metabolite (10-15%) is bound to albumin (a lower affinity carrier), and ≤1% exists unbound in the ‘free’, biologically active form.31, 32 ‘Bioavailable’ vitamin D is the sum of unbound (free) and albumin-bound: it is considered another useful estimate of biologically active vitamin D because the affinity of vitamin D for albumin is so low.31-35
Plasma 25(OH)D concentration was measured using the DiaSorin Corporation 25-hydroxyvitamin D 125I RIA Kit (Stillwater, MN, USA). No dilution was required. The intra- and inter-assay coefficients of variation were ≤10%. 1,25(OH)2D was measured by Quantitative Chemiluminescent Immunoassay at ARUP laboratories, Salt Lake City, UT, after dilution 6.25-fold. VDBP was measured using the human Vitamin D BP Quantikine ELISA kit (R&D systems, Minneapolis, MN, USA), per manufacturer’s protocol: plasma samples were diluted 10,000-fold, assayed in duplicate, and intra- and inter-assay coefficients of variation were 3% and 7%, respectively. Operators were masked to clinical status and sample order throughout and all samples from an individual were in the same assay run. Circulating albumin levels were measured at the Department of Clinical Biochemistry, Royal Victoria Hospital, Belfast, Northern Ireland. The levels of free and bioavailable 25(OH)D and 1,25(OH)2D were calculated from total measured 25(OH)D, 1,25(OH)2D, VDBP and albumin concentrations using the following equations31, 32, 36:
\begin{equation} \mathrm{\text{Calculated\ free\ }}\left[\mathrm{D}\right]\mathrm{\ =}\frac{\mathrm{\text{Total\ }}\left[\mathrm{D}\right]}{1+\left(K_{\text{Alb}}\times\left[\mathrm{\text{Albumin}}\right]\right)+\left(K_{\text{VDBP}}\times\left[\mathrm{\text{VDBP}}\right]\right)}\nonumber \\ \end{equation}\begin{equation} \mathrm{\text{Bioavailable\ }}\left[\mathrm{D}\right]\mathrm{=}\left(K_{\text{Alb}}\times\left[\mathrm{\text{Albumin}}\right]+1\right)\times\mathrm{calculated\ free\ [D]}\nonumber \\ \end{equation}\begin{equation} \mathrm{\text{The\ percentage\ of\ \ free\ }}\left[\mathrm{D}\right]\mathrm{\ =}\frac{\mathrm{\text{Free\ }}\left[\mathrm{D}\right]}{\mathrm{\text{Total\ }}\left[\mathrm{D}\right]}\nonumber \\ \end{equation}\begin{equation} \left[\mathrm{D}\right]=\mathrm{25(OH)D\ or\ }\mathrm{1,25(OH)}_{\mathrm{2}}\mathrm{D}\nonumber \\ \end{equation}\begin{equation} \left[\mathrm{\text{Albumin}}\right]=\mathrm{serum\ albumin\ in\ g/L\ \div}\mathrm{\ }\mathrm{66,430}\mathrm{\ }\mathrm{g/mol}\nonumber \\ \end{equation}\begin{equation} \left[\mathrm{\text{VDBP}}\right]=\mathrm{serum\ VDBP\ in\ g/L\ \div\ 58,000}\mathrm{\ }\mathrm{g/mol}\nonumber \\ \end{equation}
Note that the affinity constants for albumin (25(OH)D: KAlb=6×105 M−1; 1,25(OH)2D: KAlb=5.4×104 M−1) are substantially lower than those for VDBP (25(OH)D: KVDBP=7×108 M−1; 1,25(OH)2D: KVDBP=4×107M−1).31, 32
The amount of each vitamin D metabolite bound to VDBP was calculated by subtracting bioavailable from total vitamin D. Using this information, it was possible to calculate VDBP saturation for 25(OH)D and 1,25(OH)2D.