Pheochromocytomas and paragangliomas (PPGLs) are potentially lethal yet usually surgically curable causes of endocrine hypertension; therefore, once clinical suspicion is aroused it is imperative ...that clinicians choose the most appropriate laboratory tests to identify the tumors.
Compelling evidence now indicates that initial screening for PPGLs should include measurements of plasma free metanephrines or urine fractionated metanephrines. LC-MS/MS offers numerous advantages over other analytical methods and is the method of choice when measurements include methoxytyramine, the O-methylated metabolite of dopamine. The plasma test offers advantages over the urine test, although it is rarely implemented correctly, rendering the urine test preferable for mainstream use. To ensure optimum diagnostic sensitivity for the plasma test, reference intervals must be established for blood samples collected after 30 min of supine rest and after an overnight fast when measurements include methoxytyramine. Similarly collected blood samples during screening, together with use of age-adjusted reference intervals, further minimize false-positive results. Extents and patterns of increases in plasma normetanephrine, metanephrine, and methoxytyramine can additionally help predict size and adrenal vs extraadrenal locations of tumors, as well as presence of metastases and underlying germline mutations of tumor susceptibility genes.
Carried out correctly at specialist endocrine centers, collection of blood for measurements of plasma normetanephrine, metanephrine, and methoxytyramine not only provides high accuracy for diagnosis of PPGLs, but can also guide clinical decision-making about follow-up imaging strategies, genetic testing, and therapeutic options. At other centers, measurements of urine fractionated metanephrines will identify most PPGLs.
Measurements of plasma or urinary metanephrines are recommended for diagnosis of pheochromocytoma and paraganglioma (PPGL). What test offers optimal diagnostic accuracy for patients at high and low ...risk of disease, whether urinary free metanephrines offer advantages over deconjugated metanephrines, and what advantages are offered by including methoxytyramine in panels all remain unclear.
A population of 2056 patients with suspected PPGLs underwent prospective screening for disease using mass spectrometric-based measurements of plasma free, urinary deconjugated, and urinary free metanephrines and methoxytyramine. PPGLs were confirmed in 236 patients and were excluded in others on follow-up evaluation.
Measurements of plasma free metabolites offered higher (
< 0.01) diagnostic sensitivity (97.9%) than urinary free (93.4%) and deconjugated (92.9%) metabolites at identical specificities for plasma and urinary free metabolites (94.2%) but at a lower (
< 0.005) specificity for deconjugated metabolites (92.1%). The addition of methoxytyramine offered little value for urinary panels but provided higher (
< 0.005) diagnostic performance for plasma measurements than either urinary panel according to areas under ROC curves (0.991 vs 0.972 and 0.964). Diagnostic performance of urinary and plasma tests was similar for patients at low risk of disease, whereas plasma measurements were superior to both urinary panels for high-risk patients.
Diagnosis of PPGLs using plasma or urinary free metabolites provides advantages of fewer false-positive results compared with commonly measured deconjugated metabolites. The plasma panel offers better diagnostic performance than either urinary panel for patients at high risk of disease and, with appropriate preanalytics, provides the test of choice. Measurements of methoxytyramine in urine show limited diagnostic utility compared with plasma.
