Preoperative prediction of metastatic pheochromocytoma and paraganglioma using clinical, genetic, and biochemical markers: A cohort study.

BACKGROUND: The prevalence of metastatic pheochromocytoma and paraganglioma (PPGL) is approximately 15%-20%. Although there are indicators to assess metastatic risks, none of them predict metastasis reliably. Therefore, we aimed to develop and validate a scoring system using clinical, genetic, and biochemical risk factors to preoperatively predict the metastatic risk of PPGL. METHODS: In the cross-sectional cohort (n = 180), clinical, genetic, and biochemical risk factors for metastasis were identified using multivariate logistic regression analysis, and a novel scoring system was developed. The scoring system was validated and compared with the age, size of tumor, extra-adrenal location, and secretory type (ASES) score in the longitudinal cohort (n = 114). RESULTS: In the cross-sectional cohort, pseudohypoxia group-related gene variants (SDHB, SDHD, or VHL), methoxytyramine >0.16 nmol/L, and tumor size >6.0 cm were independently associated with metastasis after multivariate logistic regression. Using them, the gene variant, methoxytyramine, and size of tumor (GMS) score were developed. In the longitudinal cohort, Harrell's concordance index of the GMS score (0.873, 95% confidence interval [CI]: 0.738-0.941) was higher than that of the ASES score (0.713, 95% CI: 0.567-0.814, p = 0.007). In the longitudinal cohort, a GMS score ≥2 was significantly associated with a higher risk of metastasis (hazard ratio = 25.07, 95% CI: 5.65-111.20). A GMS score ≥2 (p < 0.001), but not ASES score ≥2 (p = 0.090), was associated with shorter progression-free survival. CONCLUSION: The GMS scoring system, which integrates gene variant, methoxytyramine level, and tumor size, provides a valuable preoperative approach to assess metastatic risk in PPGL.

Clinical Significance of Pheochromocytoma Size on the Timing and Extent of Surgery.

INTRODUCTION: Elevated metanephrine and catecholamine levels 3-fold upper limit of normal (ULN) are diagnostic for pheochromocytoma. We sought to determine whether size correlates with biochemical activity or symptoms which could guide timing of surgery. METHODS: Data from consecutive patients undergoing adrenalectomy for pheochromocytoma at our institution over a 10-year period were retrospectively collected. These included maximal lesion diameter on preoperative imaging, plasma/urine metanephrine and/or catecholamine levels, demographic variables and presence of typical paroxysmal symptoms. Receiver operating characteristic curves were used to assess predictive accuracy. RESULTS: Sixty-three patients were included in the analysis (41 females and 22 males). Median age was 56 (43, 69) years. Due to various referring practices, 31 patients had documented 24-h urine metanephrine, 26 had 24-h urine catecholamine, and 52 had fractionated plasma metanephrine levels available for review. Values were converted to fold change compared to ULN and the maximum of all measured values was used for logistic regression. Median tumor size was 3.40 (2.25, 4.55) cm in greatest dimension. Tumor size at which pheochromocytoma produced > 3-fold ULN was ≥2.3 cm (AUC of 0.84). Biochemical activity increased with doubling tumor size (odds ratio = 8, P = 0.0004) or ≥ 1 cm increase in tumor size (odds ratio = 3.03, P = 0.001). 40 patients had paroxysmal symptoms, but there was no significant correlation between tumor size/biochemical activity and symptoms. CONCLUSIONS: In our study, tumor size directly correlated with the degree of biochemical activity and pheochromocytomas ≥2.3 cm produced levels 3 times ULN. These findings may allow clinicians to adjust timing of operative intervention.

Association between excess catecholamine synthesis and polymorphic premature ventricular contraction.

