Primary aldosteronism: functional histopathology and long-term follow-up after unilateral adrenalectomy.

OBJECTIVES: To investigate the long-term outcome after unilateral adrenalectomy in patients with primary aldosteronism (PA) and to establish the role of functional pathology for the final diagnosis of aldosterone-producing adenoma (APA) or hyperplasia. DESIGN: A single-centre, retrospective cohort study in a hospital setting. PATIENTS: Consecutive patients with PA, n = 120, who underwent unilateral adrenalectomy between 1985 and 2010. Preoperative and postoperative data were analysed. To indicate the site of aldosterone secretion in the resected adrenal, we added functional methods to routine histopathology, using in situ hybridization and immunohistochemistry to detect the presence of enzymes needed for aldosterone (CYP11B2) and cortisol (CYP11B1, CYP17A1) synthesis. RESULTS: The median follow-up was 5 years and the cure rate of PA 91%. Hypertension was improved in 97% and normalized in 38%. Functional histopathology changed the final diagnosis from APA to hyperplasia in 6 cases (7%). Five of these had no expression of or staining for aldosterone synthase in the adenoma, but only in nodules in the adjacent cortex. All except one APA patient were cured of PA. They had lower preoperative serum potassium and higher 24-h urinary aldosterone than patients with hyperplasia. Among patients with hyperplasia 16 of 26 were cured. CONCLUSIONS: Most patients were cured of PA by unilateral adrenalectomy. Almost all noncured benefitted from the operation as the blood pressure improved. Functional histopathology proved helpful in the distinction between APA and hyperplasia, and we recommend that functional histopathology should be added to routine histopathology to improve the diagnostic evaluation and aid in tailoring the follow-up.

Reduced activity of 11ß-hydroxylase accounts for elevated 17α-hydroxyprogesterone in preterms.

OBJECTIVE: To characterize the urinary steroid metabolome of neonates and infants born either at term or preterm. STUDY DESIGN: We retrospectively analyzed urinary steroid hormone metabolites determined by gas chromatography-mass spectrometry of 78 neonates and infants born at term and 83 neonates and infants born preterm (median 34 weeks of gestational age). The subjects' 11ß-hydroxylase and 21-hydroxylase activities were assessed on the basis of urinary metabolite substrate-to-product ratios. RESULTS: Preterm neonates and infants had elevated urinary concentrations of 17α-hydroxyprogesterone (17OHP) metabolites (P<.001) but lower urinary concentrations of the 21-deoxycortisol metabolite pregnanetriolone (PTO) (P<.01). One reason was lower 11ß-hydroxylase activity in preterms. We could demonstrate a correlation between low 11ß-hydroxylase activity and high urinary concentrations of 17OHP metabolites (r=0.51, P<.001) but low urinary concentrations of the 21-deoxycortisol metabolite PTO (r=-0.24, P=.03) in preterms. CONCLUSIONS: Low 11ß-hydroxylase activity may explain increased 17OHP but decreased 21-deoxycortisol metabolite excretion in preterms. Our analysis clarifies, first, why preterms have higher 17OHP levels and thus higher rates of false-positive screening results for congenital adrenal hyperplasia than do term infants, and, second, why 21-deoxycortisol or its urinary metabolite PTO is more specific than 17OHP for the diagnosis of 21-hydroxylase deficiency.

Impact of immunohistochemistry on the diagnosis and management of primary aldosteronism: An important tool for improved patient follow-up.

