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Objective To explore the characteristics of steroid hormone spectrum in patients with primary aldoste- ronism (PA) and to evaluate the diagnostic efficacy of 18-oxocortisol and 18-hydroxycortisol. Methods Fifty-nine PA patients including 41 cases of aldosterone-producing adenoma (APA) and 18 cases of idiopathic hyperaldosteronism (IHA)and 42 essential hypertension (EH) patients as control group diagnosed in Department of Endocrinology of Peking Union Medical College Hospital from November 2020 to November 2021 were enrolled in this study. The basic information and clinical characteristics of patients were collected and 8 kinds of steroid hormones,including mineral corticoids (pregnenolone,progesterone, 11-deoxycorticosterone, corticosterone, 18-OH-corticoster-one) and aldosterone, and metabolites 18-oxocortisol as well as 18-hydroxycortisol were detected by liquid chromatography-tandem mass spectrometry(LC-MS/MS).The differences of steroid hormone level from different groups were compared and their diagnostic efficacy was evaluated. Results The average level of progesterone in APA group was higher than that in EH group (P＜0.05). The average level of 11-deoxycorticosterone in APA and IHA group was higher than that in EH group (both P＜0.001). The average level of 18-OH-corticosterone in APA group was higher than that in EH group (P＜0.001). The average level of aldosterone in APA and IHA group were higher than that in EH group(P＜0.01). The average level of 18-oxocortisol in APA group was higher than that in IHA group and higher than that in EH group (all P＜0.05). The average level of 18-hydroxycortisol in APA group was higher than that in EH group (P＜0.001). As for the diagnostic efficiency of 18-oxocortisol and 18-hydroxycortisol differentiating PA from EH, when the cut-off value of 18-oxocortisol was 0.06 ng/mL, the sensitivity reached 74.6% with specificity as 88.1%; when the cut-off value of 18-hydroxycortisol was 1.44 ng/mL, the sensitivity was 71.2% and the specificity was 69.0%. As for the diagnostic efficiency of 18-oxocortisol and 18-hydroxycortisol recognizing APA, when the cut-off value of 18-oxocortisol was 0.11 ng/mL, the sensitivity was 73.2% and the specificity was 95.2%; when the cut-off value of 18-hydroxycortisol was 1.77 ng/mL, the sensitivity was 68.3% and the specificity was 78.6%. Conclusions The level of various steroid hormone precursors and metabolites in PA patients is significantly higher than those in EH patients. The detection of steroid hormone profiling by LC-MS/MS plays an important role in the diagnosis and typing of PA. In particular, 18-oxocortisol and 18-hydroxycortisol show unique advantages in the recognition of APA.

Objective To explore the influence of calcium channel blocker(CCB), verapamil, on steroid hormone profile of primary cultured aldosterone producing adenoma(APA)cells. Methods The adrenal tumor tissues of 6 APA patients with hypokalemia operated in Peking Union Medical College Hospital were cultured and treated with different concentrations of verapamil. The steroid hormone profile was detected by liquid chromatography-tandem mass spectrometry(LC-MS/MS). Results The level of aldosterone in 50 μmol/L verapamil group was lower than that in 10 μmol/L verapamil group and control group(P＜0.01). The levels of aldosterone precursor, 11-deoxycorticosterone in verapamil 10 μmol/L group was lower than that in control group (P＜0.05). The levels of 18-oxocortisol(18-OXOF) and 18-hydroxycortisol(18-OHF) in verapamil 50 μmol/L group and verapamil 10 μmol/L group were lower than that in the control group (P＜0.05). In addition, verapamil also had some inhibitory effects on glucocorticoids such as cortisol and cortisone, and adrenal derived sex hormones such as dehydroepiandrosterone sulfate(DHEA-S) and 11 beta-hydroxytestosterone(11β-OHT). Conclusions In addition to inhibit-ing aldosterone secretion in primary cultured APA cells, CCB has significant inhibitory effects on a variety of other steroid hormones, such as precursor 11-deoxycorticosterone, cortisol, metabolites, 18-OXOF and 18-OHF.

