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PubMed2006

A novel polymorphism in the 17beta-hydroxysteroid dehydrogenase type 5 (aldo-keto reductase 1C3) gene is associated with lower serum testosterone levels in caucasian men.

Genetic variation in the androgen metabolizing enzymes is important to identify and feature as they may influence the risk of prostate cancer and help clarify the etiology of the disease. Human 17beta-hydroxysteroid dehydrogenase type 5 (AKR1C3) is highly expressed in the prostate gland and plays a major role in the formation and metabolism of androgens. We identified five novel polymorphisms in the AKR1C3 gene. One of those an A>G substitution in exon 2 that confers a Glu77Gly change occurred in 4.8% in Caucasians but was completely absent in Orientals. Interestingly, the testosterone level in serum was significantly lower in subjects with the Gly77 allele. A promoter A>G polymorphism was associated with significantly altered promoter activity in reporter constructs, but was not associated with any change in testosterone levels. In conclusion, the Glu77Gly polymorphism is associated with lower testosterone levels in serum.

17-Hydroxysteroid Dehydrogenases↗
PubMed1986

Inactivation of 3 alpha-hydroxysteroid dehydrogenase by superoxide radicals. Modification of histidine and cysteine residues causes the conformational change.

3 alpha-Hydroxysteroid dehydrogenase (EC 1.1.1.50) from Pseudomonas testosterone was inactivated by superoxide radicals generated by the aerobic xanthine oxidase reaction. Superoxide dismutase, NAD+, bovine serum albumin and histidine and cysteine as free amino acids partially protected the enzyme from inactivation. NADH-binding properties were determined by fluorescence spectroscopy, and no variation was found between native enzyme and the unmodified fraction of the partly inactivated one. The fluorescence emission maximum for the completely inactivated enzyme was shifted 10 nm to a longer wavelength when compared with the native one, and it seems possible that the modification of histidine and cysteine residues by superoxide radicals causes the conformational change of the enzyme and the consequent loss of catalytic activity.

3-Hydroxysteroid Dehydrogenases↗
PubMed1986

Factors responsible for the formation of different N-alkylated porphyrins in rat liver microsomal systems exposed to norethindrone. The role of 3 alpha-hydroxysteroid dehydrogenase.

Incubation of rat liver microsomes with norethindrone and a NADPH-generating system leads to the formation of one N-alkylated porphyrin (green pigment, GP1). Administration of this steroid to male rats in vivo results in the formation of three more-polar green pigments (GP2, 3 and 4). A cytosolic protein (green-pigment converting protein) has been purified from rat liver that, when added to liver microsomal mixtures containing norethindrone (0.03 mM) and a NADPH-generating system, results in the formation of all four green pigments (GP1, 2, 3 and 4). Field-desorption mass spectrometry of the purified green pigments gave protonated molecules, [M + H]+, at m/z 905 for GP1, m/z 909 for GP2, m/z 925 for GP3 and 4. The Mr of the purified cytosolic protein on SDS/polyacrylamide-gel electrophoresis or gel filtration was 37000. Polyacrylamide-gel isoelectric focusing gave a pI value of 5.9. Antibodies raised in rabbits against this protein, after preincubation with rat liver cytosol, subsequently prevented the formation of the more-polar norethindrone-induced green pigments (GP2, 3 and 4). The purified protein in the presence of either NADH or NADPH catalysed the reduction of delta 4-ring-reduced norethindrone, 5 alpha-oestran-17 alpha-ethynyl-17 beta-ol-3-one and, with the appropriate cofactor, the oxidation and reduction of steroids lacking the ethynyl function, e.g. androsterone or dihydrotestosterone. Indomethacin inhibited the reduction of dihydrotestosterone by this protein with an I50 (concn. causing 50% inhibition) value of 4.9 microM. From its physical and enzymic properties it is concluded that green-pigment converting protein is the same as 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50).

3-Hydroxysteroid Dehydrogenases↗
PubMed1987

Affinity-labelling of the anti-inflammatory drug and prostaglandin-binding site of 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol with 17 beta- and 21-bromoacetoxysteroids.

