Plasma Free Metanephrines Research Articles

Localization of an Adrenocorticotropin-Producing Pheochromocytoma Using 18F-Dihydroxyphenylalanine Positron Emission Tomography

A 67-yr-old man was evaluated for a fulminating ACTHdependent Cushing’s syndrome. The pituitary magnetic resonance imaging scan was normal. Computed tomography revealed a 2-cm left adrenal mass. Somatostatin receptor scintigraphy with In-pentreotide showed no abnormal uptake, but 6-fluoro-[F]L-dihydroxyphenylalanine positron emission tomography showed uptake in the left adrenal mass, highly suggestive of pheochromocytoma (Fig. 1). Plasma-free fractioned metanephrine and urine total fractioned metanephrine (free plus conjugated) were elevated (416.2 ng/liter and 979 g per 24 h, reference values 13.8 – 65.0 and 64.1–301.8). The patient wascured by adrenalectomy (Fig. 2). Histology of the tumor revealed a pheochromocytoma and hyperplasia of the surrounding adrenal cortex. Hydrocortisonesubstitutionwasnecessary for 5 months after surgery; afterward physiological cortisol secretion was restored. Despite extensive evaluation, including computed tomography, magnetic resonance imaging, and somatostatin receptor scintigraphy, in 12–19% of patients with ectopic ACTH

Plasma free metanephrines are superior to urine and plasma catecholamines and urine catecholamine metabolites for the investigation of phaeochromocytoma

To compare the relative diagnostic efficacy of several different tests used to establish a diagnosis of phaeochromocytoma, in patients with a proven diagnosis of phaeochromocytoma, and in hospital patients with significant disease of other types. We prospectively compared biochemical markers of catecholamine output and metabolism in plasma and urine in 22 patients with histologically proven phaeochromocytoma, 15 intensive care unit (ICU) patients, 30 patients on chronic haemodialysis and both hypertensive (n = 10) and normotensive (n = 16) controls. Receiver operating characteristic curves were plotted. At the point of maximum efficiency, plasma free metanephrines showed 100% sensitivity and 97.6% specificity, compared with plasma catecholamines (78.6% and 70.7%), urine catecholamines (78.6% and 87.8%), urine metanephrines (85.7% and 95.1%), and urine hydroxymethoxymandelic acid (HMMA or VMA) (93.0% and 75.8%). All patients with phaeochromocytoma had plasma free metanephrine concentrations at least 27% above the upper limit of the reference range. Only three other patients (two on haemodialysis and one in ICU) had PFM concentrations more than 50% above the upper limit of the reference range. In patients with phaeochromocytoma, plasma free metanephrines displayed superior diagnostic sensitivity and specificity compared with other biochemical markers of catecholamine output and metabolism.

Hormonal and metabolic evaluation of adrenal incidentalomas

The biochemical and hormonal data in patients with adrenal incidentalomas were evaluated to compare the differences between adrenal adenomas and other benign lesions and to find the relationship between metabolic parameters and adrenal hormones. Ninety two patients (29men, age 20-90 years) with incidentally discovered unilateral or bilateral adrenal masses detected on CT were included in this study for the reasons others than adrenal pathology. Glycemia, cholesterolemia, triglyceridemia, hormonal evaluation including plasma ACTH, plasma aldosterone, plasma renin acitivity, overnight dexametasone test, ACTH test, free plasma metanephrines, urinary catecholamines were determined. In the group of patients with adrenal masses the prevalence of arterial hypertension was three fold higher, the prevalence of DM was approximately five fold higher and the prevalence of the overweight and obesity two fold higher than is reported in the general population. The most frequent adrenal masses were nonfunctional masses, the occurence of functional lesions was as follows: steroid enzymopathies (an exaggerated response of 17-OHP indicating a possible 21-hydroxylase deficiency), subclinical Cushing syndrome, primary aldosteronism and pheochromocytoma (5%, 2%, 2% and 1% respectively). There were no significant differences in evaluated data between patients with adenomas and hyperplasia and also no significant difference in evaluated data between lesions smaller than 3 cm and lesions greater than 3 cm. We did not find any correlations between plasma cortisol and lipid values. In this study we confirmed a higher prevalence of symptoms characteristic for different metabolic syndromes in these patients with adrenal incidentalomas, which indicate systematic screening for the metabolic syndrome including evaluation of the insuline resistance in this patients.