Catecholamine-producing tumors of childhood include most notably neuroblastoma, but also pheochromocytoma and paraganglioma (PPGL). Diagnosis of the former depends largely on biopsy-dependent ...histopathology, but this is contraindicated in PPGL where diagnosis depends crucially on biochemical tests of catecholamine excess. Such tests retain some importance in neuroblastoma though continue to largely rely on measurements of homovanillic acid (HVA) and vanillylmandelic acid (VMA), which are no longer recommended for PPGL. For PPGL, urinary or plasma metanephrines are the recommended most accurate tests. Addition of methoxytyramine to the plasma panel is particularly useful to identify dopamine-producing tumors and combined with normetanephrine also shows superior diagnostic performance over HVA and VMA for neuroblastoma. While use of metanephrines and methoxytyramine for diagnosis of PPGL in adults is established, there are numerous pitfalls for use of these tests in children. The establishment of pediatric reference intervals is particularly difficult and complicated by dynamic changes in metabolites during childhood, especially in infants for both plasma and urinary measurements, and extending to adolescence for urinary measurements. Interpretation of test results is further complicated in children by difficulties in following recommended preanalytical precautions. Due to this, the slow growing nature of PPGL and neglected consideration of the tumors in childhood the true pediatric prevalence of PPGL is likely underappreciated. Earlier identification of disease, as facilitated by surveillance programs, may uncover the true prevalence and improve therapeutic outcomes of childhood PPGL. For neuroblastoma there remain considerable obstacles in moving from entrenched to more accurate tests of catecholamine excess.
Mass spectrometric-based measurements of the steroid metabolome have been introduced to diagnose disorders featuring abnormal steroidogenesis. Defined reference intervals are important for ...interpreting such data.
Liquid chromatography–tandem mass spectrometry was used to establish reference intervals for 16 steroids (pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone, aldosterone, 18-oxocortisol, 18-hydroxycortisol, 17-hydroxyprogesterone, 21-deoxycortisol, 11-deoxycortisol, cortisol, cortisone, dehydroepiandrosterone, dehydroepiandrosterone-sulfate, androstenedione, testosterone) measured in plasma from 525 volunteers with (n=227) and without (n=298) hypertension, including 68 women on oral contraceptives.
Women showed variable plasma concentrations of several steroids associated with menstrual cycle phase, menopause and oral contraceptive use. Progesterone was higher in females than males, but most other steroids were higher in males than females and almost all declined with advancing age. Using models that corrected for age and gender, body mass index showed weak negative relationships with corticosterone, 21-deoxycortisol, cortisol, cortisone, testosterone, progesterone, 17-hydroxyprogesterone and 11-deoxycorticosterone, but a positive relationship with 18-hydroxycortisol. Hypertensives and normotensives showed negligible differences in plasma concentrations of steroids.
Age and gender are the most important variables for plasma steroid reference intervals, which have been established here according to those variables for a panel of 16 steroids primarily useful for diagnosis and subtyping of patients with endocrine hypertension.
•Reference intervals established for LC-MS/MS-measurements of 16 plasma steroids•Reference population consisted of 525 normotensive and hypertensive volunteers.•Age and gender were the most important variables to consider for reference intervals.•Weak negative associations of several steroids with body mass index•Negligible associations of plasma steroids with blood pressure status
Abstract
Context:
Pheochromocytomas and paragangliomas (PPGLs) in children are often hereditary and may present with different characteristics compared with adults. Hereditary PPGLs can be separated ...into cluster 1 and cluster 2 tumors due to mutations impacting hypoxia and kinase receptor signaling pathways, respectively.
Objective:
To identify differences in presentation of PPGLs between children and adults.
Design:
A retrospective cross-sectional clinical study.
Setting:
Seven tertiary medical centers.
Patients:
The study included 748 patients with PPGLs, including 95 with a first presentation during childhood. Genetic testing was available in 611 patients. Other data included locations of primary tumors, presence of recurrent or metastatic disease, and plasma concentrations of metanephrines and 3-methoxytyramine.
Results:
Children showed higher (P < 0.0001) prevalence than adults of hereditary (80.4% vs 52.6%), extra-adrenal (66.3% vs 35.1%), multifocal (32.6% vs 13.5%), metastatic (49.5% vs 29.1%), and recurrent (29.5% vs 14.2%) PPGLs. Tumors due to cluster 1 mutations were more prevalent among children than adults (76.1% vs 39.3%; P < 0.0001), and this paralleled a higher prevalence of noradrenergic tumors, characterized by relative lack of increased plasma metanephrine, in children than in adults (93.2% vs 57.3%; P < 0.0001).