BACKGROUND AND AIMS: Primary aldosteronism is a common cause of secondary hypertension. Primary aldosteronism is caused by an aldosterone-producing adenoma or bilateral hyperplasia that in some cases is asymmetrical with one adrenal dominating aldosterone secretion. Most patients with aldosterone-producing adenoma are biochemically cured by unilateral adrenalectomy, but patients with bilateral hyperplasia have a significant risk of residual or recurrent disease. Here, immunohistochemistry of CYP11B1 and B2 was used to investigate whether these markers could aid in the diagnostic workup of primary aldosteronism patients. MATERIALS AND METHODS: A total of 39 patients with primary aldosteronism who underwent unilateral adrenalectomy for a presumed adenoma during 2013-2016 were included. Immunohistochemistry using monoclonal antibodies identifying the enzymes CYP11B1 and B2 was part of routine histopathological workup in 6 cases; in 33 cases, it was applied retrospectively. The hyperplasia diagnosis was suggested when there was no dominating nodule but immunoreactivity for CYP11B2 was seen in several nodules, which were also seen on routine sections. To distinguish between adenoma and hyperplasia, a ratio between the largest and second largest CYP11B2-positive nodules was calculated. RESULTS: In all, 22 patients had an aldosterone-producing adenoma, while 13 patients were judged to have hyperplasia. In four cases, a final diagnosis could not be established, thus these were judged equivocal. Among the 33 cases investigated retrospectively, the primary histopathological diagnosis was altered from hyperplasia to aldosterone-producing adenoma in 9 cases (27%) after immunohistochemistry, and the immunohistochemically rectified adenoma group displayed improved clinical cure rates compared to the routine H&E-diagnosed cohort. Moreover, the B2 ratio was significantly higher in adenoma than in hyperplasia and equivocal cases. CONCLUSION: Immunohistochemistry detecting CYP11B1 and B2 expression is of great help in establishing a final histopathological diagnosis in patients with primary aldosteronism. This procedure should be part of the histopathological routine in all operated primary aldosteronism patients.

[Comparative genetic analysis of different forms of low-renin arterial hypertension].

High level of clinical and genetic heterogeneity is a characteristic of arterial hypertension (AH) that is one of the most wide-spread cardiovascular diseases. In most cases (excluding a few monogenic forms), AH is a polygenic disease and genes of renin-angiotensin-aldosterone system play an important role in AH predisposition. 20-25% AH cases occur during low activity of renin in blood plasma (low-renin form of AH) while aldosterone production can be increased (hyperaldosteronism, HA) or normal. We examined polymorphism of genes that code the renin-angiotensin-aldosterone system components in the groups of low-renin forms of AH, namely, primary HA, idiopathic HA and AH with normal level of aldosterone. For all HA cases, the absence of chimeric CYP11B2/CYP11B1 gene that is a cause for monogenic disease--amilial HA of first type, was shown. A comparison of distributions of alleles and genotypes of polymorphous regions of genes: CYP11B2 (C-344T), REN (C-5434T, C-5312T and A BglI G), AGT (Thr174Met), ACE (I/D), CMA (G-1903A), AT2R1 (A1166C) and of their combinations is the groups described above was done. The analysis of carriership of the alleles and genotypes combinations of the polymorphous regions has shown that genes CYP11B2, REN, ACE, CMA andA T2R1 participate in development of low-renin HA. The results are evidence of similarities and some definite differences in genetic nature of the different forms of low-renin AH and, to say more widely, argue that the investigation of genetic predisposition for clinically heterogeneous forms of polygene diseases by comparison of groups of patients, separated in accordance with peculiarities of disease course, holds much promise for their hereditary background understanding.

[Congenital adrenal hyperplasia caused by 11beta-hydroxylase deficiency. A case report]. / Wrodzony przerost nadnerczy wywolany niedoborem 11beta-hydroksylazy. Opis przypadku

The authors describe a case of an adult woman with congenital adrenal hyperplasia caused by 11beta-hydroxylase deficiency. The patient presented not only typical features of this disease such as virilization and hypokaliemic hypertension but also rare pathologies, such as ectopic adrenal tissues, salt loss during infancy, and functional adrenomedullary hyperactivity. In spite of the severe disease and delays in its diagnosis and adequate treatment, significant improvement in appearance and normalization of blood pressure as well as the birth of a healthy child were achieved. The paper also discusses current opinions concerning the pathogenesis and treatment in CAH with 11beta-hydroxylase deficiency as well as difficulties in diagnostic and therapeutic management.

Non-classical 11ß-hydroxylase deficiency caused by compound heterozygous mutations: a case study and literature review.