Proficiency test of plasma free and total metanephrines: report from a study group

Proficiency test of plasma free and total metanephrines: report from a study group

Das Phäochromozytom – noch immer eine Herausforderung

Pheochromocytomas are rare, mostly benign catecholamine-producing tumors arising from the chromaffin cells of the adrenal medulla or in the paraganglia. Clinical presentation is highly variable but typically with hypertension, severe headaches, palpitations and sweating. Biochemical testing by 24 h urinary fractioned metanephrines or catecholamines and plasma free metanephrines as the most sensitive screening approach, confirms the catecholamine excess. Computed tomography scan and magnetic resonance imaging of the adrenal glands and abdomen as well as functional imaging with (123)Iod-MIBG scintigraphy and (18)F-dopa positron emission tomography are used for tumor localization. Because approximately a quarter of tumors develop secondary to germ-line mutations, screening for genetic alterations is important. The therapy of choice is the endoscopic adrenal sparing surgery following preoperative alpha-blockade. Regular follow-up remains essential due to possible recurrence and malignancy.

Cyclic Rapid Fluctuation of Hypertension and Hypotension in Pheochromocytoma

Cyclic Rapid Fluctuation of Hypertension and Hypotension in Pheochromocytoma

Isopropanol Protein Precipitation for the Analysis of Plasma Free Metanephrines by Liquid Chromatography–Tandem Mass Spectrometry

High-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS)1 analysis of plasma free metanephrines is the most diagnostically sensitive and specific screening test for the diagnosis of pheochromocytoma. We sought to develop an in-house method for this expensive test We used off-line isopropanol protein precipitation of plasma to remove interfering substances before LC-MS/MS analysis. We compared the extraction efficiency and limits of quantification of protein precipitation to those of previously reported solid-phase techniques. The new method had limits of quantification of 0.09 nmol/L and 0.17 nmol/L for metanephrine and normetanephrine, respectively. Method comparison with a previously described solid-phase extraction method revealed Deming regression slopes of 0.904 and 0.994, intercepts of 0.007 and 0.023, and SEs of the residuals (S(y/x)) of 0.071 and 0.284 for metanephrine and normetanephrine, respectively. Extraction efficiency of isopropanol protein precipitation was 66% for metanephrine and 35% for normetanephrine, results that were superior to the efficiencies of 4% and 1% for our adapted solid-phase extraction method. No ion suppression was observed at the retention times for metanephrine and normetanephrine. Isopropanol protein precipitation is a novel and effective off-line sample preparation method for metanephrines that offers a less expensive alternative to on-line solid-phase extraction for low-volume testing and requires a sample volume of only 200 microL. The mass spectrometric analysis time is equivalent to that of solid-phase techniques.

Simultaneous Determination of Free Polyamines, Catecholamines and Metanephrines in Plasma and Urine

We describe a new, rapid, and sensitive HPLC method with fluorescent detection for simultaneous analysis of plasma and urine free catecholamines, metanephrines, and polyamines without prior treatment procedures via precolumn derivatization with Fmoc-Cl. The separation gradient was 40 minutes. The results showed good linearity across the calibration range 0.05–20 µM and linearity coefficient (r 2) higher than 0.990, except for epinephrine, r 2 = 0.985. The LOQ was in range 680–1300 fmol and LOD 204–390 fmol of these analytes per injection 20 microlitres. The reproducibility of the method for the analyzed samples, expressed as R.S.D. was from 1.2 to 4.5%. Isoproterenol was used as internal standard.

Comparison of free plasma metanephrines enzyme immunoassay with 131I-MIBG scan in diagnosis of pheochromocytoma

Measurement of free plasma metanephrines (metanephrine and normetanephrine), usually performed by high-performance liquid chromatography with electrochemical detection (HPLC-ECD), has been recommended as the single biochemical test of choice for the diagnosis of pheochromocytoma. Alternatively, a widely available, simple means to measure these biomarkers with enzyme immunoassay (EIA) needs to be studied. The aim of this study was to investigate the diagnostic efficacy of such a method in comparison with (131)I-metaiodobenzylguanidine (MIBG) whole body scan (WBS) in patients with pheochromocytoma. We enrolled patients undergoing (131)I-MIBG WBS due to clinical findings suggestive of pheochromocytoma (n = 45), and patients with primary hypertension (n = 36). All subjects had blood tests for free plasma metanephrine (MN) and normetanephrine (NM) with a commercially available EIA kit. WBS was positive in 30 pheochromocytoma patients and negative in 15 refuted ones, with 100% accuracy. The sensitivity, specificity and accuracy of MN and NM in combination (either or both positive) were 96.7%, 86.3% and 90.1%, showing comparable diagnostic performance both to (131)I-MIBG WBS (all p > 0.1), and also to the same markers measured with HPLC-ECD reported in the literature. These results showed that the EIA method may be eligible as an alternative to HPLC-ECD for plasma metanephrine determination in the identification of pheochromocytoma.

