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Monovalent bile acids, such as taurine- and glycine-conjugated bile acids, are excreted into bile by bile salt export pumps (BSEP, ABCB11). Human BSEP (hBSEP) is physiologically important because it was identified as the gene responsible for the genetic disease: progressive familial intrahepatic cholestasis type 2 (PFIC-2). The evaluation of the inhibitory effect of hBSEP transport activity provides significant information for predicting toxic potential in the early phase of drug development. The role and function of hBSEP have been investigated by the examination of the ATP-dependent transport of radioactive isotopically (RI)-labeled bile acid such as a tritium labeled taurocholic acid, in membrane vesicles obtained from hBSEP-expressing cells. The chemiluminescence detection method using 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) had been developed for a simple analysis of bile acids in human biological fluids. This method is extremely sensitive and it may be applicable for the measurements of bile acid transport activities by hBSEP vesicles without using RI-labeled bile acid. The present paper deals with an application of the chemiluminescence detection method using 3alpha-HSD with enzyme cycling method to the measurement of ATP-dependent transport activities of taurocholic acid (T-CA) in membrane vesicles obtained from hBSEP-expressing Sf9 cells. Calibration curves for T-CA was linear over the range from 10 to 400 pmol/ml. The values of the kinetic parameters for hBSEP vesicles obtained by the chemiluminescence detection method were comparable with the values of that obtained by liquid chromatography-mass spectrometry method. This assay method was highly useful for the measurements of bile acid transport activities.

AKR1C20, a member of the aldo-keto reductase (AKR) superfamily, found by mouse genomic analysis, exhibits the highest sequence identity (89%) with mouse liver 17beta-hydroxysteroid dehydrogenase (HSD) type 5, but its function remains unknown. In this report, we have expressed the recombinant AKR1C20 from its cDNA, and examined its properties. The purified enzyme was a 36-kDa monomer, and showed both 17beta-HSD and 3alpha-HSD activities in the presence of NADP(H) as the coenzymes. While the Km values for testosterone and 5alpha-dihydrotestosterone were high (0.2 mM), those for 3alpha-hydroxy- and 3-keto-steroids were low (0.3-5 microM), resulting in high catalytic efficiency for the substrates. Although no significant dehydrogenase activity towards non-steroidal alcohols was observed, the enzyme highly reduced alpha-dicarbonyl compounds such as 16-ketoestrone, 9,10-phenanthrenequinone, acenaphthenequinone, 1-phenylisatin and camphorquinone. The pH optima of the dehydrogenase and reductase activities were 10.5 and 6.5-7.5, respectively. The enzyme was inhibited by sulfobromophthalein, hexestrol, indomethacin and flufenamic acid. The properties of AKR1C20 are distinct from those of previously known mouse 17beta-HSD type 5 (AKR1C6), 3alpha-HSD (AKR1C14) and other members of the AKR1C subfamily. Thus, AKR1C20 is a novel 3alpha(17beta)-HSD, which may also function as a reductase for xenobiotic alpha-dicarbonyl compounds.

In this report, we compared kinetic constants and products in the reduction of the neurosteroids, 3alpha,5alpha-tetrahydroprogesterone (3alpha,5alpha THP) and 3alpha,5alpha-tetrahydrodeoxycorticosterone (3alpha,5alpha-THDOC), and their precursors, 5alpha-dihydroprogesterone (5alpha-DHP), 5alpha-dihydrodeoxycorticosterone (5alpha-DHDOC) and progesterone, by three isoenzymes (AKR1C1, AKR1C2 and AKR1C3) of human 3alpha-hydroxysteroid dehydrogenase. AKR1C1 efficiently reduced 3alpha,5alpha-THP, 5alpha-DHP and progesterone to their 20alpha-hydroxy metabolites, and slowly converted 5alpha-DHDOC to 3alpha,5alpha-THDOC. AKR1C2 exhibited low 20-ketoreductase activity for 3alpha,5alpha-THP and moderate 3-ketoreductase activity for 5alpha-DHP and 5alpha-DHDOC. 3alpha,5alpha-THDOC was not reduced by the two isoenzymes. No significant activity for the steroids was detected with AKR1C3. The results suggest that AKR1C2 is involved in the neurosteroid synthesis, but AKR1C1 decreases the neurosteroid concentrations in human brain by inactivating 3alpha,5alpha-THP and eliminating the precursors from the synthetic pathways. In addition, we found that the several benzodiazepines inhibited the three isoenzymes noncompetitively with respect to the substrate. Although cloxazolam was a potent and specific inhibitor of AKR1C3, diazepam, estazolam, flunitrazepam, medazepam and nitrazepam, that inhibited AKR1C1 and AKR1C2, may influence the neurosteroid metabolism.

