3-Keto-50-hydroxysteroids

The enzyme 3a-hydroxysteroid dehydrogenase plays a key role in this transport across the hepatocyte. A particularly elegant experiment demonstrated the role of the 3a-hydroxysteroid dehydrogenase, by using [ H] at the 3p hydrogen to show cyclical oxidation-reduction of the 3a-hydroxyl with no accumulation of 3-keto bile acids. Confirmation was obtained by use of indo-methacin, an inhibitor of 3a-hydroxysteroid dehydrogenase, which decreased... 

The most common assay uses 3a-hydroxysteroid dehydrogenase to form the 3-keto bile acid that is trapped by, for example, hydrazine hydrate, causing the reaction to go to completion. The co-factor NAD is reduced stoichiometrically and can be measured by ultraviolet absorption or more commonly by fluorescence at an activation of 345 nm and emission of 450 nm. Use of this enzyme measures all bile acids with a 3a-hydroxyl but not cholesterol, which has a 3p-hydroxyl, and does not measure bile acids with a sulphate or glucuronide group conjugated to the 3a-hydroxyl. 

A. 17,20 lyase is required for androgen synthesis, cyclooxygenase for prostaglandin production, 11- 3-hydroxysteroid dehydrogenase-2 acts as a reductase-converting cortisol to its inactive 11-keto derivative cortisone, whereas 18-hydroxylase is required for aldosterone production. 

Some of the effects of glucocorticoids can be attributed to their binding to aldosterone receptors (ARs). Indeed, ARs bind aldosterone and cortisol with similar affinity. A mineralocorticoid effect of cortisol is avoided in some tissues by expression of llE>-hydroxysteroid dehydrogenase type 2, the enzyme responsible for biotransformation to its 11-keto derivative (cortisone), which has minimal affinity for aldosterone receptors. 

Hoog SS, Pawlowski JE, Alzari PM, Penning TM, Lewis M. Three-dimensional structure of rat liver -hydroxysteroid/dihydrodiol dehydrogenase a member of the aldo-keto reductase superfamily. Proc Natl Acad Sci USA 1994 91 2517-2521. 

Cell specificity of mineralocorticoid action is achieved in a different manner. Aldosterone, cortisol, and corticosterone bind with similar affinities to mineralocorticoid and glucocorticoid receptors. However, aldosterone activates only its own receptor in target tissues such as the kidney because of an enzyme, 110-hydroxysteroid dehydrogenase, that converts the prevalent glucocorticoids into inactive 11-keto derivatives but does not affect aldosterone. 

Oximino-17-ketosteroids (1) are reduced by zinc dust in refluxing acetic acid to 16-keto-17 8-hydroxysteroids. The reaction proceeds through the 16f-amino-17-ketone (2). A related reduction reported by Mauthner and by Windaus in 1903 is the reduction of 6-nitrocholesteryl acetate (5) to 6-ketocholestanyl acetate (6). 

Dehydration. Ehrenstein and co-workers find that 3-keto-5/3-hydroxysteroids can be dehydrated to the A -B-ketosteroids by refluxing with the reagent in acetic acid solution. 

The enzyme 3a-hydroxysteroid dehydrogenase (EC 1.1.1.50), which is isolated from Pseudomonas testosteroni, catalyzes the conversion of all 3a-hydroxycholanic acids to 3-ketochoIanic acids, with the concomitant reduction of NAD to NADH. The NADH formed in the reaction is then determined spectrophotometrically at 340 nm. To ensure complete reaction, hydrazine is usually added to bind the 3-keto products (P2). The optimum conditions for enzymatic assay include a pH of 9.0 to 9.5 and reaction temperature in the range 20 to 40 C (T13). Reaction rates for individual bile acids may not be identical, but the assay is normally carried out as an endpoint determination. Alternatively, the addition of bovine serum albumin appears to overcome the problem of variable aflinity of 3a-hy-droxysteroid dehydrogenase for different bile acids if reaction rates are to be measured (S13). 

Besides the involvement in the de novo biosynthesis of BH4, SR may also participate in the pterin salvage pathway by catalyzing the conversion of sepiapterin (Figure 14, 47) into 7,8-dihydrobiopterin (46) that is then transformed to BH4 by dihydrofolate reductase (DHFR EC 1.5.1.3). Both reactions consume NADPH. Although SR is sufficient to complete the BH4 biosynthesis, a family of alternative NADPH-dependent aldo—keto reductases, including carbonyl reductases (CR), aldose reductases (AR), and the 3a-hydroxysteroid dehydrogenase type 2 (AKR1C3) may participate in the diketo reduction of the carbonyl side chain in Moreover, based on the discover) of the autosomal recessive deficiency for SR, which presents... 

The crystal structures of 3 a, 20)ff-hydroxysteroid dehydrogenase [58], J -3-keto steroid isomerase [56], and cholesterol oxidase [57] have been reported. 

The discovery of the steroid-active isozymes of horse liver alcohol dehydrogenase (Sections II,A,l,a and II,A,l,d) also established that 3-keto-and 3 3-hydroxysteroids are substrates, but the functional significance of this is not clear. Since this activity also seems to be present in the rat enzyme (9) and in all isozymes of the human enzyme (W), it may be more important than previously realized when the horse ethanol-active isozyme was the most studied form. It may also be noted that mammalian livers contain many different specific steroid dehydrogenases (11). Structural studies have established that mammalian alcohol dehydrogenases have a distant evolutionary link to both the yeast (ff) and bacterial enzymes (IS). 

The oxidation-reduction process for the conversion of hydroxysteroids to keto-steroids (and the reverse reaction) has been studied in detail by T dalay and his collaborators with induced enzymes isolated from Pseudomonas testosterord. They purified one enzyme system which oxidized the 3o -hydroxyl group and another which oxidized both 3j3- and 17/3-hydroxyl groups. Talalay has characterized the oxidation-reduction reaction with these enzymes as NAD-linked and illustrated the general reaction as ... 

Butenandt, Dannenberg, and Suranyi (B-80) reported the reduction of A -3-keto-5a-steroids to 3/3-hydroxysteroids with yeast. 

The 3 hydroxysteroid reductase of rat liver which catalyzes the conversion of XXX to cholestanol (XXXI) was localized also in the microsomal fractions, and shown to provide the epimeric alcohols in a ratio of 10 1 (3/3 3a) in the presence of NADPH. The enzyme was not inhibited by cholestanol, but pronounced inhibition was noted with 7-keto- or 7a-hydroxycholestanoI (XXXII) or zl -cholestenone (XXVIII) (120). This enzyme differs from the Cj9 steroid reductase, since the latter utilize NADH equally well and provides predominantly the 3a-ol. 

This activity refers to the oxidation of primary alcohols to aldehydes and of secondary alcohols to ketones, as well as to the corresponding reverse reductions. Besides simple aliphatic mono and polyalcohols, such as ethanol and ethylene glycol, some steroids, such as 3-keto and 3-beta-hydroxysteroids have also been found to... 

Sensitive methods for SBA based on bioluminescence measurement have recently been deve p using hydroxysteroid dehydrogenase (3o( or 12d or 7d -HSD). The HSD catalyzes the conversion of the bile acid hydroxy g up (3o( or 7d or 12 c. ) to a keto-group in the presence of NAD(P). The resulting NAD(P)H, in presence of NAD(P)H FMN oxydoreductase (OXRED), converts FMN to its reduced form (FMNH ). This in turn reacts with decanal and oxygen in the presence dr bacterial luciferase to produce light. The intensity of the light emitted is proportional to the amount of bile acids in the initial raction. The overall scheme,for 7 -HSD for instance, is this ... 

---