Cortisol, aldosterone, cortisol precursor, androgen and endogenous ACTH concentrations in dogs with pituitary-dependant hyperadrenocorticism treated with trilostane

Domestic Animal Endocrinology 31 (2006) 63–75

Cortisol, aldosterone, cortisol precursor, androgen and endogenous ACTH concentrations in dogs with pituitary-dependant hyperadrenocorticism treated with trilostane N.S. Sieber-Ruckstuhl a,∗ , F.S. Boretti a , M. Wenger a , C. Maser-Gluth b , C.E. Reusch a a

Clinic for Small Animal Internal Medicine, Vetsuisse Faculty University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland b Steroid Laboratory, Institute of Pharmacology, Ruprecht-Karls-University, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany

Received 21 July 2005; received in revised form 7 September 2005; accepted 10 September 2005

Abstract Trilostane is thought to be a competitive inhibitor of the 3␤-hydroxysteroid dehydrogenase (3␤HSD), an essential enzyme system for the synthesis of cortisol, aldosterone and androstenedione. Due to its reliable clinical efficacy, trilostane is increasingly used to treat dogs with pituitary-dependant hyperadrenocorticism (PDH). The objective of our study was to investigate the effect of trilostane on precursor concentrations located before (17␣-OH-pregnenolone, dehydroepiandrostenedione) and after (17␣-OHprogesterone, androstenedione, 11-deoxycortisol, 21-deoxycortisol) the proposed enzyme inhibition, on end products of steroid biosynthesis (cortisol and aldosterone) and on endogenous adrenocorticotrophic hormone (ACTH) concentrations in dogs with PDH. Hormones of the steroid biosynthesis pathway were evaluated in 15 dogs before and 1 h after injection of synthetic ACTH prior to (t0 ), in weeks 1–2 (t1 ) and in weeks 3–7 (t2 ) of trilostane treatment. Endogenous ACTH concentrations were measured at the same time points before performing the ACTH stimulation test. During trilostane treatment baseline and post-stimulation cortisol concentrations decreased significantly. Baseline serum aldosterone levels showed a significant increase; post-stimulation

∗

Corresponding author. Tel.: +41 44 635 83 01; fax: +41 44 635 89 30. E-mail address: [email protected] (N.S. Sieber-Ruckstuhl).

0739-7240/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.domaniend.2005.09.004

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values decreased. Baseline and post-stimulation 17␣-OH-pregnenolone and dehydroepiandrostenedione concentrations increased significantly. 17␣-OH-progesterone and androstenedione levels did not change. Post-stimulation 21-deoxycortisol concentrations decreased significantly, baseline 11deoxycortisol concentrations increased significantly. Endogenous ACTH levels showed a significant increase. The significant increase in 17␣-OH-pregnenolone and dehydroepiandrostenedione concentrations confirms an inhibitory effect of trilostane on the 3␤-HSD. Since 17␣-OH-progesterone concentrations did not change, but cortisol concentrations markedly decreased, trilostane seems to influence additional enzymes of the hormone cascade, like the 11␤-hydroxylase and possibly the 11␤-hydroxysteroid dehydrogenase. © 2005 Elsevier Inc. All rights reserved. Keywords: Hyperadrenocorticism; Trilostane; Steroid hormone concentrations; Endogenous ACTH; Dogs

