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Stimulation of gonadal synthesis by chronic excess of adrenocorticotropin in patients with adrenocortical insufficiency.

H Vierhapper, … , P Nowotny, W Waldhäusl

J Clin Invest. 1986;77(4):1063-1070. https://doi.org/10.1172/JCI112405.

Research Article

Analysis of 24-h urinary steroid excretion was performed by capillary gas chromatography in six patients (five men, one woman) with adrenocortical insufficiency. Ten healthy subjects (five men, five women) served as controls. A complete absence of all 21-hydroxylated steroid metabolites was seen in patients with adrenocortical insufficiency, whereas the excretion of several lacking hydroxylation in the 21-position (, pregnenetriol, and 11- ketoandrosterone) was markedly increased. In addition, the presence of 11 beta-hydroxyandrosterone was confirmed by mass-spectrometry in the of three patients. This pattern of steroid excretion was unchanged in patients with adrenocortical insufficiency, both after stimulation by 1-24 adrenocorticotropin (ACTH) and after short-term (3-d) suppression with . We conclude that patients with adrenocortical insufficiency present a pattern of steroid excretion characterized by the absence of 21-hydroxylated metabolites. In the absence of functional adrenocortical tissue, long-term pathologically elevated concentrations of ACTH apparently stimulate early steps of steroid synthesis, most likely in the gonads. In addition, the presence of 11-hydroxylated steroid metabolites (11-ketoandrosterone, 11 beta- hydroxyandrosterone) in the urine of patients with adrenocortical insufficiency demonstrates that chronic ACTH excess in this disorder may induce some activity of 11 beta-hydroxylase, an not found in the gonads under physiological conditions.

Find the latest version: https://jci.me/112405/pdf Stimulation of Gonadal Steroid Synthesis by Chronic Excess of Adrenocorticotropin in Patients with Adrenocortical Insufficiency H. Vierhapper, P. Nowotny, and W. Waldhausl Division of Clinical Endocrinology and Diabetes Mellitus, I. Medizinische Universitdtsklinik, A-1090 Wien, Austria

Abstract predominantly influences the function of the adrenal cortex, an additional effect on gonadal steroid production has not yet been Analysis of 24-h urinary steroid excretion was performed by excluded. Patients with adrenocortical insufficiency provide an capillary gas chromatography in six patients (five men, one excellent model from which to study the effect of markedly in- woman) with adrenocortical insufficiency. Ten healthy subjects creased endogenous concentrations of ACTH on gonadal steroid (five men, five women) served as controls. A complete absence production in the absence of functioning adrenocortical tissue. of all 21-hydroxylated steroid metabolites was seen in patients In the present investigation we have compared steroid excretion with adrenocortical insufficiency, whereas the excretion of several rates in patients having adrenocortical insufficiency with those steroids lacking hydroxylation in the 21-position (pregnenolone, of healthy controls, both under basal conditions as well as after pregnenetriol, and 11-ketoandrosterone) was markedly increased. additional stimulation with exogenous ACTH 1-24 and after In addition, the presence of 1 1j-hydroxyandrosterone was con- suppression with dexamethasone. firmed by mass-spectrometry in the urine of three patients. This pattern of steroid excretion was unchanged in patients with ad- renocortical insufficiency, both after stimulation by 1-24 adren- Methods ocorticotropin (ACTH) and after short-term (3-d) suppression with dexamethasone. We conclude that patients with adreno- Healthy subjects. The subjects were five healthy female volunteers, aged cortical insufficiency present a pattern of steroid excretion char- 19-27 yr, who had not taken oral contraceptives for at least 6 mo, and acterized by the absence of 21-hydroxylated metabolites. In the five healthy male volunteers, aged 21-28 yr. All subjects were within absence of functional adrenocortical tissue, long-term patholog- ± 10% oftheir ideal body weight (according to Metropolitan Life Insurance ically elevated concentrations of ACTH apparently stimulate Tables). The aim and potential risks of the investigation were explained early steps of steroid most in the In to each subject and written consent was obtained in each case. In healthy synthesis, likely gonads. women, the study was begun between the fifth and the seventh day of addition, the presence of 11-hydroxylated steroid metabolites the menstrual cycle. Following the collection of a 24-h urine for the (1 1-ketoandrosterone, 1 1 fl-hydroxyandrosterone) in the urine of determination of steroid excretion, an indwelling catheter was inserted patients with adrenocortical insufficiency demonstrates that into an antecubital vein and 0.25 mg synthetic ACTH (1-24 ACTH, chronic AC1TH excess in this disorder may induce some activity Synacthen, 0.25 mg = 100 IU; CIBA-GEIGY Ltd. Basle, Switzerland) of 11,6-hydroxylase, an enzyme not found in the gonads under was administered at 8:00 a.m. as an intravenous bolus. This was followed physiological conditions. by a continuous infusion (3 ml/h; t = 8 h) containing 0.25 mg 1-24 ACTH in 24 ml of 0.9% NaCI solution. On days 3-6 of the experiment the subjects were treated with oral dexamethasone (0.5 mg four times a Introduction day; Schering A.G., Berlin, Federal Republic of Germany), but not with intravenous ACTH. A 24-h urine (8:00 a.m.-8:00 p.m.) was collected The simultaneous evaluation of major urinary steroid metab- for the determination of urinary steroid excretion on day 1 (basal), day olites by capillary gas chromatography (1, 2) comprehensively 2 (ACTH), and day 5 (dexamethasone) of the experiment. Blood samples characterizes steroid production in man. Since a differentiation for the determination of plasma concentrations of , 21 -deoxy- of metabolites derived from cortisol and 11 -ketoandrostenedione, pregnenolone, and 17-hydroxy- adrenocortical or gonadal steroid pregnenolone were obtained before the infusion of ACTH. Plasma con- synthesis is not possible from determinations performed under centrations of cortisol were also determined after the administration of basal conditions, dynamic tests of adrenocortical function (i.e., ACTH and of dexamethasone. stimulation by adrenocorticotropin [ACTH] or suppression by Patients with adrenocortical insufficiency. Six patients with adreno- exogenous ) are used to characterize the contri- cortical insufficiency (five men, aged 26-48 yr, and one postmenopausal bution of the adrenal cortex to overall steroid secretion. The woman, aged 55 yr) consented to participate in the study. '3'I- interpretation of the results thus obtained relies on the assump- scanning failed in all patients to reveal the presence of functional adre- tion that these maneuvers interfere with adrenal, but not with nocortical tissue. Adrenocortical insufficiency was of autoimmune origin gonadal, steroid synthesis. Although it is undisputed that ACTH in four patients and due to bilateral adrenalectomy for Cushing's disease in two cases. Pituitary surgery had not been performed on any of these patients. The patients had been on continuous (37.5-50.0 Please address correspondence to Dr. Vierhapper, Division of Clinical mg acetate/d divided into two doses) and Endocrinology and Diabetes Mellitus, I. Medizinische Universittitsklinik, (0.05-0.1 mg 9a-fluoro-hydrocortisone/d) replacement therapy for 2- Lazarettgasse 14, A-1090 Wien, Austria. 17 yr before this investigation. Nevertheless, supranormal concentrations Receivedfor publication 20 August 1984 and in revisedform 26 No- of ACTH (> 100-> 1,000 pg/ml; normal <60 pg/ml) were found in each vember 1985. patient when estimated during routine controls on an outpatient basis. For the purpose of this study the patients were admitted to the hospital J. Clin. Invest. and taken off their individual substitution therapy. Consecutively, elec- © The American Society for Clinical Investigation, Inc. trolyte equilibrium was monitored daily and maintained by intravenous 0021-9738/86/04/1063/08 $ 1.00 administration of 0.9% saline. 84 h after the last dose of the respective Volume 77, April 1986, 1063-1070 substitution therapy the patients received an intravenous bolus and an