BACKGROUND: The reasons for the etiology of premature ventricular contractions (PVCs) are not specifically known. Many patients are resistant to medical treatment, and a factor that would predict response to medical treatment cannot be identified. This study aims to investigate if a high catecholamine level results in polymorphic PVC. METHODS: This study was obtained by prospective data registry analysis. A total of 100 patients, 50 from the PVC group, and 50 from the control group have been evaluated. The participants who were included in the patient group had a polymorphic PVC of 5% or more in their 24-h Holter evaluations. Metanephrine showing the level of adrenaline and normetanephrine, showing the level of noradrenaline levels have been measured from these urine samples. RESULT: There was no difference between the two groups in terms of biochemical and essential characteristics. Normetanephrine level has been significantly higher in the PVC group compared to the control group (323.9 ± 208.9 µg to 129.25 ± 67.88 µg; p < 0.001). Similarly, metanephrine level has also been higher in the PVC group (124.75 ± 82.43 µg to 52.615 ± 36,54 µg; p < 0.001). A positive and moderate correlation has been identified between the number and ratio of PVC and the metanephrine and normetanephrine levels. CONCLUSION: In this study, we found that the catecholamine levels were higher in the polymorphic PVC group than in the healthy volunteers. Also, an increase in the number and rate of PVC has been observed as the catecholamine levels increased. CLINICAL TRIAL REGISTRATION: Urine Levels of Metanephrine and Normetanephrine in Patients With Frequent PVC; ClinicalTrials.gov number NCT03447002.

LC-MS based simultaneous profiling of adrenal hormones of steroids, catecholamines, and metanephrines.

Metabolic changes in adrenocortical steroids and medullary catecholamines characterize adrenal tumors, but they are measured using different analytical protocols. To increase bioanalytical validity while maintaining sample homogeneity, LC-MS-based profiling of 29 cortical steroids and 6 medullary amines, including catecholamines and metanephrines, in a single run was developed. Alkyloxycarbonylation with isobutyl chloroformate was employed together with our comprehensive steroid assay, and all adrenal hormones were separated on a reversed-phase C18 column (50 × 2.1 mm, 1.9 µm) at a flow rate of 0.3 ml/min. The lower limits of quantification for all analytes ranged from 0.1 to 2.0 ng/ml, with extraction recoveries of 58.5%-109.5%, while the imprecision and accuracy were 1.6%-14.8% and 89.2%-114.9%, respectively. The validated LC-MS assay was applied to serum samples obtained from 60 patients with adrenal Cushing syndrome, primary aldosteronism, and pheochromocytoma/paraganglioma (PPGL). In addition to the characteristic metabolic changes in glucocorticoids, mineralocorticoids, catecholamines, and metanephrine, the molecular ratios of dehydroepiandrosterone sulfate and 20α-dihydrocortisol indicated Cushing syndrome and primary aldosteronism (P < 0.01 for all compounds), respectively. Moreover, the interactive molecular ratios of 11-deoxycortisol with normetanephrine, metanephrine, norepinephrine, and epinephrine (P < 0.01 all compounds) were proposed to characterize the metabolic features of PPGL. Novel LC-MS-based quantitative profiling of steroids, catecholamines, and metanephrines in human serum was successfully established and characterized metabolic features of individual adrenal tumors that could be used for clinical purposes.

Optimising urinary catecholamine metabolite diagnostics for neuroblastoma.

INTRODUCTION: The analysis of urinary catecholamine metabolites is a cornerstone of neuroblastoma diagnostics. Currently, there is no consensus regarding the sampling method, and variable combinations of catecholamine metabolites are being used. We investigated if spot urine samples can be reliably used for analysis of a panel of catecholamine metabolites for the diagnosis of neuroblastoma. METHODS: Twenty-four-hour urine or spot urine samples were collected from patients with and without neuroblastoma at diagnosis. Homovanillic acid (HVA), vanillylmandelic acid (VMA), dopamine, 3-methoxytyramine, norepinephrine, normetanephrine, epinephrine and metanephrine were measured by high-performance liquid chromatography coupled with fluorescence detection (HPLC-FD) and/or ultra-performance liquid chromatography coupled with electrospray tandem mass spectrometry (UPLC-MS/MS). RESULTS: Catecholamine metabolite levels were measured in urine samples of 400 neuroblastoma patients (24-hour urine, n = 234; spot urine, n = 166) and 571 controls (all spot urine). Excretion levels of catecholamine metabolites and the diagnostic sensitivity for each metabolite were similar in 24-hour urine and spot urine samples (p > .08 and >.27 for all metabolites). The area under the receiver-operating-characteristic curve (AUC) of the panel containing all eight catecholamine metabolites was significantly higher compared to that of only HVA and VMA (AUC = 0.952 vs. 0.920, p = .02). No differences were observed in metabolite levels between the two analysis methods. CONCLUSION: Catecholamine metabolites in spot urine and 24-hour urine resulted in similar diagnostic sensitivities. The Catecholamine Working Group recommends the implementation of spot urine as standard of care. The panel of eight catecholamine metabolites has superior diagnostic accuracy over VMA and HVA.