The homogeneous 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol binds prostaglandins with low micromolar affinity at its active site and is competitively inhibited by the non-steroidal and steroidal anti-inflammatory drugs [Penning, Mukharji, Barrows & Talalay (1984) Biochem. J. 222, 601-611]. To examine the portion of this binding site that accommodates the glucocorticoid side chain, we have synthesized 17 beta-bromoacetoxy-5 alpha-dihydrotestosterone (BrDHT) and 21-bromoacetoxydesoxycorticosterone (BrDOC) as affinity-labelling agents. Both these agents promote rapid inactivation of the purified enzyme in a time- and concentration-dependent manner. Analyses of the inactivation progress curves gave estimates of Ki for the inactivators and half-life (t1/2) for the enzyme at saturation (tau) as follows: Ki = 33 microM and tau = 18 s for BrDHT, and Ki = 10 microM and tau = 203 s for BrDOC. Under initial-velocity conditions BrDHT and BrDOC act as competitive inhibitors, yielding Ki values identical with those measured in the inactivation experiments. Both indomethacin and prostaglandin E2 can protect the enzyme from inactivation, yielding Ki values for these ligands consistent with those measured independently by competitive-inhibition studies. These data confirm that the bromoacetoxysteroids label the active site, which is coincident with the prostaglandin- and anti-inflammatory-drug-binding site. Neither gel filtration nor extensive dialysis restores activity to the enzyme inactivated with either affinity-labelling agent. Use of radioactive BrDHT or BrDOC, in which either the steroid portion is labelled with 3H or the bromoacetate portion is labelled with 14C, indicates that inactivation is accompanied by a stoichiometric incorporation of 0.7-1.0 molecules of inhibitor per enzyme monomer. The linkage that forms between the dehydrogenase with either [14C]BrDHT or [14C]BrDOC is stable to acid and base treatment. Complete acid hydrolysis of the enzyme inactivated with [14C]BrDHT, followed by amino acid analyses, indicates that 87% of the radioactivity is eluted with carboxymethylcysteine. An almost identical result is obtained with [14C]BrDOC, where at least 75% of the radioactivity is eluted with this amino acid. Thus BrDHT and BrDOC alkylate at least one reactive cysteine residue at the active site that may be of functional importance in binding the glucocorticoid side chain.

3-Hydroxysteroid Dehydrogenases↗
PubMed1991

The kinetic mechanism catalysed by homogeneous rat liver 3 alpha-hydroxysteroid dehydrogenase. Evidence for binary and ternary dead-end complexes containing non-steroidal anti-inflammatory drugs.

Rat liver 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) (EC 1.1.1.50) is an NAD(P)(+)-dependent oxidoreductase that is potently inhibited at its active site by non-steroidal anti-inflammatory drugs (NSAIDs). Initial-velocity and product-inhibition studies performed in either direction at pH 7.0 are consistent with a sequential ordered Bi Bi mechanism in which pyridine nucleotide binds first and leaves last. This mechanism is supported by fluorescence titrations of the E-NADH complex, and by the failure to detect the binding of either [3H]androsterone or [3H]androstanedione to free enzyme by equilibrium dialysis. Dead-end inhibition studies with NSAIDs also support this mechanism. Initial-velocity studies with indomethacin show that this drug is an uncompetitive inhibitor against NAD+, but a potent competitive inhibitor against androsterone, indicating the ordered formation of an E.NAD+.indomethacin complex. Calculation of the individual rate constants reveals that the binding and release of pyridine nucleotide is rate-limiting and that isomerization of the central complex is favoured in the forward direction. Equilibrium dialysis experiments with [14C]indomethacin reveal the presence of two abortive NSAID complexes, a high-affinity ternary complex corresponding to E.NAD+.indomethacin (Kd = 1-2 microM for indomethacin) and a low-affinity binary complex corresponding to E.indomethacin (Kd = 22 microM for indomethacin). Since indomethacin has a low affinity for free enzyme, the formation of this abortive binary complex does not complicate kinetic measurements which are made in the presence of NAD+, but may contribute to the inhibition of the enzyme by NSAIDs. Using either pro-R-[4-3H]NADH or pro-S-[4-3H]NADH as cofactor, radiolabelled androsterone was formed only when the pro-R-[4-3H]NADH was used, confirming that purified 3 alpha-HSD is a Class A dehydrogenase.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Purification and characterization of a novel dimeric 20 alpha-hydroxysteroid dehydrogenase from Tetrahymena pyriformis.