Steroid Excretion in Adrenocortical Insufficiency 1063 infusion of 1-24 ACTH as described above for the healthy volunteers. of methoxyamine hydrochloride (2% in pyridine; Pierce Chemical Co., Subsequently each patient was given 0.5 mg dexamethasone four times Rockford, IL) and heated at 80'C for 60 min. After evaporation with a day and 0.05 mg 9a-fluoro-hydrocortisone/d by mouth for 3 d. A 24- dry N2 at 300C, 100 Ml of a mixture of trimethylsilylimidazole, NO- h urine (08:00-08:00 h) for the determination of steroid excretion was bis(trimethylsilyl)acetamide, and trimethylchlorosilane (3:3:2, TRI-SIL collected under basal conditions (i.e., from 60 to 84 h after withdrawal TBT; Pierce Chemical Co.) was added and the mixture was heated to of substitution therapy) during ACTH stimulation and on the third day 60C for 20 h. After evaporation to dryness by dry N2 (250C, overnight), of reinstituted glucocorticoid (dexamethasone) therapy. Blood samples 2 ml of and 1.5 ml of 0.1 N H2SO4 were added to each for the determination of plasma concentrations of cortisol were drawn sample, the mixture was shaken, and the aqueous (lighter) phase was at the beginning of each urine-collecting period and at the end of the pipetted off. The organic layer was washed once with I ml of double ACTH infusion. Concentrations of ACTH, of 2 1-deoxycortisol, of 11- distilled water. If the pH of the washing was >7, the organic layer was ketoandrostenedione, of pregnenolone, and of 1 7-hydroxypregnenolone diluted with 1 ml 0.1 N H2SO4. The organic layer was then diluted with were obtained before the' infusion of ACTH. Concentrations of ACTH 1 ml CH2Cl2 and washed with H20 until the washings were neutral. were also estimated after the administration of dexamethasone. Sodium sulfate was added to the organic solutions and the samples were Reference steroids. Reference steroids were purchased from Steraloids shaken for 20 min and centrifuged. The clear solutions were transferred Inc., Wilton, NH; and from Makor Chemicals, Jerusalem, Israel. Purity into reactivials; the solvent was evaporated with dry N2 at room tem- was checked by controlling melting point in each case. peratur: and the residue was dissolved in 100 ,l of a mixture of ethyl- Radioimmunoassays. For the simultaneous radioimmunologic de- acetate/n-hexane (9:1) containing 100 ng/Al of alkanes n-C24H50 and termination of cortisol, 2 1-desoxycortisol, and 1 1-ketoandrostenedione, n-C32H66 2.0 ml of plasma were diluted with 8.0 ml H20. To determine recoveries Recovery. 20 Ml ofeach sample were measured in a fl-counter (Packard 20,000 dpm of [3H]cortisol (NET 396, specific activity 115 Ci/mmol; Instruments Co., Delft, Netherlands). Mean recovery (n = 75) was New England Nuclear, Boston, MA), 1,000 dpm of 21-[3H]desoxycortisol 55.7+5.5%. (specific activity 43 Ci/mmol; Amersham International, Amersham, Gas chromatography. Gas chromatography was carried out using a Buckinghamshire, England), and 10,000 dpm of [3H]DOC (specific ac- Packard Instrument Co. 428 gaschromatograph equipped with a 50 m tivity 42 Ci/mmol; Amersham International) were added to each sample. OV-101 wall-coated open tubular column (0.25 mm i.d., film thickness [3HJDOC was used to estimate the recovery of 11-ketoandrostenedione, 0.25 Mm, pretested at 180.000 effective plates), a solid injection device, since the latter is not available in a labeled form and shows a chromato- and a flame ionization detector. N2 was used as a carrier gas with a flow graphic behavior in the chromatographic system we employed which is rate of -0.8 ml/min. After sample injection (300°C), the temperature identical to that of DOC. Subsequently the samples were extracted with of the oven was immediately programmed between 180°C and 265°C 150 ml of cold CH2Cl2 and the organic extract was washed once with at 0.40C/min followed by an isothermal run for 15 min at end temper- 10 ml of cold 0.1 N NaOH and twice with 10 ml ofcold H20. Separation ature. of the steroids was achieved by thin-layer chromatography (/ Steroid identification. Steroids were identified by comparison of re- = 75:25). For the simultaneous determihation of pregnenolone tention times expressed as methylene units as determined by intrapolation and 17-hydroxypregnenolone, 2 ml of plasma were diluted with 8 ml of between those of the alkanes n-C24H50 and n-C32H66; (Table I). Trivial H20. To determine recoveries 10,000 dpm of [3H]pregnenolone (specific names of steroid metabolites determined by gas chromatography and of activity 12 Ci/mmol; Amersham Corp.) and 17-[3H~hydroxypreg- the other steroids mentioned in this report are given in Table I. nenolone (specific