AN IMPROVED METHOD FOR THE DETECTION OF HETEROZYGOSITY OF CONGENITAL VIRILIZING ADRENAL HYPERPLASIA

A modified short ACTH test for the detection of heterozygote carriers of 21-hydroxylase deficiency (21-OHD) was applied to twenty-one controls and fourteen parents of children with 21-OHD. The following modifications were introduced: (1) Endogenous ACTH was suppressed by dexamethasone administration prior to the test, (2) Plasma 17-hydroxyprogesterone (17-OHP), cortisol (F), progesterone (P), corticosterone (B) and delta 4- androstenedione (A) were measured, (3) Variables studied were the ratio of plasma increments (delta) between precursors and end products, (4) Data were analysed by a step-wise discriminant analysis. Significant alterations in the metabolic pathway of F, B and A were demonstrated. The discriminant analysis showed that the addition of B pathway data did not improve the discrimination potency of the test performed on F pathway data. The combination of variates which provided the best discrimination was the logarithmic sum of delta 170HP/delta F at 15 and 30 min. It led to a 94% correct classification for normals and carriers.     

ANTIPYRINE CLEARANCE IN CONGENITAL ADRENAL HYPERPLASIA

Antipyrine (AP) metabolism was studied in four adult patients with congenital adrenal hyperplasia (CAH) due to classical 21-hydroxylase deficiency and in four (adult) patients with CAH due to non-classical 21-hydroxylase deficiency. This investigation was prompted by the known biochemical and functional similarities between drug- and steroid-metabolizing enzymes. We hypothesized that a common genetic mechanism may regulate the expression and activity of both the adrenal and hepatic cytochrome P450 enzymes. AP half-lives (T1/2, mean +/- SD) were 10.7 +/- 2.1 h in classical CAH patients, 10.1 +/- 0.4 h in the nonclassical CAH patients and 10.9 +/- 2.2 h in the control group, suggesting that there was no significant difference in AP metabolism among the three groups. Similarly, no significant differences were found in the AP apparent volume of distribution (aVd) and metabolic clearance rate (MCR) among the three groups. These results indicate that despite the deficiency of adrenal cytochrome P450 in patients with 21-hydroxylase deficiency CAH, these individuals have normal hepatic drug metabolizing activity for the biotransformation of AP.     

THE STEROID RESPONSE TO CONTROLLED ADRENAL STIMULATION IN CONGENITAL ADRENAL HYPERPLASIA

Adrenal steroidogenesis has been studied in vivo in eleven patients aged 13-68 years with 21-hydroxylase deficiency, in one patient with 11 beta-hydroxylase deficiency and in ten female control subjects. Serum levels of the delta 5 3 beta-hydroxysteroids, pregnenolone (Pe), 17 alpha-hydroxypregnenolone (17Pe), dehydroepiandrosterone (DHEA) and androstenediol (Adiol) and their delta 4 3-keto counterparts, progesterone (Po), 17 alpha-hydroxyprogesterone (17Po) androstenedione (Adione) and testosterone as well as of 11-deoxycortisol and cortisol were measured during acute adrenal suppression with dexamethasone followed by stimulation with synthetic 1-24 ACTH. In the seven patients with 21-hydroxylase deficiency who were on adequate glucocorticoid therapy, grossly exaggerated responses of 17Po and Po to ACTH were nevertheless preserved. In contrast, there was a grossly subnormal response of 17Pe, DHEA and Adiol to ACTH, and low basal levels of DHEA-sulphate. In the untreated patients the response of 17Pe and DHEA was normal. The Adione response was exaggerated in untreated and normal in treated cases. Similar findings obtained in the patient with 11 beta-hydroxylase deficiency who was studied after 6 weeks without replacement therapy. Our findings demonstrate that production of adrenal steroids that are associated with the adrenarche is not exaggerated in untreated CAH, and is grossly suppressed in treated cases. These findings are compatible with the hypothesis that intra-adrenal cortisol may initiate and/or maintain production of the delta 5 steroids by the zona reticularis that occurs in the human adrenarche.     

