Biosynthesis of hydroxyl-linked glucuronides of short-chain bile acids by rat liver 3-hydroxysteroid UDP-glucuronosyltransferase

Microsomal preparations from livers of Sprague-Dawley rats catalyze the glucuronidation of 3 alpha-hydroxy-5 beta-H (3 alpha, 5 beta) short-chain bile acids (C20-C23), predominantly at the hydroxyl group, while the glucuronidation of 3 beta, 5 beta short-chain bile acids occurs exclusively at the carboxyl group. A similar pattern of conjugation was also observed in Wistar rats having normal levels of 3-hydroxysteroid UDP-glucuronosyltransferase. Significant reductions of formation rates for hydroxyl-linked, but not carboxyl-linked, short-chain bile acid glucuronides were observed in hepatic microsomes from Wistar rats with low 3-hydroxysteroid UDP-glucuronosyltransferase activity. 3-Hydroxysteroid UDP-glucuronosyltransferase, purified to homogeneity from Sprague-Dawley liver microsomes, catalyzed the 3-O-glucuronidation of 3 alpha, 5 beta C20-23 bile acids, as well as of lithocholic and isolithocholic acids (C24). The apparent Michaelis constants (KM) for short-chain bile acids were similar to the value obtained for androsterone. 3 alpha, 5 beta-C20 and 3 beta, 5 beta-C20 competitively inhibited glucuronidation of androsterone by the purified 3-hydroxysteroid UDP-glucuronosyltransferase. Purified 17 beta-hydroxysteroid and p-nitrophenol UDP-glucuronosyltransferases did not catalyze the glucuronidation of bile acids. In addition, none of the purified transferases catalyzed the formation of carboxyl-linked bile acid glucuronides. The results show that 3-hydroxysteroid UDP-glucuronosyltransferase, an enzyme specific for 3-hydroxyl groups of androgenic steroids and some conventional bile acids, also catalyzes the glucuronidation of 3 alpha-hydroxyl (but not carboxyl) groups of 3 alpha, 5 beta short-chain bile acids.     

An enzymatic cycling method for the determination of serum total bile acids with recombinant 3alpha-hydroxysteroid dehydrogenase

A highly sensitive enzymatic cycling method was developed for the serum total bile acids assay. We constructed a prokaryotic expression system to prepare the recombinant 3alpha-hydroxysteroid dehydrogenase in place of the natural enzyme and for the first time used it in the total bile acids assay. The production rate of thio-NADH correlated with the bile acids concentration and was measured by the change of absorbance at 405/660 nm. The enzymatic cycling method could detect 0.22 micromol/L total bile acids in serum. Within-run and between-run imprecisions were 1.2-3.7% and 2.3-4.8%, respectively. The calibration curve for total bile acids in serum was linear between 0.5 and 180 micromol/L. This method was free from interference by bilirubin, hemoglobin, ascorbate, and lactate dehydrogenase. In conclusion, serum total bile acids could be measured by the enzymatic cycling method with recombinant 3alpha-hydroxysteroid dehydrogenase as the tool enzyme.     

Molecular basis of human 3β-hydroxysteroid dehydrogenase deficiency

The enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD) catalyses an essential step in the biosynthesis of all classes of steroid hormones. Classical 3β-HSD deficiency is responsible for CAHII, a severe form of congenital adrenal hyperplasia (CAH) that impairs steroidogenesis in both the adrenals and gonads. Newborns affected by 3β-HSD deficiency exhibit signs and symptoms of adrenal insufficiency of varying degrees associated with pseudohermaphroditism in males, whereas females exhibit normal sexual differentiation or mild virilization. Elevated ratios of 5-ene-to 4-ene-steroids appear as the best biological parameter for the diagnosis of 3β-HSD deficiency. The nonclassical form has been suggested to be related to an allelic variant of the classical form of 3β-HSD as described for steroid 21-hydroxylase deficiency. To elucidate the molecular basis of the classical form of 3β-HSD deficiency, we have analysed the structure of the highly homologous type I and II 3β-HSD genes in 12 male pseudohermaphrodite 3β-HSD deficient patients as well as in four female patients. The 14 different point mutations characterized were all detected in the type II 3β-HSD gene, which is the gene predominantly expressed in the adrenals and gonads, while no mutation was detected in the type I 3β-HSD gene predominantly expressed in the placenta and peripheral tissues. The finding of a normal type I 3β-HSD gene provides the explanation for the intact peripheral intracrine steroidogenesis in these patients and increased androgen manifestations at puberty. The influence of the detected mutations on enzymatic activity was assessed by in vitro expression analysis of mutant enzymes generated by site-directed mutagenesis in COS-1 cells. The mutant type II 3β-HSD enzymes carrying mutations detected in patients affected by the salt-losing form exhibit no detectable activity in intact tranfected cells, whereas those with mutations found in nonsalt-loser index cases have some residual activity ranging from 1–10% compared to the wild-type enzyme. Although in general, our findings provide a molecular explanation for the enzymatic heterogeneity ranging from the severe salt-losing form to the clinically inapparent salt-wasting form of the disease, we have observed that the mutant L108W or P186L enzymes found in a compound heterozygote male presenting the salt-wasting form of the disease, has some residual activity (1%) similar to that observed for the mutant N100S enzyme detected in an homozygous male patient suffering from a nonsalt-losing form of this disorder. Unlike the classical 3β-HSD deficiency, our study in women presenting nonclassical 3β-HSD deficiency strongly suggests that this disorder is not due to a mutant type II 3β-HSD.     

