Serum 25(OH)D.

Blood was drawn after an overnight fast by trained phlebotomists, and samples were processed and stored at −80°C until analysis at Boston University Medical Center. Serum total 25(OH)D was measured by using LC-MS/MS on a TSQ Quantum Ultra triple mass spectrometer (Thermo Finnigan Corp.). This method included fractionation of 25-hydroxyergocalciferol [25(OH)D₂], 25-hydroxycholecalciferol [25(OH)D₃], and epimer of 25(OH)D₃ in serum (28). 25(OH)D₃ and 25(OH)D₂ calibration control solutions were generated from standards provided by Calbiochem. Samples from study subjects were prepared and analyzed through a turbulent flow LC system (Cohesive Technologies) followed by traditional laminar flow chromatography. Study samples were analyzed relative to vitamin D standard references (National Institute of Standards and Technology), and the intra-assay (within-batch) coefficient of variation is 6.0%. Total 25(OH)D was calculated as the sum of the 25(OH)D₃ and 25(OH)D₂ components.