Estimation of Urinary 17-KS Fractions by Thin Layer Chromatography

The subjects study were consisted of 20 nomal healthy males, two groups of 20 women each with normal menstrual cycle, one group of proliferative phase and the other of secretory phase, 10 castrated women, 11 climacteric women and 11 of various diseases. With 24-hours urine collected carefully from these subjects, 10 fractions of 17-KS were measured. For the estimation of fractions, 2-step hydrolysis of β-glucuronidase hydrolysis and solvolysis was conducted first, then ether extraction, rinsed with water, washed again with alkali, dehydrated, dried and fixed, the separation of C(10) fractions was done by Florisil column chromatography with the methanol-chloroform system, and 10 fractions of 17-KS were finally isolated by TLC using 55 cm glass plate. For colorimetry alcoholic Zimmemann's technic was conducted and each 17-KS fraction of the 24-hour urine was estimated. By these procedures, the following results were obtained. 1) After TLC of the purified extracts prepared by the two step hydrolysis, the percentage of β-glucuronidase hydrolysate and solvolysis hydrolysate was determined. As a result, it was found that urinary 17-KS contains 67% of β-glucuronidase hydrolysate and 33% solvolysis hydrolysate. Of the fractions, Ⅳ, Ⅵ, Ⅷ and Ⅸ fractions in β-glucuronidase hydrolysate were over two-fold those in solvolysis hydrolysate. 2) By measuring 10 fractions of 17-KS in 24-hour urine from normal females at proliferative phase, secretory phase and from normal male, values and their ratios were established, The ratios of 10 fractions showed no appreciable difference due the sex gland but the values of Ⅱ, Ⅲ, Ⅳ, Ⅴ and Ⅵ fractions of normal males were more than twice those of normal females. These 11-deoxy-17-KS said to compose the so-called sex gland system show distinct sex differences. Namely, in sum total normal males showed the values 1.9 times of those of normal females. However, these could be seen no significant differences in these values between the women of proliferative phase and those of secretory phase. 3) In the comparative study of the effects of sex gland and adrenals on urinary 17-KS and on the fraction ratio in the groups of castrated women and climacteric women on one hand, and nomal women and men on the other, Ⅳ, Ⅴ and Ⅵ fractions of the 11-deoxy-17-KS groups seem to be derived from the sex glands. Especially, androsterone in normal men is six times that of castrated women, and that of normal women is 3-fold that of castrated women. On the other hand, the excretin of the 11-oxy-17-KS group is not much affected by castration, but as the fraction ratio is increased, this 11-oxy-17-KS group seems to be derived from adrenals. Opinions are divided in that these are many who contend (9)10)11)) that the values of urinary 17-KS are not affected by oophorectomy, and some (12)) claim that these can be observed no decrease in the 11-deoxy-17-KS. However, these differences in the opinions seem to be due to the methods of messurements and the development of those. By the author's own results, the values of 11-deoxy-17-KS are decreased by castration while 11-oxy-17-KS values are somewhat increased. The sum total value of the fractions in the castrated women has been found to be 87% that of normal women at proliferative phase. 4) In the fractionratios between castrated women and climacteric women Ⅲ, Ⅳ and Ⅴ fractions show higher values in the latter group but Ⅵ fraction said to belong to the sex gland system is found to show higher values in castrated women. This seems to be due to the formation etiocholanolone by passing the pathway of THS→11-deoxy-cortol. 5) A new finding that cannot be overlooked and the like of which is not seen in the available literature is the fact average value of androstanedione excreted into urine is 840 μg in normal women, 1190 μg in nomal men, 900 μg in castrated women and 470 μg in climacteric women. 6) Some discussion was made simultaneously about estimated values of urinary 17-KS fractions in several diseases with abnomality in the sex gland and adrenals, though the cases studied were only a few in number