activity 19'Ci/mmol; Amersham Corp.) were added Mass spectrometry. Positive identification of I 1-hydroxyandrosterone to each sample. Subsequently the samples were extracted with 150 ml was attempted by gas chromatography-mass spectrometry using a 5995 of cold CH2Cl2 and the organic extract was washed with 10 ml of cold gas chromatography-mass spectrometry system equipped with a 25-m H20. Separation of steroids was achieved by two-dimensional thin-layer fused silica capillary column coated with OV 101 stationary phase (Hew- chromatography (I: benzene/acetone, 75:25; II: ethylacetate/cyclohexane, lett-Packard Co., Palo Alto, CA). The GC injector was maintained at 55:45). Radioimmunoassays were performed in triplicate using antisera 300°C and the oven was programmed from 180°C to 260°C at a rate against cortisol and 2 1-desoxycortisol obtained from Dr. Vecsei, Division of I °C/min. Temperatures of the analyzer, transfer line, and ion source of Pharmacology, University of Heildelberg, Heidelberg Federal Republic were kept at 1800C, 280°C, and 150°C, respectively, and the electron of Germany (3). Antisera for the determination of pregnenolone and 17- energy was 70 eV. To increase sensitivity it was necessary to determine hydroxypregnenolone were obtained from Steranti Research Ltd., St. I 1-hydroxyandrosterone by selected ion monitoring, the instrument Albans, England; and from Radioassay Systems Laboratories, Inc., Car- having been set to monitor the ions at m/e 479 (M+), m/e 449 (M+ son, CA, respectively. Since no specific antiserum was available for the - 30), m/e 448 (M+- 31), and m/e 358 (M+- 121). determination of 1 l-ketoandrostenedione, this steroid was determined Calculation of steroid excretion. An automatic integrator (3358 A; using the cross-reactivity (12.5%) of an antibody raised against andro- Hewlett-Packard Co.) was used for the calculation of the area under the stenedione (Nr. XXA001; Steranti Research Ltd.). Cross-reactivity of respective peaks. Response factors (f) for the two n-alkanes (f C24 and this antibody against DOC is <0.1%. Furthermore, the behavior of an- fC32) and for each reference steroid standard (f St) were determined by drostenedione and 1 1-ketoandrostenedione permits complete separation their injection in varying amounts. These response factors were checked in the used chromatographic system. Urinary concentrations of cortisol and adjusted regularly (i.e., after every 15th injection of biological sam- and plasma concentrations of ACTH were determined as previously re- ples) by derivatizing and injecting defined mixtures of the reference com- ported (4). pounds. Response factors for the two n-alkanes were practically identical Urine processing. Urine samples (5 ml) of 24-h collections were acid- (fC24, 1.05±0.18; f C32, 1.07±0.20) over the complete series of injec- ified to pH 5.2 by addition of 0.2 N HCI. ,B-glucuronidase/arylsulfatase tions. It was unnecessary to introduce additional correction factors for (20,000 Fisher units, Calbiochem-Behring Corp., La Jolla, CA) was added, the increasing elution temperatures. The excreted amounts of individual the hydrolysis taking place at 37°C. Hydrolysis did not affect pH values. steroids were calculated by the following formula: a of 24 h, were After total hydrolysis period 50,000 dpm [3Hjcortisol mg/24 h = area X f C24 X f St X 100/recovery(%) X dilution, added to estimate recovery. The unconjugated steroids were extracted with three times 20 ml of ethylacetate and the combined extract was where area equals measured peak area; f C24 equals response factor for washed once with 20 ml of 0.1 N NaOH and at least twice with 20 ml hydrocarbon C24 (provides for the actually injected amount); f St equals of double distilled water until neutrality was reached. Anhydrous sodium response factor for individual steroid; recovery equals the yield of the sulfate (5-10 g) was then added to each sample. After 12 h the solvent complete treatment of the samples before gas chromatography by mea- was distilled off at 350C at reduced pressure. The residue was transferred suring recovery of [3H]cortisol; and dilution equals the aliquotization of with 3 x 1 ml ethylacetate into a centrifuging tube and the solvent was original urinary samples. evaporated in a stream of dry at 350C. Sensitivity and precision. The sensitivity of the procedure varied ac- Derivatization (5, 6). Samples were dissolved in 100 ,d of a solution cording to the gas chromatography conditions used. Applying standard 1064 H. Vierhapper, P. Nowotny, and W Waldhdusl Table I. Trivial Names and Gas-chromatographic Characteristics ofEvaluated Standard Steriod Compounds*

