Radiometric quantitation of delta 4-3-ketosteroid-5 alpha-oxidoreductase utilizing high performance liquid chromatography.

A reliable isocratic high performance liquid chromatography (HPLC) procedure has been developed for the separation and subsequent radiometric quantitation of delta 4-3-ketosteroid-5 alpha-oxidoreductase enzyme activity. The specificity of this HPLC procedure has been confirmed through the use of authentic androgen radioisotopes, linearity of detector response, and mass spectral analysis. In conjunction with this we have also developed a simple Sep-Pak sample preparation procedure which allows the uniform complete isolation of these androgens from epididymal tissue homogenates. Utilizing these procedures we have determined the regional distribution of 5 alpha - reductase in the epididymis of the CD-1 mouse. Regional differences in enzyme activity were found between the caput-corpus and cauda regions. Enzyme activity (specific activity) was higher in the caput-corpus region (28.98 pmol 5 alpha - reduced androgens formed/h/mg protein) than the cauda region (6.20 pmol 5 alpha - reduced androgens formed/h/mg protein).

Studies on neurosteroids XXII. Liquid chromatography-tandem mass spectrometric method for profiling rat brain 3-oxo-4-ene-neuroactive steroids.

A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method for the simultaneous determination of five 3-oxo-4-ene-neuroactive steroids, i.e. androstenedione, testosterone (T), progesterone (PROG), 20alpha-dihydroprogesterone and 20beta-dihydroprogesterone, in rat brain has been developed and validated. The brain steroids were extracted with methanol-acetic acid, purified using solid-phase extraction cartridges and subjected to LC-ESI-MS/MS. The method does not require derivatization. Deuterium-labeled T and PROG were used as the internal standards, and quantification was based on the selected reaction monitoring mode. This method allowed the reproducible and accurate quantification of the brain neuroactive steroids using 100 mg of tissue; the intra- and inter-assay relative standard deviations were below 4.7 and 4.3%, respectively, and the accuracy values were 97.6-103.2% for all the steroids. The limits of quantitation were 0.1 ng/g tissue for all the steroids. The application of this developed method for the analysis of changes in the brain neuroactive steroid levels by immobilization stress is also presented.

Isolation and identification of androstanediol glucuronide from human plasma.

[3H]Dihydrotestosterone (50 microCi) was infused into normal men and women for 8 h. It was previously shown that this was sufficient time for this material to reach a steady state. Venous plasma was obtained at 6 and 8 h, pooled, and the unconjugated steroids removed by ether extraction. The remaining plasma was adjusted to pH 4.9 and the steroid conjugate was extracted first with ethyl acetate and then with an ether-ethanol mixture. The extracts were combined and taken to dryness. Steroid sulfates were solvolyzed using dioxane, and the mixture partitioned between ether and 1% NaOH. The aqueous phase was acidified and added to an XAD-2 column, washed with water, and the glucuronide fraction eluted with methanol. The solvent was concentrated and the methanol extract was passed through a C18 Sep-Pak, filtered through an Acrodisc CR and then subjected to gradient high performance liquid chromatography [HPLC] (Nova-Pak C18, KH2PO4, pH 3, and methanol). The fractions containing steroid glucuronides were collected and esterified with diazomethane and then acetylated with acetic anhydride in pyridine. The glucuronide triacetyl methyl ester (GAME) derivatives were then run in a second HPLC system (3 Lichrosorb 5 mu columns, 4 mm x 25 cm) using a gradient of ethanol-heptane and heptane. We clearly established that this system separates 3 alpha-diol GAME conjugated at the 17 and 3 positions (44 vs 50 min) with authentic samples previously synthesized in our laboratory. We concluded that the pooled plasma contained only the 17-GAME conjugate. No significant activity of the 3-glucuronide was detected. The natural compound in circulation, therefore, is 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide.

Application of over-run thin-layer and gas-liquid chromatography in the separation of closely related C19O2 steroids.

An improved method for the separation of epimeric C19O2 steroids and their related allylic alcohols is described. In this method, the steroids are first separated by over-run thin-layer chromatography, and the unresolved groups are further analysed as free or as trimethylsilyl ether derivatives by gas-liquid chromatography. The behaviour of twenty-one C19O2 steroids was investigated by thin-layer chromatography in four systems and by gas-liquid chromatography in four liquid phases. All steroid pairs of similar polarity were resolved by the combination of these two fractionation procedures.