Urine steroid metabolomics as a diagnostic tool in primary aldosteronism

Primary aldosteronism (PA) causes 5-10% of hypertension cases, but only a minority of patients are currently diagnosed and treated because of a complex, stepwise, and partly invasive workup. We tested the performance of urine steroid metabolomics, the computational analysis of 24-hour urine steroid metabolome data by machine learning, for the identification and subtyping of PA. Mass spectrometry-based multi-steroid profiling was used to quantify the excretion of 34 steroid metabolites in 24-hour urine samples from 158 adults with PA (88 with unilateral PA [UPA] due to aldosterone-producing adenomas [APAs]; 70 with bilateral PA [BPA]) and 65 sex- and age-matched healthy controls. All APAs were resected and underwent targeted gene sequencing to detect somatic mutations associated with UPA. Patients with PA had increased urinary metabolite excretion of mineralocorticoids, glucocorticoids, and glucocorticoid precursors. Urine steroid metabolomics identified patients with PA with high accuracy, both when applied to all 34 or only the three most discriminative steroid metabolites (average areas under the receiver-operating characteristics curve [AUCs-ROC] 0.95-0.97). Whilst machine learning was suboptimal in differentiating UPA from BPA (average AUCs-ROC 0.65-0.73), it readily identified APA cases harbouring somatic KCNJ5 mutations (average AUCs-ROC 0.79-85). These patients showed a distinctly increased urine excretion of the hybrid steroid 18-hydroxycortisol and its metabolite 18-oxo-tetrahydrocortisol, the latter identified by machine learning as by far the most discriminative steroid. In conclusion, urine steroid metabolomics is a non-invasive candidate test for the accurate identification of PA cases and KCNJ5-mutated APAs

Urine steroid metabolomics as a diagnostic tool in primary aldosteronism

Primary aldosteronism (PA) causes 5-10% of hypertension cases, but only a minority of patients are currently diagnosed and treated because of a complex, stepwise, and partly invasive workup. We tested the performance of urine steroid metabolomics, the computational analysis of 24-hour urine steroid metabolome data by machine learning, for the identification and subtyping of PA. Mass spectrometry-based multi-steroid profiling was used to quantify the excretion of 34 steroid metabolites in 24-hour urine samples from 158 adults with PA (88 with unilateral PA [UPA] due to aldosterone-producing adenomas [APAs]; 70 with bilateral PA [BPA]) and 65 sex- and age-matched healthy controls. All APAs were resected and underwent targeted gene sequencing to detect somatic mutations associated with UPA. Patients with PA had increased urinary metabolite excretion of mineralocorticoids, glucocorticoids, and glucocorticoid precursors. Urine steroid metabolomics identified patients with PA with high accuracy, both when applied to all 34 or only the three most discriminative steroid metabolites (average areas under the receiver-operating characteristics curve [AUCs-ROC] 0.95-0.97). Whilst machine learning was suboptimal in differentiating UPA from BPA (average AUCs-ROC 0.65-0.73), it readily identified APA cases harbouring somatic KCNJ5 mutations (average AUCs-ROC 0.79-85). These patients showed a distinctly increased urine excretion of the hybrid steroid 18-hydroxycortisol and its metabolite 18-oxo-tetrahydrocortisol, the latter identified by machine learning as by far the most discriminative steroid. In conclusion, urine steroid metabolomics is a non-invasive candidate test for the accurate identification of PA cases and KCNJ5-mutated APAs.</p

Targeting CXCR4 (CXC Chemokine Receptor Type 4) for Molecular Imaging of Aldosterone-Producing Adenoma

