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.