Tetrahymena pyriformis was found to exhibit high NADPH-dependent 20-oxosteroid reductase activity that converted 17 alpha-hydroxyprogesterone into 17 alpha,20 alpha-dihydroxypregn-4-en-3-one. The enzyme was purified 400-fold from the cytosolic fraction. The purified enzyme with a specific activity of 6.4 mumol/min per mg of protein had an isoelectric point of 4.9 and M(r) of 68,000, and was composed of two subunits of equal size. The N-terminal sequence was determined to be LAKTVPLNDGTNFPIFGG. The enzyme reduced pregnanes and pregnanes possessing a 17 alpha-hydroxy group to a greater extent than those without the hydroxy group, and oxidized 20 alpha-hydroxy groups of the steroids in the presence of NADP+. The Km values for 17 alpha-hydroxyprogesterone and 17 alpha-hydroxypregnenolone were 2.9 and 3.4 microM respectively. Although the enzyme was inactive towards androgens and oestrogens with 3- or 17-oxo groups, it reduced several nonsteroidal carbonyl compounds and oxidized trans-benzene dihydrodiol. The enzyme activity was inhibited by synthetic oestrogens, barbiturates, aldose reductase inhibitors and quercitrin. Thus, this enzyme is a novel form of 20 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.149) which structurally and functionally differs from the mammalian and bacterial enzymes.

17-alpha-Hydroxypregnenolone↗
PubMed1996

Purification of 11 beta-hydroxysteroid dehydrogenase type 2 from human placenta utilizing a novel affinity labelling technique.

11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) efficiently inactivates potent glucocorticoid hormones (cortisol and corticosterone), leaving aldosterone unmetabolized. Abundant 11 beta-HSD2 activity in human placenta plays a central role in controlling fetal glucocorticoid exposure, which if excessive is harmful and may predispose to low birth weight and hypertension in adulthood. Similar 11 beta-HSD2 activity in the distal nephron protects mineralocorticoid receptors from glucocorticoids and appears to be important in normal blood pressure control. We have purified human placental 11 beta-HSD2 16000-fold, to homogeneity, and determined over 100 residues of the internal amino acid sequence. Purification was assisted by a novel technique allowing highly specific (single spot on two-dimensional electrophoresis) photoaffinity labelling of active 11 beta-HSD2 in crude tissue extracts by its glucocorticoid substrates. This work reveals that 11 beta-HSD2 is a member of the short-chain alcohol dehydrogenase superfamily (apparent monomer M(r) approximately 40,000). It is a very basic (apparent pI = 9.1) intrinsic membrane protein, requiring as yet undefined membrane constituents for full stability. Affinity chromatography and affinity labelling studies suggest that 11 beta-HSD2 has a compulsory ordered mechanism, with NAD+ binding first, followed by a conformational change allowing glucocorticoid binding with high affinity.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1996

Cloning and production of antisera to human placental 11 beta-hydroxysteroid dehydrogenase type 2.

By inactivating potent glucocorticoid hormones (cortisol and corticosterone), 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) plays an important role in the placenta by controlling fetal exposure to maternal glucocorticoids, and in aldosterone target tissues by controlling ligand access to co-localized glucocorticoid and mineralocorticoid receptors. Amino acid sequence from homogeneous human placental 11 beta-HSD2 was used to isolate a 1897 bp cDNA encoding this enzyme (predicted M(r) 44126; predicted pI 9.9). Transfection into mammalian (CHO) cells produces 11 beta-HSD2 activity which is NAD(+)-dependent, is without reductase activity, avidly metabolizes glucocorticoids (Km values for corticosterone, cortisol and dexamethasone of 12.4 +/- 1.5, 43.9 +/- 8.5 and 119 +/- 15 nM respectively) and is inhibited by glycyrrhetinic acid and carbenoxolone (IC50 values 10-20 nM). Rabbit antisera recognizing 11 beta-HSD2 have been raised to an 11 beta-HSD2-(370--383)-peptide-carrier conjugate. Recombinant 11 beta-HSD2, like native human placental 11 beta-HSD2, is detectable with affinity labelling and anti-11 beta-HSD2 antisera, and appears to require little post-translational processing for activity. 11 beta-HSD2 mRNA (approximately 1.9 kb transcript) is expressed in placenta, aldosterone target tissues (kidney, parotid, colon and skin) and pancreas. In situ hybridization and immunohistochemistry localize abundant 11 beta-HSD2 expression to the distal nephron in human adult kidney and to the trophoblast in the placenta. 11 beta-HSD2 transcripts are expressed in fetal kidney (but not lung, liver or brain) at 21-26 weeks, suggesting that an 11 beta-HSD2 distribution resembling that in the adult is established by this stage in human development.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1996

