Baseline blood samples were frozen at −80°C and stored for later analysis. 25(OH)D, serum calcium, albumin, and levels of parathyroid hormone (PTH) were measured in the Massachusetts General Hospital (MGH) clinical laboratories. 25(OH)D2 and 25(OH)D3 levels were measured by liquid chromatography–tandem mass spectrometry (LC-MS), with interassay coefficients of variation (CVs) of 9.1% for 25(OH)D2 and 8.6% for 25(OH)D3. Total 25(OH)D level was calculated as the sum of 25(OH)D2 level and 25(OH)D3 level. Intact PTH was measured by electrochemiluminescense immunoassay on the Cobas E160 automated analyzer (Roche Diagnostics, Indianapolis, IN, USA). The interassay CV for intact PTH measurement was 4.2%. Calcium and albumin levels were measured by dye-based photometric assays on an automated analyzer.
DBP was measured in duplicate by commercial enzyme-linked immunosorbent assay (ELISA; Cat. No. DVDBP0, R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. The assay was conducted after diluting serum samples 1:2000 in Calibrator Diluent RD6-11 (Part No. 895489, R&D Systems). The interassay CV was 8.5% at a concentration of 40 µg/mL. The assay recovered between 93% and 110% of a 100- to 200-µg/mL dose of exogenous DBP added to human serum samples containing between 25 and 200 µg/mL of endogenous DBP. The manufacturer reports no significant cross-reactivity with human albumin, vitamin D3, or α-fetoprotein.
In a subset of patients in whom adequate serum was available (n = 45), total 1,25(OH)2D3 was measured by LC-MS/MS in the Mayo Clinic Medical Laboratories (Rochester, MN, USA).
DBP was measured in duplicate by commercial enzyme-linked immunosorbent assay (ELISA; Cat. No. DVDBP0, R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. The assay was conducted after diluting serum samples 1:2000 in Calibrator Diluent RD6-11 (Part No. 895489, R&D Systems). The interassay CV was 8.5% at a concentration of 40 µg/mL. The assay recovered between 93% and 110% of a 100- to 200-µg/mL dose of exogenous DBP added to human serum samples containing between 25 and 200 µg/mL of endogenous DBP. The manufacturer reports no significant cross-reactivity with human albumin, vitamin D3, or α-fetoprotein.
In a subset of patients in whom adequate serum was available (n = 45), total 1,25(OH)2D3 was measured by LC-MS/MS in the Mayo Clinic Medical Laboratories (Rochester, MN, USA).
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