Characterisation of an associate 17-β-hydroxysteroid dehydrogenase activity and affinity labelling of the 3-α-hydroxysteroid dehydrogenase of Pseudomonas testosteroni

The 3-α-hydroxysteroid dehydrogenase and the 3-β-hydroxysteroid dehydrogenase of Pseudomonas testosteroni were purified to homogeneity by polyacrylamide gel electrophoresis using the following stages: DEAE cellulose chromatography, affinity chromatography on oestrone-aminocaproate sepharose and Sephadex gel filtration.The pure 3-α-hydroxysteroid dehydrogenase was completely devoid of 3-β-hydroxysteroid dehydrogenase activity but could oxidize estradiol 17-β at an appreciable rate. This activity accounts for about 40 per cent of the total 17-β-estradiol dehydrogenase of the crude bacterial extract.Affinity labelling of pure 3-α-hydroxysteroid dehydrogenase was carried out using 5-β-pregnane 3,20-dione-12-α-iodoacetate and 5-α-androstane 3-one-17-β-bromoacetate. With both reagents, inactivation was obtained only in the presence of coenzyme, the substrate protected against inactivation and the enzyme was fully inhibited with covalent binding of 1 mole of reagent per mole of subunit suggesting an active site directed inhibition. Histidine and methionine were identified as the labelled aminoacid residues.     

Demonstration of 3 alpha(17 beta)-hydroxysteroid dehydrogenase distinct from 3 alpha-hydroxysteroid dehydrogenase in hamster liver.

NAD(+)-linked and NADP(+)-linked 3 alpha-hydroxysteroid dehydrogenases were purified to homogeneity from hamster liver cytosol. The two monomeric enzymes, although having similar molecular masses of 38,000, differed from each other in pI values, activation energy and heat stability. The two proteins also gave different fragmentation patterns by gel electrophoresis after digestion with protease. The NADP(+)-linked enzyme catalysed the oxidoreduction of various 3 alpha-hydroxysteroids, whereas the NAD(+)-linked enzyme oxidized the 3 alpha-hydroxy group of pregnanes and some bile acids, and the 17 beta-hydroxy group of testosterone and androstanes. The thermal stabilities of the 3 alpha- and 17 beta-hydroxysteroid dehydrogenase activities of the NAD(+)-linked enzyme were identical, and the two enzyme activities were inhibited by mixing 17 beta- and 3 alpha-hydroxysteroid substrates, respectively. Medroxyprogesterone acetate, hexoestrol and 3 beta-hydroxysteroids competitively inhibited 3 alpha- and 17 beta-hydroxysteroid dehydrogenase activities of the enzyme. These results show that hamster liver contains a 3 alpha(17 beta)-hydroxysteroid dehydrogenase structurally and functionally distinct from 3 alpha-hydroxysteroid dehydrogenase.     

A novel 3-hydroxysteroid dehydrogenase that regulates reproductive development and longevity

Endogenous small molecule metabolites that regulate animal longevity are emerging as a novel means to influence health and life span. In C. elegans, bile acid-like steroids called the dafachronic acids (DAs) regulate developmental timing and longevity through the conserved nuclear hormone receptor DAF-12, a homolog of mammalian sterol-regulated receptors LXR and FXR. Using metabolic genetics, mass spectrometry, and biochemical approaches, we identify new activities in DA biosynthesis and characterize an evolutionarily conserved short chain dehydrogenase, DHS-16, as a novel 3-hydroxysteroid dehydrogenase. Through regulation of DA production, DHS-16 controls DAF-12 activity governing longevity in response to signals from the gonad. Our elucidation of C. elegans bile acid biosynthetic pathways reveals the possibility of novel ligands as well as striking biochemical conservation to other animals, which could illuminate new targets for manipulating longevity in metazoans.     

