Abstract
Katzman and Doisy 1 , 2 reported that the gonadotropic substance found in the urine of pregnant women (P.U.) can be concentrated conveniently by adsorption on benzoic acid or by precipitation with phosphotungstic acid. Hellbaum et al. 3 have shown that the same substance is precipitated quantitatively from the urine by tannic acid. However, the purification of the tannic acid precipitate is rather difficult.
We have found that cresol extracts P.U. efficiently and its use offers definite advantages over the methods mentioned in simplicity, ease of manipulation and recovery of reagents. It removes the active material from the urine as completely as does tannic or benzoic acid and the product is a powder which is a convenient starting material for further purification.
The urine used in these experiments was from a stock supply which had been collected and kept in the coldroom for several days. The cresol was the U.S.P. grade of J. T. Baker Co.
The routine extraction of pregnancy urine is carried out as follows: The urine, in a separatory funnel, is shaken with 1/10 volume of cresol. After allowing to separate, the cresol is drawn off and the urine is extracted a second time with 1/10 volume cresol. The second portion of cresol is then used for the first extraction of a fresh batch of urine. The second urine sample is extracted a second time with a fresh batch of cresol which is also used for the first extraction of a third sample of urine, etc. The cresol, containing the active material is diluted with 2 volumes of acetone which precipitates the active substance. The precipitate is collected by centrifugation or by allowing it to settle to the bottom of the flask and the acetone-cresol solution decanted. The precipitate is washed with acetone and ether and stored as a dry powder. Twenty liters of urine, extracted in this manner yielded 12.6 gm. of dry powder which contained 1 rat unit per milligram. Another batch of urine (24 liters) yielded 19.4 gm. of powder which assayed 1 rat unit per 1.5 mg.
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