Primary aldosteronism is the most frequent cause of secondary hypertension and is associated with increased morbidity and mortality compared with hypertensive controls. The central diagnostic challenge is the differentiation between bilateral and unilateral disease, which determines treatment options. Bilateral adrenal venous sampling, currently recommended for differential diagnosis, is an invasive procedure with several drawbacks, making it desirable to develop novel noninvasive diagnostic tools. When investigating the expression pattern of chemokine receptors by quantitative real-time polymerase chain reaction and immunohistochemistry, we observed high expression of CXCR4 (CXC chemokine receptor type 4) in aldosterone-producing tissue in normal adrenals, adjacent adrenal cortex from adrenocortical adenomas, and in aldosterone-producing adenomas (APA), correlating strongly with the expression of CYP11B2 (aldosterone synthase). In contrast, CXCR4 was not detected in the majority of nonfunctioning adenomas that are frequently found coincidently. The specific CXCR4 ligand 68Ga-pentixafor has recently been established as radiotracer for molecular imaging of CXCR4 expression and showed strong and specific binding to cryosections of APAs in our study. We further investigated 9 patients with primary aldosteronism because of APA by 68Ga-pentixafor-positron emission tomography. The tracer uptake was significantly higher on the side of increased adrenocortical aldosterone secretion in patients with APAs compared with patients investigated by 68Ga-pentixafor-positron emission tomography for other causes. Molecular imaging of aldosterone-producing tissue by a CXCR4-specific ligand may, therefore, be a highly promising tool for noninvasive characterization of patients with APAs

Steroid metabolome analysis reveals prevalent glucocorticoid excess in primary aldosteronism

BACKGROUND. Adrenal aldosterone excess is the most common cause of secondary hypertension and is associated with increased cardiovascular morbidity. However, adverse metabolic risk in primary aldosteronism extends beyond hypertension, with increased rates of insulin resistance, type 2 diabetes, and osteoporosis, which cannot be easily explained by aldosterone excess. METHODS. We performed mass spectrometry–based analysis of a 24-hour urine steroid metabolome in 174 newly diagnosed patients with primary aldosteronism (103 unilateral adenomas, 71 bilateral adrenal hyperplasias) in comparison to 162 healthy controls, 56 patients with endocrine inactive adrenal adenoma, 104 patients with mild subclinical, and 47 with clinically overt adrenal cortisol excess. We also analyzed the expression of cortisol-producing CYP11B1 and aldosterone-producing CYP11B2 enzymes in adenoma tissue from 57 patients with aldosterone-producing adenoma, employing immunohistochemistry with digital image analysis. RESULTS. Primary aldosteronism patients had significantly increased cortisol and total glucocorticoid metabolite excretion (all P < 0.001), only exceeded by glucocorticoid output in patients with clinically overt adrenal Cushing syndrome. Several surrogate parameters of metabolic risk correlated significantly with glucocorticoid but not mineralocorticoid output. Intratumoral CYP11B1 expression was significantly associated with the corresponding in vivo glucocorticoid excretion. Unilateral adrenalectomy resolved both mineralocorticoid and glucocorticoid excess. Postoperative evidence of adrenal insufficiency was found in 13 (29%) of 45 consecutively tested patients. CONCLUSION. Our data indicate that glucocorticoid cosecretion is frequently found in primary aldosteronism and contributes to associated metabolic risk. Mineralocorticoid receptor antagonist therapy alone may not be sufficient to counteract adverse metabolic risk in medically treated patients with primary aldosteronism

Etude de propriétés antioxydantes et cytoprotectrices du resvératrol et d'analogues structuraux

