A Method for the Isolation of Pregnandiol from the Urine of Pregnant Mares

Abstract

The isolation of pregnandiol from the urine of pregnant mares has been reported by Marker, et al. 1 A method for the isolation of pregnandiol from the urine of pregnant mares based on a method for the quantitative determination of pregnandiol in human pregnancy urine 2 is herewith described because of its relative simplicity and possible application in the study of the function of the corpus luteum of other animals.

The fresh urine∗ without the addition of preservative is incubated at 37°C for 96 hours. It is filtered with suction and the precipitate is collected and heated with 100 cc of 95% ethyl alcohol. The alcoholic mixture is filtered with suction. This filtrate is evaporated to dryness and the residue taken up in 10 cc of acetone and 10 cc of a 0.1 N solution of NaOH. When complete solution is effected with moderate heating, the volume is brought to 50 cc by the addition of 30 cc of 0.1 N NaOH. The solution is placed in the cold and pregnandiol completely precipitates when the solution is thoroughly chilled. The pregnandiol is collected by filtration with suction and the precipitate is washed with warm water. The precipitate is dissolved in a minimum amount of hot acetone and 2 volumes of 0.1 N NaOH added. This is put in the cold to effect precipitation. A third precipitation using a minimum amount of ethyl alcohol and two volumes of water usually give a pure product.

The essential points in the procedure are: (1) The liberation of the free form of pregnandiol from its conjugation form by enzyme action during incubation; (2) the insolubility of pregnandiol in aqueous solutions such as urine : (3) the purification of pregnandiol by precipitation from alkaline acetone.