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Background: Urine metanephrines are used to screen for phaeochromocytoma or paraganglioma (PPGL). Current reference intervals (RI) derived in healthy individuals are not age or sex-stratified, and lower than in hypertensive patients, leading to high false positive rates. This study aims to determine age and sex-stratified RI from a contingent screening population., Methods: Patients with 24-h deconjugated urine metanephrines from 3/6/2010 to 27/8/2022 were included (2936 males, 5285 females), initially by liquid chromatography-electrochemical detection (LC-ECD) then liquid chromatography-tandem mass spectrometry (LC-MS/MS). Bhattacharya analysis was used after log transformation to determine age and sex-stratified RI for metanephrine excretion, normetanephrine excretion, metanephrine/creatinine and normetanephrine/creatinine ratios., Results: Normetanephrine excretion increases with age (RI: males: 18-<30 years: <3.4 µmol/24 h, 30-<40 years: <3.7 µmol/24 h, 40+ years: <5.3 µmol/24 h; females: 18-<30 years: <2.7 µmol/24 h, 30-<40 years: <3.1 µmol/24 h, 40+ years: <3.7 µmol/24 h), while metanephrine excretion was consistent across adulthood (RI: males: 18+ years: <1.8 µmol/24 h; females: 18+ years: <1.2 µmol/24 h). However, normetanephrine/creatinine and metanephrine/creatinine increase steadily with age after early adulthood, likely due to a decrease in muscle mass, with females having higher normetanephrine/creatinine and metanephrine/creatinine ratios., Conclusions: Age and sex-stratified RI were derived for metanephrine excretion, normetanephrine excretion, metanephrine/creatinine and normetanephrine/creatinine ratios. This is expected to reduce false positives while flagging most PPGL., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Objectives: Biochemical testing of urinary metanephrines is useful in the diagnosis and monitoring of pheochromocytoma and paragangliomas. We investigated the feasibility of mixture decomposition (ie, indirect) methods in verifying clinically derived reference intervals for urinary deconjugated metanephrine metabolites., Methods: Urinary 24-hour metanephrine and normetanephrine excretion results were extracted from our data warehouse and intervals were estimated by the modern variant of the Hoffmann method, maximum likelihood estimation (MLE), and gamma mixture model using R software., Results: Hoffmann, MLE, and gamma mixture models provided metanephrine and normetanephrine intervals that closely matched those derived from clinical studies. However, three-component MLE and gamma models were required for normetanephrine in adult women because the Hoffmann method was not suitable. Some data transformations caused blending of the mixed distributions and subsequent widening of the reference interval estimation, emphasizing the importance of careful data transformation for Hoffmann and MLE analyses. Gamma mixture models gave overall good agreement without the need for data transformation., Conclusions: Indirect methods have utility in verifying reference intervals in 24-hour urine specimens collected by patients. We emphasize the benefits of applying multiple decomposition methods to corroborate findings and careful application of data transformation when using Gaussian-based models., (© American Society for Clinical Pathology, 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Catecholamines are physiological regulators of carbohydrate and lipid metabolism during stress, but their chronic influence on metabolic changes in obese patients is still not clarified. The present study aimed to establish the associations between the catecholamine metabolites and metabolic syndrome (MS) components in obese women as well as to reveal the possible hidden subgroups of patients through hierarchical cluster analysis and principal component analysis. The 24-h urine excretion of metanephrine and normetanephrine was investigated in 150 obese women (54 non diabetic without MS, 70 non-diabetic with MS and 26 with type 2 diabetes). The interrelations between carbohydrate disturbances, metabolic syndrome components and stress response hormones were studied. Exploratory data analysis was used to determine different patterns of similarities among the patients. Normetanephrine concentrations were significantly increased in postmenopausal patients and in women with morbid obesity, type 2 diabetes, and hypertension but not with prediabetes. Both metanephrine and normetanephrine levels were positively associated with glucose concentrations one hour after glucose load irrespectively of the insulin levels. The exploratory data analysis showed different risk subgroups among the investigated obese women. The development of predictive tools that include not only traditional metabolic risk factors, but also markers of stress response systems might help for specific risk estimation in obesity patients.

Since activation of the sympathetic nervous system is associated with both impaired insulin secretion and insulin resistance, or namely with diabetes, evaluation of such activation in ordinary clinical settings may be important. Therefore, we evaluated the relationships between urinary concentrations of the catecholamine metabolites, urinary normetanephrine (U-NM) and urinary metanephrine (U-M), and glucose metabolism in a general population. From 1,148 participants in the 2016 population-based Iwaki study of Japanese, enrolled were 733 individuals (gender (M/F): 320/413; age: 52.1±15.1), who were not on medication affecting serum catecholamines, not diabetic, and had complete data-set and blood glucose levels appropriate for the evaluation of insulin secretion and resistance, using homeostasis model assessment (HOMA-β and HOMA-R, respectively). Univariate linear regression analyses revealed significant correlations between both U-NM and U-M, and HOMA-β, but adjustment for multiple factors correlated with HOMA indices abolished these (β = -0.031, p = 0.499, and β = -0.055, p = 0.135, respectively). However, the correlation between U-NM and HOMA-R observed using univariate linear regression analysis (β = 0.132, p<0.001) remained significant even after these adjustments (β = 0.107, p = 0.007), whereas U-M did not correlate with HOMA-R. Furthermore, use of the optimal cut-off value of U-NM for the prediction of insulin resistance (HOMA-R >1.6) determined by ROC analysis (0.2577 mg/gCr) showed that individuals at risk had an odds ratio of 2.65 (confidence interval: 1.42-4.97) after adjustment for the same factors used above. Higher U-NM concentrations within the physiologic range are a significant risk factor for increased insulin resistance in a general Japanese population., Competing Interests: The authors have declared that no competing interests exist.