The search for mineralocorticoids to explain some cases of low renin hypertension with suppressed aldosterone levels led to the isolation of the abundant steroid 18-hydroxycortisol in human urine. 18-Hydroxycortisol proved to be inactive, but because of its similarity to precursors for the synthesis of aldosterone, bullfrog adrenals were incubated with cortisol, resulting in the discovery of 18-oxocortisol which is structurally similar to aldosterone, but with a 17α-hydroxy group like cortisol. 18-Oxocortisol is a weak mineralocorticoid. Its synthesis occurs primarily in the zona glomerulosa where co-expression of the CYP11B2 (aldosterone synthase) and the CYP17A1 (17α-hydroxylase) occurs in a variable number of cells. The clinical value of the measurement of 18-oxocortisol is that it serves to distinguish subtypes of primary aldosteronism. It is significantly elevated in patients with aldosterone-producing adenomas in comparison to those with idiopathic bilateral hyperaldosteronism and helps predict the type of somatic mutation in the aldosterone-producing adenomas, as it is higher in those with KCNJ5 mutations compared to other gene mutations. • 18-hydroxycortisol and 18-oxocortisol are produced in the zona glomerulosa of the adrenal. • 18-Oxocortisol is both a weak mineralocorticoid and glucocorticoid; 18-hydroxycortisol is inactive. • 18-Oxocortisol measurements differentiate aldosterone-producing adenomas due to KCNJ5 mutations from bilateral idiopathic hyperaldosteronism. [ABSTRACT FROM AUTHOR]

Background: Eighteen-hydroxycortisol (18-OHF) is a potential biomarker for differential diagnosis of the two major primary aldosteronism subtypes, aldosterone-producing adenoma, and idiopathic hyperaldosteronism., Methods: Urine samples were processed, and the 18-OHF in urine samples were successfully quantified by in-house established dilute-and-shoot liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Separation was accomplished on a Sigma Ascentis Express C18 column with a gradient mixture of phase (A) 0.2% formic acid in water and phase (B) 0.2% formic acid in methanol at a flow rate of 0.4 ml/min. Mass spectrometric detection was performed in positive electrospray ionization mode via a mass spectrometer., Results: The linearity of urinary 18-OHF ranged from 4.28 to 8.77 × 10 3  nmol/L, with a lower limit of quantification at 4.28 nmol/L. The intra- and inter-precision were both below 3%. The range of analytical recovery was 97.8%-109.2%. The validated dilute-and-shoot LC-MS/MS method was compared with the SPE LC-MS/MS method modified from the one reported in 2013. The results by Passing-Bablok regression analysis and Bland-Altman plotting demonstrated a good agreement between the two methods. The presented method was then applied to establish sex-specific reference intervals from 62 males and 62 females, respectively. The calculated 2.5%-97.5% reference intervals for 24-h urinary 18-OHF were 113-703 nmol/day for males and 71.2-450 nmol/day for females., Conclusion: The presented dilute-and-shoot LC-MS/MS method for 18-OHF quantification showed a good performance in the clinical application. Furthermore, the sex-specific reference intervals for 24-h urinary 18-OHF were first established and quite important for its application in primary aldosteronism subtyping., (© 2022 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Objective: Glucocorticoid-remediable aldosteronism (GRA) is caused by the presence of a chimeric gene originating from an unequal cross-over between the CYP11B1 and CYP11B2 genes. Aldosterone suppression by dexamethasone and high 18-hydroxycortisol (18-OHF) levels have been used to differentiate GRA from the other forms of primary aldosteronism. Methods: A dexamethasone suppression test including serum 18-OHF determination and the measurement of urinary excretion of aldosterone, its metabolites and 18-OHF were performed in 3 children of a family with primary aldosteronism. Polymerase chain reactions were performed to identify the chimeric gene. Results: The chimeric gene was identified in 2 children, their mother and grandmother. The affected children had an aldosterone-to-plasma renin activity ratio >30, elevated serum 18-OHF concentration and increased urinary excretion of aldosterone, its metabolites, and 18-OHF. Post-dexamethasone concentrations of serum aldosterone and 18-OHF concentrations were suppressed. Conclusion: Although very rare, the possible diagnosis of GRA should be considered in all children or young adults with low-renin hypertension. Since genetic testing is more specific than biochemical testing, a definitive diagnosis can only be obtained by identification of the CYP11B1/CYP11B2 chimeric gene. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

