Fluorometric Determination of Conjugated 3-hydroxysteroid in Human Serum using Hydroxysteroid Dehydrogenase

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Serum 3-hydroxysteroids (3-OHS) were estimated fluorometrically with hydroxysteroid dehydrogenase (HSD) coupled with diaphorase-resazurin system. The serum sample (0.5ml) was deproteinized with 1.5M HCIO4 and the supernatant was heated at 100°C for 10 minutes. The hydrolyzed steroids were extracted with methylene chloride and evaporate to dryness. The residue was incubated with NAD, HSD, diaphorase and resazurin, and resorufin, the reaction product, was measured by fluorometry.(λ1=57 Onm, λ2=580nm). The sensitivity of this method increased about 20 times compared with photometry of NADH (340nm), and 6 times compared with fluorometry of NADH. Two hydrolysis method, solvolysis and acid hydrolysis, were studied and the latter was more suitable for the application of laboratory routine work. Normal levels of 3-OHS were 1.69±0.49μg/ml (mean±2 S. D., n=14) for male and 1.41±0.64μg/ml (n=8) for female. Correlation between urinary steroids (17-KS and 17-OHCS) and serum steroids (3-OHS and 11-OHCS) were studied and high correlation was observed between urinary 17-OHCS and serum 11-0HCS (r=0.698), and between urinary 17-KS and serum 3-OHS (r=0.600). Serum 3-OHS could be an indicator of adrenal androgenic function as a substitute of urinary 17-KS.
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