11 beta-hydroxysteroid dehydrogenase type 1 expression in 2S FAZA hepatoma cells is hormonally regulated: a model system for the study of hepatic glucocorticoid metabolism.

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) is a key enzyme in glucocorticoid metabolism, catalysing the conversion of active glucocorticoids into their inactive 11-keto metabolites, thus regulating glucocorticoid access to intracellular receptors. The type 1 isoform (11 beta-HSD 1) (EC 1.1.1.146) is widely distributed, with particularly high levels in liver, where accumulating evidence suggests that it acts as an 11 beta-reductase, regenerating active glucocorticoids. Investigation of the function and regulation of 11 beta-HSD 1 in liver has been hampered by the lack of hepatic cell lines which express 11 beta-HSD 1. Here, we describe 11 beta-HSD 1 mRNA expression and activity in 2S FAZA cells, a continuously cultured rat liver cell line. In intact 2S FAZA cells 11 beta-HSD 1 acts predominantly as a reductase, with very low dehydrogenase activity. In 2S FAZA cells 11 beta-HSD 1 activity and mRNA expression are regulated by hormones, with dexamethasone increasing activity and insulin, forskolin and insulin-like growth factor 1 decreasing it. Transfection of 2S FAZA cells with a luciferase reporter gene driven by the proximal promoter of the rat 11 beta-HSD 1 gene demonstrates that sequences which can mediate the responses to insulin, dexamethasone and forskolin all lie within 1800 bp of the transcription start site.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1990

Mineralocorticoid activity of carbenoxolone: contrasting effects of carbenoxolone and liquorice on 11 beta-hydroxysteroid dehydrogenase activity in man.

1. 11-beta-Hydroxysteroid dehydrogenase is an enzyme complex consisting of 11 beta-dehydrogenase and 11-oxoreductase responsible for the interconversion of cortisol to cortisone in man. Inhibition of 11 beta-dehydrogenase (e.g. after liquorice ingestion) results in cortisol acting as a potent mineralocorticoid. We have evaluated the effect of the synthetic liquorice derivative, carbenoxolone, on this enzyme complex. 2. Carbenoxolone given to six volunteers in metabolic balance produced sodium retention with suppression of the renin-angiotensin-aldosterone system. Plasma potassium fell, although there was no kaliuresis. This was associated with inhibition of 11 beta-dehydrogenase (as measured by a rise in the plasma half-life of [11 alpha-3H]cortisol). Unlike liquorice, however, carbenoxolone also inhibited 11-oxoreductase (as measured by the generation of cortisol after oral cortisone acetate). 3. The mineralocorticoid activity of carbenoxolone, like liquorice, is mediated via cortisol by inhibition of 11 beta-dehydrogenase. Carbenoxolone, however, also inhibits 11-oxoreductase activity and this may relate to its effect on renal potassium excretion.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2002

Effect of glycyrrhetinic acid on 11 beta-hydroxysteroid dehydrogenase activity in normotensive and hypertensive subjects.