The polyphenol epigallocatechin gallate lowers circulating catecholamine concentrations and alters lipid metabolism during graded exercise in man: a randomized cross-over study.

PURPOSE: Physical exercise is shown to mitigate catecholamine metabolites; however, it is unknown if exercise-induced increases in sympatho-adrenal activity or catecholamine metabolites are influenced by ingestion of specific catechins found within green tea. This study explored the impact of epigallocatechin gallate (EGCG) ingestion on catecholamine metabolism during graded cycle exercise in humans. METHODS: Eight males (22.4 ± 3.3 years, BMI:25.7 ± 2.4 kg.m2) performed a randomised, placebo-controlled, single-blind, cross-over trial after consumption (1450 mg) of either EGCG or placebo (PLAC) and performed graded cycling to volitional exhaustion. Venous bloods were taken at rest, 2 h post-ingestion and after every 3-min stage. Blood variables were analysed for catecholamines, catecholamine metanephrines and metabolic variables at rest, 2 h post-ingestion (POST-ING), peak rate of lipid oxidation (FATpeak), lactate threshold (LT) and peak rate of oxygen consumption (VO2peak). Data were analysed using SPSS (Version 26). RESULTS: Resting catecholamine and metanephrines were similar between trials. Plasma adrenaline (AD) was lower in ECGC treatment group between trials at FATpeak (P < 0.05), LT (P < 0.001) and VO2peak (P < 0.01). Noradrenaline (NA) was lower under EGCG at POST (P < 0.05), FATpeak (P < 0.05), LT (P < 0.01) and VO2peak (P < 0.05) compared to PLAC. Metanephrines, glucose and lactate increased similarly with exercise intensity in both trials. Lipid oxidation rate was 32% lower in EGCG at FATpeak (EGCG 0.33 ± 0.14 vs. PLAC 0.49 ± 0.11 g.min-1, P < 0.05). Cycle time to exhaustion was similar (NS). CONCLUSION: Acute EGCG supplementation reduced circulating catecholamines but not; metanephrine, glucose or lactates, response to graded exercise. Lower circulating catecholamines may explain a lower lipid oxidation rate.

Correlation between the size of pheochromocytoma and the level of metanephrines.

Objective. Pheochromocytomas (PHEO) and paraganglioma (PGLs) are rare neuroendocrine catecholamine-producing tumors that arise from the chromaffin cells of either the adrenal medulla or extra-adrenal paraganglionic tissues. Despite the recent advances in imaging technologies, biochemical evidence of excessive catecholamine production by the tumor is considered the most important test for the diagnosis of these tumors. The aim of the present study is to investigate the role of the catecholamine metabolites (normetanephrine and metanephrine) levels in the diagnosis of PHEO/PGLs and to evaluate if their levels correlate with the size of these tumors. Patients and Methods. Twenty-five patients were included in the study during the time period of 10 years. Their data were compared with another set of 25 patients to obtain the sensitivity and specificity of metanephrine and normetanephrine in the diagnosis of PHEO/PGLs. The tumor size was reviewed in every patient to obtain the correlation coefficient between the tumor sizes and the plasma/24-hour urinary metanephrine levels. Results. The sensitivity and specificity rates for plasma metanephrine were 80-92% and 92-96%, respectively; while for 24-hour urinary metanephrine were 80-90% and 95-100%, respectively. We found a strong positive relationship between the tumor size and the plasma levels of normetanephrine (r=0.518, p<0.01), and metanephrine (r=0.577, p<0.01). While the relation with the 24-hour urinary concentrations of normetanephrine (r=0.384, p=0.01) and 24-h urinary meta-nephrine (r=0.138, p<0.01) was low. Conclusion. The determination of plasma and 24-hour urinary levels of metanephrines is a reliable test for the diagnosis of PHEO, as they are continuously produced by the tumor cells in contrast to catecholamines.

Reliability of Biomarkers of Physiological Stress at Rest and Post-exertional Heat Stress.