Introduction: A substantial number of patients with neuroblastoma (NB) have increased excretion of catecholamines and metanephrines. Here, we have investigated the diagnostic role of plasma free metanephrines (PFM), metanephrine (MN), normetanephrine (NMN) and 3-methoxytyramine (3MT) for NB, the most common extra-cranial solid tumour in children., Methods: PFM were quantified by using a commercial IVD-CE LC-MS/MS method on a TSQ Quantiva coupled to an Ultimate 3000. The method was further validated on 103 samples from pediatric subjects (54 patients with histologically confirmed NB and 49 age and sex matched controls). Correlations between PFM concentrations with clinical factors were tested. We directly compared MN, NMN, and 3MT concentrations in matched plasma and urine samples of NB patients (n = 29)., Results: 3MT and NMN showed an excellent diagnostic performance with very high specificity (100% and 95.8%, respectively) and sensitivity (88.2% and 80.4%). ROC curves were obtained (AUC of 0.93 and 0.91 for 3MT and NMN, respectively) and optimal cut-offs that could discriminate between controls and NB patients were defined. A positive correlation between NMN levels in urine and plasma (p = .0017) was found., Discussion: The determination of plasma 3MT and NMN should be taken in consideration as a new diagnostic tool for NB. Validation in prospective clinical studies in comparison to urinary catecholamines and metanephrines is warranted., (Copyright © 2019 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: Plasma or urinary metanephrines are recommended for screening of pheochromocytomas and paragangliomas (PPGLs). Measurements of urinary free rather than deconjugated metanephrines and additional measurements of methoxytyramine represent other developments. For all measurements there is need for reference intervals., Methods: Plasma free, urinary free and urinary deconjugated O-methylated catecholamine metabolites were measured by LC-MS/MS in specimens from 590 hypertensives and normotensives. Reference intervals were optimized using data from 2,056 patients tested for PPGLs., Results: Multivariate analyses, correcting for age and body surface area, indicated higher plasma and urinary metanephrine in males than females and sex differences in urinary normetanephrine and free methoxytyramine that largely reflected body size variation. There were positive associations of age with plasma metabolites, but negative relationships with urinary free metanephrine and methoxytyramine. Plasma and urinary normetanephrine were higher in hypertensives than normotensives, but differences were small. Optimization of reference intervals using the data from patients tested for PPGLs indicated that age was the most important consideration for plasma normetanephrine and sex most practical for urinary metabolites., Conclusion: This study clarifies impacts of demographic and anthropometric variables on catecholamine metabolites, verifies use of age-specific reference intervals for plasma normetanephrine and establishes sex-specific reference intervals for urinary metabolites., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Catecholamines can be used to evaluate neuroendocrine tumors, stress, and potentially pain, but catecholamines degrade rapidly. Their metabolites normetanephrine (NME) and metanephrine (ME) have better stability in urine. In cats, urine sampling in a home environment would be beneficial to reduce effects of clinical stress and simplify sampling. We evaluated a human urine ELISA for analysis of NME and ME in feline urine, and investigated the effects of acidification, cat tray pellets, and storage time at room temperature up to 8.5 h. In 26 feline urine samples, mean NME concentration was 192 ± 80 ng/mL, mean intra- and inter-assay CV was 6.5% and 4.2%, respectively, and spike recovery was 98-101%, but dilutional recovery was unsatisfactory. For ME, mean intra- and inter-assay CV was 10.2% and 4.1%, respectively. Mean urine ME concentration was 32.1 ± 18.3 ng/mL, close to the kit's lowest standard, and spike recovery was 65-90%; the ELISA could not be validated for ME. The stability study, performed for NME on 12 urine samples, did not identify differences between acidified and non-acidified samples, cat tray pellets, or storage time, and no interaction effects. The ME ELISA was not suitable for feline urine; performance of the NME ELISA was acceptable, except for dilution recovery. For analysis of NME, feline urine can be sampled at home using cat tray pellets and stored at room temperature up to 8.5 h without acidification.

Background After adrenalectomy, urinary fractionated metanephrine concentrations are expected to be reduced. However, there are few studies suggesting cut-offs for adrenalectomy patients. Methods Urinary metanephrine and normetanephrine concentrations in adrenalectomy patients and two controls were compared and hormonal concentrations were evaluated via time intervals after surgery. Results The median urinary metanephrine level after unilateral adrenalectomy was lower than that of the non-pheochromocytoma controls but comparable to healthy controls. Urinary normetanephrine concentrations did not differ between adrenalectomy patients and non-pheochromocytoma controls, although both group had levels higher than those of healthy controls. The median urinary normetanephrine level in the immediate postoperative period was higher than in the later period. Conclusions Urinary metanephrine concentrations were lower after adrenalectomy, but urinary normetanephrine concentrations were not changed compared with the non-pheochromocytoma controls. However, urinary normetanephrine concentrations in the patient group were higher than levels in the heathy controls.

The development of advanced imaging techniques has increased the detection of subclinical pheochromocytomas. Because of the substantial proportions of subclinical pheochromocytomas, measurement of urine metanephrine concentrations is crucial due to detect or exclude pheochromocytoma. Although urine metanephrines are elevated in symptomatic subjects, diagnostic cut-offs according to the presence of adrenergic symptoms have not been studied. Pheochromocytomas patients who underwent adrenalectomy at Samsung Medical Center and a control group were compared to determine cut-off concentrations of urine metanephrines. An independent population was analyzed for urine metanephrines with different kits to validate the improvement in diagnostic accuracy using adjusted cut-offs. Symptom-dependent cut-offs of urine metanephrines were higher for symptomatic patients (307 μg/day in males, 235 μg/day in females for urine metanephrine, and 1,045 μg/day in males and 457 μg/day in females for urine normetanephrine) than for asymptomatic patients (206 μg/day in males, 199 μg/day in females for urine metanephrine, and 489 μg/day in males and 442 μg/day in females for urine normetanephrine). Symptom-dependent cut-offs of urine metanephrines improved a specificity from 92.7 % to 96.3 % and a high sensitivity of 97.8 % was maintained. Using the Symptom-dependent cut-offs raised diagnostic accuracy by 5.5 % (p <0.001). Similar trend was also observed in an independent population using different hormone kits. Using symptom-dependent cut-offs of urine metanephrines in symptomatic patients for pheochromocytomas resulted in a significant improvement in diagnostic accuracy in two separate cohorts.

Measuring urinary metanephrines aides in the diagnosis of pheochromocytomas-catecholamine producing tumors. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) allows for greater sensitivity and simpler sample preparation as compared with other techniques. Here we describe a simple LC-MS/MS method for measuring metanephrines in urine. Each urine sample was treated with diphenylboronic acid to create boronate complexes, and then applied to a Bond-Elut Plexa cartridge. After solid phase extraction, samples were concentrated and analyzed on an Atlantis T3 column with chromatographic run time totaling 8.5 min. MS/MS was set in positive electrospray ionization mode with multiple reaction monitoring for data collection. The assay was linear from 0.2 to 27.4 μmol/L and 0.3 to 14.6 μmol/L for metanephrine and normetanephrine, respectively. Intra-assay and total precision at three concentration levels over 10 days were <5 % for metanephrine and <10 % for normetanephrine.

The article considers the technique of simultaneous detection of free catecholamines and free metanephrines in urine using inverse phase highly effective liquid chromatography with fluorimetric detection. The solid phase extraction was implemented on cartridges with 30 mg of hyper cross-linked polystyrene (Purosep-200). The simplicity, reproducibility and sufficient sensitivity of technique permit applying it in clinical practice to diagnose pheochromocytoma.

Ambulatory blood pressure variability (ABPV) is a predictor of cardiovascular risk in hypertensives. Factors that contribute to ABPV are not well described. This pilot study aimed to investigate the relationship between ABPV and urine catecholamine metabolites in order to rationalise further therapeutic intervention in the management of hypertension. Twenty individuals (14 female), aged from 23 to 71 years, with primary hypertension were identified from referrals to a regional hypertension clinic. Individuals had a 24-h ambulatory blood pressure recording (Del Mar Reynolds), and a 24-h urine collection was analysed for metadrenaline and normetadrenaline by reverse-phase chromatography (Spherisorb ODS2) and electrochemical detection (Coulochem II). Normetadrenaline concentration correlated with systolic blood pressure (SBP) variability, r=0.493, P=0.02, and with SBP coefficient of variability, r=0.490, P=0.02. Metadrenaline concentration did not correlate with either SBP variability, r=0.177, P=0.43, or coefficient of variability, r=0.185, P=0.42. A correlation was observed between 24 h urinary normetadrenaline and measures of SBP variability but not 24 h urinary metadrenaline and blood pressure variability. It is hypothesised that sympathetic activity may influence SBP variability.