La production inappropriée d espèces radicalaires et/ou la diminution des défenses anti-oxydantes sont à l origine du stress oxydant, impliqué dans la physiopathologie de maladies chroniques telles que le diabète sucré et les maladies cardiovasculaires. Le resvératrol (trans-3,5,4'-trihydroxystilbène) est un antioxydant d origine naturelle, possédant des propriétés anti-tumorale, anti-aggrégant plaquettaire, anti-inflammatoire et neuroprotectrice. L objectif de notre recherche a été d évaluer les propriétés antioxydantes et cytoprotectrices d analogues structuraux du resvératrol, le resvératrol étant considéré comme molécule de référence. Dans un premier temps, nous avons étudié les propriétés physico-chimiques du resvératrol qui existe sous deux formes stéréoisomériques trans- et cis-. Le trans-resvératrol dissous en milieu aqueux par sonication puis exposé à la lumière solaire, se transforme en isomère cis-. Nous avons participé à la mise au point d une technique de détermination du rapport trans-/cis- resvératrol par une méthode spectrophotométrique simple. Dans un second temps, nous avons évalué les propriétés anti-oxydantes in vitro de trois dérivés du stilbène, analogues structuraux du resvératrol : le PDM2 (1,3-dichloro-5-[(1E)-2-(4-chlorophényl) éthényl]-benzène), le PDM11 ((E)-5-[2-(4-chlorophényl) éthényl]-1,3-dimethoxyphényl-éthène) et le picéatannol (trans-3,5,3 ,4 -tetrahydroxystilbène). Des micelles d acide linoléique (10-2 M) renfermant les molécules à tester ont été soumises à l action de radicaux hydroxyles générés par radiolyse gamma de l eau en milieu aéré. Les marqueurs de peroxydation lipidique étudiés après irradiation des micelles ont été les diènes conjugués (mesure de l absorbance à 234 nm) et les hydroperoxydes (CLHP en phase inverse avec détection par chimiluminescence).Nos résultats montrent une activité antioxydante in vitro croissante en fonction de la concentration de picéatannol et de resvératrol (10-5 à 10-4 M). Le picéatannol présente, à concentrations égales, un pouvoir protecteur vis-à-vis de la peroxydation lipidique supérieur à celui du resvératrol, probablement en relation avec la présence d un groupement hydroxyle supplémentaire comparativement au resvératrol. Le schéma réactionnel proposé pour le mécanisme de protection antioxydante du picéatannol et du resvératrol serait une compétition avec l acide gras vis-à-vis de la capture des radicaux libres peroxyles LOO de l acide gras, la constante de vitesse de réaction des radicaux LOO étant supérieure dans le cas du picéatannol à celle du resvératrol : k (LOO + picéatannol) >= 1,4x105 M-1.s-1 et k (LOO + resvératrol) >= 0,3x105 M-1.s-1. Aucune protection significative des micelles vis-àvis de l attaque radicalaire n a été observée avec les dérivés PDM2 et PDM11, et l évaluation des propriétés antioxydantes de ces deux molécules a donc été interrompue. Dans un troisième temps et à la lumière des résultats obtenus en milieu acellulaire, nos travaux ont été orientés vers l évaluation biologique de l effet cytoprotecteur de l apport de groupements hydroxyles sur le noyau de base stilbène constitutif du resvératrol, en milieu cellulaire aortique. Les dérivés tétra- (picéatannol) et hexa-hydroxylés du resvératrol ont été synthétisés (collaboration Dr Jaeger, Vienne, Autriche). Les capacités cytoprotectrices de ces molécules, comparées à celles du resvératrol, ont été évaluées sur le modèle cellulaire des cellules endothéliales aortiques bovines (BAEC). Ces cellules ont été soumises à un stress oxydant chronique (glucose à forte concentration 25 mM) après un prétraitement (ou un co-traitement) par les molécules d intérêt (1 à 50 M). Un effet anti-apoptotique, lié à une réduction de la production radicalaire intracellulaire, a été recherché par la mesure des activités de la caspase-3 et de la Poly (ADP ribose) Polymérase (PARP). La modulation de l effet cytoprotecteur des dérivés tétra- (picéatannol) et hexa-hydroxylés du resvératrol est apparue partielle et non corrélée à l enrichissement de la molécule de référence en groupements hydroxyles. L activité antioxydante in vitro ne paraît pas correspondre à un effet cytoprotecteur dans les conditions de stress oxydant étudiées.Oxidative stress is implicated in chronic diseases such as diabetes mellitus and cardiovascular disease. Resveratrol (trans-3,5,4'-trihydroxystilbene) is a natural antioxidant displaying anti-tumor, anti-platelet aggregation, anti-inflammatory and neuroprotective effects. Our work aimed to study the antioxidative and cytoprotective properties of resveratrol s structural analogues. We participated in the development of a simple spectrophotometrical technique to determine the trans-/cis- resveratrol ratio. We also studied the in vitro protective effects of three derivatives of resveratrol, i.e., piceatannol(trans-3,5,3_,4_-tetrahydroxystilbene), PDM2 (1,3-dichloro-5-[(1E)-2-(4-chlorophenyl)ethenyl]-benzene) and PDM11 ((E)-5-[2-(4-chlorophenyl)ethenyl]-1,3-dimethoxyphenyl-ethene), compared with resveratrol as reference compound, against oxidation of linoleate micelles (10-2 M) initiated by radiolysisgenerated hydroxyl radicals. Lipid peroxidation was monitored by conjugated dienes (differential absorbance at 234 nm), and by hydroperoxides (reverse phase HPLC with chemiluminescence detection). The higher the concentration of resveratrol or piceatannol (from 10-5M to 10-4 M), the stronger the antioxidant ability. Piceatannol, with the presence of an additional hydroxyl group, showed a better antioxidant effect than resveratrol for a given concentration (competition with the fatty acid to scavenge lipid peroxyl radicals LOO ), whereas PDM2 and PDM11, without any hydroxyl group, did not exhibit any significant protective effect. A lower limit for the LOO rate constant has been estimated for piceatannol (>= 1.4.105M-1 s-1) and for resveratrol (>= 0.3.105 M-1 s-1). The cytoprotective effect of two hydroxylated analogues, piceatannol and hexahydroxystilbene (collaboration with Dr. Jaeger, Vienna, Austria) compared with resveratrol, was evaluated by measuring the activities of caspase-3 and Poly (ADP ribose) polymerase (PARP) in bovine aortic endothelial cells (BAEC) undergoing high glucose (25 mM) stress conditions and pre-treated or co-treated with the studied analogues (1-10-50 M). Only a partial cytoprotective effect of these molecules seemed to be observed under our experimental conditionsPARIS-BIUP (751062107) / SudocSudocFranceF