Methylation units No. Steroid Trivial name (as MO-TMS derivatives) n-C24-Alkane 24.00 2 Sa-Androstan-3a-ol- 17-one 25.09 3 5,B-Androstan-3a-ol- 17-one 25.28 4 5-Androsten-3,B-ol- 17-one 25.68 5 Sa-Androstan-3,B-ol- 17-one 25.83 6 5-Androsten-313,173-diol 25.92 7 5a-Androstan-3a-ol-1 1, 17-dione 1 l-Ketoandrosterone 26.01 8 1,3,5(10)-Estratrien-3-ol- 17-one 26.01 9 5,B-Androstan-3a-ol- 11, 17-dione 1 1-Ketoetiocholanolone 26.14 10 4-Androsten-3,17-dione 26.19/26.24 11 1,3,5( 10)-Estratrien-3,173-diol 26.38 12 4-Androsten-173-ol-3-one 26.47 13 5,B-Pregnan-3a-ol-20-one 26.87 14 5a-Androstan-3a- 1 3-diol- 17-one 1 IB-Hydroxyandrosterone 26.96 15 5,B-Androstan-3a- If3-diol- 17-one 1 lB-Hydroxyetiocholanolone 27.13 16 50-Pregnan-3,3-ol-20-one 27.15 17 5a-Pregnan-3a,20a-diol Allo- 27.42 18 5,B-Pregnan-3a,20a-diol Pregnanediol 27.62 19 5-Pregnen-30-ol-20-one Pregnenolone 27.67 20 5(3-Pregnan-3a, 17a,20a-triol 27.95 21 5-Androsten-3,B-16a, 17a-triol 17a-Androstenetriol 28.16 22 4-Pregnen-3,20-dione 28.20/28.25 23 5-Androsten-30,16a, 17(-triol 17,B-Androstenetriol 28.41 24 503-Pregnan-3a, 17a,2 1-triol-20-one TH-"-S")' 28.67 25 5,B-Pregnan-3a,2 1-diol-20-one TH-"DOC" 28.76 26 1,3,5( 10)-Estratrien-3, 16a,1 7/3-triol 28.76 27 5a-Pregnan-3a,2 1-diol-20-one Allo-TH-"DOC" 28.91 28 5a-Pregnan-3a, 17a,2 I-triol-20-one Allo-TH-"S" 29.02 29 53-Pregnan-3a, 17a,20a-triol- 1l-one 29.02 30 5-Pregnen-3fl, 17a,20a-triol Pregnenetriol 29.47 31 5,B-Pregnan-3a, 17a,2 I-triol-I 1 ,20-dione TH-"E" 29.66 32 5(3-Pregnan-3a,2 1-diol-1 1,20-dione TH-"A" 29.75 33 5(3-Pregnan-3a, 1 1(,2 1 -triol-20-one oTH-"B" 30.00 34 5a-Pregnan-3a, 1 1,,2 1-triol-20-one Allo-TH-"B" 30.12 35 Sa-Pregnan-3a, 17a,2 1-triol-I 11,20-dione Allo-TH-"E" 30.25 36 50-Pregnan-3a, 1 1(, 17a,2 I-tetrol-20-one TH-(4'F" 30.27 37 Sa-Pregnan-3a, 1 1(, 17a,2 I-tetrol-20-one Allo-TH"F" 30.37 38 5,B-Pregnan-3a, 17a,20a,2 1-tetrol- l-one1 a-Cortolone 30.49 39 5,B-Pregnan-3a, 1 7a,20,B,2 I-tetrol- 1 -one ,B-Cortolone 30.77 40 5,B-Pregnan-3a, 11(, 17a,20,B,21-pentol (3-Cortol 30.77 41 5-Cholesten-3,B-ol Cholesterol 30.89 42 5/3-Pregnan-3a, 113, 17a,20a,2 1-pentol a-Cortol 31.22 43 n-C32-Alkane 32.00

