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

National Animal Disease Center, Science and Education Administration, Agricultural Research US Department of Agriculture, P. O. Box 70 Ames, IA 50010

ABSTRACT

A sensitive, precise assay for vitamin D in plasma is described. Three to five milliliters of plasma were extracted with methanol:methylene chloride (2:1). The lipid extract was chromatographed on Sephadex LH-20¹ 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 ultraviolet absorbance.

We used this assay system for monitoring daily changes of vitamin D₃ in plasma of two Jersey cows after four intramuscular doses (15 x 10⁶ IU) of vitamin D₃ administered at weekly intervals. Basal vitamin D in plasma was 3.2 ± .99 ng/ml with a range of 1.7 to 4.9 ng/ml. Vitamin D₃ in plasma remained relatively low (10 to 45 ng/ml) the week after the first vitamin D₃ injection. Vitamin D3 was high (130 to 234 ng/ml) after the second, third, and fourth injections. Vitamin D₃ decreased steadily to 88 ng/ml by 38 days after the fourth vitamin D₃ injection.

Phosphorus in plasma increased sharply to a plateau at 9.5 mg/100 ml during the week after the second vitamin D₃ injection and returned to normal (4.5 mg/100 ml) at the end of the experiment. Calcium, however, gradually increased to 14.0 mg/100 ml 20 days after the fourth vitamin D₃ injection. Both animals remained hypercalcemic (calcium 11.5 mg/100 ml) during the experiment.