• Plasma 18-OHB, 18-OHF and 18-OXOF are able to be quantified simultaneously based on our newly established LC-MS/MS based assay. • The LC-MS/MS assay is able to quantify the 3 hormones within 3minutes per sample. • Nearly 700 apparently healthy people were recruited to establish the reference intervals. 18-hydroxycorticosterone (18-OHB), 18-hydroxycortisol (18-OHF) and 18-oxocortisol (18-OXOF) are important biomarkers for the diagnosis of subtypes of primary aldosteronism. The detection of these three analytes by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is free from structurally similar compounds. The aim of this study was to develop and validate a new LC-MS/MS assay for the simultaneous quantification of 18-OHB, 18-OHF and 18-OXOF in plasma and to establish a reference intervals for apparently healthy population. Plasma samples were prepared by solid phase extraction and separated in an ultra-high performance reversed phase column. MS detection was achieved using a triple quadrupole mass spectrometer in both positive and negative ionization modes. The developed assay was then validated against standard guidelines. We collected 691 plasma samples from apparently healthy individuals (M:398, F:293) to establish the reference intervals. The analytes were separated and quantified within 5 min. The newly developed method demonstrated linearity for the detected steroid concentration in range of 5 to 3000 pg/ml for 18-OXOF (r2 = 0.999) and 20 to 3000 pg/ml for 18-OHB (r2 = 0.997) and 18-OHF (r2 = 0.997). The lower limit of quantification (LLOQ) was 2.5 pg/ml, 20 pg/ml and 20 pg/m for 18-OXOF, 18-OHB and 18-OHF respectively. Specificity, precision, accuracy and stability were tested, and met the requirements of the guidelines. 18-OHB was higher in females than in males, but 18-OHF were higher in males than females. The reference intervals of 18-OHB, 18-OHF and 18-OXOF for both genders together were 90.5–1040.6 pg/ml, 224.4–1685.2 pg/ml, 4.0–70.5 pg/ml, respectively. Age was also an important factor influencing the levels of these three hormones. We have developed a sensitive and reliable method for the simultaneous quantification of 18-OHB, 18-OHF, and 18-OXOF. Our work provides a reference interval for the clinical application of these three steroid hormones. [ABSTRACT FROM AUTHOR]

Background: In patients with glucocorticoid remediable aldosteronism (GRA), a rare hypertensive disorder caused by the presence of a chimeric aldosterone synthase (CYP11B2) and 11β-hydroxylase (CYP11B1) gene, high level of urinary 18-hydroxycortisol (18OHF) excretion are observed. In some patients with hypertension, increased urinary 18OHF secretion is also found in the absence of the hybrid CYP11B1/CYP11B2 gene. We hypothesised that gene variants of CYP11B1 or CYP11B2 may be linked to this abnormal glucocorticoid production. Methods: The urinary steroid profile was analysed by gas chromatography/mass spectrometry in 429 hypertensive patients and 98 (23%) thereof tested positive for increased 18OHF excretion. After correction for total cortisol excretion, 12 subjects showed an abnormally high 18OHF excretion. For genotyping DNA was obtained from six of these patients. All were tested negative for the hybrid CYP11B1/CYP11B2 gene and were further analysed for mutations in all exons and promoter regions of both CYP11B1 and CYP11B2 by single strand conformation polymorphism (SSCP) and sequencing when appropriate. Results: The genetic analysis of the two genes revealed the presence of nine molecular variants in CYP11B2 and three in CYP11B1. In addition to published polymorphic sites, we identified two new variants in CYP11B2 but no new variants in CYP11B1. The newly identified CYP11B2 mutations are a C/T single nucleotide exchange located in the first intron and a double nucleotide exchange at the 3′-splice site of exon 8. The mutated sequence corresponds to the sequence of CYP11B1 indicating a gene conversion. This suggests that the mutant is not likely to affect splicing. Thus, none of the genetic variants identified explains the high urinary excretion of 18OHF. Conclusions: We present here a complete method for the genetic analysis of the CYP11B1 and CYP11B2 genes. By this method we could not identify genetic variants responsible for a GRA-like phenotype. The presence of high levels of 18OHF should not be used alone as a diagnosis tool for GRA. [Copyright &y& Elsevier]