This work discusses the clinical performance of deconjugated metanephrine (MN), normetanephrine (NMN) and 3-methoxytyramine (3MT) determined in the basal first morning urine using a chromatographic method with electrochemical detection for the clinical diagnosis of pheochromocytoma (PHEO) and paraganglioma (PGL). Urine samples were collected from 44 patients (36 with PHEO, 8 with PGL) aged 54+/-17 (20-78) years (22 females, 22 males). A sampling of biological materials was performed preoperatively and about one week, six months and one year after adrenal gland surgery. The control group consisted of 34 PHEO/PGL patients more than 4 months after adrenal gland surgery. All subjects in the control group were without a diagnosis of PHEO or PGL. Clinical sensitivity was 55 % for MN, 64 % for NMN, 80 % for combination of both MN and NMN, and only 23 % for 3TM. Clinical specificity calculated from the control group was 93 % for MN, 95 % for NMN, 95 % for the combination MN and NMN, and 97 % for 3TM. Cut-off values for deconjugated metanephrines in the basal urine were 310 (MN), 690 (NMN) and 250 microg/l (3MT). Chromatographic determination of deconjugated urinary metanephrines, which is simple without the necessity of special laboratory material, can serve for the screening of PHEO or PGL patients. Urine NMN and 3MT exerts an association to malignity, and all markers are associated with tumor mass. However, the principal laboratory diagnosis of PHEO or PGL must be based on plasma-free metanephrines and plasma chromogranin A with better performance in the laboratory diagnosis of PHEO or PGL.

Background: The purpose of this case-control study is to investigate the association between stress and periodontitis by determining stress biomarkers in saliva and urine and to determine whether oral hygiene, gingival inflammation, and tooth loss are correlated with stress biomarkers in patients with periodontitis., Methods: A total of 77 patients (41 cases [with periodontitis] and 36 controls) participated in this study. Periodontal examination findings included probing depth, clinical attachment loss, bleeding on probing (BOP), plaque index (PI), and tooth loss. Secretory immunoglobulin (sIg)A and cortisol were determined in saliva. Cortisol, creatinine-adjusted cortisol, metanephrine, normetanephrine, and total metanephrines were measured in urine., Results: Urinary metanephrine (P = 0.013) and total metanephrine (P = 0.023) levels were higher in the case group. In cases, salivary cortisol was correlated with PI (r = 0.464, P <0.01), BOP (r = 0.401, P <0.05), and tooth loss (r = 0.245, P <0.05). Urinary metanephrine levels above the median were associated with a 3.4-fold higher risk of periodontitis (95% confidence interval [CI] = 1.1 to 10.2; P = 0.029), with an 82% increase in risk for each increment of 0.05 µg/24 hours. Urinary total metanephrine levels above the median were associated with a five-fold higher risk of periodontitis (95% CI = 1.6 to 15.7; P = 0.006)., Conclusions: The present results offer new evidence of the association between urinary concentrations of catecholamine metabolites (metanephrine and total metanephrines) and chronic periodontitis. Salivary IgA level showed no statistical difference between the cases and controls. Salivary cortisol levels in the patients with periodontitis were correlated with worse PI, higher gingival inflammation, and greater tooth loss.

Objective: To document the influences of blood sampling under supine fasting versus seated nonfasting conditions on diagnosis of phaeochromocytomas and paragangliomas (PPGL) using plasma concentrations of normetanephrine, metanephrine and methoxytyramine., Design and Methods: Biochemical testing for PPGL was performed on 762 patients at six centres, two of which complied with requirements for supine sampling after an overnight fast and four of which did not. Phaeochromocytomas and paragangliomas were found in 129 patients (67 noncompliant, 62 compliant) and not in 633 patients (195 noncompliant, 438 compliant)., Results: Plasma concentrations of normetanephrine and methoxytyramine did not differ between compliant and noncompliant sampling conditions in patients with PPGL but were 49-51% higher in patients without PPGL sampled under noncompliant compared with compliant conditions. The 97·5 percentiles of distributions were also higher under noncompliant compared with compliant conditions for normetanephrine (1·29 vs 0·79 nmol/l), metanephrine (0·49 vs 0·41 nmol/l) and methoxytyramine (0·42 vs 0·18 nmol/l). Use of upper cut-offs established from seated nonfasting sampling conditions resulted in substantially decreased diagnostic sensitivity (98% vs 85%). In contrast, use of upper cut-offs established from supine fasting conditions resulted in decreased diagnostic specificity for testing under noncompliant compared with compliant conditions (71% vs 95%)., Conclusions: High diagnostic sensitivity of plasma normetanephrine, metanephrine and methoxytyramine for the detection of PPGL can only be guaranteed using upper cut-offs of reference intervals established with blood sampling under supine fasting conditions. With such cut-offs, sampling under seated nonfasting conditions can lead to a 5·7-fold increase in false-positive results necessitating repeat sampling under supine fasting conditions., (© 2013 John Wiley & Sons Ltd.)

Measuring the levels of the plasma free metanephrines (PFMs) represents a recently developed and promising test for the diagnosis of pheochromocytoma in the United States and Europe. As this test has not yet been evaluated in Japan, it is necessary to evaluate the diagnostic efficacy of measuring the levels of PFMs compared with the standard measurement of the urinary excretion of metanephrines (uMNs) whose reliability is well established to detect of pheochromocytoma. A total of 101 Japanese subjects clinically suspected of having pheochromocytoma in were included in this study. Subsequently, we prospectively measured the PFMs levels in all patients, compared with those of biochemical markers of the catecholamine secretion and metabolisms in the plasma and urine. All subjects with adrenal tumors underwent tumor excision. Data were available for 84 of the 101 patients, 47 of whom had histopathologically proven pheochromocytoma and 37 were finally diagnosed with non-pheochromocytoma. The results of comparisons in the accuracy of measurement for diagnosis of pheochromocytoma between PFMs and the urinary excretion of metanephrines (uMNs) were 0.980 VS 0.951 for AUC of receiver operatorating characteristic (ROC) curve, 0.957 VS 0.894 for sensitivity, and 0.973 VS 0.946 for specificity, respectively. Although the differences were small, the results of our study definitely demonstrated that measurement of PFMs was not inferior to standard urinary metanephrines (uMNs) measurement, which is established to be the most reliable biochemical method to detect pheochromocytoma. This study clearly shows measuring the PFMs levels to be a reliable and efficient method for diagnosing pheochromocytoma in Japanese patients, as demonstrated in previous reports.

Background & Objectives: Plasma and urinary metanephrines are used as screening tests for the diagnosis of phaeochromocytoma. The recommended cut-off levels are not standardized. This study was conducted to identify a cut-off level for 24 h urinary fractionated metanephrines viz. metanephrine (uMN) and normetanephrine (uNMN) using enzyme immunoassay for the diagnosis of phaeochromocytoma., Methods: Consecutive patients suspected to have phaeochromocytoma were included in the study. uMN and uNMN in 24 h urinary sample were measured using a commercial ELISA kit., Results: Overall, 72 patients were included over a period of 18 months. Twenty patients had histopathologically confirmed phaeochromocytoma and in 52 patients phaeochromocytoma was ruled out. Using the upper limit of normal stated by the assay manufacturer as the cut-off, uMN >350 μg/day had a low sensitivity and uNMN >600 μg/day had a poor specificity. By increasing the cut-off value of uNMN to twice the upper limit, specificity increased significantly without much loss in sensitivity. Combining uMN and uNMN using a cut-off twice the upper limit improved the diagnostic performance - sensitivity (95%); specificity (92.3%); positive predictive value (PPV - 82.6%); negative predictive value (NPV - 98%). In subsets of patients with a variable pretest probability for phaeochromocytoma, the PPV correlates well with the occurred of these tumors decreased, while the NPV remained at 100 per cent., Interpretation & Conclusions: ELISA is a simple and reliable method for measuring uMN and uNMN. The test has a good NPV and can be used as an initial screening test for ruling out phaeochromocytoma. Each hospital will have to define the cut-off value for the assay being used, choosing a proper control population.