Current Progress and Future Challenges in the Biochemical Diagnosis and Treatment of Pheochromocytomas and Paragangliomas

Findings from five independent studies - with close to 350 patients with pheochromocytoma and more than 2,500 in whom the tumor was excluded - indicate that measurements of plasma free metanephrines provide an overall diagnostic sensitivity of 98% and specificity of 92%. The recommendation that initial testing for the tumor should always include measurements of either plasma or urinary fractionated metanephrines results from recognition of the high diagnostic sensitivity of measurements of plasma metanephrines. The few patients with pheochromocytoma in whom the test may not yield a positive result include those with very small tumors or microscopic disease and others with tumors that do not produce norepinephrine and epinephrine. Such patients are typically normotensive and do not exhibit symptoms of catecholamine excess. Additional measurements of methoxytyramine can be useful for detecting those tumors that produce only dopamine. Suboptimal diagnostic specificity and difficulties in distinguishing true- from false-positive elevations of plasma metanephrines remain challenges for diagnosis. Improvements in analytical technology (e.g., liquid chromatography with tandem mass spectrometry) and new strategies for follow-up testing provide possible solutions to these problems. The single most important remaining clinical care challenge is the development of effective cures for patients with malignant disease. Current treatments, none of which are truly satisfactory, include chemotherapy and radiopharmaceutical therapy with (131)I-labelled M-iodobenzylguanidine or radioactive somatostatin analogues. Improvements in treatment may in the future come from several fronts, but proof of efficacy ideally will require well-coordinated multicenter prospective trials in larger numbers of patients than in previous studies.

High-Throughput, Automated, and Accurate Biochemical Screening for Pheochromocytoma: Are We There Yet?

Biochemical testing for pheochromocytoma is a diagnostic challenge due both to the highly variable nature of this rare catecholamine-producing tumor and the technical demands required for accurate and reliable laboratory tests of catecholamine excess. In this issue of Clinical Chemistry , de Jong et al. (1) describe a high-throughput automated liquid–chromatography-tandem mass spectrometry (LC-MS/MS) method enabling simultaneous extraction, concentration, separation, and mass-selective detection of plasma free normetanephrine and metanephrine, the respective O-methylated metabolites of norepinephrine, and epinephrine. This method for measurements of plasma free metanephrines (Note: the term “metanephrines” refers to both normetanephrine and metanephrine) also allows quantification of methoxytyramine, the O-methylated derivative of dopamine and a metabolite normally present in plasma at lower concentrations (<0.1 nmol/L) than free normetanephrine (<0.6 nmol/L) or metanephrine (<0.3 nmol/L). Recognition that catecholamines are metabolized to free metanephrines within pheochromocytoma tumor cells, and that this process is independent of catecholamine release, provides a rationale for use of these metabolites in the diagnosis of pheochromocytoma (2). The diagnostic superiority of metanephrines over catecholamines has led to the recommendation that initial testing for pheochromocytoma should always include measurements of metanephrines in plasma or urine or both (3). Metanephrines in urine are usually measured after an acid hydrolysis step that converts the high concentrations of sulfate-conjugated metabolites into free metanephrines. In part because of this step, concentrations of metanephrines in urine are 2–3 orders of magnitude higher than those for the free metanephrines in plasma. These higher concentrations, along with the simpler matrix, make urinary metanephrines easier to measure than plasma free metanephrines. However, the enzyme responsible for sulfate conjugation is present mainly in gastrointestinal tissues (2). Thus, the free metanephrines produced within tumor cells should provide …

Plasma Free Metanephrine Measurement Using Automated Online Solid-Phase Extraction HPLC–Tandem Mass Spectrometry