The purpose of this study was to assess the reliability of blood biomarkers that can signify exercise-induced heat stress in hot conditions. Fourteen males completed two heat stress tests separated by 5-7 days. Venous blood was drawn pre- and post- heat stress for the concentration of normetanephrine, metanephrine, serum osmolality, copeptin, kidney-injury molecule 1, and neutrophil gelatinase-associated lipocalin. No biomarker, except copeptin, displayed systematic trial order bias (p≥0.05). Normetanephrine, copeptin and neutrophil gelatinase-associated lipocalin presented acceptable reliability (CV range: 0.9-14.3%), while greater variability was present in metanephrine, osmolality and kidney-injury molecule 1 (CV range: 28.6-43.2%). Normetanephrine exhibited the largest increase (p<0.001) in response to heat stress (trial 1=1048±461 pmol. L-1; trial 2=1067±408 pmol. L-1), whilst kidney-injury molecule 1 presented trivial changes (trial 1=-4±20 ng. L-1; trial 2=2 ± 16 ng. L-1, p>0.05). Normetanephrine, copeptin, and neutrophil gelatinase-associated lipocalin demonstrated good reliability and sensitivity to an acute bout of heat stress. These biomarkers may be suitable for application in laboratory and field research to understand the efficacy of interventions that can attenuate the risk of thermal injury whilst exercising in the heat.

[Pheochromocytoma associated with primary hyperparathyroidism and type 1 neurofibromatosis]. / Feokhromotsitoma v sochetanii s pervichnym giperparatireozom i neirofibromatozom tipa 1.

Neurofibromatosis type 1 (NF-1) is a predominantly inherited genetic disease with prevalence of 1 per 2500-3000 newborns and based on clear clinical criteria. In addition to common neurofibromas and gliomas of the visual pathways, these patients have a higher risk of various benign and malignant tumors throughout their lives including tumors of the central nervous system, membranes of peripheral nerves, gastrointestinal stromal tumors and leukemia. Endocrine diseases and neoplasms also occur in patients with NF-1 and can include extrarenal paraganglioma, primary hyperparathyroidism, gastroenteropancreatic neuroendocrine tumors, thyroid tumors and other adrenal neoplasms. We demonstrate multiple neuroendocrine neoplasia (MEN 2A) of neurofibromatosis type 1 combined with pheochromocytoma and primary hyperparathyroidism in a woman with a long history of palpitations, paroxysmal hypertension and osteoporosis. Biochemical analysis revealed severe hypercalcemia with elevated parathyroid hormone indicating primary hyperparathyroidism, as well as high fractionated normetanephrine and metanephrine in the urine as a sign of catecholamine-releasing pheochromocytoma/paraganglioma. Further scintigraphy revealed signs of solitary parathyroid adenoma causing primary hyperparathyroidism and right-sided pheochromocytoma. Clinical diagnosis of MEN-2 syndrome is based on combination of at least two major MEN-2-associated endocrine tumors. Resection of parathyroid adenoma and pheochromocytoma normalized biochemical parameters and blood pressure. Combination of pheochromocytoma with primary hyperparathyroidism and type 1 neurofibromatosis is discussed.

Addition of 3-methoxytyramine or chromogranin A to plasma free metanephrines as the initial test for pheochromocytoma and paraganglioma: Which is the best diagnostic strategy.

OBJECTIVE: Measurements of plasma free metanephrines (MNs), including MN and normetanephrine, provide high sensitivity and specificity for the diagnosis of pheochromocytoma and paraganglioma (PPGL). 3-Methoxytyramine (3-MT) and chromogranin A (CgA) may allow the detection of dopamine-producing or biochemically silent PPGL. The aim of this study was to evaluate whether measurements of plasma 3-MT or CgA as a supplement of plasma MNs offer a better diagnostic strategy for initial testing of PPGL. PATIENTS AND DESIGN: We enroled 125 patients who underwent surgery from 2015 to 2016 for our study and identified 33 patients with PPGL and 92 patients with non-PPGL masses. MEASUREMENT: The levels of plasma free MNs and 3-MT were measured for all 125 patients using liquid chromatography-tandem mass spectrometry. Plasma CgA concentrations were determined using a radioimmunoassay. To evaluate the diagnostic performance of plasma free MNs, 3-MT and CgA, sensitivity and specificity were determined, and receiver operating characteristic curves were constructed. RESULTS: We found that combining 3-MT and MNs increased the diagnostic sensitivity from 93.9% (95% confidence interval [CI]: 78.4%-98.9%) to 97.0% (95% CI: 82.5%-99.8%). In contrast, addition of plasma CgA test reduced the diagnostic specificity significantly from 91.3% (95% CI: 83.1%-95.9%) to 75.0% (95% CI: 64.7%-83.2%). CONCLUSION: Here, we demonstrated that 3-MT represents a valuable supplementary test to plasma MNs, which can further enhance the sensitivity of the assay, while plasma CgA added no additional diagnostic value to MNs due to the lowered diagnostic specificity.