1. Introduction Trilostane is known as a competitive inhibitor of the 3␤-hydroxysteroid dehydrogenase/
5,4 -isomerase enzyme system (3␤-HSD) [1–4]. This enzyme system catalyses the conversion of the
5 -3␤-hydroxysteroids, pregnenolone, 17␣-OH-pregnenolone and dehydroepiandrostenedione (DHEA) to the
4 -3-ketosteroids, progesterone, 17␣-OHprogesterone, and androstenedione. Therefore, the enzymatic action of the 3␤-HSD is essential for the biosynthesis of all classes of steroid hormones, namely glucocorticoids, mineralocorticoids, progesterone, androgens, and estrogens (Fig. 1). The inhibitory effect of trilostane on cortisol, aldosterone, or androstenedione synthesis has been shown in humans and various other species [2–9]. Its influences on steroid precursor concentrations have been documented incompletely, but seem to confirm the proposed mechanism of action. In rats and guinea pigs an increase in pregnenolone and 17␣-OH-pregnenolone concentrations, respectively, was seen during trilostane administration [2,4]. Observed precursor alterations in humans during trilostane therapy were increased DHEA and pregnenolone and decreased progesterone, 17␣-OH-progesterone, and
4 -androstenedione concentrations [5,7,8,10]. In dogs with pituitary-dependant hyperadrenocorticism (PDH) trilostane treatment has been shown to significantly decrease serum cortisol and to a lesser extent aldosterone concentrations [11–13]. One study reported two dogs with PDH who showed an increase in 17␣-OH-progesterone concentrations during trilostane treatment [14]. In another study 17␣-OH-progesterone concentrations were measured in dogs with alopecia X treated with trilostane. Again, trilostane lead to an increase in 17␣-OH-progesterone concentrations [15]. Other precursor concentrations during trilostane treatment in dogs with PDH have not been evaluated so far. The purpose of our study was to investigate the effect of trilostane on precursor concentrations located before (17␣-OH-pregnenolone, dehydroepiandrostenedione) and after (17␣-OH-progesterone, androstenedione, 11-deoxycortisol, 21-deoxycortisol) the proposed enzyme inhibition, on end products of steroid biosynthesis (cortisol and aldosterone) and on

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Fig. 1. Biosynthesis pathway of steroid hormones in the adrenal gland. Illustration of the biosynthetic pathways and enzymes for mineralocorticoids, glucocorticoids, and androgens in the adrenal cortex. Enzymes involved are as follows: 1, cholesterol desmolase (CYP11A); 2, 21-hydroxylase (CYP21); 3, 11␤-hydroxylase (CYP11B1); 4, 18-hydroxylase; 5, 18-oxidase; 6, 17␣-hydroxylase (CYP17); 7, 17,20-lyase (CYP17); 8, 17␤-hydroxysteroid dehydrogenase; 9, 11␤-hydroxysteroid dehydrogenase; 3␤-HSD: 3␤-hydroxysteroid dehydrogenase/
5 -
4 isomerase; DHEA: dehydroepiandrosterone.

endogenous adrenocorticotrophic hormone (ACTH) concentrations to better characterize its mechanism of action in dogs with PDH. 2. Material and methods 2.1. Dogs Fifteen client-owned dogs with PDH were included in the study. Age ranged from 7 to 14 years (median, 11 years) and body weight from 3.7 to 33.2 kg (median, 10.9 kg). Five were female (three spayed) and 10 were male (three castrated). Breeds represented included Dachshund (n = 4), Poodle (2), Maltese (1), Silky Terrier (1), Shih-Tzu (1), Coton de Tul´ear (1), Lakeland Terrier (1), English Bulldog (1), Golden Retriever (1), and two mixed-breed dogs. Diagnosis of hyperadrenocorticism was confirmed on the basis of results of an ACTH stimulation test, low-dose dexamethasone suppression test, and the urine cortisol-to-urine creatinine ratio. In addition, the adrenal glands were examined ultrasonographically, and PDH was confirmed on the basis of a bilateral symmetric appearance or bilateral enlargement of the adrenal glands. Dogs were included in the study when at least three clinical signs consistent with hyperadrenocorticism (e.g. polyuria–polydipsia [PU/PD], polyphagia, dermatologic problems, decreased activity, panting, pendulous abdomen) were detected, at least two of the three screening tests yielded positive results, and the dog’s owner agreed to return the dog to our clinic facility for regularly scheduled reevaluations throughout a 7-week period. Informed consent was obtained from the owners of all dogs.