BACKGROUND: 11ß-hydroxylase deficiency (11OHD) is extremely rare, and reports of non-classical 11OHD are even rarer. Non-classical 11OHD usually presents as premature adrenarche, hyperandrogenism, menstrual disorders, and hypertension. Because the symptoms of non-classical 11OHD are mild, delayed diagnosis or misdiagnosis as polycystic ovary syndrome or primary hypertension is common. CASE PRESENTATION: This paper introduces a case of a young female patient presenting hypertension and menstrual disorders. Laboratory examination revealed increased androgen levels, mild adrenal hyperplasia, mild left ventricular hypertrophy, and mild sclerosis of the lower limb arteries. 11OHD was confirmed by genetic testing, and the patient was found to carry compound heterozygous mutations in CYP11B1 (c.583 T > C and c.1358G > A). The mutation Y195H is located in exon 3 and has not been reported previously. In silico studies indicated that this mutation may cause reduced enzymatic activity. After treatment with hydrocortisone and spironolactone, blood pressure was brought under good control, and menstruation returned to normal. We also conducted a retrospective review of previously reported cases in the literature (over 170 cases since 1991). CONCLUSIONS: Early diagnosis of non-classical 11OHD is difficult because its symptoms are mild. The possibility of this disease should be considered in patients with early-onset hypertension, menstrual disorders, and hyperandrogenism to provide early treatment and prevent organ damage due to hypertension and hyperandrogenism. CYP11B1 mutations are known to be race-specific and are concentrated in exons 3 and 8, of which mutations in the former are mostly associated with non-classical 11OHD, whereas mutations in the latter are mostly found in classical 11OHD, characterized by severe loss of enzymatic activity.

Testicular tumors in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency show functional features of adrenocortical tissue.

CONTEXT: In male patients with congenital adrenal hyperplasia (CAH), testicular adrenal rest tumors are frequently found that may interfere with gonadal function. OBJECTIVE: Our objective was to determine steroid-producing features of testicular adrenal rest tumors. DESIGN AND SETTING: The study is descriptive and took place at a university medical center. PATIENTS: Eight adult CAH patients with bilateral testicular adrenal rest tumors were treated with testis-sparing surgery. INTERVENTIONS: In all but one patient, spermatic veins were cannulated during surgery and blood samples collected to measure the adrenal-specific steroid 21-deoxycortisol (21DF) and 17-hydroxyprogesterone (17OHP) and androstenedione (A). The same parameters were measured in simultaneously taken peripheral blood. mRNA concentrations of adrenal-specific enzymes CYP11B1 and CYP11B2 and ACTH and angiotensin II (AII) receptors were measured in tumor tissue. MAIN OUTCOME MEASURES: Adrenal-specific steroids/enzymes were assessed. RESULTS: 21DF, 17OHP, and A levels were measurable in all spermatic vein samples. The ratio (mean +/- SD) between spermatic vein and simultaneously taken peripheral blood samples was 37.8 +/- 56.3 (21DF), 132.0 +/- 249 (17OHP), and 57.0 +/- 68.2 (A). CYP11B1, CYP11B2, and ACTH and AII receptor mRNAs were detected in all tumors with a strong correlation between ACTH receptor mRNA in tumors and 21DF (r = 0.85; P = 0.015), 17OHP (r = 1; P = 0.01) and A (r = 0.89; P = 0.007) concentrations in peripheral blood. CONCLUSION: Testicular adrenal rest tumors produce adrenal-specific steroids and express adrenal-specific enzymes and ACTH and AII receptors, confirming the strong resemblance with adrenal tissue. Because AII receptors are present in tumor tissue, it can be hypothesized that AII may be an additional factor responsible for testicular adrenal rest tumor growth.

Histopathological and genetic characterization of aldosterone-producing adenomas with concurrent subclinical cortisol hypersecretion: a case series.