Luteolytic action of RU486: Modulation of luteal 3β-hydroxysteroid dehydrogenase and 20α-hydroxysteroid dehydrogenase activities in late pregnant rats

The effect of the synthetic antiprogestin RU486 on luteal function in late pregnant rats was studied by evaluating the activities of the enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD) and 20α-hydroxysteroid dehydrogenase (20α-HSD). RU486 (2 mg/kg) administered to rats on day 18 of pregnancy at 10.00 h induced preterm delivery 26.4 ± 0.35 h (n = 8) after treatment. Luteal 3β-HSD activity increased 24 and 34 h after RU486 injection, but a significant and progressive decrease started at 48 h with the maximal reduction 72 h after RU486 treatment, when compared with controls. Serum progesterone concentration decreased at the time of 3β-HSD activity reduction. Interestingly, 20α-HSD activity started to increase 58 h after RU486 injection. The administration of the cyclooxygenase inhibitor, diclofenac (1.3 mg/kg), on days 17–19 of pregnancy to RU486-treated rats, delayed abortion and the duration of delivery, and prevented the decrease in 3β-HSD and the increase in 20α-HSD activities observed 58 h after antiprogesterone treatment. RU486 administered intrabursally (1 μg per ovary) on day 20 (14.00–15.00 h) increased 3β-HSD and decreased 20α-HSD luteal activities at 18.00 h on day 21 of pregnancy, without modifying serum progesterone concentration, when compared with normal pregnant rats. In conclusion, the luteolytic process after preterm delivery induced by RU486 administration in late pregnant rats is characterized by a decrease in luteal 3β-HSD activity and circulating progesterone, which may trigger the increase in luteal 20α-HSD activity. Prostaglandins seems to be involved in the increase of 20α-HSD activity and therefore, in the demise of corpora lutea.     

Heterogeneity of 11β-hydroxysteroid dehydrogenase in rat tissues

Using specific antisera to purified rat liver 11β-hydroxysteroid dehydrogenase (11-HSD), we showed that the antigen is widely distributed in rat organs. Enzyme activity and immunoreactivity generally corresponded. Highest by both criteria were liver, testis, kidney and lung. In some tissues (epididymis, pancreas and duodenum) activity was found, but antigen corresponding to 11-HSD at a M_w of 34 kDa was absent. It is suggested that these tissues have alternate enzyme forms. The 11-HSD of brain and liver were compared. Brain enzyme may control selective binding of aldosterone to Type I receptors in the hippocampus and other regions. Rat brain 11-HSD resembled that of liver or kidney in most characteristics. It differed in (a) its steroid specificity: cortisol was a good substrate for liver 11-HSD, and a poor substrate for brain enzyme; (b) stability of 11-oxoreductase (11-OR) component. Brain 11-OR was not readily inactivated; 11-OR from other tissues lost activity rapidly and spontaneously. The variations in properties of 11-HSD in specific tissues may reflect aspects of its various specific functions.     

Total fasting and postprandial serum bile acids determination by 3 alpha-hydroxysteroid dehydrogenase in normal subjects

Total serum bile acids (SBA) have been evaluated in 28 healthy subjects by an enzymatic-fluorimetric method using the enzyme 3 alpha-hydroxysteroid dehydrogenase. The healthy subjects showed a value of 2.78 +/- 1.84 microM/1 (mean +/- SD), ranging 0.5 to 4.7 microM/1. In addition, two hours postprandial evaluation of SBA was carried out in five of the above subjects. The mean value of SBA before and after the meal was 2.58 +/- 1.4 and 8.73 +/- 1.7 microM/1 respectively and the postprandial increase was found statistically significant (P less than 0.001). The present method for quantitative determination of SBA seems to be sensitive, reliable and useful as a routine clinical aid.     