* Trivial names of other steroids mentioned in this paper: 4-Androsten- 1,B-ol-3,17-dione 1 1,B-Hydroxyandrostenedione 4-Androsten-3,11,17-trione 1 l-Ketoandrostenedione 5-Pregnen-3j, 17a-diol,20-one 17a-Hydroxypregnenolone 4-Pregnen- I3,1 17a-diol-3,20-dione 2 1-Desoxycortisol 4-Pregnen- I7a-ol-3,20-dione 17a-Hydroxyprogesterone (" 170H-P") 4-Pregnen-17a,2 I-diol-3,11,20-trione Cortisone ("E") 4-Pregnen- 113, 17a,2 I-triol-3,20-dione Cortisol ("F") 4-Pregnen-1 1(3,2 1-diol-3,20-dione ("B") 4-Pregnen,2 1-ol-3,20-dione Desoxycorticosterone ("DOC") 4-Pregnen-17a,2 I-diol-3,20-dione 1 1-Desoxycortisol ("S")

Steroid Excretion in Adrenocortical Insufficiency 1065 conditions sensitivity was -1 ng per injected sample corresponding to spectrometry using selected ion monitoring. As shown in Fig. 2 about 0.02 mg of steroid per 24 h. The coefficient of variation for multiple for one single case the chosen characteristic ions were readily determinations (n = 8) of the whole spectrum of standard compounds identified, both in a mixture of derivatized reference compounds was 12±3% (range, 10-20%). and in the patient's urine. Statistics. Data are presented as mean±SD. Student's t test (two- tailed) for matched and for unmatched pairs was used for statistical eval- uation. Discussion

Results The prerequisite for the simultaneous estimation of a large number of urinary steroid metabolites has been provided by the development of glass capillary gas chromatography (1, 2). The In unsubstituted patients with adrenocortical insufficiency, variety of techniques and hydrolytic procedures used by different plasma concentrations of cortisol were 1. 1±0.5 ug/dl (controls, laboratories implies that the only valid reference material is a 6.8±2.2 ug/dl). Concentrations of 1 1-ketoandrostenedione and material obtained using that very method. In order to study the of 21-deoxycortisol were 64±18 ng/dl (controls, 279±139 ng/ steroid excretion rates in patients with adrenocortical insuffi- dl) and 2.0±0.3 ng/dl (controls, 5.1 ± 1.0 ng/dl) respectively, and ciency, it was therefore pivotal to establish reference data in a the concentrations of pregnenolone and 17-hydroxypregneno- group of healthy subjects. These data are included in this report. lone were 38.2±14.2 ng/dl (controls, 53.3±6.2 ng/dl) and Although the relative share of the gonads and the adrenal 12.9±6.4 ng/dl (controls, 45.0±18.9 ng/dl), respectively. Thus, cortex in overall steroid production can easily be studied in pa- when compared with healthy controls, mean estimated concen- tients with adrenocortical insufficiency, few such data have been trations of cortisol, 1 I-ketoandrostenedione, 21 -deoxycortisol, reported as yet in this disorder (7-9). pregnenolone, and 17-hydroxypregnenolone in patients with Under physiological conditions the 21-hydroxylase, adrenocortical insufficiency were 13, 23, 39, 72, and 29%, re- 1 lB-hydroxylase, and 1 8-hydroxylase are unique to the adrenal spectively. In patients with adrenocortical insufficiency, plasma cortex, whereas other enzymes, such as 3,B-steroid dehydrogenase, concentrations of cortisol were unchanged by the administration 17a-hydroxylase, and the desmolases are shared by the adrenals of ACTH (1.5±0.7 Atg/dl; controls, 24.0±10.6 gg/dl) and of and by the gonads and probably are controlled by a single gene dexamethasone (0.6±0.2 ,ug/dl; controls, 0.8±0.2 ,g/dl). Plasma (10). The lack of 2 1-hydroxylated steroid metabolites observed concentrations of ACTH in patients with adrenocortical insuf- in the urine of our patients can therefore be easily deduced from ficiency were 968±410 pg/ml (range, 462-1670 pg/ml) after the differences in the enzymatic equipment ofthe adrenal cortex withdrawal of substitution therapy and 151 ± 144 pg/ml (range, as compared with that of the gonads, whereas the presence of 54-429 pg/ml) after 3 d of dexamethasone by mouth. two 1 1-hydroxylated metabolites, 1 I11B-hydroxyandrosterone and Steroid excretion rates of healthy men, healthy women, and 1 1-ketoandrosterone, and the elevated excretion of pregneno- of patients with adrenocortical insufficiency are given in Table lone, pregnanolone, and pregnenetriol requires further discus- II. Comparable excretion rates of cholesterol were found in pa- sion. tients with adrenocortical insufficiency and in healthy controls. Although adrenal scanning is believed to be superior to other Urine samples of patients were characterized by a lack of all 21- noninvasive techniques in the detection of adrenal rest tissue hydroxylated metabolites (TH-"A", TH-"B", allo-TH-"B", TH- (11), the negative outcome of '311-cholesterol scanning per se "DOC", allo-TH-"DOC", TH-"S", allo-TH-"S", TH-"E", allo- may not completely rule out this possibility in our patients. Ad- TH-"E", TH-"F", allo-TH-"F", a-cortol, f3-cortol, a-cortolone, ditional evidence against a residual adrenal function is, however, and fl-cortolone, Fig. 1). In addition, patients with adrenocortical provided by the lack of urinary cortisol and by plasma cortisol insufficiency presented with a reduced excretion of androsterone, concentrations close to the lower limit of the used radioim- etiocholanolone, dehydroepiandrosterone, androstenediol, 11 (,- munological method. Finally, the increased excretion of some hydroxyetiocholanolone, 1 3l-hydroxyandrosterone, 11 -keto- metabolites not requiring 2 1-hydroxylation (e.g., pregnanetriol etiocholanolone, pregnanediol, and , whereas and pregnenetriol) in the absence of all urinary 2 1-hydroxylated the mean excretion of pregnanetriol (3.92±3.07 mg/24 h), preg- metabolites and a similarly distorted relationship among the es- nenetriol (2.22±2.47 mg/24 h), pregnenolone (1.85±2.47 mg/ timated plasma steroids implies that the site of 24 h), and 1 1-ketoandrosterone (1.34±1.64 mg/24 h) was in- production in our patients is characterized by a pattern of ste- creased as compared with healthy controls. In regard to the low roidogenesis different from that of normal adrenal tissue. excretion rates, both in patients with adrenocortical insufficiency In the absence of functional adrenal tissue the presence of and in healthy controls, a comparison of the excreted amounts 1 1-hydroxylated steroid metabolites must be due to an extra- was not possible for the remaining compounds listed in Tables adrenal 1 1-hydroxylase. Such a speculation may be based on I and II. Finally, the excretion rates of cortisol in patients with the previous demonstration of an extraadrenal 21 -hydroxylase adrenocortical insufficiency were <0.5 ,ug/24 h after withdrawal (12) and of a hepatic 313-steroid dehydrogenase (10). However, of substitution therapy, during stimulation by ACTH, and after in regard to the increased excretion of early steroid metabolites 3 d of dexamethasone by mouth. Whereas the administration mentioned above, a gonadal origin of 1 l3-hydroxylated steroids of ACTH and of dexamethasone induced the expected changes in our patients appears more likely. The minor alterations in in the excretion of steroid metabolites in healthy men and gonadotropin secretion seen in patients with adrenocortical in- women, the respective excretion rates remained unchanged in sufficiency (13) are not likely to account for these pronounced patients with adrenocortical insufficiency (Table II). changes in steroid excretion. Gas-chromatographic analysis indicated the presence of 11- Under the appropriate experimental conditions, ACTH, the hydroxyandrosterone in the urine of three patients with adre- tropic hormone of the adrenal, may stimulate both rat and hu- nocortical insufficiency. This was confirmed by additional mass man testicular cells at the molecular level in terms of RNA syn-