The 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) isoenzymes catalyse the interconversion of cortisol and cortisone. Type 1 11 beta-HSD mainly converts cortisone into active cortisol. Type 2 11 beta-HSD inactivates cortisol in mineralocorticoid target tissues, and its activity can be inhibited by glycyrrhetinic acid (GA). Inactivation of cortisol to cortisone is impaired in a subgroup of patients with primary hypertension. To study where this defect is located, we measured cortisol and cortisone concentrations in arterial plasma, in saliva and across the forearm at baseline and after administration of GA in normotensive and hypertensive subjects. GA (500 mg) or placebo was administered orally to 20 normotensive subjects in a placebo-controlled double-blind fashion. Further, we compared the effect of GA in 20 patients with primary hypertension with that in 20 normotensive subjects. Cortisol and cortisone were measured in plasma from the brachial artery and vein and in saliva. Samples were obtained at 0, 90 and 150 min. Forearm blood flow (FBF) was measured simultaneously. Forearm production of corticosteroid hormones was assessed by multiplying the arteriovenous difference in corticosteroid concentration by FBF. The cortisol/cortisone ratio in arterial plasma remained at baseline levels after placebo (4.9 +/- 1.2; mean +/- S.D.), while after GA the ratio increased similarly in normotensive subjects (12.3 +/- 3.4) and in hypertensive patients (12.2 +/- 3.7). A similar effect of GA on the salivary cortisol/cortisone ratio was found. In both normotensive subjects and hypertensive patients no forearm production of cortisol or cortisone could be demonstrated, either at baseline or after administration of GA. Thus, both before and after GA administration, we did not find any difference in systemic and salivary 11 beta-HSD type 2 activity between subjects with primary hypertension and normotensive controls. Further, both at baseline and after GA administration we were not able to demonstrate net inactivation or re-activation of cortisol and cortisone by the 11 beta-HSD isoenzymes in the forearm in either normotensive or primary hypertensive subjects.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1996

Urinary free cortisone and the assessment of 11 beta-hydroxysteroid dehydrogenase activity in man.

OBJECTIVE: Two isoforms of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) catalyse the interconversion of cortisol to hormonally inactive cortisone; defects in the 11 beta-HSD2 isoform result in hypertension. The kidney, expressing high levels of 11 beta-HSD2, is the principal source of cortisone in man. We have validated the measurement of urinary free cortisone (UFE) excretion in normals and in patients with disorders of the pitultary-adrenal axis in an attempt to more accurately measure the activity of 11 beta-HSD2 in vivo. SUBJECTS: Forty-one normal adults, 12 normal children < 12 years of age, 15 patients with Cushing's syndrome, 12 with hypopitultarism on replacement hydrocortisone, 12 with the syndrome of apparent mineralocorticoid excess (AME) and 7 volunteers consuming liquorice. MEASUREMENTS: A complete 24-hour urine collection was analysed by gas chromatography/mass spectrometry for "A-ring' reduced cortisol and cortisone metabolites, i.e. tetrahydrocortisols (THF and allo-THF) and tetrahydrocortisone (THE). In addition, urinary free cortisol (UFF) and urinary free cortisone were quantified using deuterium-labelled internal standards. RESULTS: In normal adults and children, UFE excretion exceeded that of UFF (UFF 30.4 +/- 2.4 micrograms/24h (mean +/- SE), UFE 54.6 +/- 4.1 micrograms/24h, adults) (for conversion to nmol/24h multiply E by 2.78 and F by 2.76 respectively). Thus the normal UFF/UFE ratio was 0.54 +/- 0.05 in contrast to the (THF + allo-THF)/THE ratio of 1.21 +/- 0.06. UFE excretion was normal in hypopituitary patients on replacement hydrocortisone. Although UFE was elevated in all forms of Cushing's syndrome, the UFF/UFE ratio was grossly elevated in patients with the ectopic ACTH syndrome (14.0 +/- 6.7, n = 6). UFE was below the lower limit of the assay (< 1 microgram/24h) in most patients with the so-called type 1 variant of AME and significantly reduced in 4 patients described as having the type 2 variant of AME (10.5 +/- 3.5 micrograms/h, P < 0.05) and in 7 volunteers consuming liquorice (26.8 +/- 10.0 micrograms/24h, P < 0.01). In ectopic ACTH syndrome, AME, and liquorice ingestion the UFF/UFE ratio was more deranged than the (THF + allo-THF)/THE ratio. CONCLUSION: In normals the discrepant THF + allo-THF/ THE and UFF/UFE ratio suggests that much more of the UFE is derived from the kidney. Reduction in UFE excretion is seen following liquorice ingestion and in both variants of AME, though it is more profound in AME1. The high UFF/UFE ratio in the mineralocorticoid excess state seen in the ectopic ACTH syndrome is compatible with substrate-saturation of renal 11 beta-HSD2. The measurement of UFE and the UFF/UFE ratio is a significant advance in the analysis of human 11 beta-HSD activity in vivo; in particular, the UFF/UFE ratio appears to be a more sensitive index than the (THF + allo-THF)/THE ratio of renal 11 beta-HSD2 activity.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1996

