Norman F. Taylor

Hyperandrogenism in Polycystic Ovary Syndrome -- Evidence of Dysregulation of 11β-Hydroxysteroid Dehydrogenase

Background Hyperandrogenemia is the hallmark of the polycystic ovary syndrome, yet the relative contributions of the adrenal cortex and ovary to the overproduction of androgen remain unclear. To identify possible causes of adrenocortical overactivity, we studied the metabolism of adrenal and ovarian steroid hormones in women with this disorder. Methods We measured 24-hour urinary excretion of steroid hormone metabolites by high-resolution capillary gas chromatography in 65 women with the polycystic ovary syndrome and 45 normal women matched for body-mass index. Results After adjustment for body-mass index, the urinary excretion of testosterone and androstenedione metabolites was 1.9 times higher in the women with the syndrome than in the normal women, and the excretion of dehydroepiandrosterone metabolites (C19 steroid sulfates) and cortisol metabolites was 1.5 and 1.3 times higher, respectively (P<0.01 for all comparisons). The affected women also had significantly higher ratios of 11-oxo (oxygenated) me...

The effect of growth hormone replacement therapy on cortisol–cortisone interconversion in hypopituitary adults: evidence for growth hormone modulation of extrarenal 11β‐hydroxysteroid dehydrogenase activity

Growth hormone (GH) replacement therapy in hypopituitary adults has been associated with a decreased urinary ratio of 11‐hydroxy/11‐oxo‐cortisol metabolites (CoM). This could result from GH regulation of the activity of hepatic or renal 11β‐hydroxysteroid dehydrogenase (11β‐HSD1 and 2), the enzymes responsible for cortisol–cortisone interconversion, or alternatively it might reflect decreased cortisol availability. To elucidate this, we examined the effect of GH on urinary cortisol, cortisone and cortisol metabolites in hypopituitary adults at increasing doses of hydrocortisone replacement.

Sexual dimorphism in 11 β hydroxysteroid dehydrogenase activity and its relation to fat distribution and insulin sensitivity; a study in hypopituitary subjects

Sexual dimorphism of 11 β hydroxysteroid dehydrogenase activity (11 β HSD) as measured by the urinary 11‐OH/11‐oxo cortisol metabolite ratio has been documented in healthy subjects. Since body composition, fat distribution and insulin sensitivity vary between the sexes we have investigated whether these factors may account for the observed difference. Studies were performed in ACTH deficient hypopituitary subjects to eliminate the effect of feedback modulation of cortisol secretion.

The relationship between the effects of metyrapone treatment on depressed mood and urinary steroid profiles

In order to investigate mechanisms by which the adrenal 11 beta-hydroxylase inhibitor metyrapone might exert its antidepressant effect, we used gas chromatography to analyse the 24 h urinary steroid profiles from six females with major depression taking part in a trial of metyrapone (2-4 g/day) as an antidepressant. Due to concurrent administration of hydrocortisone (30 mg/day), plasma cortisol levels were not significantly reduced. Treatment with metyrapone resulted in greatly increased urinary excretion of 11-deoxy corticosteroids, including the GABA-modulatory steroid tetrahydro-11-deoxycorticosterone (from 68 +/- 34 to 219 +/- 75 micrograms/24 h, p < .05). Metyrapone also had multiple extra-adrenal effects on corticosteroid metabolism, including inhibition of the peripheral conversion of cortisone to cortisol as demonstrated by a significant decrease in the ratio of 11 beta-hydroxy/11-oxo metabolites of cortisol (from 0.81 +/- 0.08 to 0.46 +/- 0.04, p < .01). The decreased Montgomery-Asberg Depression Rating Scale scores seen during treatment with metyrapone did not correlate with changes in plasma cortisol, but did correlate significantly with total 11-deoxycortisol metabolites (r = 0.778, n = 12, p < .01). We conclude that, in addition to decreased cortisol synthesis, increased secretion of cortisol precursors and reduced local bioavailability of cortisol may play a role in the antidepressant effect of metyrapone.

Modulation of Cortisol Metabolism during Treatment of Acromegaly Is Independent of Body Composition and Insulin Sensitivity

Objectives: The set point of cortisol-cortisone conversion is shifted in the direction of cortisone by the inhibition of the activity of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) during adult GH replacement and in active acromegaly. Additionally, both fat mass and insulin may modulate 11β-HSD1 and are both influenced by changes in GH status. This study examined the relative direct contribution of GH/IGF1 in modulating cortisol metabolism. Methods: Overall cortisol/cortisone conversion (ratio of urine 11-hydroxy-/11-oxo-cortisol metabolites; Fm/Em), insulin sensitivity (homeostatic model assessment; HOMA %S) and fat mass (DXA) were examined in parallel in 6 patients (mean age 53 years, range 42–76; 4 males, 2 females) with previously untreated active acromegaly during 6 months of therapy with Sandostatin® LAR® (20–30 mg i.m. 4 weekly). All but 1 patient had normal ACTH reserve. Results: At baseline, Pearson correlation demonstrated an inverse relationship between serum GH (mean of a 5-point day curve) and Fm/Em (r = –0.83, p = 0.04) and a trend towards an inverse relationship between HOMA %S and Fm/Em (r = –0.79, p = 0.06) but no other patterns were evident. During the course of treatment, serum GH decreased from 9.9 ± 6.4 (mean ± SD) to 3.5 ± 3.1 ng/ml (p < 0.01) and serum IGF-1 from 785 ± 268 to 431 ± 156 ng/ml (p < 0.005). Fm/Em increased from 0.52 ± 0.1 to 0.75 ± 0.08 (p < 0.03) consistent with increased 11β-HSD1 activity. There were no significant changes in truncal fat percentage (33.0 ± 9.0 vs. 33.0 ± 8.2) or insulin sensitivity (HOMA %S: 37.1 ± 8.6 vs. 52.8 ± 33.7). Conclusions: Modulation of cortisol metabolism during treatment of active acromegaly is dependent on changes in GH/IGF-1 status and is not influenced by any individual change in body composition or insulin sensitivity.