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Steroid Excretion in Adrenocortical Insufficiency 1067 CHOLESTEROL (41) PREGNENETRIOL (30)

PREGNENOLONE (19) '-l177-HYDROXY- - DHEA (4) ANDRO;TENEDIOL (6) I PREGNENOLONE

PREGNANOLONE (16)4' PROGESTERONE (22) * 17o(-HYDROXY- - R ANDROTENEDIONE (10) - TESTOSTERONE (12) PROGESTERONE [21 - HYDROXYLASE]

CORTICOSTERONE ..21-DESOXYCORTISOL I DESOXYCORTICOSTERRONEC 1L\X21-DESOX PREGNANEDIOL (18) TH-A' (32) ..-. 'I\ TH-'B' (33), allo-TH-'B'(34) .TH-'S' (24) 1113-HYDROXY-ANDRO- TH-'DOC' (25) allo-TH-'S' (28) STENEDIONE o~lo-TH-'DOC' (27) .I 1-KETO-ANDRO- CORTISONE CORTISOL STENEDIONE PREGNANETRIOL (20) TH-'E' (31) allo-TH-'E' (35) TH-'F' (36) allo-TH-'F' (37) d.-Cortolone (38) B-Corto)one (39) oL -Cortol (42) B-Cortol (40) ETIOCHOLANOLONE ANDROSTERONE e (3) 4 4 (2) I1-KETO 11-KETO 11 B-HYDROXY I 1 -HYDROXY ETIOCHOLANOLONE ANDROSTERONE ANDROSTcRONE ETIOCHOLANOLONE (9) (7) (14) (15) t ~~t t t Figure 1. Simplified scheme of steroidogenesis in patients with adrenocortical insufficiency. The numbers in brackets refer to the numbering sys- tem of steroid standards given in Table I. thesis and of steroid hormone synthesis ( 14). It appears possible In the adrenal cortex the presence of elevated concentrations that concentrations of ACTH elevated, as in our patients, for of ACTH for prolonged periods of time has been shown to alter periods of several years may influence gonadal steroidogenesis the relative activities of the various steroid hydroxylases (15). in adrenocortical insufficiency. Chronic overexposure to ACTH apparently modifies gonadal

ST. P. SUM OF MONITORED ION ABUNDANCES SUM OF MONITORED ION ABUNDANCES Full Scale - t00 Full Scale 500

.1 : : A. ~ ~~~~~~~~~~ ; %% M~~~~~~~~~~~~~~ .

ION 479.30 ION 479.30 F.S.w 5 F.S. ' I

ION *449.30 ION ' 449.30 F.S.= 13M+ -30 F.S.= M+-30

ION 448.30 - M+3M-3 ION 448.30 M+3 F.S.m 34 F.S.= 1 -31

ION *358.20 M-2 ION z 358.20 F.. 28 F.S. * 2 M +-121f\, 091%~~~~~~~~~~~~~1

37 38 39 40 41 42 43 44 45 46 47 48 36 37 38 39 40 41 42 43 44 45 46 47 48 MINUTES MINUTES Figure 2. Identification of I 1-hydroxyandrosterone by gas chromatography-mass spectrometry (selected ion monitoring) in a mix- ture of derivatized reference compounds (St.) and in the urine of a patient with adrenocortical insufficiency (P.). Plots of the fol- lowing four ions are shown: m/e 479 (M+), m/e 449 (M+- 30), m/e 448 (M+- 31), and m/e 358 (M+ - 129). The abscissa represents the retention time (minutes).