Adrenal pheochromocytomas are neoplasms characterized by catecholamine excess. Determination of metanephrines by high-pressure liquid chromatography has been well established for the diagnosis of pheochromocytomas, demonstrating high sensitivity and specificity. This study evaluates the diagnostic value of newly available enzyme immunoassays for metanephrines in plasma and urine. Chromogranin A was studied as a potential additional diagnostic tool. Spontaneous blood samples and 24-h urine samples were collected in 149 subjects, including 24 histologically proven pheochromocytomas, 17 aldosterone-secreting and 21 cortisol-secreting adrenal adenomas, 30 nonfunctioning adrenal masses, 15 patients with essential hypertension and 42 healthy normotensive volunteers. Plasma and urinary metanephrine and normetanephrine as well as chromogranin A were determined and putative thresholds were calculated by ROC analysis. Plasma free normetanephrine was found to be the best single parameter with the highest sensitivity (89.5%) and specificity (98.3%) using a threshold of 167 pg/ml. Analysis of the combination of plasma free metanephrines revealed a similar sensitivity with lower specificity of 90.0%. Considering both urinary parameters demonstrated a slightly higher sensitivity (92.9%) with lower specificity (77.6%). ROC analysis revealed a threshold of 215 µg/l for chromogranin A with rather low sensitivity (73.9%) and specificity (74.2%). A weak positive correlation was found between the tumor size of pheochromocytomas and plasma metanephrine (r = 0.53, p ≤ 0.05) as well as chromogranin A (r = 0.60, p ≤ 0.01). In conclusion, plasma free and urinary metanephrines measured by enzyme immunoassays are convenient and reliable parameters for the diagnosis of pheochromocytoma. In contrast, CgA demonstrated poor sensitivity and specificity., (© J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York.)

The diagnosis of pheochromocytoma depends on the documentation of catecholamine overproduction. The use of urinary fractionated metanephrines has recently become common for the diagnosis of pheochromocytoma. In order to avoid false positive and false negative results, optimal cut-off levels are necessary; however, there have been few published reports on whether different cut-off levels are needed to diagnose pheochromocytoma according to sex. We reviewed the medical records of 815 subjects (including 103 pheochromocytoma patients) whose of 24-h urinary fractionated metanephrine was measured using high-performance liquid chromatography methods and adrenal imaging at Samsung Medical Center. Receiver operating characteristic (ROC) curves were used to determine cut-off values according to sex. The upper limit values of fractionated metanephrine in healthy volunteers and the control group were significantly higher in male subjects compared with females. When we applied cut-off values according to sex, the diagnostic efficacies (defining a positive test as either metanephrine or normetanephrine levels above the cut-off value) were a sensitivity of 96% in male subjects and 98.1% in female subjects and a specificity of 88.6% in male subjects and 94.1% in female subjects. However, when we applied cut-off values without considering sex, the specificity decreased from 88.6% to 77.8% in male subjects. In this study, urinary fractionated metanephrines had a high level of sensitivity and specificity for the diagnosis of pheochromocytoma. However, diagnostic cut-off values were higher in male subjects than in female subjects. Therefore, different cut-off values may be needed according to sex to diagnose pheochromocytoma in Koreans.

Determination of urinary metanephrines is requested frequently for the differential diagnosis and monitoring of pheochromocytoma. Although numerous methods have been developed, interferences are common and hinder most available assays. This study describes the development, validation and implementation of a reliable high-throughput LC-MS/MS method for the measurement of metanephrine and normetanephrine in urine. Metanephrine and normetanephrine were isolated from urine samples subjected to acid hydrolysis using solid phase extraction on a mixed mode cation exchange sorbent in 96-well format. The extracts were injected directly onto a Restek perfluorophenyl column and separated isocratically in 0.2% formic acid in 5% methanol with a gradient cleanout step to 50% methanol. Detection was accomplished using an API 3200 triple quadrupole mass spectrometer with electrospray ionization in positive mode. Data were acquired in multiple reaction monitoring mode. Three transitions were monitored for metanephrine and its deuterated internal standard; two transitions were monitored for normetanephrine and its deuterated internal standard. Two quantification methods were used to address metanephrine interferences without reducing throughput. The method was linear to 15,000 nmol/L. The limits of detection and quantification were 2.5 and 10 nmol/L, respectively. Within run, between-day and total imprecision values were at or below 1.9%, 2.5% and 2.7% for both analytes. The method correlated well with our previously used GC-MS method. Injection-to-injection time was 6 min. The validated LC-MS/MS method for measurement of metanephrine and normetanephrine in urine specimens was placed into service in August 2010 and has performed exceptionally well., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Measuring urinary fractionated metanephrines is one of the initial tests in the diagnosis of pheochromocytoma. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) represents the most specific and accurate technology for this purpose. The goal of this work was to develop a simple LC-MS/MS method for measuring metanephrines in urine., Methods: Each urine sample was complexed with diphenylboronic acid, and purified on a Bond-Elute Plexa cartridge. The extract was concentrated and analyzed on a short Atlantis T3 column in 8.5 min. Metanephrines and their deuterated internal standards were monitored in positive electrospray ionization mode by multiple reaction monitoring., Results: Ion suppression was observed, but was compensated for by the respective internal standard. The analytical measurement range was 0.2-27.4 μmol/L and 0.3-14.6 μmol/L for metanephrine and normetanephrine, respectively. The intra-assay and total coefficient of variation throughout the linear ranges was 2.03%-2.11% and 2.20%-3.80% for metanephrine, and 4.50%-8.09% and 9.00%-10.00% for normetanephrine, respectively. Comparison with a commercial HPLC method using patient samples (n=65) by Passing-Bablok regression showed a slope of 1.000 and 1.014, y-intercept of -0.080 and -0.067, a correlation coefficient of 0.8830 and 0.9022, and a mean difference of 14.0% and -0.43% for metanephrine and normetanephrine, respectively., Conclusions: This simple method for urine metanephrines is suitable for clinical use.

Background: Pheochromocytomas are rare catecholamine-producing tumors derived in more than 30% of cases from mutations in 9 tumor-susceptibility genes identified to date, including von Hippel-Lindau tumor suppressor (VHL); succinate dehydrogenase complex, subunit B, iron sulfur (Ip) (SDHB); and succinate dehydrogenase complex, subunit D, integral membrane protein (SDHD). Testing of multiple genes is often undertaken at considerable expense before a mutation is detected. This study assessed whether measurements of plasma metanephrine, normetanephrine, and methoxytyramine, the O-methylated metabolites of catecholamines, might help to distinguish different hereditary forms of the tumor., Methods: Plasma concentrations of O-methylated metabolites were measured by liquid chromatography with electrochemical detection in 173 patients with pheochromocytoma, including 38 with multiple endocrine neoplasia type 2 (MEN 2), 10 with neurofibromatosis type 1 (NF1), 66 with von Hippel-Lindau (VHL) syndrome, and 59 with mutations of SDHB or SDHD., Results: In contrast to patients with VHL, SDHB, and SDHD mutations, all patients with MEN 2 and NF1 presented with tumors characterized by increased plasma concentrations of metanephrine (indicating epinephrine production). VHL patients usually showed solitary increases in normetanephrine (indicating norepinephrine production), whereas additional or solitary increases in methoxytyramine (indicating dopamine production) characterized 70% of patients with SDHB and SDHD mutations. Patients with NF1 and MEN 2 could be discriminated from those with VHL, SDHB, and SDHD gene mutations in 99% of cases by the combination of normetanephrine and metanephrine. Measurements of plasma methoxytyramine discriminated patients with SDHB and SDHD mutations from those with VHL mutations in an additional 78% of cases., Conclusions: The distinct patterns of plasma catecholamine O-methylated metabolites in patients with hereditary pheochromocytoma provide an easily used tool to guide cost-effective genotyping of underlying disease-causing mutations.