Conclusions:
The higher prevalence of hereditary, extra-adrenal, multifocal, and metastatic PPGLs in children than adults represents interrelated features that, in part, reflect the lower age of disease presentation of noradrenergic cluster 1 than adrenergic cluster 2 tumors. The differences in disease presentation are important to consider in children at risk for PPGLs due to a known mutation or previous history of tumor.
This study establishes the link between extraadrenal, multifocal, metastatic, reccurent, hereditary PPGLs to a higher prevalence of noradrenergic and cluster 1 tumors in pediatric than adults.
Diagnosis of primary aldosteronism (PA) for many patients depends on positive results for the saline infusion test (SIT). Plasma aldosterone is often measured by immunoassays, which can return ...inaccurate results.
This study aimed to establish whether differences in aldosterone measurements by immunoassay versus mass spectrometry (MS) might impact confirmatory testing for PA.
This study, involving 240 patients tested using the SIT at 5 tertiary care centers, assessed discordance between immunoassay and MS-based measurements of plasma aldosterone.
Plasma aldosterone measured by Liaison and iSYS immunoassays were respectively 86% and 58% higher than determined by MS. With an immunoassay-based SIT cutoff for aldosterone of 170 pmol/L, 78 and 162 patients had, respectivel, negative and positive results. All former patients had MS-based measurements of aldosterone < 117 pmol/L, below MS-based cutoffs of 162 pmol/L. Among the 162 patients with pathogenic SIT results, MS returned nonpathologic results in 62, including 32 under 117 pmol/L. Repeat measurements by an independent MS method confirmed nonpathogenic results in 53 patients with discordant results. Patients with discordant results showed a higher (P < 0.0001) prevalence of nonlateralized than lateralized adrenal aldosterone production than patients with concordant results (83% vs 28%). Among patients with nonlateralized aldosterone production, 66% had discordant results. Discordance was more prevalent for the Liaison than iSYS immunoassay (32% vs 16%; P = 0.0065) and was eliminated by plasma purification to remove interferents.
These findings raise concerns about the validity of immunoassay-based diagnosis of PA in over 60% of patients with presumed bilateral disease. We provide a simple solution to minimize immunoassay inaccuracy-associated misdiagnosis of PA.
Differentiating patients with primary aldosteronism caused by aldosterone-producing adenomas (APAs) from those with bilateral adrenal hyperplasia (BAH), which is essential for choice of therapeutic ...intervention, relies on adrenal venous sampling (AVS)-based measurements of aldosterone and cortisol. We assessed the utility of LC-MS/MS-based steroid profiling to stratify patients with primary aldosteronism.
Fifteen adrenal steroids were measured by LC-MS/MS in peripheral and adrenal venous plasma from AVS studies for 216 patients with primary aldosteronism at 3 tertiary referral centers. Ninety patients were diagnosed with BAH and 126 with APAs on the basis of immunoassay-derived adrenal venous aldosterone lateralization ratios.
Among 119 patients confirmed to have APAs at follow-up, LC-MS/MS-derived lateralization ratios of aldosterone normalized to cortisol, dehydroepiandrosterone, and androstenedione were all higher (P < 0.0001) than immunoassay-derived ratios. The hybrid steroids, 18-oxocortisol and 18-hydroxycortisol, also showed lateralized secretion in 76% and 35% of patients with APAs. Adrenal venous concentrations of glucocorticoids and androgens were bilaterally higher in patients with BAH than in those with APAs. Consequently, peripheral plasma concentrations of 18-oxocortisol were 8.5-fold higher, whereas concentrations of cortisol, corticosterone, and dehydroepiandrosterone were lower in patients with APAs than in those with BAH. Correct classification of 80% of cases of APAs vs BAH was thereby possible by use of a combination of steroids in peripheral plasma.