PURPOSE: Aldosterone-producing adenomas with concurrent subclinical cortisol hypersecretion are reported in an increasing number of patients. Five aldosterone-producing adenomas from patients with primary aldosteronism and subclinical hypercortisolism were examined. THE AIMS OF OUR STUDY WERE: (1) to analyze pathological features and immunohistochemical expression of CYP11B1 (11ß-hydroxylase) and CYP11B2 (aldosterone synthase) in these tumors; (2) to investigate somatic mutations involved in adrenal steroid hypersecretion and/or tumor growth. METHODS: Archival micro-dissected paraffin-embedded slides from tumor specimens were used for histological and molecular studies. Immunohistochemistry was performed using monoclonal anti-CYP11B1 and anti-CYP11B2 antibodies. Cellular composition was determined by examining for known features of zona fasciculata and zona glomerulosa, and immunoreactivity for CYP11B1 and CYP11B2 by McCarty H-score. Spot regions for mutations in KCNJ5, ATP1A1, ATP2B3, CACNA1D, PRKACA, and CTNNB1 gene sequences were evaluated. RESULTS: Four APAs showed a predominant (≥50%) zona fasciculata-like cell pattern: one tumor had CYP11B1 H-score = 150, no detectable CYP11B2 expression, and harbored a PRKACA p.Leu206Arg mutation (that we have reported previously elsewhere), one had no CYP11B1 expression, CYP11B2 H-score = 40, and no mutations; the remaining two adenomas had high CYP11B1 H-score (160 and 240, respectively) and low CYP11B2 H-score (30 and 15, respectively), with the latter harboring a CTNNB1 p.Ser45Phe activating mutation. One of five aldosterone-producing adenomas had a predominant zona glomerulosa-like pattern, CYP11B1 H-score = 15, CYP11B2 H-score = 180, and no mutations. CONCLUSIONS: The majority of aldosterone-producing adenomas with concurrent subclinical cortisol hypersecretion were composed mainly of zona fasciculata-like cells, while CYP11B1 and CYP11B2 immunostaining demonstrated clear heterogeneity. In a subset of cases, different somatic mutations may be involved in hormone excess and tumor formation.

Altered 11 beta-hydroxylase activity in glucocorticoid-suppressible hyperaldosteronism.

Corticosteroid 11 beta-hydroxylation is catalyzed by 11 beta-hydroxylase and aldosterone synthase. Using plasma steroid ratios, the level of this process in patients with glucocorticoid-suppressible hyperaldosteronism (GSH) was compared with that in unaffected control subjects and patients with Conn's syndrome. Based on both 11-deoxycortisol/cortisol (S:F) and 11-deoxycorticosterone/corticosterone (DOC:B) ratios, patients with GSH showed impaired resting 11 beta-hydroxylase activity. In GSH, but not in the other groups, the S:F ratio was significantly correlated with the basal plasma aldosterone concentration. ACTH infusion increased the S:F ratio in all of these patient groups, suggesting a common partial deficiency. The results also indicate that 11 beta-hydroxylation may be rate limiting in normal subjects. In control subjects and patients with Conn's syndrome, the DOC:B ratio was not affected by ACTH. However, in GSH patients, this ratio fell markedly, indicating an increased efficiency of 11 beta-hydroxylation of DOC (but not S). This may be due to the activation by ACTH of the zona fasciculata chimeric aldosterone synthase characteristic of this disease. Plasma aldosterone, corticosterone, and DOC concentrations appeared to be more sensitive to ACTH in GSH than in the other groups. The defect in 11 beta-hydroxylation in GSH accounts for the increased levels of DOC reported in this condition and may contribute to the phenotype variability.

Age-related differences in corticosterone secretion in female rats.