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 (11\u3b2-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 ( 6550%) zona fasciculata-like cell pattern: one tumor had CYP11B1 H-score\u2009=\u2009150, 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\u2009=\u200940, 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\u2009=\u200915, CYP11B2 H-score\u2009=\u2009180, 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

Histological characterization of aldosterone-producing adrenocortical adenomas with different somatic mutations

CONTEXT Aldosterone-producing adrenocortical adenomas (APAs) are mainly composed of clear (lipid rich) and compact (eosinophilic) tumor cells. The detailed association between these histological features and somatic mutations (KCNJ5, ATP1A1, ATP2B3 and CACNA1D) in APAs is unknown. OBJECTIVE To examine the association between histological features and individual genotypes in APAs. METHODS Examination of 39 APAs subjected to targeted next-generation sequencing (11 KCNJ5, 10 ATP1A1, 10 ATP2B3, and 8 CACNA1D) and quantitative morphological and immunohistochemical (CYP11B2 and CYP17A1) analyses using digital imaging software. RESULTS KCNJ5- and ATP2B3-mutated APAs had clear cell dominant features [KCNJ5: clear 59.8% (54.4%-64.6 %) vs. compact 40.2% (35.4%-45.6 %), P=0.0022; ATP2B3: clear 54.3% (48.2%-62.4 %) vs. compact 45.7% (37.6%-51.8 %), P=0.0696]. ATP1A1- and CACNA1D-mutated APAs presented with marked intratumoral heterogeneity. A significantly positive correlation of immunoreactivity was detected between CYP11B2 and CYP17A1 in tumor cells of KCNJ5-mutated APAs (P=0.0112; ρ=0.7237) , in contrast, significantly inverse correlation was detected in ATP1A1-mutated APAs (P=0.0025; ρ=-0.8667). CONCLUSION KCNJ5-mutated APAs, co-expressing CYP11B2 and CYP17A1, were more deviated in terms of zonation-specific differentiation of adrenocortical cells compared with ATP1A1- and ATP2B3- mutated APAs