Quantification of plasma free metanephrine (MN) and normetanephrine (NMN) is considered to be the most accurate test for the clinical chemical diagnosis of pheochromocytoma and follow-up of pheochromocytoma patients. Current methods involve laborious, time-consuming, offline sample preparation, coupled with relatively nonspecific detection. Our aim was to develop a rapid, sensitive, and highly selective automated method for plasma free MNs in the nanomole per liter range. We used online solid-phase extraction coupled with HPLC-tandem mass spectrometric detection (XLC-MS/MS). Fifty microliters plasma equivalent was prepurified by automated online solid-phase extraction, using weak cation exchange cartridges. Chromatographic separation of the analytes and deuterated analogs was achieved by hydrophilic interaction chromatography. Mass spectrometric detection was performed in the multiple reaction monitoring mode using a quadrupole tandem mass spectrometer in positive electrospray ionization mode. Total run-time including sample cleanup was 8 min. Intra- and interassay analytical variation (CV) varied from 2.0% to 4.7% and 1.6% to 13.5%, respectively, whereas biological intra- and interday variation ranged from 9.4% to 45.0% and 8.4% to 23.2%. Linearity in the 0 to 20 nmol/L calibration range was excellent (R(2) > 0.99). For all compounds, recoveries ranged from 74.5% to 99.6%, and detection limits were <0.10 nmol/L. Reference intervals for 120 healthy adults were 0.07 to 0.33 nmol/L (MN), 0.23 to 1.07 nmol/L (NMN), and <0.17 nmol/L (3-methoxytyramine). This automated high-throughput XLC-MS/MS method for the measurement of plasma free MNs is precise and linear, with short analysis time and low variable costs. The method is attractive for routine diagnosis of pheochromocytoma because of its high analytical sensitivity, the analytical power of MS/MS, and the high diagnostic accuracy of free MNs.

Comparison of Diagnostic Accuracy of Urinary Free Metanephrines, Vanillyl Mandelic Acid, and Catecholamines and Plasma Catecholamines for Diagnosis of Pheochromocytoma

Recent evidence suggests that plasma-free metanephrines provide a highly sensitive test in patients requiring exclusion of pheochromocytoma. The diagnostic efficacy of urinary free metanephrines, however, has not been evaluated. OBJECTIVE, DESIGN, SETTING, PATIENTS, AND OUTCOME MEASURES: We compared retrospectively the diagnostic efficacy of 24-h urinary free metanephrines with our currently available measurements of 24-h urinary vanillyl mandelic acid (VMA), urinary catecholamines, and plasma catecholamines in 159 outpatients tested in a tertiary referral center for pheochromocytoma over a 4-yr period. The sensitivity of urinary free metanephrines was 100% [25 of 25 patients; 95% confidence interval (CI) 86-100%)] compared with the sensitivity of 84% (21 of 25; 95% CI 64-95%) for urinary catecholamines; 72% (18 of 25; 95% CI 51-88%) for urinary VMA; and 76% (16 of 21; 95% CI 53-92%) for plasma catecholamines. The specificity of urinary free metanephrines was 94% (116 of 123; 95% CI 89-98%), compared with the specificity of 99% (127 of 129; 95% CI 96-100%) for urinary catecholamines; 96% (130 of 134; 95% CI 91-98%) for urinary VMA; and 88% (66 of 75; 95% CI 78-94%) for plasma catecholamines. Receiver operating characteristic curves for all test groups were generated. Pairwise comparisons of the area under the receiver operating characteristic curve for urinary free metanephrines with that of each of the other three test groups individually were: 0.993 (95% CI 0.962-0.999) vs. 0.919 (95% CI 0.862-0.957, P = 0.032) for urine catecholamines; 0.993 (95% CI 0.962-0.999) vs. 0.846 (95% CI 0.778-0.900, P = 0.002) for urine VMA; and 0.992 (95% CI 0.945-0.998) vs. 0.852 (95% CI 0.762-0.918, P = 0.009) for plasma catecholamines. Testing with urinary free metanephrines failed to misidentify a single case of pheochromocytoma, compared with four missed cases for urinary catecholamines, seven missed cases for urinary VMA, and five missed cases for plasma catecholamines. Urinary free metanephrines were superior to urinary VMA, urinary catecholamines, and plasma catecholamines and can provide a valuable test for diagnosis of pheochromocytoma in adults.