1068 H. Vierhapper, P. Nowotny, and W. Waldhdusl steroidogenesis in an analogous manner, thus explaining the in- In conclusion, patients with adrenocortical insufficiency creased excretion of early steroid metabolites not requiring 21- present a pattern of steroid excretion characterized by the com- hydroxylation. This would also imply that the attempt to qualify plete absence of 2 1-hydroxylated, but not of 1 1-hydroxylated, the origin of any given steroid as gonadal or adrenal by its absence metabolites. The obtained data suggest that chronic ACTH ex- or presence in patients with adrenocortical insufficiency (9) is posure may result in the activation of 1 1f-hydroxylase in the an oversimplification. Steroid excretion rates remained un- human gonad. changed in our unsubstituted patients during the administration of 1-24 ACTH and of dexamethasone, indicating that the ad- Acknowledgment ditional administration of ACTH or the short-term partial The valuable technical assistance of Ms. E. Nowotny is gratefully ac- suppression of endogenous ACTH by 2 mg dexamethasone for knowledged. 3 d is apparently of little relevance in this pathological situation. Concerning the presence of 1 1-hydroxylated metabolites, i.e., of References 11 3-hydroxyandrosterone and 11 -ketoandrosterone, in the urine of patients with adrenocortical insufficiency, it should be re- 1. Luyten, J. A., and G. A. F. M. Rutten. 1974. Analysis of steroids membered that some testicular tumors in patients with congen- by high resolution gas-liquid chromatography. II. Application of urinary ital adrenal hyperplasia and in adrenalectomized patients with samples. J. Chromatogr. 91:393-406. 2. Pfaffenberger, C. D., and E. C. Horning. 1975. High resolution Cushing's syndrome possess both 1 13-hydroxylase and 2 1-hy- biochemical gas-chromatography. Determination of human steroid me- droxylase activity in vitro ( 16-19). The question of whether these tabolites using glass open tubular capillary columns. J. Chromatogr. 1 12: tumors arise from regular testicular tissue (20) or from adrenal 581-594. remnants within the testes (21) is as yet unsettled (19), but pro- 3. Milewicz, A., P. Vecsei, S. Korth-Schutz, D. Haack, A. Rosler, K. longed exposure to markedly elevated concentrations of ACTH Lichtwald, S. Lewicka, and G. von Mittelstaedt. 1984. Development of has generally been implied in their pathogenesis (17). Thus, dur- plasma 2 1-deoxycortisol radioimmunoassay and application to the di- ing prolonged overexposure to ACTH, the human gonad is ca- agnosis of patients with 2 1-hydroxylase deficiency. J. Steroid Biochem. pable of producing 1 1-hydroxylated steroids. 21:185-191. Since the side chain at C1 7 found in C21 steroids orients the 4. Waldhausl, W., K. Herkner, P. Nowotny, and P. Bratusch-Marrain. reduction of the double bond at C4/C5 in the Sf direction (22), 1978. Combined 17a- and 18-hydroxylase deficiency associated with steroids with the 1 complete male pseudohermaphroditism and hypoaldosteronism. J. Clin. C1903 5:l-configuration (i.e., 1-ketoetiochol- Endocrinol. Metab. 46:236-246. 1 anolone, and Il3-hydroxyetiocholanolone) are metabolites of 5. Chambaz, E. M., and E. C. Horning. 1969. Conversion of steroids C21 precursors such as cortisol. Compounds with the 5a config- to trimethylsilyl derivatives for gas phase analytical studies: reaction of uration (i.e., 11-ketoandrosterone, 1 (3-hydroxyandrosterone) are silylating reagents. Anal. Biochem. 30:7-24. derived from 1 l/3-hydroxyandrostenedione and from 1 I-ke- 6. Shackleton, C. H. L., and J. W. Honour. 1976. Simultaneous toandrostenedione (22-24) via side-chain cleavage of 2 1-deoxy- estimation of urinary steroids by semiautomated gas chromatography. cortisol. The synthesis of these steroids, therefore, requires the Investigation of neo-natal infants and children with abnormal steroid activity of 1 l3-hydroxylase, but not of 2 1-hydroxylase. The as- synthesis. Clin. Chim. Acta. 69:267-283. sumed presence of 11 3-hydroxylase in the gonads of our patients 7. Abraham, G. E., and Z. H. Chakmakjian. 1973. Serum steroid thus not only explains the observed excretion of C 1 -oxygenated levels during the menstrual cycle in a bilaterally adrenalectomized woman. steroids, but also the preponderance of the 5a- vs. the 5(-con- J. Clin. Endocrinol. Metab. 37:581-587. 8. Worgul, T. J., R. J. Santen, W. Samojlik, and S. A. Wells. 1982. figurated metabolites. How effective is surgical adrenalectomy in lowering steroid hormone The estimated plasma concentrations of 21 -desoxycortisol concentrations? J. Clin. Endocrinol. Metab. 54:22-26. and 1 -ketoandrosterone are also in keeping with the presence 9. Urban, M. D., P. A. Lee, J. P. Gutai, and C. J. Migeon. 1980. of 1 l-hydroxylase activity in our patients, and it is of note that in pubertal males with Addison's disease. J. Clin. Endocrinol. the reduction in the plasma concentrations of both steroids was Metab. 51:925-929. less pronounced than that of cortisol. In regard to the elevated 10. Bongiovanni, A. M. 1981. Acquired adrenal hyperplasia: with excretion rates of 1 -ketoandrosterone, pregnanetriol, pregne- special reference to 3,B-hydroxysteroid dehydrogenase. Fertil. Steril. 35: netriol, and pregnenolone, elevated plasma concentrations of 599-607. the respective precursor steroids (i.e., 1 -ketoandrostenedione, 11. Beierwaltes, W. H., J. C. Sisson, and B. Shapiro. 1984. Diagnosis 21-desoxycortisol, pregnenolone, and 17-hydroxypregnenolone) of adrenal tumors with radionuclide imaging. Spec. Top. Endocrinol. might have been expected. This was clearly not the case and Metab. 6:1-54. there 12. Winkel, C. A., M. L. Casey, R. J. Worley, J. D. Madden, and is no ready explanation for this observation, although one P. C. MacDonald. 1983. Extraadrenal steroid 21 -hydroxylase activity in might point out that plasma concentrations of steroids do not a woman with congenital adrenal hyperplasia due to steroid 2 1-hydrox- necessarily reflect the excretion rates of their metabolites, since ylase deficiency. J. Clin. Endocrinol. Metab. 56:104-107. both values also depend on the metabolic clearance rate of the 13. Vierhapper, H., W. Waldhausl, and P. Nowotny. 1982. Gonad- respective precursor (25). otrophin-secretion in adrenocortical insufficiency: impact of glucocor- Under physiological conditions both the adrenal cortex and ticoid substitution. Acta Endocrinol. 101:580-585. the gonads contribute to the urinary excretion of 11 -desoxy- 17- 14. Villee, D. B., and J. Fisher. 1975. The influence of ACTH on ketonic steroid metabolites (etiocholanolone, androsterone), al- steroid synthesis in normal and abnormal testicular tissues. Adv. Enzyme though direct glandular secretion of both compounds is small Regul. 13:317-328. (26). In our patients with adrenocortical insufficiency the lack 15. Ney, R. L., R. N. Dexter, W. W. Davis, and L. D. Garren. 1967. of adrenal precursors apparently accounts for the diminished A study of the mechanism by which adrenocorticotroic hormone main- tains adrenal steroidogenic responsiveness. J. Clin. Invest. 46:1916-1924. excretion of etiocholanolone and androsterone. The latter is in 16. Dominguez, 0. V. 1961. Biosynthesis of steroids by testicular with the 65% keeping reduction in plasma androsterone as re- tumors complicating congenital adrenocortical hyperplasia. J. Clin. En- ported by others (26). docrinol. Metab. 21:663-674.

