Steroid Analysis by means of Combination of Ultravioletspectrophotometry and Gas-chromatography

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1) Serum lipid of normal human subjects presented the maximal absorption at the region of 235mμ, which was assumed to be due to occurrence of 7-ketocholesterol in it. When this lipid was boiled with 10% methanolic potassium hydroxide, the absorption at 235mμ disappeared and a new absorption maximum at 277mμ appeared. As cholesta-3, 5-dien-7-one has the maximal absorption at 277mμ, it was supposed that 7-ketocholesterol was converted to cholesta-3, 5-dien-7-one by being treated with methanolic potassium hydroxide. It was proved that synthetic pure 7-ketocholesterol was oxidized to form cholesta-3, 5-dien-7-one by being boiled with methanolic potassium hydroxide, and the pure 7-ketocholesterol was isolated from normal serum lipid by preparative thin-layer chromatography, after removal of serum cholesterol by digitonin precipitation ; its melting point 168°C, [α] -108°C, and εmax at 235mμ was 1,340. On gas-chromatogram, 7-ketocholesterol isolated from serum lipid had the same retention time as the pure synthetic preparation. Thus, occurrence of 7-ketocholesterol in normal serum lipid was elucidated.<BR>2) Brain white matter lipid of Schilder's disease presented the maximal absorption at 230mμ and 277mμ. The latter was proved to be due to occurrence of cholesta-3, 5-dien-7-one, by the same method as described in the preceding section and by isolating pure cholesta-3, 5-dien-7-one from the white matter lipid of Schilder's disease. The isolation method mainly used was silicic acid and alumina column chromatography. The former substance, which had the maximal absorption at 230mμ, was assumed by Fieser's formula to be cholesta-3, 5-dien. The lipid was analyzed by gas-chromatography with QF-1 as thestationary liquid phase, and a peak, the retention time of which coincided with that of the synthetic cholesta-3, 5-dien, was observed. Cholesta-3, 5-dien was purified partially from the white matter lipid by preparative thin-layer chromatography and was hydrogenated catalytically with paladium chloride and active carbon. The major product was cholestane and smaller amounts of coprostane was obtained at the same time. These saturated hydrocarbonswere identified on gas-chromatography.<BR>3) It has long been assumed that cholesta-3, 5-dien-7-one and cholesta-3, 5-dien, especially the latter, might be the autoxidation products of cholesterol. Occurrence of cholesta-3, 5-dien-7-one was, however, limited in the peculiar tissues, such as red blood cellsand demyelinated brain white matter. Thus it is presumable that this oxidation product is a metabolite of cholesterol in those tissues. On the other hand, occurrence of cholesta-3, 5-dien was pervading in all tissues. Thus it might be possible that cholesta-3, 5-dien is formed by some oxidation mechanism during extraction and isolation from cholesterol or some metabolite of it. However, further investigation on the dehydroxylation mechanism of cholesterol to form cholesta-3, 5-dien would raise an interesting problem in the cholesterol metabolism.
Japan Society of Clinical Chemistryの論文

Steroid Analysis by means of Combination of Ultravioletspectrophotometry and Gas-chromatography

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