Urinary Gonadotropins from Goats

I. Extraction by the Kaolin Method

Abstract

The method for extraction of gonadotropins from the urine of goat was investigated. Specimens of human urine collected from postmenopausal and normally menstruating women were used for a preliminary study. Twenty-four-hour urine samples were collected from cyclic and pregnant goats of the Japanese Saanen breed. Crude urinary gonadotropic substances (crude UGS) were extracted from these urine samples by means of a modification by the authors of the kaoline-absorption method developed by Bradbury, Brown and Brown. Biological assays of crude UGS were conducted with the enlargement of the uterus in injected immature mice as criterion. When the uterine weight was increased by at least 100% of that of the control animal, it was considered that a positive response was exhibited. A mouse uterine unit (MUU) was defined as the smallest amount of crude UGS which made 50% of the injected mice exhibit a positive response.
The kaolin method used by the present authors is as follows.
1. Add 5 volume percent of 20% kaolin suspension to acidified-filtrated urine (pH4.5) and stir thoroughly for 5 minutes.
2. Discard the supernatant and collect the precipitated kaolin fraction. Add accidified water (pH4.5) and stir vigorously. Decant the supernatant and wash the precipitate repeatedly with pH-4.5 water so that the kaolin may be free from urine.
3. Add 1N ammonia water and stir thoroughly for 20 minutes. After centrifugation, add ammonia water again to the precipitate, stir, and centrifuge. Combine the second ammonia eluate with the first.
4. Adjust the combined eluates to pH8.5 with 2N acetic acid and centrifuge. Adjust the supernatant to pH5.5 and centrifuge. Add 5 volumes of cold acetone to the supernatant and keep it in the refrigerator overnight. Collect and dry the precipitate.
Gonadotropic activity could be determined in crude UGS derived from human urine by means of biological assay, but could not in those derived from goat urine due to the toxic effect of contamination found in them. No positive response was given by mice injected with crude UGS derived from goat urine and purified by some simple procedures.
It was concluded that the kaolin-extract from goat urine had a toxic effect which would affect the general condition of animals used for assay and thereby influenced the responsiveness of the end-point in the biological assay. Therefore, it was required to purify further crude UGS derived from goat urine.