Clonidine suppression test for a reliable diagnosis of pheochromocytoma: When to use.

OBJECTIVE: In clinical practice, false-positive results in biochemical testing for suspected pheochromocytoma/paraganglioma (PPGL) are not infrequent and may lead to unnecessary examinations. We aimed to evaluate the role of the clonidine suppression test (CST) in the era of analyses of plasma-free metanephrines for the diagnosis or exclusion of PPGL in patients with adrenal tumours and/or arterial hypertension. DESIGN AND METHODS: This single-centre, prospective trial investigated the use of CST in 60 patients with suspected PPGL associated with out-patient elevations of plasma normetanephrine (NMN) and/or metanephrine (MN), in most cases accompanied with hypertension or an adrenal mass. Measurements of plasma catecholamines and free metanephrines were performed by liquid chromatography with electrochemical detection and tandem mass spectrometry, respectively. RESULTS: Forty-six patients entered final analysis (n = 20 with PPGL and n = 26 with a nonfunctional adrenal mass and/or hypertension). CST reliably excluded false-positive baseline NMN results with a specificity of 100%. The sensitivity of CST improved from 85% to 94% when tumours with isolated MN increase (n = 3) were not considered. In patients with elevated baseline NMN (n = 24), CST correctly identified all patients without PPGL. Patients with falsely elevated baseline NMN results (n = 7, 26.9%) exhibited increases of baseline NMN up to 1.7-fold above the upper reference limit. CONCLUSION: CST qualifies as a useful diagnostic tool for differential diagnosis of borderline elevated plasma-free NMN in patients with suspected PPGL. In this context, CST helps to correctly identify all false-positive NMN screening results.

Application of Metanephrine and Normetanephrine in Evaluating the Selectivity of Adrenal Vein Sampling.

The aim of the study was to investigate the usefulness of metanephrine (MN) and normetanephrine (NMN) in bilateral simultaneous adrenal vein sampling (AVS) with and without ACTH stimulation. The study was conducted in a single referral center. Prospective recruited patients with PA were treated with AVS. The effects of cortisol, MN and NMN on selectivity catheterization were assessed and determined by lateralization. We enrolled 54 patients with PA who were treated with simultaneous bilateral AVS with ACTH. The Selectivity Index (SI) calculated by MN was higher than that calculated by other indicators (p<0.001), the catheterization success rate of MN at baseline was the same as that of cortisol after ACTH stimulation, and in lateralization diagnosis, MN was not inferior to cortisol. In conclusion, among the studied indicators, MN is the best index for determining the catheterization selectivity in AVS, especially in the absence of ACTH stimulation.

An automated magnetic bead extraction method for measuring plasma metanephrines and 3-methoxytyramine using liquid chromatography tandem mass spectrometry.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) is used routinely in clinical diagnostics; however, automating the sample pretreatment is challenging. We established and evaluated an automated method based on the magnetic bead extraction principle (MBE) to measure normetanephrine (NMN), metanephrine (MN), and 3-methoxytyramine (3-MT). The target analytes were extracted, purified, and concentrated using different solvents and chemical bond-modified magnetic beads transferred via a magnetic bar. The linearity, recovery, matrix effect, and precision of MBE were evaluated thoroughly, and compared with traditional solid-phase extraction (SPE) using 131 plasma samples. The chromatography peaks of metanephrines and 3-MT, extracted via MBE, are symmetrical, without interfering peaks. The linearity was excellent with correlation coefficient (r) > 0.99. The MBE exhibited good reproducibility with within-run coefficient variations (CVs) of 1.96-2.00%, 4.06-5.75%, and 3.89-4.90% for MN, NMN, and 3-MT, respectively. The total CVs for MN, NMN, and 3-MT were 1.96-2.80%, 5.12-5.75%, and 5.44-6.27%, respectively. The relative recoveries for MN, NMN, and 3-MT varied between 93.5 and 107.4%, whereas their biases were all within 10%. The results for MN, NMN, and 3-MT extracted via MBE compared with SPE exhibited excellent correlation, with r > 0.99; the mean bias% for MN, NMN, and 3-MT were small (-2.9%, -3.2%, and -3.2%, respectively). In conclusion, the automated MBE method for measuring plasma metanephrines and 3-MT can be applied in future routine clinical diagnostics, and the MBE principle may indicate a new era for LC-MS/MS in clinical application.