Genotype-Specific Steroid Profiles Associated With Aldosterone-Producing Adenomas

Primary aldosteronism comprises 2 main subtypes: unilateral aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia. Somatic KCNJ5 mutations are found in APA at a prevalence of around 40% that drive and sustain aldosterone excess. Somatic APA mutations have been described in other genes (CACNA1D, ATP1A1, and ATP2B3) albeit at a lower frequency. Our objective was to identify genotype-specific steroid profiles in adrenal venous (AV) and peripheral venous (PV) plasma in patients with APAs. We measured the concentrations of 15 steroids in AV and PV plasma samples by liquid chromatography-tandem mass spectrometry from 79 patients with confirmed unilateral primary aldosteronism. AV sampling lateralization ratios of steroids normalized either to cortisol or to DHEA+androstenedione were also calculated. The hybrid steroid 18-oxocortisol exhibited 18- and 16-fold higher concentrations in lateralized AV and PV plasma, respectively, from APA with KCNJ5 mutations compared with all other APA combined together (P<0.001). Lateralization ratios for the KCNJ5 group were also generally higher. Strikingly, we demonstrate that a distinct steroid signature can differentiate APA genotype in AV and PV plasma. Notably, a 7-steroid fingerprint in PV plasma correctly classified 92% of the APA according to genotype. Prospective studies are necessary to translate these findings into clinical practice and determine if steroid fingerprinting could be of value to select patients with primary aldosteronism who are particularly suitable candidates for adrenal venous sampling because of a high probability of having an APA

PRKACA Somatic Mutations Are Rare Findings in Aldosterone-Producing Adenomas

CONTEXT Somatic mutations have been found causative for endocrine autonomy in aldosterone-producing adenomas (APAs). Whereas mutations of PRKACA (catalytic subunit of protein kinase A) have been identified in cortisol-producing adenomas, the presence of PRKACA variants in APAs is unknown, especially in those that display cosecretion of cortisol. OBJECTIVE The objective of the study was to investigate PRKACA somatic variants identified in APA cases. DESIGN Identification of PRKACA somatic variants in APAs by whole-exome sequencing followed by in vitro analysis of the enzymatic activity of PRKACA variants and functional characterization by double immunofluorescence of CYP11B2 and CYP11B1 expression in the corresponding tumor tissues. SETTING AND PATIENTS APA tissues were collected from 122 patients who underwent unilateral adrenalectomy for primary aldosteronism between 2005 and 2015 at a single institution. RESULTS PRKACA somatic mutations were identified in two APA cases (1.6%). One APA carried a newly identified p.His88Asp variant, whereas in a second case, a p.Leu206Arg mutation was found, previously described only in cortisol-producing adenomas with overt Cushing's syndrome. Functional analysis showed that the p.His88Asp variant was not associated with gain of function. Although CYP11B2 was strongly expressed in the p.His88Asp-mutated APA, the p.Leu206Arg carrying APA predominantly expressed CYP11B1. Accordingly, biochemical Cushing's syndrome was present only in the patient with the p.Leu206Arg mutation. After adrenalectomy, both patients improved with a reduced number of antihypertensive medications and normalized serum potassium levels. CONCLUSIONS We describe for the first time PRKACA mutations as rare findings associated with unilateral primary aldosteronism. As cortisol cosecretion occurs in a subgroup of APAs, other molecular mechanisms are likely to exist