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2.2. Endocrinologic analyses Before performing the ACTH stimulation test, blood was collected into chilled EDTAcoated tubes placed on ice for measurement of plasma endogenous ACTH concentration. After centrifugation at 4 ◦ C plasma was stored at −80 ◦ C until assayed. Measurement was performed at the University of Utrecht, The Netherlands by use of a commercially available two-site immunoradiometric assay (IRMA) (Nichols Institute, Wijchen, The Netherlands) as described elsewhere [16]. An ACTH stimulation test was performed by obtaining samples for determination of serum concentrations of cortisol, aldosterone, 17␣-OH-pregnenolone, DHEA, 17␣-OHprogesterone, androstenedione, 11-deoxycortisol and 21-deoxycortisol before and 1 h after IM injection of 0.25 mg of synthetic ACTH (Synacthen® , Novartis Pharma Schweiz AG, Bern, Switzerland). Cortisol concentrations were determined by use of a chemiluminescence assay (ADVIA Centaur® System, Bayer (Schweiz) AG, Zurich, Switzerland), and aldosterone concentrations were measured by use of radioimmunoassay (DPC Coat-a-CountTM aldosterone kit, Diagnostic Products Corporation, Los Angeles, CA, USA) [13]. Sensitivity of the aldosterone assay was 4 pg/ml. Intra- and inter-assay coefficients of variation for aldosterone were 4.7 and 11%, respectively. 17␣-OH-pregnenolone, DHEA, 17␣-OH-progesterone, androstenedione, 11deoxycortisol and 21-deoxycortisol were measured by specific in-house radioimmunoassay (RIA) established at the Steroid Laboratory of the University of Heidelberg, Germany, using titrated steroid (Amersham Biosciences, Freiburg, Germany) and antibodies, raised and characterized in the steroid laboratory, as described elsewhere [17–19]. Prior to RIA a recovery-corrected extraction was performed for all hormones and chromatographic purification was performed for 17␣-OH-progesterone and 21-deoxycortisol, thereby efficiently removing cross-reacting steroids. In more detail, the chromatographic separation on microcolumns modified from the method previously described [20] was carried out using Celite (Celite 545 AW, Sigma–Aldrich, Taufkirchen, Germany) as an inert support for partition chromatography. Standard curves of all steroids measured ranged from 2 to 1000 pg/tube and sensitivities were 0.07, 0.3, 0.13, 0.18 ng/ml, 0.02 ␮g/dl, and 5 ng/dl for 17␣-OH-pregnenolone, DHEA, 17␣-OH-progesterone, androstenedione, 11-deoxycortisol and 21-deoxycortisol, respectively. Intra- and inter-assay coefficients of variation were 6.2 and 13, 6.6 and 12.4, 8.6 and 11.3, 5.4 and 13.7, 7 and 12.6 and 8.2 and 11.6% for 17␣-OH-pregnenolone, DHEA, 17␣-OH-progesterone, androstenedione, 11-deoxycortisol and 21-deoxycortisol, respectively. A low-dose dexamethasone suppression test and a urine cortisol-to-urine creatinine ratio were performed as reported previously [11,13]. 2.3. Experimental design The prospective study was performed between January 2001 and June 2004 at our facility. The initial dose of trilostane for dogs with PDH was determined on the basis of three categories of body weight (<5 kg, 30 mg, PO, q 24 h; 5–20 kg, 60 mg, PO, q 24 h; and >20 kg, 120 mg, PO, q 24 h). ACTH stimulation tests were performed prior to trilostane treatment (t0 ), 1–2 weeks (t1 ) and 3–7 weeks (t2 ) thereafter. At t1 and t2 the test was

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performed 2–6 h after the daily dose of trilostane according to previously described protocol [11,13]. The treatment goal was to achieve a serum cortisol concentration of 1–2.5 ␮g/dl in samples obtained after ACTH stimulation. In dogs with serum cortisol concentrations <1 or >2.5 ␮g/dl after ACTH stimulation, the dose of trilostane was decreased or increased, respectively [11]. 2.4. Statistical analysis Results were analyzed by use of nonparametric statistical methods (SPSS 11.0 for Windows, SPSS Inc., Chicago, IL, USA and GraphPad Prism 2, San Diego, CA, USA). Ranges and median values are reported. Differences were tested by use of Friedman’s repeated measures test and Dunn’s post-test. Differences were considered significant at values of p ≤ 0.05. For values below detection limit the mean between 0 and the detection limit was entered for statistical analysis.