The effects of age on steroidogenesis in rat zona fasciculata-reticularis (ZFR) cells were studied. Young, adult, and middle-aged rats were ovariectomized (Ovx) and received replacement therapy with oil or estradiol benzoate ([EB] 25 microg/mL/kg). Rat ZFR cells were incubated with corticotropin (ACTH), prolactin (PRL), or forskolin at 37 degrees C for 1 hour. The effects of age on the activity of steroidogenic enzymes of ZFR cells were measured by the amount of intermediate steroidal products separated by thin-layer chromatography. Plasma levels were higher for PRL (54% to 254%) and corticosterone (179% to 257%) in middle-aged versus young rats. In oil-treated Ovx rats, basal and ACTH-stimulated corticosterone release by ZFR cells were also greater in middle-aged compared with young rats. Replacement with EB in Ovx rats increased the ACTH-stimulated release of corticosterone. Administration of ovine PRL in vitro resulted in a dose-dependent increase of corticosterone production. In oil-treated middle-aged rats, ovine PRL-stimulated corticosterone release was higher than in young rats. Forskolin-induced production of cyclic adenosine 3',5'-monophosphate (cAMP) was greater in middle-aged versus young rats and correlated with the increase of corticosterone production. The activity of steroidogenic enzymes in rat ZFR cells was unchanged by age. These results suggest that the age-related increase of corticosterone production in female rats is associated with the stimulatory effect of PRL on ZFR cells and is due in part to an increase of cAMP generation.

A prevalent and three novel mutations in CYP11B1 gene identified in Chinese patients with 11-beta hydroxylase deficiency.

UNLABELLED: 11ß-Hydroxylase deficiency (11ß-OHD), caused by CYP11B1 mutations, is characterized by hyporeninemic, hypokalemic hypertension and hyperandrogenism. We identified a prevalent and three novel mutations of CYP11B1 gene in nine patients with classic 11ß-OHD. SUBJECTS AND METHODS: Nine patients with 11ß-OHD from unrelated families were recruited. The complications of 11ß-OHD occurred in three patients who never received glucocorticoid treatment. CYP11B1 gene was sequenced and 11ß-hydroxylase enzymatic activities were assessed in vitro. A haplotype analysis was performed to determine a common ancestor for those subjects who carried the same p.R454C mutation. RESULTS: CYP11B1 gene mutations were identified in all patients, with a prevalent (p.R454C) and three novel mutations (p.V148G, IVS7-9C>A, c.1359_1360insG). The p.R141X, p.V148G, c.1359_1360insG and p.R454C mutations retained 4.9%, 3.9%, 3.7%, 4.5% of residual enzymatic activity, respectively. Five of nine patients carried p.R454C mutation, which was only reported in Chinese 11OHD patients. Haplotype analysis showed that this mutation might be inherited from a common ancestor. CONCLUSION: The enzymatic activities for p.R141X, p.V148G, c.1359_1360insG and p.R454C mutants were almost completely abolished, which corresponds to classic form of 11ß-OHD. The observations of a prevalent mutation and three novel mutations might have potential clinical utility for genetic counseling and prenatal diagnosis in Chinese 11ß-OHD patients.

Three novel CYP11B1 mutations in congenital adrenal hyperplasia due to steroid 11Beta-hydroxylase deficiency in a moroccan population.

BACKGROUND/AIMS: Steroid 11beta-hydroxylase deficiency (11OHD), the second cause of congenital adrenal hyperplasia (CAH), accounts only for 5% of all CAH. To date, only 51 different mutations have been reported with poor clinical and biological data. Most of them could be considered as private mutations except one, p.R448H, identified especially in Moroccan Jews but also in Caucasian patients. As two other CYP11B1 mutations have a high incidence in Tunisian patients, we report from another Maghreb population the clinical, follow-up and molecular genetics of 5 Moroccan patients with classical 11OHD. METHODS: Patients belonging to 3 families were recruited on clinical data. The diagnosis was confirmed by 11-deoxycortisol determination. Sequencing of the CYP11B1 gene and molecular modeling were performed. RESULTS: Clinical, hormonal and follow-up data were consistent with a severe form of 11OHD. Gender reassignment and evolution of hypertension were discussed. Three novel mutations, p.Ala259Asp, p.Gly446Val and IVS5+2T>G were identified. As each patient was homozygous for one mutation, we could deduce from their phenotype and our modeling studies that the p.Gly446Val mutation was more severe than p.Ala259Asp. CONCLUSION: This study shows a good correlation between phenotype and genotype. Each CYP11B1 mutation is new and private, contrasting with the high incidence of two Tunisian mutations.

