A METHOD FOR THE DETERMINATION OF STEROID 5-ALPHA-REDUCTASE ACTIVITY BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH A FLUORESCENCE DETECTOR

A method for the determination of the activity of steroid 5 alpha-redu ctase in rat ventral prostate has been newly developed. It includes th e enzyme reaction with cortexolone as a substrate, and the process of derivatization of androgens with fluorescent agent and their separatio n by high-performance liquid chromatography with fluorescence detectio n: After the enzyme reaction at 35 degrees C and pH 6.6 in the presenc e of 100 mu M NADPH, cortexolone and reduction products (5 alpha-pregn ane-17 alpha,21-diol-3,20-dione and 5 alpha-pregnane3 alpha,17 alpha,2 1-triol-20-one) were derivatized by treatment with 1-anthroyl nitrile. The resulting fluorescent esters of the three compounds were separate d on an ODS column using 84% (v/v) methanol in water as a mobile phase , and the enzyme activity was estimated from the amounts of the produc ts. The detection limit was 0.2 pmol as an injection amount of each de rivative. The applicability of this method was successfully demonstrat ed in the measurement of the inhibition of the 5 alpha-reductase activ ity by certain steroids.