3. Results Changes of cortisol, aldosterone, cortisol precursor, androgen and endogenous ACTH concentrations during trilostane treatment are summarized in Table 1 and Figs. 2–6. 3.1. Effect on end products of steroid biosynthesis (cortisol and aldosterone) Serum cortisol concentrations before ACTH stimulation were significantly lower at t1 compared to t0 . Post-stimulation cortisol levels decreased significantly at t1 and t2 compared to t0 . Baseline serum aldosterone levels showed a significant increase at t1 and t2 compared to t0 . Post-stimulation aldosterone levels were significantly lower at t1 compared to t0 (Fig. 2). 3.2. Effect on precursors located before the proposed enzymatic blockage (17α-OH-pregnenolone and DHEA) Serum 17␣-OH-pregnenolone and DHEA concentrations before and after ACTH stimulation showed a significant rise at t1 and t2 compared to t0 (Fig. 3). In all dogs these hormone values increased during therapy. 3.3. Effect on precursors located after the proposed enzymatic blockage (17α-OH-progesterone, androstenedione, 21-deoxycortisol and 11-deoxycortisol) No significant changes were seen for 17␣-OH-progesterone and androstenedione concentrations before and after ACTH stimulation (Fig. 4). Baseline 21-deoxycortisol concentrations showed no significant changes during trilostane therapy. Post-stimulation 21deoxycortisol levels were significantly lower at t1 and t2 compared to t0 (Fig. 5). Baseline, but not post-stimulation 11-deoxycortisol concentrations showed a significant increase at t1 and t2 compared to t0 (Fig. 5).

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Hormones

Cortisol (␮g/dl) Aldosterone (pg/ml) 17␣-OH-pregnenolone (ng/ml) DHEA (ng/ml) 17␣-OH-progesterone (ng/ml) Androstenedione (ng/ml) 21-Deoxycortisol (ng/dl) 11-Deoxycortisol (␮g/dl) Endogenous ACTH (pg/ml)

t2

t1

t0 0h

1h

0h

1h

0h

1h

4.9 (1.5–9.4) <4 (<4–290) 1.82 (<0.07–5.1) 2.14 (0.4–15.1) 0.75 (0.2–6.4) 0.18 (<0.18–7) 13 (<5–332) 0.25 (0.04–0.7) 79.5 (16–260)

30.6 (10.6–46.8) 185 (25–2092) 3.33 (0.9–10.1) 1.96 (<0.3–11.3) 2.71 (0.7–6.1) 0.45 (<0.18–8.8) 142.8 (14.6–684) 1.7 (0.2–6.6) nd

0.9* (0.1–3.3) 69.5* (<4–377) 36.13* (5.6–84.7) 8.33* (3.04–39.2) 0.87 (<0.13–2.3) 0.63 (<0.18–1.9) 18.2 (<5–38.6) 0.67* (0.5–7.7) 112.5 (17–634)

1.9* (0.2–11.1) 75* (22–1491) 72.62* (19.8–137.1) 10.25* (2.7–31.5) 2.33 (0.6–7.9) 0.8 (<0.18–1.96) 25.9* (<5–49.5) 1.08 (0.4–2.6) nd

1.2 (0.4–10.3) 52* (<4–208) 36.95* (4.5–82.0) 8.69* (2.56–43.71) 1.05 (0.5–3.1) 0.85 (<0.18–3.8) 15.3 (<5–30) 0.81* (<0.02–1.6) 182* (62–495)

3.6* (0.7–19.5) 130 (44–724) 72.14* (12.6–113.6) 10.88* (3.2–47.5) 2.62 (<0.13–6.6) 0.81 (<0.18–3.8) 25* (<5–75.4) 1.03 (0.5–2.3) nd

Median (range) cortisol, aldosterone and precursor concentrations before (0 h) and after (1 h) ACTH stimulation and median (range) endogenous ACTH concentrations in 15 dogs prior to (t0 ), in weeks 1–2 (t1 ) and in weeks 3–7 (t2 ) of trilostane treatment. * Significant change in relation to the corresponding value before treatment.