Congenital adrenal hyperplasia 11beta-hydroxylase deficiency: two cases managed with bilateral adrenalectomy.

This series describes two patients with congenital adrenal hyperplasia due to 11beta-hydroxylase deficiency. The first patient, a ten-year-old with XX genotype, reared as a male, presented with resistant hypokalaemia and hypertension. The second patient, a 23-year-old with XY genotype, presented with bilateral adrenal masses and resistant hypertension. Both the patients were offered bilateral adrenalectomy. These two patients are described with a discussion on the role of bilateral adrenalectomy in the management of difficult cases of congenital adrenal hyperplasia. The association of myelolipoma and testicular rests with this condition is also discussed.

[18F]FETO: metabolic considerations.

PURPOSE: 11beta-Hydroxylase is a key enzyme in the biosynthesis of adrenocortical steroid hormones and is a suitable target for the imaging of the adrenal cortex. [(11)C]Metomidate (MTO), [(11)C]etomidate (ETO) and desethyl-[(18)F]fluoroethyl-etomidate (FETO) are potent inhibitors of this enzyme and are used for PET imaging of adrenocortical pathologies. The aims of this study were (1) to evaluate and compare the metabolic stability of MTO, ETO and FETO against esterases and (2) to investigate the metabolic pattern of FETO in vivo. METHODS: In vitro assays were performed using different concentrations of MTO, ETO and FETO with constant concentrations of carboxylesterase. Human in vivo studies were performed with human blood samples drawn from the cubital vein. After sample clean-up, the serum was analysed by HPLC methods. RESULTS: In vitro assays showed Michaelis-Menten constants of 115.1 mumol for FETO, 162.0 mumol for MTO and 168.6 mumol for ETO. Limiting velocities were 1.54 mumol/min (FETO), 1.47 mumol/min (MTO) and 1.35 mumol/min (ETO). This implies insignificantly decreased esterase stability of FETO compared with MTO and ETO. In vivo investigations showed a rapid metabolisation of FETO within the first 10 min (2 min: 91.41%+/-6.44%, n=6; 10 min: 23.78%+/-5.54%, n=4) followed by a smooth decrease in FETO from 20 to 90 min (20 min: 11.23%+/-3.79% n=4; 90 min: 3.68%+/-3.65%, n=4). Recovery rate was 61.43%+/-3.19% (n=12). CONCLUSION: In vitro experiments demonstrated that FETO stability against esterases is comparable to that of ETO and MTO. The metabolic profile showed that FETO kinetics in humans are fast.

Novel missense mutations, GCC [Ala306]- > GTC [Val] and ACG [Thr318]- > CCG [Pro], in the CYP11B1 gene cause steroid 11beta-hydroxylase deficiency in the Chinese.

OBJECTIVE: Steroid 11beta-hydroxylase (CYP11B1) deficiency, an autosomal recessive inherited disease, accounts for 5-8% of congenital adrenal hyperplasia (CAH). It is mainly caused by mutations of nucleotide substitutions in the coding region. PATIENTS AND METHODS: The study reports on a 9-year-old Chinese boy who presented with a bone age of 16 years, an enlarged penis, an accelerated growth rate since early childhood and hypertension (160-170/100-110 mmHg) for 3 years. Because it shares 95% sequence homology with aldosterone synthetase (CYP11B2), we developed gene-specific primers for differential PCR amplification of the CYP11B1 gene. The secondary PCR products of nine exons of the CYP11B1 gene were then subjected to single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. The serum hormone levels were also determined. RESULTS: We found that the boy diagnosed with CAH due to 11beta-hydroxylase deficiency carried mutations of A306V (GCC- > GTC) and T318P (ACG- > CCG) in two respective chromosomes. The hormone assay showed that the 11-deoxycortisol level was higher (667 nmol/l) than normal and was further increased after ACTH stimulation (1206 nmol/l). CONCLUSIONS: These two mutations have not previously been described in the CYP11B1 gene. The discovery of these two novel mutations increases our knowledge of CAH caused by 11beta-hydroxylase deficiency.