Introduction: Normetanephrine and metanephrine are intermediate metabolites of noradrenaline and adrenaline metabolism. To assess whether normetanephrine and metanephrine analysis may aid in the diagnosis of Neuroblastoma, a reference interval for these metabolites must first be established., Aim: The overall aim of this study was to establish a paediatric age-related reference interval for the measurement of total fractionated metanephrines., Methods: A total of 267 urine samples were analysed following acid hydrolysis. This releases the metanephrines from their sulphate-bound metabolites. The samples were analysed using reverse phase high-performance liquid chromatography with electro-chemical detection on a Gilson automated sequential trace enrichment of dialysate sample system., Results: Data were analysed using Minitab Release version 14. Outliers were removed using the Dixon/Reed one-third rule. Partitioning of the age groups was achieved using Harris and Boyd's standard normal deviate test. Non-parametric analysis of the data was performed, followed by the establishment of the 2.5th and the 97.5th reference limits., Conclusions: The established reference intervals are described in Table 2.

Background: Measurement of metadrenalines has been recommended in the investigation of phaeochromocytoma. Urinary assays remain the most common; however, drug interference is still one of the main challenges for analytical systems. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of total urinary normetadrenaline and total urinary metadrenaline, which does not require extraction procedures prior to analysis., Method: Total urinary normetadrenaline and total urinary metadrenaline were individually quantified by electrospray ionization in the multiple-reaction monitoring mode. Deuterated internal standards were used and an acid hydrolysis step was used to convert conjugated metabolites into free metadrenalines. Chromatographic separation was achieved using a Phenomenex Luna 3 micro PFP column followed by analysis on an API 3200 LC-MS/MS., Results: Linearity was exhibited across the calibration range for both normetadrenaline (r = 1, P < 0.0001) and metadrenaline (r = 1, P < 0.0001) with the limit of quantification of 0.05 and 0.02 micromol/L, respectively. Intra-assay imprecision for both normetadrenaline and metadrenaline was less than 5.5% with % coefficient of variations of less than 4%. Inter-assay imprecision was less than 13%. Neither noradrenaline or adrenaline interfere with the assay as determined by the spiking of samples with high concentrations of noradrenaline or adrenaline (P > 0.05). Acceptable analytical performance was seen with comparison to a high-performance liquid chromatography method and on External Quality Assessment returns., Conclusions: An analytically simple and sensitive method has been developed and evaluated for the analysis of total urinary normetadrenaline and total urinary metadrenaline which is now in routine use.

The silica monolith with ionizable silanol groups and large surface area was found able to function as an offline cation exchange solid phase extraction (SPE) cartridge for extracting polar analytes. The prepared cartridge was housed in a 2-mL syringe fixed over a SPE vacuum manifold. The unique property of this silica monolithic cartridge was demonstrated by extracting epinephrine, normetanephrine and metanephrine from urine samples. These analytes were chosen as model compounds for testing because of their high hydrophilicity, and being candidates monitored for clinical diagnosis. The extracted analytes, after concentration and reconstitution were then quantitated by high-performance liquid chromatography coupled to mass spectrometer (HPLC/ESI/MS). Multiple reactions monitoring was carried out with transitions: 184-->107, 184-->134 and 198-->148 for analyzing epinephrine, normetanephrine and metanephrine, respectively. The limit of detection was 3 ng/mL for metanephrine and 5 ng/mL for normetanephrine and epinephrine. The relative standard deviations of measurements ranged from 2 to 10%. The sorbent offered good linearity with coefficient of determination (r(2))>0.99, over a concentration range of 20-200 ng/mL. The relative recoveries ranged from 60 to 67%, 55 to 59% and 99 to 105% for epinephrine, normetanephrine and metanephrine, respectively. The prepared cartridge had shown potential and was found robust in extracting the polar analytes repeatedly without any significant loss in efficiency., (Copyright 2010 Elsevier B.V. All rights reserved.)

Background: Up to 2007 there was no formal external quality assurance programme for plasma free metanephrines. A pilot programme was conceived by the AACB (Australian Association of Clinical Biochemists) Working Party on biogenic amines. With support from the AACB and Royal College of Pathologists of Australasia Quality Assurance programmes, a pilot study was developed. Data from this study are presented for the first time., Methods: Twelve lyophilized plasma samples were distributed to 15 centres. Samples were spiked with metanephrine (metadrenaline), normetanephrine (normetadrenaline) and 3-methoxytyramine, all derived from human urine. Concentrations were arranged in a linear relationship. The analytes were present at six levels and samples were duplicated., Results: High-pressure liquid chromatography and tandem mass spectrometry methods showed acceptable precision but in general enzyme immunoassay displayed a higher degree of imprecision as well as a negative bias., Conclusions: Differences in calibration and matrix effects are likely to have been responsible for the discrepancy between chromatographic and immunoassay methods. These differences need to be further examined although efforts at standardization between different methods have been hampered by the lack of a universal calibrator for plasma metanephrines. Meanwhile, a laboratory's performance characteristics can be monitored and enhanced by participation in suitable external quality assurance programmes.

Introduction: We report the results of a laboratory evaluation of the BioRad Urinary Metanephrines Reagent Kit. The test was designed for the quantitative measurement of normetanephrine and metanephrine in urine by high performance liquid chromatography. The kit was evaluated in view of improving assay reliability and specificity as compared to the manual method based on cation exchange chromatography and spectrophotometry., Methods: Performance was evaluated for precision, linearity, accuracy, sensitivity and detection limit based on National Committee on Clinical Laboratory Standards (NCCLS) protocols. Analytical precision was evaluated using commercial controls and patient sample. Accuracy was evaluated by assessing recovery. Linearity was determined using aqueous standards., Results: The within-run coefficient of variation (CV) for metanephrine and normetanephrine were 1.9 percent and 2.4 percent (low control); 4.2 percent and 3.5 percent (high control); 3.8 percent and 3.3 percent (patient sample), respectively. The between-day precisions were 3.8 percent and 4.3 percent (low control); and 5.5 percent and 3.7 percent (high control) for metanephrine and normetanephrine, respectively. The linearity curve showed metanephrine and normetanephrine to be linear with concentrations, to at least 1,600 microgramme per litre and 2,000 microgramme per litre, respectively. Analytical recovery averaged 102 percent for metanephrine and 95 percent for normetanephrine. Levels as low as 23 microgramme per litre normetanephrine and 10 microgramme per litre metanephrine were measured with this method. The detection limit was 3.3 microgramme per litre for metanephrine., Conclusion: The performance characteristics of automated sample preparation and auto-injection facilitate handling of larger number of samples as well as improve assay reliability.

Background: Measurements of plasma free metanephrines (normetanephrine and metanephrine) provide a useful test for diagnosis of pheochromocytoma and may provide other information about the nature of these tumors., Methods: We examined relationships of tumor size, location, and catecholamine content with plasma and urinary metanephrines or catecholamines in 275 patients with pheochromocytoma. We then prospectively examined whether measurements of plasma free metanephrines could predict tumor size and location in an additional 16 patients., Results: Relative proportions of epinephrine and norepinephrine in tumor tissue were closely matched by relative increases of plasma or urinary metanephrine and normetanephrine, but not by epinephrine and norepinephrine. Tumor diameter showed strong positive relationships with summed plasma concentrations or urinary outputs of metanephrine and normetanephrine (r = 0.81 and 0.77; P <0.001), whereas relationships with plasma or urinary catecholamines were weaker (r = 0.41 and 0.44). All tumors in which increases in plasma metanephrine were >15% of the combined increases of normetanephrine and metanephrine either had adrenal locations or appeared to be recurrences of previously resected adrenal tumors. Measurements of plasma free metanephrines predicted tumor diameter to within a mean of 30% of actual diameter, and high plasma concentrations of free metanephrine relative to normetanephrine accurately predicted adrenal locations., Conclusions: Measurements of plasma free metanephrines not only provide information about the likely presence or absence of a pheochromocytoma, but when a tumor is present, can also help predict tumor size and location. This additional information may be useful for clinical decision-making during tumor localization procedures.