Apparent mineralocorticoid excess due to 11 beta-hydroxysteroid dehydrogenase deficiency: a possible cause of intrauterine growth retardation.

A Japanese boy with apparent mineralocorticoid excess (AME) is described. He was born with intrauterine growth retardation (IUGR) and elevated serum level of creatine phosphokinase (CPK). He was studied at 2 years of age because of polyurea and polydipsia of one year's duration and was found to have hypokalaemic alkalosis and sustained hypertension. His plasma renin activity and aldosterone levels were always low and his ratio of urinary tetrahydrocortisol plus allo-tetrahydrocortisol to that of tetrahydrocortisone was very high. Therefore, AME due to 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) deficiency was diagnosed. He was successfully treated with a combination of spironolactone and nifedipine for at least 16 months. His blood pressure, plasma pH and serum potassium levels were normalized by this treatment, but serum CPK level remained high. We researched the birth records of previously reported AME cases and found that IUGR is a characteristic feature of AME. The mechanism by which IUGR occurs in AME is discussed and we speculate that 11 beta-HSD might be deficient in the placenta and/or fetal tissues, as well as in the kidney, in AME. An explanation for the elevated CPK could not be found.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1997

Placental 11 beta-hydroxysteroid dehydrogenase: a key regulator of fetal glucocorticoid exposure.

OBJECTIVE: Placental 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), which converts active cortisol to inactive cortisone, has been proposed to be the mechanism guarding the fetus from the growth retarding effects of maternal glucocorticoids; however, other placental enzymes have also been implicated. Placental 11 beta-HSD is unstable in vitro, and enzyme activity thus detected may not be relevant to the proposed barrier role. We have therefore examined placental glucocorticoid metabolism in dually perfused freshly isolated intact human placentas. DESIGN: Placentas were obtained from randomly selected normal term deliveries. The maternal circuit was perfused with physiological concentration of cortisol, the fetal effluent collected and steroid metabolites separated and quantified using silica columns (Sep-pak Plus) and HPLC. RESULTS: Most of the maternally administered cortisol was metabolized to cortisone, and no conversion of cortisone to cortisol was detected. Cortisone was the only product of cortisol metabolism. Inhibition of 11 beta-HSD with glycyrrhetinic acid allowed cortisol to gain direct access to the fetal circulation. CONCLUSION: We conclude that human placental 11 beta-HSD plays a crucial role in controlling glucocorticoid access to the fetus. Other enzymes are not significant contributors at physiologically relevant cortisol concentrations.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1997

Additional value of measurement of urinary cortisone and unconjugated cortisol metabolites in assessing the activity of 11 beta-hydroxysteroid dehydrogenase in vivo.