CUSHING'S SYNDROME, NODULAR ADRENAL HYPERPLASIA AND VIRILIZING CARCINOMA

A 48-year-old hypertensive diabetic woman rapidly became virilized. Urine 17-oxo-and oxogenic steroids and plasma testosterone, androstenedione, DHEA, DHEA-sulphate and androstenediol were greatly elevated. Plasma cortisol was constantly high and was not suppressed by dexamethasone. Circulating immunoreactive ACTH was consistently detectable at 18–24 ng/l. A 450 g carcinoma arising from a nodular hyperplastic right adrenal gland was resected. Production by the tumour of 17α-hydroxypregnenolone, 17α-hydroxyprogesterone and five C-19 steroids, but very little prenenolone, progesterone or cortisol, was shown by blood sampling, tumour culture and dramatic falls after operation. The plasma cortisol fell to half, with no diurnal variation, consistent with persistent Cushing's syndrome, and the plasma ACTH rose to 55 ng/l. She died 3 months later from a myocardial infarction. Autopsy revealed a pituitary basophil adenoma at a site where radiologically there had been an indentation in the fossa floor for at least 7 years. The left adrenal gland showed nodular hyperplasia. Therefore we conclude that mild pituitary-dependent Cushing's syndrome may have been present for many years before development of a virilizing carcinoma. This case demonstrates that adrenal carcinoma in man can sometimes develop as a consequence of nodular adrenal hyperplasia which may in turn be due to long-standing trophic hyper-stimulation.     

CIRCADIAN RHYTHM OF PLASMA CORTICOSTEROIDS IN CONGENITAL ADRENAL HYPERPLASIA

Circadian rhythm and dexamethasone suppression of plasma fluorogenic corticosteroids and plasma progesterone were studied in six patients with the simple virilizing form of congenital adrenal hyperplasia. Basal levels of plasma fluorogenic steroids were normal or high in the untreated patients. A definite circadian variation was demonstrated, suggesting a similar variation in ACTH secretion. Dexamethasone administration in the form of the overnight suppression test was followed by a virtually complete disappearance of plasma fluorescence. Plasma progesterone levels were high in the morning hours and showed a circadian variation and dexamethasone suppression similar to those of fluorogenic steroids. The diagnostic and therapeutic importance of these findings is discussed.     

SALIVARY 17α-HYDROXYPROGESTERONE IN CONGENITAL ADRENAL HYPERPLASIA

A specific radioimmunoassay having the sensitivity (4 pg/tube) and precision required for the routine determinations of 17α-hydroxyprogesterone concentrations in mixed whole saliva, parotid fluid and plasma has been developed. The correlation between 17α-hydroxyprogesterone concentrations in matched samples of saliva and parotid fluid was excellent (r = 0.98): therefore saliva, being easier to collect, was used exclusively in later studies. The median 17α-hydroxyprogesterone concentration in mixed whole saliva collected from thirty-two healthy children was 390 pmol/litre ranging from 90–1520 pmol/litre. In a group (n=14) of treated CAH patients having a C21 hydroxylase deficiency, 17α-hydroxyprogesterone showed a 20 fold greater variation, ranging from 67 pmol/litre in patients receiving excessive glucocorticoid dosage to 26300 pmol/litre in inadequately treated patients. A close correlation (r = 0.91) in 17α-hydroxyprogesterone levels was observed in matched samples of saliva and plasma collected between 09.00 and 10.00 h from these patients. Concentrations of 17α-hydroxyprogesterone in saliva therefore could well replace those in plasma for monitoring treatment of these patients. Matched samples of mixed whole saliva, parotid fluid and plasma were also collected from one inadequately treated female patient at frequent intervals over a 24 h period. Two other patients, one male and one female, collected matched samples at 30 min intervals for 4 h following Synacthen stimulation. The pattern of 17α-hydroxyprogesterone in these samples suggests that salivary steroid concentrations are of potential value in assessing endocrine function in conditions such as CAH, where frequent sampling over prolonged periods is required.     

