Assay for Vitamin D2 and Vitamin D3 in Plasma of Dairy Cows: Changes after Massive Dosing of Vitamin D3

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Abstract
A sensitive, precise assay for vitamin D in plasma is described. Plasma (3-5 ml) was extracted with methanol:methylene chloride (2:1). The lipid extract was chromatographed on Sephadex LH-20 and then on Lipidex-5000 columns. After high pressure liquid chromatography with a reverse phase chromatographic system, vitamin D2 and vitamin D3 were quantitated by UV absorbance. This assay system was used for monitoring daily changes of vitamin D3 in plasma of 2 Jersey cows after 4 i.m. doses (15 .times. 106 IU) of vitamin D3 administered at weekly intervals. Basal vitamin D in plasma was 3.2 .+-. 0.99 ng/ml with a range of 1.7-4.9 ng/ml. Vitamin D3 in plasma remained relatively low (10-45 ng/ml) the week after the 1st vitamin D3 injection. Vitamin D3 was high (130-234 ng/ml) after the 2nd, 3rd and 4th injections. Vitamin D3 decreased steadily to 88 ng/ml by 38 days after the 4th vitamin D3 injection. P in plasma increased sharply to a plateau at 9.5 mg/100 ml during the week after the 2nd vitamin D3 injection and returned to normal (4.5 mg/100 ml) at the end of the experiment. C gradually increased to 14.0 mg/100 ml 20 days after the 4th vitamin D3 injection. Both animals remained hypercalcemic (C 11.5 mg/100 ml) during the experiment.