When incubated with isolated rat hepatocytes, pravastatin sodium (PS) yielded a small amount of a metabolite in addition to two major metabolites that have already been reported. The previously uncharacterized metabolite was found to be formed by at first being enzymatically dehydrogenated to 6'-keto intermediate (R-104), followed by decomposition to give the aromatized metabolite (R-195), through spontaneous deesterification with accompanying aromatization. The PS-6'beta-hydroxydehydrogenase activity was localized in cytosolic fraction and required NADP, preferentially over NAD, as a cofactor. The formation of R-195 by rat liver cytosol was strongly inhibited by indomethacin, 3 alpha-hydroxysteroids (but not 3 beta-isomers) and 3-ketosteroids. The results and high substrate specificity of purified PS-6'beta-hydroxydehydrogenase toward 3 alpha-hydroxysteroids suggested that the enzyme is identical to 3 alpha-hydroxysteroid dehydrogenase.

Glycyrrhetic acid (GA), an aglycone of glycyrrhizin (GL), is a potent inhibitor of 11 beta- and 3 alpha-hydroxysteroid dehydrogenases. 11 beta-Hydroxysteroid dehydrogenase activity of rat liver microsomes was potently inhibited by GA, 3-deoxyglycyrrhetic acid (3-deoxyGA), 3-ketoglycyrrhetic acid (3-ketoGA), 3-epiglycyrrhetic acid (3-epiGA) and 11-deoxoglycyrrhetic acid (11-deoxoGA), with I50 values of 2-4 x 10(-7) M. However, 18 alpha-stereoisomers (I50 = 3-7 x 10(-6) M) of GA, 3-deoxyGA and 11-deoxoGA were one tenth less inhibitory on the enzyme activity than the corresponding 18 beta-isomers. On the other hand, 18 alpha-stereoisomers of GA, 3-deoxyGA and 11-deoxoGA inhibited 3 alpha-hydroxysteroid dehydrogenase activity of rat liver cytosol more potently than the corresponding 18 beta-isomers. I50 values of 18 alpha- and 18 beta-isomers were 2 and 7 x 10(-6) M, respectively, in the case of GA, 8 and 20 x 10(-6) M in 3-deoxyGA, 3 and 20 x 10(-6) M in 11-deoxoGA. These results indicate that the 18 beta-conformation of oleanane is important for the inhibition of 11 beta-hydroxysteroid dehydrogenase but on the contrary the 18 alpha-conformation is important for the inhibition of 3 alpha-hydroxysteroid dehydrogenase.

A 3 alpha-reducing activity of 5 alpha-dihydrotestosterone (5 alpha-DHT) was found in pig adrenal cytosol. The enzyme (3 alpha-hydroxysteroid dehydrogenase: 3 alpha-HSD) has been purified to homogeneity from pig adrenal cytosol by ammonium sulfate precipitation followed by DEAE-cellulose, 2', 5'-adenosine diphosphate-Sepharose and Sephadex G-100 column chromatographies. The molecular weight was estimated to be 33,000 and 39,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric point was estimated to be 8.5 by isoelectric focusing. The Km and Vmax values for 5 alpha-DHT in the reduction were 10.2 microM and 10.6 nmol/min/mg. The enzyme utilized reduced nicotinamide adenine dinucleotide phosphate (NADPH) or reduced nicotine amide adenine dinucleotide (NADH) in the reduction as a cofactor, but it preferentially required NADPH rather than NADH. Furthermore, the purified enzyme catalyzed not only 3 alpha-reduction of 5 alpha-DHT (9.65 nmol/min/mg), but also catalyzed 20 alpha-reduction of 17 alpha-hydroxyprogesterone (0.58 nmol/min/mg). The enzyme activity of 3 alpha-HSD was strongly inhibited by Hg2+, but it was not inhibited by medroxyprogesterone acetate and some anti-inflammatory agents. No remarkable differences was demonstrated between 3 alpha-HSD and 20 alpha-HSD activity under the influence of heat treatment, divalent cation, anti-inflammatory agents and some inhibitory steroids. These results strongly suggest that 3 alpha-HSD purified from pig adrenal cytosol is a bi-functional enzyme catalyzing 3 alpha- and 20 alpha-HSD activities.

A method for the analysis of androsterone sulfate in serum by means of an enzymatic cycling reaction was developed. Androsterone sulfate was solvolyzed, extracted and purified on Sephadex LH-20, then androsterone was oxidized by 3α-hydroxysteroid dehydrogenase from Pseudomonas tetstosteroni and the resulting NADH was determined by an enzymatic cycling method. The cycling system consisted of two enzyme, alcohol dehydrogenase from yeast and diaphorase from Clostridium kluyverii, and resazurin was used as an electron acceptor for diaphorase. Subsequently, resorfin formed from the resazurin was determined by the rate assay method at Ex560, Em580 and 25°C. A linear relationship was obtained between the amount of androsterone and ΔF/min in the range from 0.035 to 3.5 μg/ml. The recovery, the reproducibility and the correlation between the proposed method and the conventional method using gas-liquid chromatography were also good. It appears that this simple and sensitive method could be useful as a clinical diagnostic test.