This Hypertension Grand Rounds shows how applying new clinical laboratory techniques helped to diagnose pheochromocytoma in a difficult case. In the setting of long-standing, sustained hypertension, the patient had a hypertensive paroxysm during anesthesia induction for surgery, leading to suspicion of a pheochromocytoma. Conventional testing, including CT scanning and fractionated urinary metanephrine test, was not diagnostic. The patient had another hypertensive paroxysm during subsequent anesthesia induction, requiring intensive care. Consistently elevated plasma levels of free normetanephrine provided the first and only biochemical evidence for a pheochromocytoma in this case. 6-[18F]Fluorodopamine positron emission tomography and 123I-metaiodobenzylguanidine scintigraphy subsequently agreed on the existence of a small left adrenal mass, which when removed surgically proved to be a pheochromocytoma. Postoperatively, plasma levels of normetanephrine normalized, and there were no further hypertensive paroxysms, although the patient remained hypertensive. This case illustrates the superiority of plasma levels of free (unconjugated) metanephrines, compared with other biochemical tests, to detect pheochromocytoma. It also confirms that functional imaging by 6-[18F]fluorodopamine or 123I-metaiodobenzylguanidine scanning can localize pheochromocytoma in difficult cases in which other imaging tests are not diagnostic.

Background: Metanephrines are biochemical markers for tumors of the adrenal medulla (e.g., pheochromocytoma) and other tumors derived from neural crest cells (e.g., paragangliomas and neuroblastomas). We describe a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the measurement of urinary conjugated metanephrines., Methods: We added 250 ng of d3-metanephrine (d3-MN) and 500 ng of d3-normetanephrine (d3-NMN) to 1 mL of urine samples as stable isotope internal standards. The samples were then acidified, hydrolyzed for 20 min in a 100 degree C water bath, neutralized, and prepared by solid-phase extraction. The methanol eluates were analyzed by LC-MS/MS in the selected-reaction-monitoring mode after separation on a reversed-phase amide C16 column., Results: Multiple calibration curves for the analysis of urine MN and NMN exhibited consistent linearity and reproducibility in the range of 10-5000 microg/L. Interassay CVs were 5.7-8.6% at mean concentrations of 90-4854 microg/L for MN and NMN. The detection limit was 10 microg/L. Recovery of MN and NMN (144-2300 microg/L) added to urine was 91-114%. The regression equation for the LC-MS/MS (x) and colorimetric (y) methods was: y = 0.81x - 0.006 (r = 0.822; n = 110). The equation for the HPLC (x) and LC-MS/MS (y) methods was: y = 1.09x + 0.05 (r = 0.998; n = 40)., Conclusions: The sensitivity and specificity of the MS/MS method for urinary conjugated metanephrines offer advantages over colorimetric, immunoassay, HPLC, and gas chromatography-mass spectrometry methods because of elimination of drug interferences, high throughput, and short chromatographic run time.

Background: Widely used HPLC methods for quantification of metanephrine and normetanephrine in urine often have long analysis times and are frequently plagued by drug interferences. We describe a gas chromatography-mass spectrometry method designed to overcome these limitations., Methods: Metanephrine and normetanephrine conjugates were converted to unconjugated metanephrine and normetanephrine by acid hydrolysis. To avoid the rapid decomposition of the deuterated internal standards (metanephrine-d(3) and normetanephrine-d(3)) under hydrolysis conditions, the internal standards were added after hydrolysis. Solid-phase extraction was used to isolate the hydrolyzed metanephrines from urine. Samples were concentrated by evaporation, then derivatized simultaneously with N-methyl-N-(trimethylsilyl)trifluoroacetamide and N-methyl-bis-heptafluoro-butryamide at room temperature., Results: The assay was linear from 25 to 7000 microg/L. The intraassay CVs were < 5 % and the interassay CVs < 12%. Comparison with a routine HPLC method (n = 192) by Deming regression yielded a slope of 1.00 +/- 0.02 microg/L, an intercept of -5.8 +/- 7.8 micro/L, and S(y/x) = 50.6 microg/L for metanephrine and a slope of 0.94 +/- 0.03, intercept of 19 +/- 11 microg/L, and S(y/x) = 60 microg/L for normetanephrine. The correlation coefficients (r) were calculated after log transformation of the data and gave r = 0.97 for metanephrine and r = 0.97 for normetanephrine. Interference from common medications or drug metabolites was seen in <1% of samples. The time between sequential injections was < 7 min., Conclusions: This new gas chromatography-mass spectrometry assay for total fractionated metanephrines is rapid, compares well with a standard HPLC assay, and avoids most drug interferences that commonly affect HPLC assays for urine metanephrines.

A method for the determination of metanephrine (MN; also known as metadrenaline), normetanephrine (NMN; also known as normetadrenaline) and 3-methoxytyramine (3-MT) in human urine using high-performance liquid chromatography followed by electrochemical detection (ECD) was validated primarily by comparing the results with those obtained by a gas chromatography mass spectrometry (GC-MS) reference method. Correlation coefficients of 0.93, 0.94 and 0.91 were obtained for MN, NMN and 3-MT, respectively, in a group of healthy controls consisting of 30 women and 30 men. A systematic difference was detected only for 3-MT (-16%). Further tests of accuracy (linearity and recovery) and precision demonstrated that the described method must be considered to be a reliable approach to assess urinary metanephrines in the diagnosis of phaeochromocytoma. At lower concentrations (MN, 248 nmol/L; NMN, 434 nmol/L; 3-MT, 402 nmol/L), within-assay coefficients of variation were close to 5% or less (5.3, 4.6 and 2.2%, respectively) and between-assay coefficients of variation were 8.9, 11.2 and 12.3%, respectively, for the same low levels. This raises the possibility that this method can also be applied to assess urinary free, unconjugated metanephrines. Sex differences were detected for MN and NMN excretion when expressed in nmol per 24h and nmol/mmol creatinine, respectively, by both ECD and GC-MS methods.

Objective: To audit the performances of the analytes used in the diagnosis of pheochromocytoma and to present a graphical guideline to help the diagnosis., Design: A 5 year retrospective study., Settings: Laboratory and departments of a university hospital., Participants: In-patients, suspected of bearing a pheochromocytoma, were investigated for urinary metanephrines and catecholamines (photometric method) and vanillylmandelic acid, fractionated catecholamines and metanephrines [high pressure liquid chromatography (HPLC) coupled to electrochemical detection (ED)] urinary excretion., Main Outcome: Patients with a pheochromocytoma (24 out of 2003 patients) were diagnosed by the combination of normetanephrine and metanephrine determination., Results: All analytes but dopamine were significantly elevated in patients with a pheochromocytoma. The area under the receiver operating characteristics (ROC) curves were the highest for total metanephrines, normetanephrine and metanephrine determinations. Because of analytical interferences in the metanephrines determination, the normetanephrine and metanephrine performed better. It is noteworthy that all pheochromocytomas had either normetanephrine or metanephrine levels above their respective optimal threshold (sensitivity 100%). The best optimal threshold performance was reached by the mean of three daily samples. Total or fractionated catecholamines or vanillylmandelic acid were less accurate tools., Conclusion: Amongst urinary tests, the combined use of HPLC/ED determination of normetanephrine and metanephrine seems the most effective screening strategy for the diagnosis of pheochromocytoma. The older total metanephrine photometric assay is grieved by analytical interferences.

