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We previously showed that a single high dose of oral (po) cholecalciferol (D3) sharply increases serum 25-hydroxyvitamin D [25(OH)D].We evaluated the long-term bioavailability and metabolism of a single po or intramuscular (im) high dose of ergocalciferol (D2) or D3.This was a prospective intervention study.The study was conducted in an ambulatory care setting.Participants were 24 subjects with hypovitaminosis D.A single dose of 600,000 IU of po or im D2 or D3 was administered.Serum 25(OH)D and 1,25-dihydroxyvitamin D [1,25(OH)2D] were measured at baseline and at days 30, 60, 90, and 120 by RIA. Serum 1,25(OH)2D2, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], 24,25-hydroxyvitamin D2 [24,25(OH)D2], and 24,25-hydroxyvitamin D3 [24,25(OH)D3] were measured by liquid chromatography-tandem mass spectrometry in a subgroup of patients receiving the po formulations.The areas under the curve of 25(OH)D after D3 were significantly higher than those after D2 (P < .0001). Serum 25(OH)D basal difference significantly increased at day 30 with po D2 and D3 (P < .01 and P < .0001) and up to day 90 with po D3 (P < .01). The im formulations produced a slow increased, and values peaked at day 120 relative to the other time points (P < .0001). We found a decrease in 1,25(OH)2D at day 30 (P < .05) and up to day 120 (P < .001) and an increase in 1,25(OH)2D2 at day 30 (P < .01) and up to day 120 (P < .01) after po D2. Oral D2 and D3 produced increases in 24,25(OH)D2 and 24,25(OH)D3, respectively, at day 30 (P < .001).A po dose of 600,000 IU of D2 or D3 is initially more effective in increasing serum 25(OH)D than the equivalent im dose and is rapidly metabolized. Our RIA assay for 1,25(OH)2D may not recognize 1,25(OH)2D2.