EFFECT OF CORTISOL TREATMENT ON HORMONAL RELATIONSHIPS IN CONGENITAL ADRENAL HYPERPLASIA

The temporal relationship between administration of cortisol and serum 17α-hydroxyprogesterone was investigated in five patients aged 9-19 years with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. There was marked variability in the 17α-hydroxyprogesterone response (determined hourly for 24 h) of individual patients to administration of cortisol. Mean concentration was less than 0.030 μmol/l in one patient but 0.519μ mol/l in another. Levels were higher in all patients while off treatment, and were greatest in those with salt-losing adrenal hyperplasia. Growth hormone secretion was not suppressed by treatment with cortisol. Withdrawal of cortisol for 3 days resulted in a significant decrease in the mean serum FSH/LH ratio and a rise in serum testosterone in all subjects. Episodic release of gonadotrophins persisted in the adolescent patients.     

PREVALENCE OF SALT-LOSING AMONG CONGENITAL ADRENAL HYPERPLASIA PATIENTS

Among clinic patients with congenital adrenal hyperplasia (CAH) the observed frequency of salt-losing has increased significantly over the past 30 years. Comparing estimates from various clinics, older estimates of the frequency of salt-losing are lower than estimates cited in more recent literature. Within the patient population of a single long-established clinic, an increase in the frequency of salt-losing can also be observed. It is likely that the increase in the frequency of salt-losing CAH reflects improved case finding and survival of salt-losing patients. Based on reports of recent populations of CAH patients, the frequency of salt-losing among CAH patients is approximately 66%.     

REVERSIBLE MALE INFERTILITY DUE TO CONGENITAL ADRENAL HYPERPLASIA

We have studied a patient who was azoospermic and infertile. The cause of his infertility was unusual and curable: untreated congenital adrenal hyperplasia resulting from a partial 21-hydroxylase deficiency. The diagnosis was suggested by the combination of small testes, elevated levels of testosterone and suppressed levels of gonadotrophins.     

AN ACTH-SECRETING PITUITARY TUMOUR ARISING IN A PATIENT WITH CONGENITAL ADRENAL HYPERPLASIA

The case reported is of a 46-year-old woman who had congenital adrenal hyperplasia due to a 21-hydroxylase deficiency, and in whom there was the development of an ACTH secreting pituitary tumour. The patient was untreated with glucocorticoids until the age of 32 years when she presented with infertility. She next presented with amenorrhoea at the age of 44 years when she was found to have an enlarged pituitary fossa. Despite treatment with bromocriptine and adequate doses of dexamethasone, the tumour enlarged and required operative treatment 1 year later. Before and after operation, plasma ACTH levels were between 300 and 400 ng/l, immunocytochemistry showed staining for ACTH and other structurally related pro-opiocortin peptides but for no other hormones, and the tumour secreted large amounts of ACTH in vitro. The report of this case is to our knowledge the first account of a feedback tumour in congenital adrenal hyperplasia and provides yet another reason why patients with this condition should be treated, and good control achieved.     

A COMPARISON OF THREE GLUCOCORTICOID SUPPRESSIVE REGIMES IN ADULTS WITH CONGENITAL ADRENAL HYPERPLASIA

We have compared three glucocorticoids, hydrocortisone (HC) (20 mg mane & 10 mg nocte), cortisone acetate (CA) (25 mg mane & 12.5 mg nocte), dexamethasone (DXM) (0.5 mg mane & 0.25 mg nocte), for their effect on the biochemical control of adult patients with congenital adrenal hyperplasia (CAH). Twenty-four-hour profiles of plasma concentrations of ACTH, 17-hydroxyprogesterone (170HP) and androstenedione (delta 4A), and 09.00 h dehydroepiandrosterone sulphate (DHAS) plasma concentrations were used to assess control. The patients were studied after 2 weeks on each glucocorticoid. The areas under the curves, the heights of the morning peaks of each hormone, the midnight concentrations, and the concentrations of hormones just before the evening dose were analysed. The results show that all the indices, except the midnight concentrations which were uniformly low, were significantly lower on DXM than on either HC or CA. There were no significant differences between HC and CA for any of the indices. The DHAS concentrations were low on all three glucocorticoids but again significantly lower on DXM. DXM (0.5 mg mane & 0.25 mg nocte) is therefore, in the short term, a better suppressor of the pituitary-adrenal axis in adults with CAH than either HC or CA, and in the dose used did not suppress ACTH to undetectable levels, nor the steroids to below levels found in normal subjects.     