Extra‐adrenal effects of metyrapone include inhibition of the 11‐oxoreductase activity of 11β‐hydroxysteroid dehydrogenase: a model for 11‐HSD I deficiency

BACKGROUND AND OBJECTIVE Previous studies suggesting effects of metyrapone on extra‐adrenal cortico‐steroid metabolism have involved significant alterations in plasma cortisol. We have therefore studied effects of metyrapone on urinary excretion of steroids in a group of patients treated concurrently with hydrocortisone so that changes in plasma cortisol were minimized.

The effects of growth hormone deficiency and replacement on glucocorticoid exposure in hypopituitary patients on cortisone acetate and hydrocortisone replacement

objective  11β‐hydroxysteroid dehydrogenase type 1 (11βHSD1) converts inactive cortisone to active cortisol. 11βHSD1 activity is increased in GH deficiency and inhibited by GH and IGF‐I in acromegaly. However it is not known whether these changes in cortisol metabolism exert significant effects during hydrocortisone therapy, and the effect has not been studied in patients taking cortisone acetate. We have studied the effect of GH induced 11βHSD1 inhibition in hypopituitary adults with severe GH deficiency to determine whether this inhibition has a different magnitude of effect when patients are taking different forms of glucocorticoid replacement therapy.

Urinary Steroid Profiling

Urinary steroid profiling is a long-established technique which has wide application in the study of disorders of human steroid biosynthesis and catabolism. The method divides into extraction of free and conjugated steroids, steroid conjugate hydrolysis, free steroid re-extraction, derivatization and analysis by GC or GC-MS. Several developments have led to better understanding of these processes and of ways to simplify and speed them up. The core method is described, together with some alternative options.

Sodium ascorbate improves yield of urinary steroids during hydrolysis with Helix pomatia juice.

Urinary steroid profile analysis requires enzymatic hydrolysis of glucuronide and sulfate conjugates and this is achieved simultaneously using Helix pomatia juice (HPJ), but steroids with 3beta-hydroxy-5-ene structure undergo transformation and yield of 5alpha-reduced corticosteroid glucuronides is poor. We describe the use of sodium ascorbate to solve these problems and provide a basis for its mode of action. Steroid conjugates were extracted from urine, hydrolyzed in acetate buffer with HPJ and sodium ascorbate and analyzed as methyloxime-trimethylsilylether derivatives by gas chromatography-mass spectrometry. Ranges of temperature, pH and ascorbate, substrate and HPJ concentrations were compared for urine and pure standards. Activity of other antioxidants and that of bacterial cholesterol oxidase were examined. Helix pomatia enzyme preparations from different commercial sources were compared. Loss of 3beta-hydroxy-5-ene steroids was enzyme-dependant, since it required HPJ, was saturable, subject to substrate competition and heat-inactivated. Products were 3-oxo-4-ene steroids and 4,6-diene and 6-oxy derivatives of these but the latter were not formed from 3-oxo-4-ene precursors. Ascorbate, other antioxidants or oxygen exclusion diminished activity. These characteristics were shared by cholesterol oxidase. Yield of 5alpha-reduced steroids was diminished by pre-incubation of HPJ before ascorbate addition and this was reversed if ascorbate was added to the pre-incubation mixture. We conclude that transformation of 3beta-hydroxy-5-ene steroids by HPJ is due to cholesterol oxidase and is diminished by antioxidants or oxygen denial. Yield of 5alpha-reduced steroids is low due to oxidative damage of beta-glucuronidase during hydrolysis, prevented by ascorbate. These features are shared by most commercial Helix pomatia enzyme preparations tested.

Steroids excreted in urine by neonates with 21-hydroxylase deficiency: characterization, using GC-MS and GC-MS/MS, of the D-ring and side chain structure of pregnanes and pregnenes.

Steroid metabolites in urine from neonates with 21-hydroxylase deficiency are predominantly polyhydroxylated 17-hydroxyprogesterone and androgen metabolites, and most have incompletely defined structure. This study forms part of a comprehensive project to characterize and identify these in order to enhance diagnosis and to further elucidate neonatal types of steroid metabolism. Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra, obtained with constant excitation conditions, were used together to determine the structure of the D-ring and the side chain of 20-oxo and 20-hydroxy pregnane(ene)s without oxo groups on the A-, B-, and C-ring. All possible combinations of D-ring and side chain configuration were considered. Most fragmentations could be interpreted as partial or complete D-ring cleavages with loss of the side chain, aided by comparison with spectra of deuterated derivatives and of borohydride reduced metabolites. Possible rearrangement ions are also discussed. More than 140 endogenous metabolites were characterized. GC-MS/MS was especially beneficial for characterization of compounds with 16,17-dihydroxy-20-oxo structure, interpreted as markers of intra-uterine enzyme induction. It also assisted the differentiation of 16-hydroxy-20-oxo metabolites, present in urine of non-affected neonates, from the diagnostic 17-hydroxy-20-oxosteroids and enabled the detection of 15,17-dihydroxy-20-oxo compounds in low concentrations. The presence of 17,21-dihydroxylated pregnane(ene)s despite the deficit in CYP21A2 is discussed. We conclude that GC-MS combined with GC-MS/MS allows reliable identification of the structure of the D-ring and side chain of pregnane(ene)s without prior isolation, even when in low concentrations in urine.