Patients with primary aldosteronism and with glucocorticoid-suppressible aldosteronism excrete in the urine excessive amounts of the hybrid steroids 18-hydroxycortisol and 18-oxocortisol. The measurement of these steroids aids in the differential diagnosis of various adrenal disorders. We have produced mouse monoclonal antibodies against 18-oxocortisol and polyclonal antibodies against 18-hydroxycortisol and describe a time-resolved fluoroimmunoassay (TR-FIA) technique for the measurement of these steroids in the urine. We have also compared this assay with an ELISA technique for these compounds. We also describe the preparation of in-house Eu(III)-labeled avidin and an enhancement solution and compared to a commercially available Eu(III)-labeled streptavidin and enhancement solutions. The monoclonal antibodies against 18-oxocortisol are sensitive and have a high level of specificity. The TR-FIA technique using in-house prepared reagents or commercial ones were indistinguishable from each other, but at a significant saving. The TR-FIA technique was more sensitive and had a greater precision than the ELISA technique for both steroids. [Copyright &y& Elsevier]

Primary aldosteronism (PA) is characterized by autonomous aldosterone production by renin-independent mechanisms and is most commonly sporadic. While 60-70% of sporadic PA can be attributed to bilateral hyperaldosteronism, the remaining 30-40% is caused by a unilateral aldosterone-producing adenoma (APA). Somatic mutations in or near the selectivity filter the KCNJ5 gene (encoding the potassium channel GIRK4) have been implicated in the pathogenesis of both sporadic and familial PA. Several studies using tumor tissue, peripheral and adrenal vein samples from PA patients have demonstrated that along with aldosterone, the hybrid steroids 18-hydroxycortisol (18OHF) and 18-oxocortisol (18oxoF) are a hallmark of APA harboring KCNJ5 mutations. Herein, we review the recent advances with respect to the molecular mechanisms underlying the pathogenesis of PA and the steroidogenic fingerprints of KCNJ5 mutations. In addition, we present an outlook toward the future of PA subtyping and diagnostic work-up utilizing steroid profiling., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare., (2020 Gland Surgery. All rights reserved.)

Primary aldosteronism (PA) is the most common form of secondary arterial hypertension. In patients with PA, more so than in the general population, there is a prevalence of insulin resistance, diabetes mellitus, metabolic syndrome, osteoporosis, and symptoms of depression; these conditions are more likely to manifest a gluco- rather than mineralocorticoid excess. This fact is of particular importance in light of recent studies that have shown that PA is often associated with glucocorticoid excess. Since the first reports of cases of combined secretion of aldosterone and cortisol in 1979, the number of cases of so-called Connshing syndrome has increased. An analysis of data from recent studies suggests that hypercortisolism in PA is closely associated with an increased risk of cardiovascular complications, metabolic disorders and post-surgical adrenal insufficiency. The most important diagnostic problem in adenomas with combined secretion is the risk of false interpretation of the results of adrenal venous sampling (AVS). The indications that suggest aldosterone-and-cortisol-co-producing adenoma are the lack of suppression of cortisol levels following a night test with 1mg of dexamethasone, and an adrenal tumo of over 2.5cm. As an alternative test capable of differentiating this type of tumor, a number of researchers have proposed measuring the level of so-called hybrid steroids in the peripheral plasma and urine. Taking into account the high prevalence and potential risks, ruling out of excess corisol secretion is obligatory in all cases of PA before AVS and when planning surgery.