EFFECTS OF LONG TERM DEXAMETHASONE TREATMENT IN ADULT PATIENTS WITH CONGENITAL ADRENAL HYPERPLASIA

We have followed nine adult patients with congenital adrenal hyperplasia (CAH) for between 7-77 months on dexamethasone (DXM) 0.5 mg mane and 0.25 mg nocte, reducing to 0.5 mg mane. Twenty-four hour profiles of ACTH, 17-hydroxyprogesterone (17OHP), and androstenedione were performed; the areas under the curves (AUC) and the heights of the morning peaks were used to assess biochemical control. Comparisons were made between treatment before DXM (pre-DXM), 0.75 mg for 2 weeks (DXM-ST), 0.75 mg for at least 3 months (DXM-LT), and 0.5 mg for at least 3 months (DXM-0.5). None of the three males suffered any significant side-effects. All women had menstrual disturbance but in three ovulation was induced. One female developed Cushing's syndrome and two developed hirsutism which resolved on stopping DXM. Overall there was no significant difference between DXM-ST and DXM-LT (mean AUCs for ACTH: DXM-ST 660, DXM-LT 383, for 17OHP: DXM-ST 1177, DXM-LT 587, for androstenedione DXM-ST 232, DXM-LT 121). Reduction of the dose from 0.75 mg to 0.5 mg led to significant deterioration in control (Mean AUC's for ACTH DXM-0.5, 1123 (P less than 0.02), for 17OHP DXM-0.5, 2068 (P less than 0.002), for androstenedione DXM-0.5, 213 (P less than 0.5). We conclude that DXM is a satisfactory regime but the dose must be adjusted for each patient.     

FAMILIAL CONGENITAL CUSHING'S SYNDROME DUE TO BILATERAL NODULAR ADRENAL HYPERPLASIA

Two siblings with congenital Cushing's syndrome due to bilateral nodular adrenal hyperplasia are described. The older, a boy, presented with severe hypertension and died soon after subtotal adrenalectomy. His sister, who had clitoral enlargement and showed persistent hyponatraemia, had a two-stage total adrenalectomy and is still alive. Investigations in the second case showed grossly elevated urinary cortisol metabolites, 17-oxosteroids and 3β-hydroxy-5-ene-steroids. These were not suppressed by dexamethasone, and plasma ACTH was undetectable, indicating that the disorder was not due to excessive ACTH secretion. Cell culture studies on the resected adrenals failed to demonstrate an abnormal pattern of steroid synthesis in vitro, and normal trophic responses were obtained with 1–24 ACTH and monobutyryl cyclic AMP. No stimulation of steroid synthesis was obtained with a range of polypeptide hormones, and the cause of the adrenal hyperplasia remains unknown.     

BILATERAL TESTICULAR TUMOURS IN CONGENITAL ADRENAL HYPERPLASIA: A CONTINUING DIAGNOSTIC AND THERAPEUTIC DILEMMA

Bilateral testicular tumours are a rare but well described complication of congenital adrenal hyperplasia (CAH). It can be extremely difficult to distinguish histologically between Leydig cell tumours and adrenocortical rest hyperplasia which may lead in some cases to unnecessary orchidectomy in young men. A 26-year-old male in whom CAH had been diagnosed neonatally, was referred for further investigation of a craggy mass in the left testis. Six months earlier, right orchidectomy had been performed after presentation with bilateral enlarged, hard testes and azoospermia. The tumour was interpreted to be a malignant interstitial cell tumour of the testis. Our investigations revealed that his CAH was inadequately controlled and that the hormonal secretion from the remaining left testicular mass was ACTH dependent. Percutaneous venous sampling revealed high concentrations of cortisol and other adrenocortical hormones coming from the gonadal vein. Dexamethasone suppressed these adrenal hormones and reduced the size and softened the consistency of the testicular mass. Total disappearance of the mass was achieved by using dexamethasone, given in a reverse circadian rhythm regimen. Spermatogenesis returned and the patient's wife became pregnant after 6 months; she gave birth to a normal male infant. To our knowledge, this is the first time in this clinical setting that fertility has been achieved. This case highlights the need for both a functional and histological assessment of such 'tumours' in CAH prior to orchidectomy.     