Background: The detection of pheochromocytomas in patients at risk for these tumors, such as patients with von Hippel-Lindau disease or multiple endocrine neoplasia type 2 (MEN-2), is hindered by the inadequate sensitivity of commonly available biochemical tests. In this study we evaluated measurements of plasma normetanephrine and metanephrine for detecting pheochromocytomas in patients with von Hippel-Lindau disease or MEN-2., Methods: We studied 26 patients with von Hippel-Lindau disease and 9 patients with MEN-2 who had histologically verified pheochromocytomas and 50 patients with von Hippel-Lindau disease or MEN-2 who had no radiologic evidence of pheochromocytoma. Von Hippel-Lindau disease and MEN-2 were diagnosed on the basis of germ-line mutations of the appropriate genes. The plasma concentrations of normetanephrine and metanephrine were compared with the plasma concentrations of catecholamines (norepinephrine and epinephrine) and urinary excretion of catecholamines, metanephrines, and vanillylmandelic acid., Results: The sensitivity of measurements of plasma normetanephrine and metanephrine for the detection of tumors was 97 percent, whereas the other biochemical tests had a sensitivity of only 47 to 74 percent. All patients with MEN-2 had high plasma concentrations of metanephrine, whereas the patients with von Hippel-Lindau disease had almost exclusively high plasma concentrations of only normetanephrine. One patient with von Hippel-Lindau disease had a normal plasma normetanephrine concentration; this patient had a very small adrenal tumor (<1 cm). The high sensitivity of measurements of plasma normetanephrine and metanephrine was accompanied by a high level of specificity (96 percent)., Conclusions: Measurements of plasma normetanephrine and metanephrine are useful in screening for pheochromocytomas in patients with a familial predisposition to these tumors.

Urinary catecholamines (CAs) and their metabolites are usually measured during the process of diagnosing pheochromocytoma (pheo), but a 24-hour urine collection is not convenient for outpatients. Since 1987 we have utilized "spot" urine metanephrine (MN) and normetanephrine (NMN) assays for management of patients with pheo or adrenal incidentaloma. MN and NMN were measured by radioimmunoassay in 82 patients with surgically proved pheo and 15 patients with incidentaloma. In 10 patients with pheo, MN and NMN were measured with fractional every-3-hour urine samples, which were accumulated and then measured as a 24-hour urinary specimen. Fractions of 3-hour MN and NMN excretion were constant (MN 98.5 +/- 9.6%, NMN 97.6 +/- 10.8%; 24-hour MN and NMN 100%). The average levels of MN and NMN in patients with pheo were 6801 ng/mg creatinine (Cr) (range 93-88,248, median 1426) and 5627 ng/mg Cr (range 219-31,528, mean 3190), whereas the MN and NMN levels in patients with incidentaloma were 123 ng/mg Cr (range 36-246, mean 133) and 251 ng/mg Cr (range 84-472, mean 220), respectively. When we selected a cutoff value for MN + NMN of 1000 ng/mg Cr, the sensitivity was 97.6% and the specificity 100% for diagnosing pheo. When the standard was set as > 500 ng/mg Cr for either MN or NMN, both the sensitivity and specificity were 100%. The assay for MN and NMN is simple and effective, not only for screening but for diagnosing pheo and managing incidentaloma.

Determination of urinary 3-O-methylated catecholamines (metanephrines) is generally considered a principal test for the clinical chemical diagnosis of pheochromocytoma and is currently performed predominantly with chromatographic techniques such as gas-liquid chromatography and HPLC. Enzyme immunoassays based on microtiter plate technology have recently been developed for the quantitative determination of urinary metanephrine (M) and normetanephrine (NM). We compared the results for urinary M and NM determined by these ELISA methods with those obtained by a recently developed isotope dilution mass spectrometric method. From this comparative study we can conclude that the investigated ELISA methods are applicable in the quantification of urinary M and thus can be successfully used to establish the diagnosis of pheochromocytoma. These relatively simple methods can be executed in any clinical laboratory and in time may replace the present, more complicated, chromatographic techniques.

Autonomic nervous system dysfunction is a common complication of severe intracranial disease. The aim of this study was to reveal the autonomic changes in patients suffering from acute intracerebral haemorrhage (ICH). 25 patients with spontaneous ICH within 24 hours of onset of symptoms were included. All patients were treated with standardised medical management and the meta- and normetanephrines were detected by high performance liquid chromatography (HPLC) in 24-hour urine every day. The mean level of normetanephrine (709 +/- 579 micrograms/day) and metanephrine (244 +/- 161 mg/day) were significantly elevated in comparison with a control group, p < or = 0.01. The norepinephrine elevation was of greater diagnostic and prognostic importance. Maximum urinary catecholamine metabolite levels occurred between day 3 to 10 after the bleeding. Normetanephrines correlated with the prognosis and the complications of ICH: intraventricular involvement resulted in significantly elevated normetanephrine levels (896 +/- 520 micrograms/day versus 311 +/- 78 micrograms/day) p < or = 0.01. Patients with a great volume of haematoma developed severe autonomic dysregulation (normetanephrines 1114 +/- 493 micrograms/day), whereas patients with smaller haematoma did not (339 +/- 125 micrograms/day) p < or = 0.0001; patients with bad outcome (1014 +/- 620 mg/day) had higher levels of normetanephrines than those with a good prognosis (322 +/- 110 micrograms/day) p < or = 0.001. A close relationship to elevated intracranial pressure was established. This study demonstrated the feasibility of detecting autonomic nervous system dysfunction in neurological intensive care patients by means of examination of the metabolites of the catecholamines in the urine.(ABSTRACT TRUNCATED AT 250 WORDS)

We describe a procedure for the direct measurement of metanephrine (MN) and normetanephrine (NMN) in hydrolyzed urine, using HPLC with coulometric array detection. Acid-hydrolyzed samples were diluted and filtered before separation by isocratic reversed-phase ion-pair chromatography. Eight serial coulometric sensors, set at incrementally increasing anodic potentials, were used to screen lower-oxidizing interferences and provide stepwise oxidation of the metanephrines. Voltammetric behavior across three adjacent sensors was used to assess resolution and aid in peak identification. Values obtained in commercial controls were consistently within the specified target range. Variability, expressed as CV, was 5.45-9.22% between runs and 1.60-4.52% within-run for both compounds. The limit of detection was 2.6 micrograms/L for MN and 2.8 micrograms/L for NMN, with a linear response to 15.0 mg/L for both analytes. Results from patients' samples correlated well with those by a method involving dual ion-exchange extraction (r = 0.963, n = 82 for MN; r = 0.9768, n = 83 for NMN). This procedure provided high selectivity and objective peak purity information while greatly simplifying sample preparation.

We developed a method for simultaneous quantification of the urinary 3-O-methylated catecholamine metabolites 3-methoxytyramine, normetanephrine and metanephrine by stable isotope-dilution ammonia chemical ionization mass fragmentography. Prepurification of lyophilized samples was done by simultaneous deconjugation and pentafluoropropionylation, followed by extraction and rederivatization. Compared with our previously described method, based on acid hydrolysis, alkaline extraction, derivatization and electron-impact mass fragmentography, the present method was found to be less laborious, more sensitive and presumably more accurate. New urinary excretion values were established for apparently healthy adults. The present prepurification method may prove applicable for profiling of a variety of naturally occurring mono-, di- and polyamines in biological samples.