Steroid Excretion in AdrenocorticalInsufficiency 1069 17. Hamwi, G. J., G. Gwinup, J. H. Mostow, and P. K. Besch. 1963. 22. Keutmann, E. H., and W. B. Mason. 1967. Individual urinary Activation of testicular adrenal rest tissue by prolonged excessive ACTH 17-ketosteroids of healthy persons determined by gas chromatography: production. J. Clin. Endocrinol. Metab. 23:861-869. biochemical and clinical considerations. J. Clin. Endocrinol. Metab. 27: 18. Fore, W. W., T. Bledsoe, D. M. Weber, R. Akers, and R. T. 406-420. Brooks. 1972. Cortisol production by testicular tumors in adrenogenital 23. Bradlow, H. L., and T. F. Gallagher. 1959. Metabolism of 11- syndrome. Arch. Intern. Med. 130:59-63. hydroxy-A4-androstene-3,17-dione in congenital adrenal hyperplasia. J. 19. Radfar, N., F. C. Bartter, R. Easley, J. Kolins, N. Javadpour, Clin. Endocrinol. Metab. 19:1575-1580. and R. J. Sherins. 1977. Evidence for endogenous LH suppression in a 24. Goldzieher, J. W., and S. C. Beering. 1969. Metabolism of 1 Ij3- man with bilateral testicular tumors and congenital adrenal hyperplasia. hydroxyandrostenedione, and hydrocortisone to urinary J. Clin. Endocrinol. Metab. 45:1194-1204. 1 1-oxy 17-ketosteroids. J. Clin. Endocrinol. Metab. 29:171-178. 20. Landing, B. H., and E. Gold. 1951. The occurrence and signifi- 25. Samojlik, E., M. A. Kirschner, D. Silber, G. Schneider, and cance of Leydig cell proliferation in familial adrenal cortical hyperplasia. N. H. Ertel. 1984. Elevated production and metabolic clearance rates of J. Clin. Endocrinol. Metab. 11:1436-1453. androgens in morbidly obese women. J. Clin. Endocrinol. Metab. 59: 21. Wilkins, L., W. Fleischmann, and J. E. Howard. 1940. Macro- 949-953. genitosomia precox associated with hyperplasia of the androgenic tissue 26. Meikle, A. W., J. D. Stringham, D. E. Wilson, and L. I. Dolman. of the adrenal and death from corticoadrenal insufficiency. Endocrinology. 1979. Plasma Sa-reduced androgens in men and hirsute women: role of 26:385-395. adrenals and gonads. J. Clin. Endocrinol. Metab. 48:969-975.

1070 H. Vierhapper, P. Nowotny, and W. Waldhausl