尿中Pregnanediol, Pregnanetriol, Pregnanetriolone, PregnanetetrolのGas-Chromatographyによる測定

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A method for the gas-chromatographic estimation of urinary pregnanediol (5β-pregnane-3α, 20a-diol, P-diol), pregnanetriol (5β-pregnane-3α, 17α, 20α-triol, P-triol), pregnanetriolone (58-pregnane-3a, 17a, 20a-triol-11-one, P-triolone) and pregnanetetrol (58-pregnane-3a, Hp, 17β, 20α-tetrol, P-tetrol) is described. A certain amount of 24-hour urine of a healthy person or a patient of congenital adrenal hyperplasis (CAH), was adjusted to pH 4.5 with acetic acid, and added with 300 units of beef liver 8-glucuronidase per 1 ml urine. After incubation at 37°C for 48 hours, it was extracted twice with chloroform, which was washed with 4% NaOH and distilled water, dried with anhydrous Na2SO4, then evaporated to dryness. The dried residue was chromatographed through alumina column and the each fraction containing P-diol and P-triol was brought to gas-chromatography. Alumina column chromatography was performed with the following solvents : (1) 0.5% Ethanol in benzene, 50 ml prewash for P-diol, (3) 2% Ethanol in benzene, 20 ml P-diol fraction, (3) 2% Ethanol in benzene, 30 ml prewash for P-triol, (4) 6% Ethanol in benzene, 40 ml P-triol fraction, containing also P-triolone and P-tetrol. The volumes of solvents suitable for alumina we used, were determined by a preliminary experiment with standard steroids. P-diol and P-triol fractions were dried and the materials were made trimethylsilyl ether (TMSi) through the following procedure : dissolved in 0.2 ml pyridine, added with 0.1 ml hexamethyldisilazane and 0.1 ml trimethylchlorosilane, capped tightly, and shaken in ten minutes or left in 37°C for a few hours. The above solution was dried under nitrogen stream and the residue was dissolved in tetrahydrofuran. A portion of the tetrahydrofuran was served for gas-chromatographic analysis. In gas-chromatography, a 3% XE-60 colum was used for P-diol fraction and a 1.5 % SE-30 column for P-triol fraction, installed in a Model GC-1B Shimazu Gas-chromatograph with hydrogen flame ionization detector (about detailed conditions, see Tables and Figures). Retention time of standard steroids relative to cholestane is shown in Table 1. As the results (Table 2), neither P-triolone nor P-tetrol were detected in the urine of healthy persons. In cases of CAH a remarkable increase in P-triol was noticed, and P-triolone and P-tetrol were detected in good amounts. By an administration of metopirone to a case of CAH, a more increase of P-triol with the disappearance of P-triolone and P-tetrol was discovered.
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尿中Pregnanediol, Pregnanetriol, Pregnanetriolone, PregnanetetrolのGas-Chromatographyによる測定

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