This report describes a high-performance liquid chromatographic method with electrochemical detection for the simultaneous quantitation of urinary metanephrine, normetanephrine and 3-methoxytyramine. This method, which involves manual dual-column purification steps for the routine determination of urinary metanephrines, is compared with the previously used spectrophotometric Pisano method and an on-line sample preparation procedure, where the automated sequential trace enrichment (ASTED) apparatus is used for the column-switching procedure. In order to automate the metanephrine assay, the enrichment technique was evaluated against the reference chromatographic method. Bio-Rad urine controls gave coefficients of variation of less than 9% at all levels for the reference method. Values of less than 19% were found in the reference range with the enrichment method, and the recovery of 3-methoxytyramine was also too poor to be measured in normal concentrations. The linearity of both methods is sufficient to determine pathological levels of these biogenic amines. Future developments should be focused on decreasing the variation of between-day assays in an on-line, automated procedure.

The urinary excretion of normetanephrine, metanephrine, and 3-methoxy-4-hydroxymandelic acid was quantified by HPLC in hypertensive patients who were being routinely investigated for the exclusion of pheochromocytoma. The data were used to calculate reference intervals for these analytes. Age- and sex-specific 95% reference intervals for 24-h urinary excretion were as follows (mumol/24 h): normetanephrine in men ages 16-35 years (n = 17) 0.7-3.4, in men older than 35 years (n = 121) 0.8-5.1, in women ages 16-35 years (n = 41) 0.5-2.1, and in women older than 35 years (n = 144) 0.6-3.3 mumol/24 h; metanephrine in men (n = 138) 0.3-2.0, and in women (n = 185) 0.2-1.3; and 3-methoxy-4-hydroxymandelic acid in men (n = 142) 10-54, and in women (n = 184) 9-38. Normetanephrine or metanephrine excretion rates or both exceeded the proposed reference intervals in seven of the eight patients with pheochromocytoma. Determination of 3-methoxy-4-hydroxymandelic acid excretion did not yield any additional diagnostic information.

Assays of urinary catecholamines and their metabolites (HVA, VMA, dopamine) permit biochemical diagnosis of neuroblastoma in approximately 80% of patients. The urinary methylated catecholamine metabolites normetanephrine (NMN), metanephrine (MN), and 3-methoxytyramine (3-MT) were analyzed in 18 patients with neuroblastoma and compared with reference values established for 69 healthy pediatric controls. All 18 neuroblastoma patients had raised urinary excretion of at least one of the three commonly assayed metabolites (HVA, VMA, dopamine). Similarly, raised urinary excretion of a methylated metabolite was noted in all but one of the neuroblastoma patients. The 3-MT level was pathologic in 16 of the 18 patients (89%). In this series, 3-MT assay sensitivity was sufficient to warrant trials on a larger population including comparison with patients considered nonsecretors by routine assay procedures.

Urinary methoxyamine determination is considered as the most sensitive and specific parameter for the diagnosis of pheochromocytoma. Since blood sampling is easier to perform, we developed a new HPLC method to assay metanephrine (MN) and normetanephrine (NMN) in plasma. We now report the results for total (free and conjugated) MN and NMN in 22 cases of pheochromocytoma compared to 26 healthy subjects, 33 patients with essential hypertension, 14 with miscellaneous diseases and 4 patients with renal failure. The mean normal values (mean +/- SD) were 0.40 +/- 0.10 ng/ml for MN and 0.85 +/- 0.25 ng/ml for NMN. The sum of MN+NMN was 1.25 +/- 0.28 and the range 0.9-1.9. In essential hypertension, the range of NMN+MN was 1.2-6.0. In the 4 renal failures, both MN and NMN were drastically increased. In 49 samples drawn from 22 pheochromocytomas, MN was elevated over the hypertensive range in 34 samples and NMN in 47 samples. The total MN+NMN ranged from 6.2 to 436 ng/ml; this figure was observed whatever the clinical presentation even in silent tumors or in paroxysmal forms between the crisis. After tumor removal, the values dropped rapidly. In conclusion, plasma determination of MN and NMN provides a highly sensitive and specific biological pointer for the diagnosis of pheochromocytoma in patients without renal failure.

Results of 1414 measurements of urinary excretion of free and conjugated (total) metanephrine and normetanephrine (total metanephrines, tMN) and of 1630 measurements of vanilmandelic acid (VMA) excretion were analysed according to the month of sample collection. These collections had been carried out as a part of a study of hypertension at out institute over the 5-year period between 1970 and 1975. The means and scatters of tMN were significantly varied depending on the season of the year; the mean during 3 months of summer was 380 mug/day and the mean for 3 winter months 800 mug/day. The scatter of tMN in winter months was wider. A minor seasonal variation was observed for VMA: the means of summer and winter months were 3.8 mg/day and 4.5 mg/day, respectively. The results suggest a seasonal difference of catecholamine catabolism.

Hydrolyzed urine with added ring-trideuterated normetanephrine and metanephrine is applied to wet C18-reversed-phase minicolumns. The "metanephrines" are eluted, dried, derivatized with pentafluoropropionic anhydride, and analyzed with the gas chromatograph-mass spectrometer. Ions for the nondeuterated and trideuterated compounds are monitored at ml z 458 and 461, respectively. For both normetanephrine and metanephrine, the standard curve is linear over the range 10-2000 micrograms/L and the procedure has adequate precision both within-run (CV less than 3%) and between-day (CV less than 7%). Alkaline pH in the extraction is important for optimal analytical recovery. We have examined the potential value of untimed urine specimens for screening purposes and compared 24-h urine concentrations of these analyses in normotensive and hypertensive persons.

A sensitive and specific direct analysis of urinary normetanephrine (NMN) and metanephrine (MN) was achieved utilizing reversed-phase high performance liquid chromatography and electrochemical detection. Individual specimens from "control" subjects and those with pheochromocytoma were hydrolyzed and the metanephrines separated from other urinary constituents by elution with ammonia from a Dowex CG-50 resin. Chromatographic peaks were identified by retention behavior, co-chromatography with reference compounds, ratio of responses at various oxidation potentials and stopped-flow UV spectra of the collected fractions. The NMN and MN content for the control subjects was between 0.086 and 0.21 (mean - 0.14) microgram/mg creatinine and 0.012 and 0.092 (mean = 0.039) microgram/mg creatinine, respectively. The values for subjects with pheochromocytoma varied from 1.5 to 27.5 (mean = 9.9) microgram/mg creatinine for NMN and 0.10 to 1.60 (mean = 0.86) microgram/mg creatine for MN. The patient with ganglioneuroma had an NMN of 4.1 and an MN of 0.80 microgram/mg creatinine. While this method permits discrimination between those patients with pheochromocytoma and the overwhelming majority of hypertensive patients, it may ultimately be further extended to separate normal subjects from those with more subtle derangements in catecholamine metabolism.

A simple method for the simultaneous analysis of normetanephrine, metanephrine and 3-methoxytyramine (both free and conjugated) in human urine by reversed-phase ion-pair high-performance liquid chromatography with electrochemical detection has been developed. Existing methods have been optimized for extraction by study of analytical parameters. The hydrolysed urines are purified and concentrated by successive passages on two ion-exchange resins and ammoniacal elution to eliminate interference from pigments or related chemical compounds. The methoxyamines are separated by high-performance liquid chromatography on a reversed-phase column. Detection and quantitation are achieved with an electrochemical detector using a vitreous carbon electrode. Samples can be injected at 25-min intervals. Reference values of adults and children are given.