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A CYTOCHEMICAL SECTION BIOASSAY FOR PLASMA TRILOSTANE: AN ORALLY ACTIVE INHIBITOR OF 3β‐HYDROXYSTEROID DEHYDROGENASE ACTIVITY

Trilostane is a competitive inhibitor of 3β‐hydroxysteroid dehydrogenase activity in vitro and an orally active inhibitor of steroidogenesis in vivo. A cytochemical section bioassay for this drug (4α, 5‐epoxy‐17β‐hydroxy‐3‐oxo‐5α‐androstane‐2α‐carbonitrile) in human plasma has been developed. The assay is based upon the inhibition of 3β‐hydroxysteroid dehydrogenase (3β‐HSD) activity in the corpus luteum of unfixed tissue sections (6 μm) of the mature dioestrous rat ovary. Trilostane is extracted from plasma by a simple chloroform procedure. The stanuard curve (0·15–2·5 μmol/I pure trilostane) exhibits a four‐fold change in 3β‐HSD activity and is constructed in the presence of extract from control plasma pools. The detection limit of the plasma assay is 2μmol/I trilostane after allowing for dilution effects and recovery losses. This level gives a significantly different response from the zero quality control (P<0·05) and has an inter‐assay coefficient of variation of < 15%. No false positives have been recorded to date and the majority of samples from subjects receiving therapeutic doses of the drug have levels of > 2 μmol/1. Intra‐ and inter‐assay coefficients of variation using both plasma quality controls and unknown samples are 4% (n= 15) and 13·9% (n= 27) respectively. Recovery is 80·9% (n= 13) and 79·10% (n= 10) for plasma quality controls containing 12 and 4·8 μmol/1 trilostane respectively. Dilutions (up to 1:5) of plasma extracts are parallel to the standard curve and the assay throughput is approximately 16 samples/week technician. Testosterone, oestradiol, oestriol, dehydroepiandros‐terone (DHEA), DHEA‐sulphate, progesterone, cortisol, androstenedione and dexamethasone (all at 1 μmol/1) were without effect on the 3β‐HSD assay. It is concluded that the bioassay is suitable for assaying trilostane in human plasma.