The occurrence and subcellular localization of 3 -hydroxysteroid dehydrogenase in adult rat brain

The subcellular localization of 3α-hydroxysteroid dehydrogenase, the dependency of the rate of reaction on time, concentration of protein, cofactor requirements and the substrate stereospecificity were investigated in the adult rat brain. The $\text{in vitro}$ conversion of 3-keto-5β-cholanoic-24-¹⁴C acid to lithocholic acid was shown to occur in the cytosol without added cofactors. Incubation of ¹⁴C labeled 3α,7α-dihydroxy-5β-cholanoic and 3α,12α-dihydroxy-5β-cholanoic acids with adult rat brain cell-free preparations resulted in the production of less polar metabolites identified as 3-keto-7α-hydroxy-5β-cholanoic and 3-keto-12α-hydroxy-5β-cholanoic acids by TLC, GLC combined with a radioactive monitoring detection system and by cocrystallization to constant specific activity.     

Differential estrogenic 17β-hydroxysteroid dehydrogenase activity and type 12 17β-hydroxysteroid dehydrogenase expression levels in preadipocytes and differentiated adipocytes

Estradiol (E2) is produced locally in adipose tissue and could play an important role in fat distribution and accumulation, especially in women. It is well recognized that aromatase is expressed in adipose tissue; however the identity of its estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD) partner is not identified. To gain a better knowledge about the enzyme responsible for the conversion of estrone into estradiol, we determined the activity and expression levels of known estrogenic 17β-HSDs, namely types 1, 7 and 12 17β-HSD in preadipocytes before and after differentiation into mature adipocytes using an adipogenic media. Estrogenic 17β-HSD activity was assessed using [¹⁴C]-labelled estrone, while mRNA expression levels of types 1, 7 and 12 17β-HSD were quantified using real-time PCR and protein expression levels of type 12 17β-HSD was determined using immunoblot analysis. The data indicate that there is a low conversion of E1 into E2 in preadipocytes; however this activity is increased 5-fold (p < 0.0001) in differentiated adipocytes. The increased estrogenic 17β-HSD activity is consistent with the increase in protein expression levels of 17β-HSD12.     

NMR studies of the stereospecificity of 20 -hydroxysteroid dehydrogenase

We (3,4) previously observed the reduction of 21-dehydrocorticosteroids in the presence of 20β-hydroxysteroid dehydrogenase proceeded at a faster rate than the reduction of the corresponding corticosteroids. The presence of adjacent carbonyl groups suggested the possibility that the increased rate of reduction of the 20-one,21-a1 steroid analogs resulted from a lack of specificity of the enzyme 20β-hydroxysteroid dehydrogenase for either the aldehyde or ketone group. Nuclear magnetic resonance spectroscopy indicated that the angular methyl groups of the steroid were sensitive probes for the constituents on the basic steroid skeleton. The C₁₈ methyl resonance of 17,21-dihydroxy-4-pregnene-3,20-dione and 17-hydroxy-3,20-dioxo-4-pregnene-21-a1 were 0.722 ppm and 0.728 ppm respectively. The magnitude and sign of the change in chemical shift of the C₁₈ methyl resonance for the enzymatic products of 17,21-dihydroxy-4-pregnene-3,20-dione and 17-hydroxy-3,20-dioxo-4-pregnene-21-a1 (+0.135 ppm and +0.144 ppm respectively) were consistent with a stereochemical assignment of 20β-hydroxyl.     

Placental 11β-hydroxysteroid dehydrogenase and the programming of hypertension

Excessive foetal exposure to glucocorticoids retards growth and “programmes” adult hypertension in rats. Placental 11β-hydroxysteroid dehydrogenase (11β-HSD), which catalyses the conversion of corticosterone and cortisol to inert 11 keto-products, normally protects the foetus from excess maternal glucocorticoids. In both rats and humans there is considerable natural variation in placental 11β-HSD, and enzyme activity correlates with birth weight. Moreover, inhibition of placental 11β-HSD in the rat reduces birth weight and produces hypertensive adult offspring, many months after prenatal treatment with enzyme inhibitors; these effects are dependent upon maternal adrenal products. These data suggest that placental 11β-HSD, by regulating foetal exposure to maternal glucocorticoids, crucially determines foeto-placental growth and the programming of hypertension. Maternal protein restriction during pregnancy also produces hypertensive offspring and selectively attenuates placental 11β-HSD activity. Thus, deficiency of the placental barrier to maternal glucocorticoids may represent a common pathway between the maternal environment and foeto-placental programming of later disease. These data may, at least in part, explain the human epidemiological observations linking early life events to the risk of subsequent hypertension. The recent characterization, purification and cDNA cloning of a distinct human placental 11β-HSD (type 2) will aid the further study of these intriguing findings.