Computerized tomography texture analysis of pheochromocytoma: relationship with hormonal and histopathological data.

OBJECTIVES: Pheochromocytomas are rare tumors which can present with heterogeneous secretion profiles, clinical manifestations, and radiologic appearance. Under a histopathological point of view, they can be characterized as more or less aggressive with the Pheochromocytoma of the Adrenal gland Scaled Score (PASS) and the Grading system for Adrenal Pheochromocytoma and Paraganglioma (GAPP) score. The aim of this study is to analyze the texture analysis characteristics of pheochromocytoma and identify whether the texture analysis can yield information aiding in the diagnosis and the characterization of those tumors. METHODS: Radiological, biochemical, and histopathological data regarding 30 consecutive patients with histologically confirmed pheochromocytoma were analyzed. Images obtained in the unenhanced, late arterial, venous, and delayed phases were used for the texture analysis. RESULTS: Urinary epinephrine and metanephrine levels showed a significant correlation (R2 = 0.946; R2 = 699) in the multivariate linear model with texture features, as well as Ki-67 (R2 = 0.397), PASS score (R2 = 0.182), GAPP score (R2 = 0.705), and cellularity showed a significant correlation (R2 = 0.389). The cluster analysis based on radiomic features resulted in 2 clusters, with significative differences in terms of systolic and diastolic blood pressure values at the time of diagnosis (p = 0.025), GAPP score (4 vs 6, p = 0.05), histological pattern (1-2, p = 0.039), and comedonecrosis (0% vs 50%, p = 0.013). CONCLUSION: In conclusion, our study provides the proof of concept for the use of texture analysis on contrast-enhanced CT images as a noninvasive, quantitative tool for helping in the characterization of the clinical, biochemical, and histopathological features of pheochromocytoma.

The effect of acid use as a preservative on the results of biochemical tests measured in 24-h urine.

Twenty-four-hour urine measurements play a crucial role in the diagnosis, follow-up and treatment of various diseases. There are different approaches to the collection of urine in patients who need to collect multiple urine samples at a time, especially in hospitals with heavy workloads. In this study, we compared the sodium, potassium, chloride, amylase, calcium, creatinine, phosphorus, microalbumin, protein, magnesium, urea, uric acid, adrenaline, noradrenaline, dopamine, metanephrine, normetanephrine, vanillylmandelic acid, 5-hydroxyindoleacetic acid and homovanillic acid results of 24-h urine samples analyzed immediately without acid addition, which we accepted as the reference and baseline measurement, with the results of the samples analyzed after waiting for 24 h without acid addition, analyzed immediately with acid addition and analyzed after waiting for 24 h with acid addition. Chloride, microalbumin, amylase and protein tests, which are recommended to be measured in the sample without preservatives, are affected by acid addition. Adrenaline, noradrenaline and dopamine, which are the tests recommended to be measured in acid-added urine are degraded in the samples without acid, and the levels of metanephrine and normetanephrine were not significantly degraded in the absence of preservatives.

Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase.