OBJECTIVE: It has been shown recently that 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) is expressed as at least 2 isozymes. In the liver, 11 beta-HSD1 converts cortisone to cortisol; in the kidney, 11 beta-HSD2 converts cortisol to cortisone. Conventional assessment of 11 beta-HSD activity in vivo has relied on gas chromatographic measurement of the ratios of conjugated cortisol and cortisone metabolites. However, these do not permit distinction between the tissue-specific activities of the enzymes and do not reflect all forms of 11 beta-HSD deficiency. In this report, we have assessed the usefulness of measuring unconjugated cortisol metabolites and free cortisol and cortisone in urine as indices of renal 11 beta-HSD activity in man. DESIGN: Six healthy male subjects established in sodium balance were given either glycyrrhetinic acid (170 mg t.d.s., to inhibit 11 beta-HSD2), carbenoxolone (100 mg t.d.s., to inhibit both 11 beta-HSD1 and 11 beta- HSD2) or both inhibitors in combination. MEASUREMENTS: Urinary electrolytes were measured and the concentrations of total and unconjugated urinary cortisol and its metabolites were determined by gas chromatography mass spectrometry. RESULTS: Glycyrrhetinic acid and carbenoxolone inhibited renal 11 beta-HSD2 to a similar degree, as judged by similar sodium retention. As previously reported, conventional measurement of ratios of total cortisol to cortisone metabolites were influenced to a greater extent by glycyrrhetinic acid (100-200% increase in ratio from baseline) than by carbenoxolone (< 30% increase). However, the effect of carbenoxolone was readily detected by measurement of urinary unconjugated cortisol/cortisone (130-480% increase of ratio from baseline) and also by measurement of ratios of unconjugated cortisol metabolites (60-130% increase). CONCLUSIONS: Measurement of free cortisol and cortisone in urine provides the most sensitive index of renal 11 beta-HSD activity. Measurement of total and conjugated urinary steroids is insensitive in circumstances where 11 beta-HSD activity in liver or elsewhere may be abnormal.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1998

Growth hormone replacement inhibits renal and hepatic 11 beta-hydroxysteroid dehydrogenases in ACTH-deficient patients.

OBJECTIVES: The commonest side-effects of GH replacement therapy relate to sodium retention but its mechanism is unclear. In rats, GH inhibits renal and hepatic 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) activities. In man, this action might impair inactivation of cortisol to cortisone in the distal nephron thereby allowing cortisol to activate mineralocorticoid receptors. In this study, we examined the effects of GH replacement on cortisol metabolism. DESIGN: A randomized double-blind study comparing 6 months GH replacement with placebo in adults with GH deficiency due to pituitary or hypothalamic disease. PATIENTS: 6 men and 8 women received placebo and 8 men and 9 women received GH. MEASUREMENTS: Cortisol and its metabolites were measured in overnight urine samples and in a fasting morning plasma sample at baseline and at 6 months. RESULTS: Five patients withdrew from the study, 4 because of adverse effects of GH. Amongst those who completed the study, the effects of GH on cortisol metabolism differed between patients with and without ACTH deficiency. Amongst those receiving hydrocortisone replacement (n = 18), GH had no effect on plasma cortisol/cortisone ratio, or urine tetrahydrocortisols/tetrahydrocortisone ratio, but produced a substantial reduction in total urinary cortisol metabolites (from 1326 +/- 191 to 777 +/- 229 micrograms/mmol creatinine; P < 0.01) and an increase in urinary free cortisol/cortisone ratio (from 0.88 +/- 0.10 to 2.57 +/- 0.74; P < 0.02). By contrast, GH had none of these effects in patients with normal ACTH secretion. There were no changes in blood pressure or plasma electrolytes, aldosterone, or renin activity with GH, and no changes in any variables with placebo. CONCLUSIONS: The increase in cortisol/cortisone ratio in urine but not in plasma, and the lack of effect on hepatic cortisol metabolites, suggests that GH inhibits both the conversion of cortisol to cortisone by renal 11 beta-HSD2 and the conversion of cortisone to cortisol by hepatic 11 beta-HSD1. It is unclear why this action of GH only affects patients with ACTH deficiency. The reduction in total cortisol metabolite excretion suggests that GH also affects bioavailability of hydrocortisone tablets. These observations suggest that glucocorticoid replacement therapy may need to be adjusted in hypopituitary patients who are commenced on GH replacement.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2000

Endogenous inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 do not explain abnormal cortisol metabolism in polycystic ovary syndrome.