Free plasma metanephrines as a screening test for pheochromocytoma in low-risk patients

Detection of free plasma metanephrines seems to be the most exact method for biochemical diagnosis of pheochromocytoma, but their diagnostic efficacy in the common low-risk clinical settings is debated. A cross-sectional multicentre study including 1260 subjects assessed the diagnostic efficacy of free plasma metanephrine and normetanephrine in low-risk patients screened for resistant or markedly accelerated hypertension, paroxysmal hypertension, 'flushes' and, in a small proportion, for adrenal incidentaloma or genetic predisposition to pheochromocytoma. Pheochromocytoma was identified and verified by histology in 25 subjects (2%), with the diagnosis not confirmed by long-term follow-up or use of imaging techniques in the remaining 1235 individuals. The combined assay of free plasma metanephrines was a highly sensitive (100%) and specific (96.7%) measure, yielding a negative predictive value of 100%. The satisfactory diagnostic efficacy of free plasma metanephrines allows their use as a single screening test in cases of suspected pheochromocytoma in the population with a low pretest probability.

Diagnostic Management of Benign and Malignant Pheochromocytoma

As rare and thus often overlooked hormone-secreting tumors, pheochromocytomas pose a particular diagnostic challenge. Difficulties involve biochemical confirmation, localizing, and detection of malignancy. Measurement of free plasma metanephrines, genetic testing and specific imaging procedures--such as MIBG and octreotide scintigraphy or fluorodopamine PET--represent a considerable progress, and the management of benign pheochromocytomas has become very effective. However, a comparable improvement in the prognosis of malignant chromaffin cell tumors, which occur in approximately 10-15% of all cases, has not yet been achieved. Here, telomerase catalytic subunit (hTERT) activity and heat shock protein 90 expression could serve both as molecular markers allowing an earlier diagnosis of malignancy and as therapeutic targets. Familial syndromes should be considered both in benign and malignant pheochromocytoma, and should be tested for prior to surgery in selected patient groups.

Is Supine Rest Necessary before Blood Sampling for Plasma Metanephrines?

The impact of blood sampling in sitting vs supine positions on measurements of plasma metanephrines for diagnosis of pheochromocytoma is unknown. We compared plasma concentrations of free metanephrines in samples from patients with primary hypertension obtained after supine rest with those obtained in the sitting position without preceding rest. We also assessed the effects on diagnostic test performance retrospectively in patients with and without pheochromocytoma, and we calculated cost-effectiveness for pheochromocytoma testing. Upper reference limits of plasma free metanephrines were higher in samples obtained from seated patients without preceding rest than from supine patients with preceding rest. Application of these higher upper reference limits to samples from supine patients with pheochromocytoma decreased the diagnostic sensitivity from 99% to 96%. In patients without pheochromocytoma, adjusting the plasma concentration for the effects of sitting while preserving the 99% sensitivity by use of the supine upper reference limits increased the number of false-positive test results from 9% to 25%. To preserve high diagnostic sensitivity we recommend the use of upper reference limits determined from blood samples collected in the supine position. Under these conditions, negative test results for blood samples obtained with patients sitting are as effective for ruling out pheochromocytoma as negative results from samples obtained after supine rest. Repeat testing with samples obtained in the supine position offers a cost-effective approach for dealing with the increased numbers of false-positive results expected after initial sampling in the sitting position.

Multicenter Study on the Diagnostic Value of a New RIA for the Detection of Free Plasma Metanephrines in the Work‐Up for Pheochromocytoma

Available laboratory test methods for the detection of elevated concentrations of catecholamines and their metabolites in urine and/or plasma are not always sensitive enough for the detection of pheochromocytoma. High-quality immunoassays for these compounds appear to be as accurate as high-pressure liquid chromatography (HPLC) or gas chromatography/mass spectrometry (GC-MS). Therefore, the current project aims to establish a new sensitive radioimmunoassay (RIA) for the measurement of free metanephrines in the plasma of patients in the work-up for pheochromocytoma. We report first results of an ongoing multicenter clinico-chemical evaluation study in hypertensive patients and normotensive volunteers. After an overnight fast plasma samples were collected on ice in EDTA- and heparin-coated tubes after insertion of an indwelling venous line and resting in the supine (patients) or sitting position (normal volunteers) for 30 min. Plasma metanephrines were measured by a newly developed RIA from IBL, Hamburg, Germany. Good agreement of the assay with the tandem mass spectrometry (LC-MS/MS) method for normetanephrine (r2=0.975) and for metanephrine (r2=0.985) could be demonstrated. Both specimens, EDTA and heparin plasma, can be used with the same results. The RIA has a good precision of <15% in the normal range and of <10% in the elevated concentration range. Our preliminary data suggest a high validity of the newly developed RIA for measuring free metanephrine and normetanephrine in hypertensive subjects in both EDTA and heparin plasma. Further work is required to determine the accuracy of the test in larger patient populations and in patients with pheochromocytoma.