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Table 1 Steroid hormone and endogenous ACTH concentrations in dogs during trilostane treatment

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Fig. 2. Serum cortisol and aldosterone concentrations during trilostane treatment. Point plots of baseline (0 h) and post-ACTH stimulation (1 h) serum cortisol and aldosterone concentrations before (t0 ), 1–2 weeks (t1 ) and 3–7 weeks (t2 ) after starting trilostane treatment in 15 dogs. * Significant difference.

3.4. Effect on plasma endogenous ACTH levels At t2 plasma endogenous ACTH levels were significantly elevated compared to t0 (Fig. 6). 3.5. Clinical signs After starting trilostane treatment all dogs clinically improved within the first 1–3 weeks. In no dog the observed hormone alterations seemed clinically significant. 4. Discussion It is already known that cortisol and to a lesser extent aldosterone concentrations decrease during trilostane treatment [11–13]. Concomitant with these alterations in cortisol and

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Fig. 3. Serum 17␣-OH-pregnenolone and DHEA concentrations during trilostane treatment. Point plots of baseline (0 h) and post-ACTH stimulation (1 h) serum 17␣-OH-pregnenolone and DHEA concentrations before (t0 ), 1–2 weeks (t1 ) and 3–7 weeks (t2 ) after starting trilostane treatment in 15 dogs. * Significant difference.

aldosterone levels, our investigations show a significant increase in 17␣-OH-pregnenolone and DHEA concentrations. This is evidentiary of an inhibitory effect of trilostane on the 3␤-hydroxysteroid dehydrogenase enzyme system in dogs. At first glance it seems surprising that 17␣-OH-progesterone and androstenedione levels do not decrease during therapy. However, trilostane is known as a competitive inhibitor [2]. Competitive inhibitors are similar in shape to the substrate and therefore fit into the active site of an enzyme. A competitive inhibitor has a greater affinity to the enzyme than to the substrate, therefore, the substrate is out-competed. The maximal rate (Vmax ) of the enzymatic reaction, however, stays unchanged. The degree of inhibition depends on the ratio of the substrate to the inhibitor and to achieve the same Vmax an increase in substrate concentration is necessary. Giving a competitive inhibitor, product concentration (in the present situation 17␣-OH-progesterone and androstenedione) will at first decrease. If the organism reacts

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Fig. 4. Serum 17␣-OH-progesterone and androstenedione concentrations during trilostane treatment. Point plots of baseline (0 h) and post-ACTH stimulation (1 h) serum 17␣-OH-progesterone and androstenedione concentrations before (t0 ), 1–2 weeks (t1 ) and 3–7 weeks (t2 ) after starting trilostane treatment in 15 dogs.

by increasing the substrate concentration the reaction rate will increase. Depending on the extent of the increase in substrate concentration and the amount of inhibitor, the product concentration can stay unchanged with a competitive inhibition. In our model two mechanisms could lead to increased substrate concentrations. First, trilostane out-competes the substrate, which accumulates. Second, trilostane could influence the hypothalamo–pituitary–adrenal axis (HPA) thereby also causing increased substrate concentrations. Our results show that endogenous ACTH concentrations increase significantly during trilostane therapy. This is in agreement with the results found by Witt and Neiger [21]. The increase is thought to be due to the decreased cortisol concentrations within the negative feedback control on the pituitary gland and the hypothalamus. An increased endogenous ACTH concentration causes increased conversion of cholesterol to pregnenolone and further conversion to 17␣-OH-pregnenolone and DHEA, the two substrates for the inhibited enzyme system. By decreasing cortisol concentrations trilostane influences the HPA and increases the substrate concentrations. Elevated 17␣-OH-pregnenolone and DHEA levels compete against trilostane and increase the enzyme reaction rate and the 17␣-OH-progesterone and

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Fig. 5. Serum 21-deoxycortisol and 11-deoxycortisol concentrations during trilostane treatment. Point plots of baseline (0 h) and post-ACTH stimulation (1 h) serum 21-deoxycortisol and 11-deoxycortisol concentrations before (t0 ), 1–2 weeks (t1 ) and 3–7 weeks (t2 ) after starting trilostane treatment in 15 dogs. * Significant difference.

