A new method for simple fractional simultaneous determination of 6 fractions of urinary 17-KS (androstanedione, androsterone, Δ4-androstenedione, etiocholanolone, dehydroepiandrosterone and 11-oxy-17-Ketosteroids) was established and excretion values in normal adult females and several cases with ovarian dysfunction were measured by this method and several fraction ratios were evaluated as necessary informations on their androgenisity. The new method consisted of the following procedures: 170ml of 24 hours urine was adjusted to pH 4.7 with acetic acid and then 6.8ml of 0.2M acetate buffer, 40,800 Fishman units of β-glucuronidase (240 units/ml) and 5,100 units of crystalline penicillin were added, then the urine was incubated in a water bath at 37℃ for 24 hours. Then 20,400 Fishman units of β-glucuronidase were added and it was successively incubated for another 24 hours. The hydrolysed urine was extracted with ethyl acetate three times, 170, 85 and 85ml of ethyl acetate were used. Then, alkali washing with 12.75ml of cold 2N-NaOH twice and water washing with 17ml of distilled water twice were treated to the organic phase, and alkali and water used for washing were reextracted with ethyl acetate. After drying with sodium sulfate anhydrous, the extract was evaporated to residue in vacuo at 60℃. The urine layer after the extraction of β-glucuronidase hydrolysate was saturated with 34g of NaCl, and 17ml of 50% sulfuric acid, 3.4ml of 20% formalin and 170ml of ether were added, and then it was kept standing at room temperature for 4 days being shaken twice a day. After 4 days extraction, another 170ml of ether was used for the reextraction. Each 170ml of ether which extracted solvolysate was washed with 17ml of 1N-NaOH twice and then washed with 21ml of distilled water twice. Alkali and water were reextracted in the same way with the extraction of β-glucuronidase hydrolysate. The layer of ether was dried with sodium sulfate anhydrous and was evaporated to residue at 45℃. Two step hydrolysate was chromatographed as follows; 1: Florisil column chromatography. A glass column 28cm long, of lcm internal diameter was packed with 4g of florisil using chloroform. Steroid residue was absorbed on the column with 3ml×4 of chloroform. The development was carried out with 10ml of chloroform and 80ml of 0.8% v/v methanol-chloroform. The chloroform fraction was discarded as prewash. The fraction of 0.8% methanol-chloroform was evaporated to residue in vacuo at 60℃. The steroid residues obtained thus contained C(19) steroids, and were chromatographed on the following impregnant thin layer chromatography. 2: Impregnant thin layer chromatography. * Preparation of thin layer 20g of Kieselgel GF(254) was mixed vigorously with 40ml of 30% ethyleneglycol/methanol for 3 minutes, and the mixture was spread on a glass plate, 20×20cm, 0.45mm thick. And the plate was allowed to stand at room temperature for few minutes and then it was warmed at 37℃ for 10 minutes. * Spotting and covering Immediately after warming the plate, each steroid residue obtained by column chromatography was spotted on the thin layer and the mixture of 5 authorized pure steroids (11-deoxy-17-KS) was paralelly spotted by 50% methanol-chloroform. After spotting, spotting areas were covered with 50% ethyleneglycol-methanol to keep those areas impregnated. Otherwise, tailing and remained steroid on the spotting area were remarkable. The plate was warmed again at 37℃ for 10 minutes to evaporate methanol. * Development and detection of spots A chromatographic chamber was saturated with organic solvent (10% dichlormethane, 90% n-hexane). The spotted plate was developed in this chamber by the ascending method. When the front of the organic solvent reached 15cm distant from the spotting line, the plate was taken out from the chamber and dried in the air and sulfuric acid was sprayed on the strip of the development of the authorized steroids and then the plate was heated at 110℃ for 20 minutes. Comparing with these spots of standard steroids each residue was separated into the following 6 fractions; androstanedione, androsterone, Δ4-androstenedione, etiocholanolone, dehydroepiandrosterone and 11-oxy-17-KS (from the original spotting area). * Extraction from the impregnant thin layer Each spot was scraped into the centrifuge tube with top and shaked vigorously with 10ml of 50% methanol-chloroform for 5min. Then the tube was centrifuged for 10min. at 3,000 r.p.m. After the organic phase was transfered into the test tube, another 10ml of 50% methanol-chloroform was added to the centrifuge tube and shaked and centrifuged again in the same way. The eluates were collected in the test tube and evaporated at 100℃ in the water bath. Thus 6 fractions of urinary 17-KS were separated easily and recovered satisfactorily. Zimmerman reaction was treated on each sample, simultaneously on 100μg of authorized DHA. After colorimetry at 460-520-580mμ, each fraction was estimated comparing with DHA following correction by Allen's formula.
Urinary samples of 5 fractions of 11-deoxy-17-KS were identified comparing with authorized pure steroids by other thin layer chromatographies, gas liquid chromatography, absorption spectra of Zimmerman reaction, Oertel-Eik-Nes reaction, absorption spectra in methanol and in the concentrated sulfuric acid. Excretion values of 6 fractions of urinary 17-KS were determined by this method through a menstrual cycle of 3 adult normal females and through the treatment for induction of ovulation by gonadotropin on 5 patients with ovarian dysfunction. Following determination, fraction ratios were calculated as the rate of each estimated value divided by sum total of urinary 17-KS. It was confirmed that the androsterone ratio and DHA ratio were evaluated as the indicators of androgenicity. By those experiments, the new simple method for fractional determination of urinary 17-KS was established and would be available at every laboratory to get informations of androgenicity such as androsterone ratio and DHA ratio.