Pheochromocytoma Catecholamine Phenotypes and Prediction of Tumor Size and Location by Use of Plasma Free Metanephrines

Pheochromocytoma Catecholamine Phenotypes and Prediction of Tumor Size and Location by Use of Plasma Free Metanephrines

Diagnosis and Management of Tumors of the Adrenal Medulla

The adrenal medulla consists of chromaffin cells, the site of catecholamine biosynthesis. Pheochromocytomas are chromaffin-cell tumors; 80-85 % arise from the adrenal medulla and 15-20 % arise from extra-adrenal chromaffin tissues (paragangliomas). Neuroblastomas are primitive tumors that derive from the same blastic precursor as in pheochromocytomas, and are distributed along the sympathetic nervous system. Pheochromocytomas account for 6.5 % of incidentally discovered adrenal tumors; they are found in 50 % of patients with multiple endocrine neoplasia 2A (MEN 2A) and 5-25 % of patients with von Hippel-Lindau (VHL) syndrome. Neuroblastomas are the most common solid extra-cranial tumors in children, and account for 7-10 % of all tumors. The diagnosis of pheochromocytoma should first be established biochemically by measuring plasma free metanephrines (the measurement of urinary fractionated metanephrines is the second choice). Measurements of homovanillic acid (HVA), norepinephrine and vanilmandelic acid (VMA) in urine are a necessity in patients with suspected neuroblastoma. Anatomical (radiological) imaging with computed tomography (CT) or magnetic resonance imaging (MRI) is necessary for both pheochromocytomas and neuroblastomas. Functional (nuclear medicine) methods are useful for both tumors. Scintigraphy with [123I]-metaiodobenzylguanidine is the specific functional imaging test of first choice; if this is not available, scintigraphy with [131I]-MIBG is the second choice. Other newer specific modalities that have been used for evaluating pheochromocytomas include positron emission tomography (PET) with [18F]-F-fluorodopamine (F-DA) and [18F]-F-dihydroxyphenylalanine (DOPA). These should be used when MIBG scintigraphy is negative. Primary treatment for both types of tumor is surgical; chemotherapy is used for inoperable disease. After successful surgery, survival of patients with benign, sporadic pheochromocytomas is believed to be equal to that of the general population. Depending on the extent of disease and age, patients with neuroblastomas have cure rates of 15-90 %.

Circulating EM66 is a highly sensitive marker for the diagnosis and follow‐up of pheochromocytoma

We have previously demonstrated that measurement of tissue concentration of the novel secretogranin II-derived peptide EM66 may help to discriminate between benign and malignant pheochromocytomas. The aim of the present study was to characterize EM66 in plasma and urine of healthy volunteers and pheochromocytoma patients, in order to further evaluate the usefulness of this peptide as a circulating marker for the management of the tumors. HPLC analysis of plasma and urine samples demonstrated that the EM66-immunoreactive material coeluted with the recombinant peptide. In healthy volunteers, plasma and urinary EM66 levels were, respectively, 2.6 (1.9-3.7) ng/ml and 2.9 (1.9-4.6) ng/ml. In patients with pheochromocytoma, plasma EM66 levels were 10-fold higher than those of healthy volunteers (26.9 (7.3-44) ng/ml), and returned to normal values after removal of the tumor. In contrast, urinary EM66 levels were not significantly different from those of healthy volunteers (3.2 (2.2-3.9) ng/ml). Measurement of total or free plasma metanephrines and 24 hr urinary metanephrines in our series of patients revealed that these tests, taken separately, are less sensitive than the EM66 determination. Pheochromocytes in primary culture secreted high levels of EM66, suggesting that the chromaffin tumor was actually responsible for the increased plasma peptide concentrations in the patients. These data indicate that EM66 is secreted in the general circulation and that elevated plasma EM66 levels are correlated with the occurrence of pheochromocytoma. Thus, EM66 is a sensitive plasma marker that should be considered as a complementary tool in the management of pheochromocytoma.