androstenedione production. With a stable enzyme concentration, the 17␣-OH-progesterone and androstenedione concentrations therefore depend on the ratio of inhibitor (trilostane) and substrate (17␣-OH-pregnenolone and DHEA). Since 17␣-OH-progesterone concentrations do not change during trilostane therapy, but cortisol levels decrease significantly, trilostane must have additional effects more distal in the hormone metabolism. Several possibilities exist how trilostane could accomplish this. The biochemical structure of all steroid hormones is similar. Concurrent effects of trilostane on other enzymes of the steroid hormone biosynthesis pathway are therefore likely. Two enzymes are involved in the conversion of 17␣-OH-progesterone to cortisol. Inhibition of the 21-hydoxylase should decrease 11-deoxycortisol concentrations. This seems unlikely due to the increased baseline 11-deoxycortisol levels in our study. Blocking of the 11␤-hydroxylase should increase 11-deoxycortisol and decrease 21-deoxycortisol concentrations, alterations similar to the ones observed in our study. A concurrent inhibition of the 3␤-HSD and the 11␤-HSD by trilostane would best explain the observed precursor changes.

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Fig. 6. Endogenous ACTH concentrations during trilostane treatment. Point plots of endogenous ACTH (cACTH) concentrations before (t0 ), 1–2 weeks (t1 ) and 3–7 weeks (t2 ) after starting trilostane treatment in 15 dogs. * Significant difference.

Few studies exist evaluating these enzymes specifically. In vitro studies using adrenal cells of different species did not show an effect of trilostane on the 11␤-hydroxylase [2,4,22]. In sheep adrenal cells trilostane demonstrated, in addition to the inhibitory effect on the 3␤-HSD, an accelerating effect on the oxidation of cortisol to cortisone by the 11␤-HSD [22]. This effect was not demonstrated in human adrenal cells [22]. Concurrent effects of trilostane therefore exist and either differences between in vitro and in vivo effects or species dependent effects of trilostane could explain the discrepancy between our results and the in vitro studies. An influence of trilostane on the 11␤-HSD activity, like seen in sheep adrenal cells, could contribute to the significant decrease in the cortisol concentrations. In humans two isoforms of 11␤-HSD have been recognised, 11␤-HSD1 and 11␤-HSD2. 11␤-HSD1 reduces mainly cortisone to cortisol and is widely distributed in human tissues (liver, adipose, gonads, bone, ocular tissues, vascular smooth muscle and skin), whereas 11␤-HSD2 inactivates cortisol to cortisone and is predominantly located in kidney, colon, and salivary glands [23]. However, tissue distribution of these two isoforms varies between species [23]. An effect of trilostane on the 11␤-HSD in dogs could add to the decrease in cortisol concentrations, but cannot explain the changes in 11-deoxycortisol and 21-deoxycortisol concentrations. Besides in vivo effects, factors during test performance could affect hormone concentrations. Cross-reactivity of antibodies is one of the most encountered problems with RIAs [24]. Cross-reactivity can be markedly decreased by purification methods (like chromatography) preceding the measuring step [24]. In trilostane treated dogs with alopecia X increased 17␣OH-progesterone levels were observed [15]. Our differing results are possibly due to the fact that we performed chromatographic purification before measuring 17␣-OH-progesterone concentrations which was not done in the study of Cerundolo. The results of this study are consistent with an inhibitory effect of trilostane on the 3␤HSD enzyme system in dogs. In addition trilostane seems to influence other enzymes of the steroid biosynthesis pathway. We postulate that the observed precursor alterations are

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due to a competitive inhibition of the 3␤-HSD enzyme system, together with an additional influence on the 11␤-hydroxylase and possibly the interconversion of cortisol and cortisone by the 11␤-HSD. To completely understand the activity pattern of trilostane further studies evaluating cortisol and cortisone concentrations as well as determination of 11␤-hydroxylase and 11␤-HSD activities are needed.

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