MALIGNANT HYPERTENSION IN CONGENITAL ADRENAL HYPERPLASIA DUE TO 11β-HYDROXYLASE DEFICIENCY

Two brothers, aged 20 and 12, with the 11 beta-hydroxylase deficient form of congenital adrenal hyperplasia, are presented. They had refused treatment for the previous four years until the younger was admitted with malignant hypertension, partial blindness and evidence of myocardial damage. The elder was therefore admitted for assessment and found to have less severe hypertension. Gas liquid chromatographic analysis of the steroids in urine from each revealed a possible difference in the enzyme defect between the two brothers--the younger having a complete, while the elder had only a partial block. Family studies have confirmed no association with the HLA locus.     

BLOOD-SPOT 17α-HYDROXYPROGESTERONE RADIOIMMUNOASSAY IN THE FOLLOW-UP OF CONGENITAL ADRENAL HYPERPLASIA

The value of plasma 17α-hydroxyprogesterone (17α-OHP) concentration in monitoring the treatment of congenital adrenal hyperplasia (CAH) was studied by using a capillary blood micromethod. The blood-spot 17-OHP radioim- munoassay method involves serial sampling throughout the day and sending the samples into a centre by mail. Follow-up of seven children treated for CAH due to 21-hydroxylase deficiency showed that a single measurement of plasma 17-OHP concentration cannot be relied upon to determine adequacy of control, since circadian variation and timing of the sample in relation to the last dose of glucocorticoid may influence the plasma level of 17α-OHP. Our data confirm the value of sequential 17α-OHP assays throughout the day in the follow-up of CAH. With the blood-spot method the 17α-OHP determinations can be used on a wide scale for monitoring therapy.     

LINEAR GROWTH AND PUBERTAL DEVELOPMENT IN TREATED CONGENITAL ADRENAL HYPERPLASIA DUE TO 21-HYDROXYLASE DEFICIENCY

21 years experience with management of seventeen cases of congenital adrenal hyperplasia due to 21-hydroxylase deficiency has been analysed with respect to growth, bone maturation and related events at puberty: age at menarche and the occurrence of menstrual irregularities, this study showed that growth retardation is still a problem; that irregular treatment and prolonged exposure to adrenal androgens or oestrogens, may lead to disturbance in hypothalamo-pituitary-gonadal function and may be the cause fo delayed menarche, or menstrual irregularities in the case of the female. In males the start of puberty and its completion was within the normal range.     

IS SALT-WASTING IN CONGENITAL ADRENAL HYPERPLASIA DUE TO THE SAME GENE AS THE FASCICULATA DEFECT?

Clinical studies in patients with 21-hydroxylase deficiency congenital adrenal hyperplasia (CAH) were designed to ascertain the genetics of the salt-wasting component of the disorder. The gene controlling aldosterone biosynthesis may not be the same gene that controls 21-hydroxylase in the adrenal zona fasciculata. This we infer from the following clinical observations: (1) concordance for salt-wasting is not observed in all HLA-identical sibs with CAH; (2) the defect in aldosterone biosynthesis does not persist throughout life as does the fasciculata defect; (3) there is a significantly increased gene frequency of B40 and Bw47 in salt-wasting CAH; (4) obligate heterozygote parents of patients with salt-wasting CAH do not express a partial defect in aldosterone biosynthesis, as they do in the fasciculata. These observations cast doubt on the accepted concept of the autosomal recessive transmission of the glomerulosa 21-hydroxylase deficiency.