OBJECTIVE: The aetiology of enhanced adrenal androgen secretion in polycystic ovary syndrome is poorly understood. Previous reports suggest that enhanced peripheral metabolism of cortisol results in decreased negative feedback suppression of ACTH secretion, either by enhanced inactivation of cortisol by 5alpha-reductase or impaired reactivation of cortisol by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). Endogenous inhibitors of hepatic 11beta-HSD1 can be extracted from urine. We have tested the hypothesis that these are increased in patients with polycystic ovary syndrome. DESIGN: A case-control study. PATIENTS: 57 patients with polycystic ovary syndrome and 27 healthy control women. MEASUREMENTS: Aliquots from 24 h urine samples were extracted with Sep-Paks and incubated with rat liver microsomes in which 11beta-HSD1 activity was quantified by conversion of 3H-corticosterone to 3H-11-dehydrocorticosterone. RESULTS: Inhibition of 11beta-HSD1 activity was not different in extracts from patients compared with controls (40.8 +/- 18.9 arbitrary units in patients vs. 42.7 +/- 16.6 in controls, mean (+/- SEM, P > 0.60) and did not correlate with ratios of cortisol metabolites in urine or with body mass index. CONCLUSIONS: The altered cortisol metabolism in polycystic ovarian syndrome, which is consistent with impaired 11beta-HSD1 activity, cannot be accounted for by increased production of measurable endogenous inhibitors of this enzyme.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2000

Lack of relationship between 11beta-hydroxysteroid dehydrogenase setpoint and insulin sensitivity in the basal state and after 24h of insulin infusion in healthy subjects and type 2 diabetic patients.

OBJECTIVES: To test whether insulin resistance in type 2 diabetes mellitus is associated with an altered overall setpoint of the 11beta-hydroxysteroid dehydrogenase (11betaHSD) mediated cortisol to cortisone interconversion towards cortisol, and to evaluate whether changes in insulin sensitivity induced by antecedent hyperinsulinaemia are related to changes in the 11betaHSD setpoint. PATIENTS AND MEASUREMENTS: The urinary ratio of (tetrahydrocortisol + allo-tetrahydrocortisol)/tetrahydrocortisone ((THF + allo-THF)/THE) and of free cortisol/free cortisone (UFF/UFE), as well as the plasma cortisol/cortisone ratio were measured in 8 male type 2 diabetic patients and 8 healthy male subjects without and after 24 h of insulin infusion. Insulin was infused at a rate of 30 mU/kg/h with blood glucose being clamped at euglycaemic levels in healthy subjects and at isoglycaemic levels in diabetic patients. Insulin sensitivity was assessed by measurement of whole body glucose uptake (M-value) during a 3-4 h euglycaemic clamp, directly after the 24 h insulin infusion and compared to the M-value on a control day, at least 1 week apart from the 24 h insulin infusion. RESULTS: Despite impaired insulin sensitivity (M-value, 11.6 +/- 7.7 vs. 28.5 +/- 11.6 micromol/kg/minutes, in type 2 diabetic and healthy subjects, respectively, P < 0.05), urinary (THF + allo-THF)/THE ratio and baseline plasma cortisol/cortisone ratio at 0800 h were similar in type 2 diabetic patients (0.82 +/- 0.07 and 3. 77 +/- 0.70, respectively) and healthy subjects (0.76 +/- 0.14 and 3. 81 +/- 0.88, respectively, ns). Insulin sensitivity was not correlated with urinary (THF + allo-THF)/THE ratio nor with baseline plasma cortisol/ cortisone. In type 2 diabetic patients, insulin sensitivity was further impaired by antecedent hyperinsulinaemia (P < 0.05), but the urinary (THF + allo-THF)/THE ratio (0.80 +/- 0.14, ns) and the plasma cortisol/cortisone at 0800 h (3.66 +/- 0.72, ns) did not change. In healthy subjects, insulin sensitivity did not change significantly (M-value, 22.5 +/- 9.7 micromol/kg/minutes, ns), although the urinary (THF + allo-THF)/THE ratio (0.92 +/- 0.25, P < 0.05) and the plasma cortisol/cortisone (4.59 +/- 0.63, P < 0.05) increased. Insulin did not affect the UFF/UFE ratio in either group. CONCLUSION: The present study does not support the hypothesis that insulin resistance in type 2 diabetes mellitus is associated with an overall change in the 11betaHSD set point towards cortisol. In view of the stimulatory effects of insulin and cortisol on adipogenesis, long-term stimulation of 11betaHSD reductase activity by insulin could aggravate visceral obesity.

11-beta-Hydroxysteroid Dehydrogenases↗