Catechol-O-methyltransferase (COMT) has been involved in a number of medical conditions including catechol-estrogen-induced cancers and a great range of cardiovascular and neurodegenerative diseases such as Parkinson's disease. Currently, Parkinson's disease treatment relies on a triple prophylaxis, involving dopamine replacement by levodopa, the use of aromatic L-amino acid decarboxylase inhibitors, and the use of COMT inhibitors. Typically, COMT is highly thermolabile, and its soluble isoform (SCOMT) loses biological activity within a short time span preventing further structural and functional trials. Herein, we characterized the thermal stability profile of lysate cells from Komagataella pastoris containing human recombinant SCOMT (hSCOMT) and enzyme-purified fractions (by Immobilized Metal Affinity Chromatography-IMAC) upon interaction with several buffers and additives by Thermal Shift Assay (TSA) and a biological activity assessment. Based on the obtained results, potential conditions able to increase the thermal stability of hSCOMT have been found through the analysis of melting temperature (Tm) variations. Moreover, the use of the ionic liquid 1-butyl-3-methylimidazolium chloride [C4mim]Cl (along with cysteine, trehalose, and glycerol) ensures complete protein solubilization as well as an increment in the protein Tm of approximately 10 °C. Thus, the developed formulation enhances hSCOMT stability with an increment in the percentage of activity recovery of 200% and 70% when the protein was stored at 4 °C and -80 °C, respectively, for 12 h. The formation of metanephrine over time confirmed that the enzyme showed twice the productivity in the presence of the additive. These outstanding achievements might pave the way for the development of future hSCOMT structural and biophysical studies, which are fundamental for the design of novel therapeutic molecules.

Dual-Template Magnetic Molecularly Imprinted Polymer for Simultaneous Determination of Spot Urine Metanephrines and 3-Methoxytyramine for the Diagnosis of Pheochromocytomas and Paragangliomas.

A novel dual-template magnetic molecularly imprinted polymer (MMIP) was synthesized to extract normetanephrine (NMN), metanephrine (MN) and 3-methoxytyramine (3-MT) from spot urine samples. As the adsorbent of dispersive solid-phase extraction (d-SPE), the MMIP was prepared using dopamine and MN as dual templates, methacrylic acid as the functional monomer, ethylene glycol dimethacrylate as the crosslinking reagent and magnetic nanoparticles as the magnetic core. NMN, MN, 3-MT and creatinine (Cr) in spot urine samples were selectively enriched by d-SPE and detected by HPLC-fluorescence detection/ultraviolet detection. The peak area (A) ratios of NMN, MN and 3-MT to Cr were used for the diagnosis of pheochromocytomas and paragangliomas (PPGLs). The results showed that the adsorption efficiencies of MMIP for target analytes were all higher than 89.0%, and the coefficient variation precisions of intra-assay and inter-assay for the analytes were within 4.9% and 6.3%, respectively. The recoveries of the analytes were from 93.2% to 112.8%. The MMIP was still functional within 14 days and could be reused at least seven times. The d-SPE and recommended solid-phase extraction (SPE) were both used to pretreat spot urine samples from 18 PPGLs patients and 22 healthy controls. The correlation coefficients of ANMN/ACr and AMN/ACr between d-SPE and SPE were both higher than 0.95. In addition, the areas under the receiver operator curves for spot urine ANMN/ACr, AMN/ACr and plasma free NMN and MN were 0.975, 0.773 and 0.990, 0.821, respectively, indicating the two methods had the similar performances. The d-SPE method took only 20 min, which was effective in clinical application.

Harmonization of LC-MS/MS Measurements of Plasma Free Normetanephrine, Metanephrine, and 3-Methoxytyramine.

BACKGROUND: Plasma-free normetanephrine and metanephrine (metanephrines) are the recommended biomarkers for testing of pheochromocytoma and paraganglioma (PPGL). This study evaluated the status of harmonization of liquid chromatography-tandem mass spectrometry-based measurements of plasma metanephrines and methoxytyramine and clinical interpretation of test results. METHODS: 125 plasma samples from patients tested for PPGLs were analyzed in 12 laboratories. Analytical performance was also assessed from results of a proficiency-testing program. Agreement of test results from different laboratories was assessed by Passing-Bablok regression and Bland-Altman analysis. Agreement in clinical test interpretation based on laboratory specific reference intervals was also examined. RESULTS: Comparisons of analytical test results by regression analysis revealed strong correlations for normetanephrine and metanephrine (R ≥ 0.95) with mean slopes of 1.013 (range 0.975-1.078), and 1.019 (range 0.963-1.081), and intercepts of -0.584 (-53.736 to 54.790) and -3.194 (-17.152 to 5.933), respectively. The mean bias between methods was 1.2% (-11.6% to 16.0%) for metanephrine and 0.1% (-18.0% to 9.5%) for normetanephrine. Measurements of 3-methoxytyramine revealed suboptimal agreement between laboratories with biases ranging from -32.2% to 64.0%. Interrater agreement in test interpretation was >94% for metanephrine and >84% for normetanephrine; improvements in interrater agreement were observed with use of harmonized reference intervals, including age-specific cut-offs for normetanephrine. CONCLUSIONS: Analytical methods for metanephrines are well harmonized between laboratories. However, the 16% disagreement in test interpretation for normetanephrine suggests use of suboptimal method-dependent reference intervals for clinical decision-making for this metabolite. Improved analytical methods and reference interval harmonization are particularly required for 3-methoxytyramine.