LC-MS/MS-based steroid profiling during AVS achieves higher aldosterone lateralization ratios in patients with APAs than immunoassay. LC-MS/MS also enables multiple measures for discriminating unilateral from bilateral aldosterone excess, with potential use of peripheral plasma for subtype classification.
The hypothalamus-pituitary-adrenal (HPA) axis is activated in response to inflammation leading to increased production of anti-inflammatory glucocorticoids by the adrenal cortex, thereby representing ...an endogenous feedback loop. However, severe inflammation reduces the responsiveness of the adrenal gland to adrenocorticotropic hormone (ACTH), although the underlying mechanisms are poorly understood. Here, we show by transcriptomic, proteomic, and metabolomic analyses that LPS-induced systemic inflammation triggers profound metabolic changes in steroidogenic adrenocortical cells, including downregulation of the TCA cycle and oxidative phosphorylation, in mice. Inflammation disrupts the TCA cycle at the level of succinate dehydrogenase (SDH), leading to succinate accumulation and disturbed steroidogenesis. Mechanistically, IL-1β reduces SDHB expression through upregulation of DNA methyltransferase 1 (DNMT1) and methylation of the
promoter. Consequently, increased succinate levels impair oxidative phosphorylation and ATP synthesis and enhance ROS production, leading to reduced steroidogenesis. Together, we demonstrate that the IL-1β-DNMT1-SDHB-succinate axis disrupts steroidogenesis. Our findings not only provide a mechanistic explanation for adrenal dysfunction in severe inflammation, but also offer a potential target for therapeutic intervention.
•LC–MS/MS based quantification of 15 steroids in peripheral and adrenal venous samples.•Sensitivity sufficient for reliable aldosterone measurements in peripheral plasma.•Utility for clinical work-up ...of primary aldosteronism due to included hybrid steroids.•AV/pV step-ups for most other steroids higher than for cortisol.•Cosyntropin stimulation during AVS changes relative adrenal secretion of steroid.
Steroid profiling for diagnosis of endocrine disorders featuring disordered production of steroid hormones is now possible from advances in liquid chromatography with tandem mass spectrometry (LC–MS/MS). Adrenal venous (AV) measurements of aldosterone and cortisol are a standard practice in the clinical work-up of primary aldosteronism, but do not yet take advantage of steroid profiling.
A novel LC–MS/MS based method was developed for simultaneous measurement of 15 adrenal steroids: aldosterone, corticosterone, 11-deoxycorticosterone, progesterone, pregnenolone, cortisone, cortisol, 11-deoxycortisol, 17-hydroxyprogesterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone-sulfate, 21-deoxycortisol, 18-oxocortisol and 18-hydroxycortisol. These were compared in peripheral venous (pV) and AV plasma from 70 patients undergoing AV sampling with and without cosyntropin stimulation. Aldosterone and cortisol levels measured by LC–MS/MS were compared with those measured by immunoassay.
Reproducibility of measurements with coefficients of variation ≤10% as well as analytical sensitivity sufficient to measure low pV levels particularly of aldosterone demonstrate the utility of the assay for profiling adrenal steroids in primary aldosteronism. Method comparisons indicated assay and concentration dependent differences of cortisol and aldosterone concentrations measured by immunoassay and LC–MS/MS. Median AV/pV ratios of 11-deoxycortisol (53.0), 17-hydroxyprogesterone (33.4), pregnenolone (62.4), androstenedione (40.6) and dehydroepiandrosterone (33.3) were 2.9- to, 5.4-fold larger than those for cortisol (11.6), with additionally generally larger increases than for cortisol with than without cosyntropin stimulation.
Our LC–MS/MS assay, in addition to improvements over existing immunoassay measurements of aldosterone and cortisol, offers profiling of 13 other adrenal steroids, providing a potentially useful method for the clinical work-up of patients with primary aldosteronism. In particular, the larger AV/pV ratios of several steroids compared to cortisol suggest more sensitive alternatives to the latter for assessing positioning of AV sampling catheters.
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