Diagnostic Accuracy of Salivary Metanephrines in Pheochromocytomas and Paragangliomas.

BACKGROUND: Measurements of plasma free metanephrines are recommended for diagnosing pheochromocytomas and paragangliomas (PPGL). Metanephrines can be detected in saliva with LC-MS/MS with sufficient analytical sensitivity and precision. Because collecting saliva is noninvasive and less cumbersome than plasma or urine sampling, we assessed the diagnostic accuracy of salivary metanephrines in diagnosing PPGL. METHODS: This 2-center study included 118 healthy participants (44 men; mean age: 33 years (range: 19--74 years)), 44 patients with PPGL, and 54 patients suspected of PPGL. Metanephrines were quantified in plasma and saliva using LC-MS/MS. Diagnostic accuracy; correlation between plasma and salivary metanephrines; and potential factors influencing salivary metanephrines, including age, sex, and posture during sampling, were assessed. RESULTS: Salivary metanephrines were significantly higher in patients with PPGL compared with healthy participants (metanephrine (MN): 0.19 vs 0.09 nmol/L, P < 0.001; normetanephrine (NMN): 2.90 vs 0.49 nmol/L, P < 0.001). The diagnostic sensitivity and specificity of salivary metanephrines were 89% and 87%, respectively. Diagnostic accuracy of salivary metanephrines was 88%, with an area under the ROC curve of 0.880. We found a significant correlation between plasma and salivary metanephrines (Pearson correlation coefficient: MN, 0.86, P < 0.001; NMN, 0.83, P < 0.001). Salivary NMN concentrations were higher when collected in a seated position compared with supine (P < 0.001) and increased with age (P < 0.001). CONCLUSIONS: Salivary metanephrines are a promising tool in the biochemical diagnosis of PPGL. Salivary metanephrines correlate with plasma free metanephrines and are increased in patients with PPGL. At this time, however, salivary metanephrines cannot replace measurement of plasma free metanephrines.

Quantitation of plasma metanephrines using isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS): a candidate reference measurement procedure and its application to evaluating routine ID-LC/MS/MS methods.

Accurate measurement of plasma metanephrines (MNs) including metanephrine (MN) and normetanephrine (NMN) is crucial for the screening and diagnosis in pheochromocytomas and paragangliomas (PPGLs). Although the number of laboratories using liquid chromatography tandem mass spectrometry (LC-MS/MS) method to measure MNs has been increasing rapidly, those laboratory-developed assays showed incomparable results. There are no reference measurement procedures (RMPs) or reference materials (RMs) for MNs in Joint Committee for Traceability in Laboratory Medicine (JCTLM), which hindered the standardization of MNs measurement. We established a candidate RMP (cRMP) based on isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) method for plasma MNs measurement. Plasma samples were spiked with MN-D3 and NMN-D3 as internal standards; protein precipitation and ion-exchange solid phase extraction (SPE) were performed to extract samples, eventually analyzed by LC-MS/MS. The cRMP was applied to evaluate two routine ID-LC/MS/MS methods through split-sample comparisons. Fifty-three individual patient samples were determined by cRMP and two routine ID-LC/MS/MS methods; results were analyzed by ordinary linear regression and Bland-Altman plots. The cRMP exhibited desirable imprecision, with intra-run and total imprecision (coefficient variation, CV) for MN being 0.79-1.36% and 1.53-1.87% and for NMN being 1.10-1.34% and 1.15-1.64%. The analytical recoveries of MN and NMN ranged from 98.3 to 101.7% and from 98.5 to 101.9%, respectively. Significant calibrator biases and sample-specific deviations were observed in method comparison. An accurate, precise, and reliable cRMP for plasma MNs was developed, and RMs with value assigned following the cRMP would help minimize the calibration bias and improve the comparability of different measuring systems.